ES2988598T3 - Therapeutic protein - Google Patents

Abstract

A deflamin polypeptide composition for use in a method of treating the human or animal body by therapy, wherein the therapy preferably consists of preventing or treating inflammation or cancer, or providing a nutraceutical. (Automatic translation with Google Translate, no legal value)

Description

DESCRIPTION

Therapeutic protein

Field of invention

The invention relates to methods of extracting deflamin, deflamin polypeptide compositions, nucleic acid vectors and associated products.

Background of the invention

Over the past few decades, there has been extensive research into developing new anticancer drugs, both prophylactic and therapeutic. However, despite significant advances in diagnosis, detection, and treatment, the overall long-term outcome in patients has not changed significantly over the past few decades. Against this background, there has been an intense search into various biological sources to develop new anticancer drugs to combat this disease, which can be used in prevention, chemotherapy support, or in preventing recurrence. In addition, there are currently no prescription drugs that specifically target chronic inflammation (there are, of course, over-the-counter drugs that treat minor, temporary inflammation and accompanying pain caused by injuries or procedures, such as surgery. However, these are not intended to treat chronic inflammation). Some drugs, such as hydroxychloroquine, used to combat malaria, are useful in treating some patients with lupus, but they do not cure the disease. Aspirin and statins have shown promise in reducing inflammation in some people, but researchers are unsure how widely useful such drugs are in that role. With the exception of anti-inflammatory drugs, which are far from perfect, such as prednisone, a corticosteroid that carries a host of side effects, scientists are still researching how best to contain inflammation. Known are EP2333086A1, WO2006003110A1, WO2009144278A2 and W02012049215A1. EP2333086A1 provides for the use of a Lupinus polypeptide as a plant growth promoter, fertilizer, or human or animal nutritional supplement. W02006003110A1 describes a process of grinding and transforming Lupinus albus into flakes. WO2009144278A2 describes an enriched type of conglutin protein for use as a medicament, food supplement or dietary supplement. W02012049215 discloses an antimicrobial polypeptide comprising Blad or an active variant thereof for use in a method of treating the human or animal body by therapy or prophylaxis, such as for use in a method of treating or preventing an infection in a subject, caused by a microorganism.

Summary of the invention

The invention is defined in the claims.

The inventors have discovered 'deflamin', a new method for its extraction and the related composition, which comprises new polypeptides that have anticancer and anti-inflammatory properties.

Deflamin can be considered in one embodiment as a mixture of storage protein fragments, present in many (but not all) seeds (p- and 8-conglutins, in the case of plants of the genus Lupinus), typically purified by a specific procedure and exhibiting a number of unique biological/bioactive properties, namely anti-inflammatory and anticancer activities, as well as other biological activities derived therefrom.

Deflamin can be obtained from many seeds, such as lupin and other species. As described herein, a specific methodology was developed to extract and purify deflamin from seeds (lupin and others) that is suitable for scale-up, allowing mass production in industrial facilities. The invention also includes recombinant production of deflamin.

The invention includes the preventive and curative use of deflamin in all diseases that develop as a direct or indirect result (i.e. inflammation produced by a given treatment) of inflammation and/or that involve the activity of matrix metalloproteinases (MMP).

Definition of deflamina

Deflamin may be defined by its origin, bioactivities, how it is produced, and in some cases structurally. Deflamin is present in the seeds of many, but not all, species. Deflamin produced or used in the invention may exhibit one or more of the physical or therapeutic properties mentioned herein. Such properties include one or more bioactivities as measured in any of the assays (including animal models) described herein and physical properties as measured by techniques based on electrophoresis, HPLC, and mass spectrometry assays described herein. Deflamin may comprise naturally occurring sequence(s) or related (homologous or rearranged) artificial sequence(s).

Properties of deflamina

Deflamin is in the form of a mixture of soluble small polypeptides/proteins. It typically possesses one or more of the following characteristics: a) it is easily edible (non-toxic to humans); b) it is found in seeds; c) it is soluble in water; d) it is composed of one or any combination of a mixture of low molecular mass small polypeptides/proteins; e) its bioactivities are resistant to boiling, to a wide range of pH values, to ethanol and/or to digestive proteases (i.e. it resists the digestive process); f) it strongly inhibits matrix metalloproteinase (M<m>P)-9 and/or MMP-2, i.e. it is an MMP inhibitor (MMPI) at low concentrations; g) it reduces the migratory capacity of the human colon adenocarcinoma cell line HT29 without inducing significant cytotoxicity; h) it exhibits at least the following important bioactivities: (i) potent anti-inflammatory; (ii) potent antitumor (antimigratory and antimetastatic); (iii) no significant cytotoxicity.

When administered orally, deflamin does not trigger significant immunogenic (i.e. IgG) or allergic (i.e. IgE) responses. Furthermore, it is bioactive at low concentrations.

In one embodiment, polypeptides comprising deflamin have been identified as fragments of B-conglutin and large chain 5-conglutin (e.g., in the case of deflamin from Lupinus seeds). See SEQ ID NO: 192 and SEQ ID NO: 193.

As described in detail below, deflamin displays bioactivity in animal models when administered orally, intraperitoneally, intravenously or topically. In particular, deflamin exhibits the following properties: a) anti-inflammatory activity, as measured in animal models (i.e., mice) when administered by any of the following routes: oral, intraperitoneal, intravenous and topical; b) antitumor activity, as studied by potent inhibition of matrix metalloproteinase activities (i.e., MMP-9 and MMP-2), antiproliferative activity of cancer cells and inhibition of tumor cell invasion.

Relationship with other vegetable proteins

Blad is a known bioactive plant polypeptide. Blad, as well as the oligomer containing Blad (BCO), comprise fragments of p-conglutin. However, they are not related to deflamin, which typically comprises other fragments of B-conglutin and/or large chain fragments of 5-conglutin.

Functionally, both are very different, with deflamin being completely devoid of antimicrobial activity (as regards bacteria and fungi), whereas the oligomer containing Blad does not inhibit gelatinases. Blad corresponds to a fixed fragment of p-conglutin, i.e. Blad comprises residues 109 to 281 of the p-conglutin precursor (i.e. pro-p-conglutin). In addition to the large 5-conglutin chain, deflamin typically corresponds to other B-conglutin fragments, e.g. spanning the entire polypeptide.

Activity against matrix metalloproteinase (MMP) and cancers

Deflamin is a novel type of MMP-9 and/or MMP-2 inhibitor discovered in seeds of Lupinus albus and also present in other seeds such as Cicer arietinum and Glycine max. It is generally established that patient death in certain cancers, for example colorectal cancer patients, is usually caused by metastatic disease rather than the primary tumour itself. Metastasis involves the release of cancer cells from the primary tumour and their attachment to other tissues or organs. Cancer cell invasion is therefore a key element in metastasis and requires integrins for adhesion/deadhesion and matrix metalloproteinases (MMPs) for targeted proteolysis.

Targeted proteolysis is necessary to open the pathway in the extracellular matrix for cancer cells to travel. Wound healing assays provide an estimate of the capacity of cells for cell invasion. Typically, MMP-9 activities are highly correlated with cancer cell invasion, therefore, reduction in MMP-9 activity inhibits cell invasion and the two activities are often paired. This is why MMP-9 inhibition is so desired, because it directly blocks/limits cell invasion, thus inhibiting metastatic death.

On the other hand, cells adhere to a substrate through specific proteins called integrins, transmembrane receptors that are the bridges for cell-cell and cell-extracellular matrix (ECM) interactions. An important function of integrins in cells in tissue culture is their role in cell migration. Integrins are modulated by tumor progression and metastasis and are closely related to both MMP-9 and MMP-2 activities. Targeting and deactivation of integrins on cancer cell membranes may also be desirable because when cells are released from the tumor, they lack the ability to adhere to other tissue. This will also inhibit metastasis and may even help disaggregate the primary tumor.

Another goal for many studies is specific cytotoxicity against cancer cells. Many bioactive compounds, such as phenolic compounds, strongly reduce cell growth and impair metabolism, reaching toxic levels at moderately high doses. Measuring cell growth and cell metabolism in the presence of a bioactive compound is a direct measure of its toxicity to the cell. The MTT assay uses a specific dye that must be taken up by living cells and then metabolized by them accordingly.

In brief, the MTT assay is a colorimetric assay that measures the reduction of soluble, yellow MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] by mitochondrial succinate dehydrogenase. MTT enters cells and passes into the mitochondria where it is reduced to an insoluble, colored (deep purple) product [(E,Z)-5-(4,5-dimethylthiazol-2-yl)-1,3-diphenylformazan]. The cells are then solubilized with an organic solvent (e.g., isopropanol) and the released solubilized formazan reagent is quantified spectrophotometrically. Since MTT reduction can only occur in metabolically active cells, the level of activity is a measure of cell viability. Therefore, if the cell is dead or metabolically impaired, it will not produce the coloring agent. Higher levels of color are therefore indicative of a greater number of living and metabolically active cells. If a compound reduces cell growth or kills cells, there will be a lower level of color.

Targeting and killing cancer cells is, in theory, a good approach. However, it can only work if there is a high specificity towards cancer cells and not towards normal, healthy (i.e., non-cancerous) cells. Most compounds that kill cancer cells will also kill normal, healthy cells at a given dose, and while many studies focus only on the ability of a metabolite (e.g., phenolic compounds) to reduce cancer cell growth, they often fail to consider their effects on control, healthy cells. One reason this is quite common relates to the fact that, unlike normal, healthy cells, it is relatively easy to grow cancer cells in laboratory conditions. Consequently, downstream, at the level of preclinical or even clinical trials, the use of these compounds is often hampered by dose-limiting toxicity, insufficient clinical benefits, and extremely adverse side effects. Therefore, a bioactive agent that reduces cell invasion but does not affect normal cell metabolism (as is the case with deflamin) will produce fewer (or even negligible) side effects, and will be safer to use in long-term preventive administrations, because it will not exert cytotoxicity towards regular cells.

Certain embodiments of the invention contemplate curative and/or preventative methods and/or approaches. For example, deflamin may be administered to healthy individuals to prevent disease. Certain embodiments of the invention contemplate specific routes of administration including one or more of the following: oral, anal, injected, and topical.

Properties of other MMP inhibitors

Other plant-derived MMP inhibitors present one or more of the following disadvantages: a) toxicity; b) chemical inactivation (e.g. denaturation) or degradation/destruction (e.g. proteolysis) during the digestive process; c) absorption into the bloodstream, with or without triggering immunogenic (i.e. IgG) or allergenic (i.e. IgE) responses; d) destruction during boiling (e.g. during cooking); e) they do not possess specificity towards gelatinases; f) they require the use of high doses; g) they do not have a suitable, effective and low-cost isolation procedure.

This largely explains why there is not yet a single plant-derived biological compound that has found successful application in the fields of human health and nutrition at the level of MMP inhibition.

Conglutins

Typically, deflamin polypeptides have sequences that are identical to conglutin fragments, or have strong homologies to conglutin fragments. In one embodiment such conglutin sequences are from specific conglutins mentioned herein or from naturally occurring homologs of those specific conglutins.

In lupins, conglutins have been classified into four families: a-, p-, y-, and 6-conglutin. P-conglutin, the major seed globulin in lupins, is vicilin or the 7S member of the seed storage proteins, whereas a-conglutin is legumin or the 11S member of the seed storage proteins. In narrow-leaf lupins (Lupinus angustifolius), a total of three a-conglutin-encoding genes, seven p-conglutin-encoding genes, two Y-conglutin-encoding genes, and four 6-conglutin-encoding genes have been previously identified. These genes have been named alpha-conglutin 1, 2, and 3, beta-conglutin 1, 2, 3, 4, 5, 6, and 7, gamma-conglutin 1 and 2, and delta-conglutin 1, 2, 3, and 4, respectively.

5-Conglutin belongs to the 2S sulfur-rich albumin family. Lupinus 2S seed albumin, also called 5-conglutin, is a monomeric protein comprising two small polypeptide chains linked by two interchain disulfide bridges: a smaller polypeptide chain consisting of 37 amino acid residues resulting in a molecular mass of 4.4 kDa, and a larger polypeptide chain containing 75 amino acid residues with a molecular mass of 8.0 kDa. The unique amino acid sequence of 5-conglutin from L. albus has been inferred from the gene sequence. The larger polypeptide chain contains two intrachain disulfide bridges and one free sulfhydryl group. This protein exhibits some unique features among L. albus proteins: in addition to its high cysteine content, it exhibits a low absorbance at 280 nm.

Concerning the physiological role of 5-conglutin, a storage function has been proposed for this class of proteins. The structural similarity to the family of plant cereal inhibitors, which includes the bifunctional trypsin/alpha-amylase inhibitors, may suggest a defensive function for this protein in addition to its storage function. Its presence in L. albus seeds was evaluated to be approximately 10 to 12%, but more recent data suggest a lower content of approximately 3 to 4%. Albumin from Lupinus seeds is typically present in both the albumin fraction and the globulin fraction.

Inflammation

Deflamin can be used to prevent or treat inflammation. Inflammation is the body's immediate response to damage to its tissues and cells caused by pathogens, noxious stimuli such as chemicals, or physical injury. Acute inflammation is a short-term response that usually results in healing: leukocytes infiltrate the damaged region, remove the stimulus, and repair the tissue. In contrast, chronic inflammation is a prolonged, dysregulated, maladaptive response involving active inflammation, tissue destruction, and attempts at tissue repair. Deflamin can be used to prevent or treat acute or chronic inflammation.

Deflamina can be used to prevent or treat inflammatory processes of the skin or mucosa, such as dermatitis, melanoma, periodontitis and gum inflammation. It can be used to treat generalized and chronic digestive inflammations.

It is now widely accepted that chronic inflammation plays a role in a host of common and often fatal diseases, including a) inflammatory bowel disease (IBD), heart disease, stroke, cancer, chronic respiratory diseases, neurological diseases, obesity and diabetes; b) atherosclerosis, arthritis, diabetes, human immunodeficiency virus-mediated acquired immunodeficiency syndrome (AIDS), asthma, neoplasia, degenerative and cardiovascular diseases; c) allergies and autoimmune diseases; d) obesity and metabolic diseases; e) Alzheimer's and other neurodegenerative diseases; f) depression.

Deflamin can be used to prevent or treat any of these conditions.

Cancer

Cancer is a term for diseases in which abnormal cells divide without control and can invade nearby tissues in the same body. Cancer cells can also spread to other parts of the body through the blood and lymph systems. There are several main types of cancer: a) carcinoma is a cancer that begins in the skin or in the tissues that line or cover internal organs; b) sarcoma is a cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supporting tissue; c) leukemia is a cancer that begins in blood-forming tissue, such as bone marrow, and causes large numbers of abnormal blood cells to be made and enter the blood; d) lymphoma and multiple myeloma are cancers that begin in cells of the immune system; e) central nervous system cancers are cancers that begin in tissues of the brain and spinal cord; f) Melanoma is a disease in which malignant (cancer) cells form in melanocytes (cells that give color to the skin).

Cancer-related conditions: ductal carcinoma in situ, male breast cancer, breast cancer, pancreatic cancer, exocrine pancreatic cancer, prostate cancer, colon cancer, rectal cancer, colorectal cancer, cervical cancer, melanoma of the skin, carcinoma, basal cell carcinoma, skin cancer, squamous cell carcinoma, testicular cancer, thyroid cancer, ovarian cancer, ovarian germ cell tumor, lung cancer, bladder cancer, esophageal cancer, stomach cancer, uterine cancer, endometrial cancer, hepatocellular carcinoma, liver cancer, oropharyngeal cancer, hypopharyngeal cancer, laryngeal cancer, nasopharyngeal cancer, pharyngeal cancer, oral cavity cancer, brain tumors, lymphoma, Hodgkin's lymphoma, acute myeloid leukemia, kidney cancer, renal cell cancer, non-Hodgkin's lymphoma, non-small cell lung cancer, urethral cancer, lung cancer small cell, osteosarcoma, sarcoma, carcinoid tumor, carcinoid syndrome, chronic lymphocytic leukemia, Wilms tumor, retinoblastoma, pituitary tumors, hairy cell leukemia, penile cancer, leukemia, vaginal cancer, Ewing sarcoma, Kaposi sarcoma, malignant fibrous histiocytoma, Paget's disease of the nipple, gallbladder cancer, acute lymphoblastic leukemia, lymphoma of the eye, adrenocortical carcinoma, adenocarcinoma, parathyroid cancer, pancreatic neuroendocrine tumors, gastrinoma, Merkel cell carcinoma, salivary gland cancer, vulvar cancer, gastrointestinal stromal tumor, anal cancer.

The invention provides deflamin for preventing or treating any of the above-mentioned types of cancer or specific cancers. Deflamin is effective during the early stages of tumor genesis, inhibiting the formation of metastases, as part of chemotherapy therapy and preventing cancer recurrence after surgery.

Deflamina production methods

Deflamin for use in the therapeutic aspects of the invention may be obtained by any suitable method, such as any method described herein. It is preferably produced by recombinant expression or by extraction from plant material. Deflamin may be obtained from any plant material expressing deflamin or deflamin precursors, typically seeds, such as mature seeds, for example, of any plant genus or species mentioned herein.

From plant material such as seeds

Typically, deflamin is obtained by a method that follows a sequential precipitation scheme. The method can be based on the resistance of deflamin to high temperatures, low pH and high ethanol concentrations. If flour is used as a starting point for the method, it can be obtained by grinding a suitable seed.Method 1

In one embodiment the method comprises extracting deflamine from suitable seeds, comprising:

- at least one high temperature stage, preferably at least 80 °C or boiling; and

- at least one low pH stage, preferably pH 4 or lower;

- at least one stage of contacting the extract with high concentrations of ethanol, preferably at least 70% (v/v) or at least 90% (v/v) ethanol.

Method 2

In another embodiment the method comprises the following steps:

(a) boiling the intact seeds in water, followed by extraction in water or buffer, and removal of fat, or reducing the intact seeds to flour, extraction in water or buffer followed by removal of fat and boiling, or removal of fat from flour followed by extraction in water or buffer and boiling;

(b) the soluble fraction is exposed to a pH value low enough (e.g., pH 4.0 or lower) to allow precipitation of most of the remaining proteins/polypeptides;

(c) The precipitated fraction is resuspended in approximately 40% (v/v) ethanol, with the solution also optionally containing 0.4 M NaCl. The supernatant contains deflamin;

Optionally, the following steps can also be performed:

(d) the soluble fraction is brought to 90% (v/v) ethanol to precipitate deflamin and stored at 20 °C, or the precipitation of deflamin can be achieved by other means, such as lyophilization;

(e) the precipitated deflamine may be cleaned of any contaminants by repeating steps (c) and (d); (f) the precipitated deflamine may then be dissolved, for example, in water, and desalted by any suitable technique to remove low molecular weight contaminants and/or stored frozen (liquid or dry) until required.

Method 3

In one embodiment the method comprises the following steps:

(a) providing a suitable seed meal; (b) defatting said meal; (c) boiling for a period the remaining sample from said defatting step; (d) centrifuging said sample for a given period; (e) thereafter discarding the resulting pellet and further processing the resulting supernatant to precipitate polypeptides while lowering its pH; (f) centrifuging further to obtain an additional pellet; and discarding said supernatant; and (g) further processing said additional pellet with ethanol and centrifuging to obtain a pellet which is then discarded; the remaining supernatant comprising deflavin.

Any of steps (a) through (g) may be replaced with the more specific equivalent steps listed for Methods 4 and 5.

Method 4

In one embodiment the method comprises the following steps:

(a) The flour is defatted. It may be defatted with an organic solvent and then suspended in water (typically 1:10 to 1:50 w/v; pH typically adjusted to pH 8.0-8.5), under agitation, typically for 1 to 6 hours, e.g. at room temperature, or the fat is simply skimmed off the top of the suspension after suspending the flour in water (typically 1:10 to 1:50 w/v; pH typically adjusted to pH 8.0-8.5), under agitation typically for 1 to 6 hours at room temperature. As an alternative to water, typically on a laboratory scale, the flour may be suspended in Tris-HCl buffer typically 30 to 70 mM, at pH 7.5.

(b) After shaking for at least 2 to 6 hours, for example about 4 hours or overnight, typically at 4 °C, the soluble proteins are obtained by centrifugation, at 10,000 to 20,000 g, such as 13,500 g for 10 to 90 min, for example for 30 min, at 4 degrees Celsius. The pellet is discarded.

(c) The protein solution is boiled, typically at 100 °C for 5 to 20 min, such as 10 min, and centrifuged at 10,0000 or 20,000 g, e.g. at 13,500 g, typically for 20 min at 4 degrees Celsius. The pellet is discarded. The supernatant is collected and provides the heat-treated (HT) extract. (d) Polypeptides/proteins in the supernatant are precipitated by addition of dilute HCl to approximately pH 4.0. Following centrifugation at 10,000 to 20,000 g, e.g. at 13,500 g, typically for 20 min at 4 degrees Celsius, the supernatant is discarded.

(e) The pellet is resuspended in 30 to 50%, e.g. 40% (v/v) ethanol containing 0.2 to 0.6 M NaCl, e.g. 0.4 M NaCl, shaken, typically for 1 h at room temperature, and centrifuged at 10,000 or 20,000 g, e.g. 13,500 g, for 30 min at 4 degrees Celsius. This treatment brings the deflamin into solution, unlike the vast majority of seed storage proteins. The pellet is discarded.

(f) the supernatant is made up to 80 or 95%, e.g. 90% (v/v) ethanol and stored at less than -10 degrees Celsius, e.g. -20 degrees Celsius, for at least 4 hours, e.g. overnight, thus precipitating the deflamin, followed by centrifugation at 10,000 or 20,000 g, e.g. 13,500 g, typically for 30 min at 4 degrees Celsius. The supernatant is discarded.

Optionally, the differential ethanol precipitation from 40 to 90% (v/v) should be repeated. Thus, the following additional steps can be performed:

(g) The deflamine present in the pellet is redissolved in 40% (v/v) ethanol containing 0.4 M NaCl, shaken for 1 h at room temperature and centrifuged at 13,500 g for 30 min at 4 °C. This second wash cleans the deflamine of any final contaminants. The pellet is again discarded. (h) The supernatant is again brought to 90% (v/v) ethanol and stored at -20 degrees Celsius overnight, to precipitate the deflamine, followed by centrifugation at 13,500 g, for 30 min at 4 degrees Celsius. The supernatant is discarded.

(i) The final pellet contains pure deflamin, which is subsequently dissolved in the smallest possible volume of Milli-Q water. The deflamin solution is finally desalted in water on Sephadex G-25 columns (e.g. NAP-10 columns, GE Healthcare Life Sciences) to remove low molecular mass contaminants.

(j) The obtained extract can optionally be stored at -20 degrees Celsius.

Method 5

In one embodiment, deflamine is obtained using the following method:

(a) flour from a seed is

- degreased with n-hexane and then resuspended in water (1:20 w/v; pH adjusted to pH 8.0-8.5), with stirring for 2 to 3 h at room temperature, or

- the fat is simply skimmed off from the top of the suspension after the flour has been suspended in water (1:20 w/v; pH adjusted to pH 8.0-8.5), under stirring for 2 to 3 h at room temperature - as an alternative to water, typically on a laboratory scale, the flour can be suspended in 50 mM Tris-HCl buffer, pH 7.5. Stirring is for 4 h (or overnight) at 4 degrees Celsius

(b) Soluble proteins are obtained by centrifugation, at 13,500 g for 30 min, at 4 degrees Celsius. The sediment is discarded.

(c) The protein solution is boiled at 100 degrees Celsius for 10 min, and centrifuged at 13,500 g, for 20 min at 4 degrees Celsius. The pellet is discarded. The supernatant is collected and provides the heat-treated (HT) extract.

(d) Polypeptides/proteins in the supernatant are precipitated by addition of dilute HCl to approximately pH 4.0. After centrifugation at 13,500 g, for 20 min at 4 degrees Celsius, the supernatant is discarded.

(e) The pellet is resuspended in 40% (v/v) ethanol containing 0.4 M NaCl, shaken for 1 h at room temperature, and centrifuged at 13,500 g for 30 min at 4 degrees Celsius. This treatment brings deflamin into solution, unlike the vast majority of seed storage proteins. The pellet is discarded.

(f) The supernatant is made up to 90% (v/v) ethanol and stored at -20°C, for at least 4 hours, e.g. overnight, to precipitate the deflamine, followed by centrifugation at 10,000 to 20,000 g, e.g. 13,500 g, typically for 30 min at 10°C. The supernatant is discarded.

Optionally, the differential precipitation from 40 to 90% (v/v) ethanol should be repeated. Therefore, the following additional steps can be carried out:

(g) The deflamin present in the pellet is dissolved again in 40% (v/v) ethanol containing 0.4 M NaCl, shaken for 1 h at room temperature and centrifuged at 13,500 g for 30 min at 4 degrees Celsius. This second wash cleans the deflamin of final contaminants. The pellet is discarded again.

(h) The supernatant is once again made up to 90% (v/v) ethanol and stored at -20 degrees Celsius overnight, precipitating deflamine, followed by centrifugation at 13,500g for 30 min at 4 degrees Celsius. The supernatant is discarded.

(i) The final pellet contains pure deflamin, which is subsequently dissolved in as small a volume of Milli-Q water as possible. On a laboratory scale, the deflamin solution is finally desalted in water on Sephadex G-25 columns (e.g. NAP-10 columns, GE Healthcare Life Sciences) to remove low molecular mass contaminants.

(j) The obtained extract is optionally stored at -20 degrees Celsius.

The flour used in any of the above methods is optionally obtained by grinding a seed, such as grinding approximately 100 g ± 0.1 g of dry seed (typically without embryo and tegument) to obtain flour.Method 6

Methods for producing recombinant proteins are well known in the art. Such methods as applied in the present context involve inserting the polynucleotide encoding a deflamin polypeptide into a suitable expression vector - allowing juxtaposition of said polynucleotide with one or more promoters (e.g., an inducible promoter, such as T7lac) and with other polynucleotides or genes of interest - introducing the expression vector into a suitable cell or organism (e.g., Escherichia coli), expressing the polypeptide in the transformed cell or organism, and removing the expressed recombinant polypeptide from that cell or organism. To aid in such purification, the expression vector may be constructed such that the polynucleotide additionally encodes, for example, a terminal tag that may aid in purification: for example, a histidine residue tag for affinity purification. Once the recombinant polypeptide is purified, the purification tag may be removed from the polypeptide, for example, by limited proteolytic cleavage.

Method 7

Simpler, cheaper and faster methodologies are possible, leading to a fairly pure deflamin, but not as pure as that achieved with the procedures described above. These methodologies will necessarily involve extraction, boiling, exposure to low pH values and treatment with ethanol.

Physical structure of deflamin

Deflamin is a composition comprising a mixture of polypeptides obtainable from seed storage proteins by the method according to any one of claims 1 to 3. It typically comprises a mixture of at least 2 to 200 different polypeptides, such as 20 to 150, 30 to 100 or 50 to 80 different polypeptides having one or more of the following characteristics:

(a) have a length of 5 to 250 amino acid residues, such as 5 to 200, 50 to 200, 75 to 150, or 100 to 180 or 120 to 170 amino acid residues, and/or;

(b) comprise or consist of a sequence that is a portion and/or sequence homolog of a conglutin, such as any conglutin mentioned herein or a portion represented by any of SEQ ID NOs: 8 to 190, and/or;

(c) each of them comprises a sequence that is:

(i) a portion of a conglutin protein, wherein said portion is at least 5, 10, 20, 30 or 50 amino acid residues in length, and/or;

(ii) a homologue of the portion defined in (i), preferably having at least 70% identity with said portion, wherein said conglutin protein is optionally a beta 1, 2, 3, 4, 5, 6 or 7 conglutin protein or a delta 2 conglutin protein; and/or;

(d) at least 20, 30 or 50 of the polypeptides comprise a sequence that is a rearrangement derived from a conglutin.

Deflamin polypeptides may exhibit microheterogeneity. Thus, in the case of lupins, deflamin polypeptides correspond to conglutin sequences that are mostly overlapping, but which have variable lengths.

Deflamin polypeptides with a rearranged sequence typically comprise sequence portions from different conglutins or from different parts of the same conglutin molecule; or homologs of such portions. Such portions (including homologs of portions) may be at least 5, 10, 20, 30 or 50 amino acid residues in length. Portions that are derived from different parts of the same conglutin may be separated by at least 10, 20, 50 or 200 amino acids from the original conglutin in which they are found. A deflamin polypeptide with a rearranged sequence may comprise at least 2, 3, 4, 5 or 6 different portions of the conglutin sequence, which are from different conglutin molecules and/or from different parts of the same conglutin molecule. Such portions may be the same as, be portions of, and/or be homologous to any specific sequence mentioned herein, including any of SEQ ID NOs: 8 to 190.

In one embodiment the deflamin composition comprises at least 10%, 20%, 30%, 50% and/or 80%, or all of the sequence of SEQ ID NO: 8 to 55 and/or 56 to 75 and/or 76 to 190, as a part of all polypeptides that are present; or homologs of any of these specific sequences, or any other sequence specified herein.

Deflamin typically comprises a mixture of at least 2 to 200 different polypeptides, such as 20 to 150, 30 to 100, or 50 to 80 different polypeptides, each of which comprises a sequence that is:

(a) the same as any of SEQ ID NOs: 8 to 190, or is a portion of any of SEQ ID NOs: 8 to 190 that is at least 5, 10, 20, 30, or 50 amino acid residues in length, and/or

(b) a homologue of the sequence defined in (a), which preferably has at least 70% homology with (a).

In one embodiment the deflamin composition does not comprise any polypeptides other than those defined in this section, or said other polypeptides represent less than 30%, such as less than 10% of the total mass of the polypeptides in the composition.

When groups of polypeptides defined as SEQ ID NOs are referred to herein, Category I polypeptides (SEQ ID NOs: 8 to 55) are most preferred, followed by Category II (SEQ ID NOs: 56 to 75), followed by Category III (SEQ ID NOs: 76 to 190).

In one embodiment the deflamin composition comprises in the form of sequences within all of its polypeptides, sequence portions of a conglutin (such as any conglutin mentioned herein) "span" the conglutin. Typically, there are at least 3, 4, 5 or 6 portions where at least 1, 2 or 3 of the portions occur in the first half and the second half of the conglutin polypeptide (where the N-terminus of the conglutin represents the start of the molecule). Preferably in this situation at least one portion is from each of the first, second and third parts of the conglutin polypeptide if the polypeptide is assumed to be divided into sections of three equal lengths.

In certain embodiments the deflamin comprises polypeptides derived from 6- and 6- conglutins, for example, at least 1, 2, 3, 5 or 10 peptides from β- and 8- conglutins (preferably L. albus 13- and 8- conglutins). The deflamin may comprise 2 groups of such peptides corresponding to molecular masses of 13 kDa and 17 kDa. Their lengths may be at least, or in a range defined by any two or more of the following: 100, 110, 120, 130, 140, 150 or 160 amino acid residues. In certain embodiments, the deflamin is composed of a mixture of polypeptides originating from two polypeptide peaks (13 and 17 kDa).

Deflamin: variants, homologs and portions

In any embodiment of deflamin described herein, one or more of the deflamin polypeptides may be replaced by "variants" and thus typically naturally occurring sequences may be replaced with homologous sequences or one or more portions of the naturally occurring sequence. Preferably, such variants are homologs and/or portions of the sequence shown by SEQ ID NOs: 8 to 190. Percent identity levels for such homologs are described below. Portions of the sequence will consist of at least 50, 80, or 90% of the original sequence, and may be at least 5, 10, 20, or 30 amino acid residues in length. The variant will preferably retain the activity of the original polypeptide/sequence, e.g., as measured using any assay or test described herein.

Homologous sequences typically exhibit at least 40% identity, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97%, and most preferably 99% identity, for example, over the entire sequence or over a region of at least 20, preferably at least 30, preferably at least 40, preferably at least 50, preferably at least 60, preferably at least 80, preferably at least 100, preferably at least 120, preferably at least 140, and most preferably 160 or more contiguous amino acid residues. Methods for measuring protein homology are well known in the art, and those skilled in the art will appreciate that, in the present context, homology is calculated on the basis of amino acid identity (sometimes referred to as "strict homology").

The homologous sequence typically differs from the parent sequence by substitution, insertion or deletion, for example by 1, 2, 3, 4, 5 to 8, 9, 15 or more substitutions, deletions or insertions. The substitutions are preferably "conservative", i.e. one amino acid can be substituted by a similar amino acid, whereby similar amino acids share one of the following groups (with respect to their side chain R): aromatic residues (F/H/W/Y), non-polar aliphatic residues (G/A/P/I/L/V), uncharged polar aliphatic residues (C/S/T/M/N/Q) and polar charged aliphatic residues (D/E/K/R). Preferred subgroups comprise: G/A/P; I/LN; C/S/T/M; N/Q; DEL; and K/R.

Homology (identity) can be measured using known and available methods. For example, the UWGCG package provides the BESTFIT program which can be used to calculate homology (e.g. used in its default settings) (Devereux et al. (1984) Nucleic Acids Res. 12, 387–395). The PILEUP and BLAST algorithms can be used to calculate homology or align sequences (typically in their default settings), for example as described in Altschul (1993) J. Mol. Evolution. 36, 290–300, and Altschul et al. (1990) J. Mol. Biol. 215, 403–410.

Software to perform BLAST analysis is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or satisfy some positive-valued threshold score T when aligned to a word of the same length in a database sequence. T is called the neighbor word score threshold (Altschul et al., supra). These initial neighbor word matches act as seeds to initiate searches to find HSPs containing them. Word matches are extended in both directions along each sequence as far as the cumulative alignment score can be increased. Extensions for word matches in each direction are stopped when: the cumulative alignment score drops by an amount X from its maximum attained value; the cumulative score reaches zero or less, due to the accumulation of one or more negative-scoring residue alignments; or when the end of either sequence is reached. The parameters W, T, and X of the BLAST algorithm determine the sensitivity and speed of the alignment. The BLAST program uses by default a wordlength (W) of 11, the BLOSU M62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915–10919) alignments (B) of 50, expectation (E) of 10, M5, N-4, and a comparison of both strands. The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see, for example, Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90, 5873–5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probability comparing the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

Deflamina forms

A composition comprising, consisting of, or essentially consisting of deflamin is typically in an isolated or purified form (e.g., extracted from a plant or cellular source). This typically comprises less than 50% or less than 20%, or 10%, or 5% dry mass of non-deflamin.

A deflamin composition may also be a formulation comprising other compound(s) added to the composition by the person skilled in the art. In preferred embodiments, such a formulation is a pharmaceutical formulation comprising deflamin and a pharmaceutically acceptable carrier or diluent. The person skilled in the art will be able to identify, by routine methods, a suitable concentration at which to use deflamin in any particular setting, for example, when administered in therapy. Preferably, for example, it is used in a concentration of at least 1 pg/mL, at least 5 pg/mL, at least 10 |jg/mL, at least 20 pg/mL, at least 50 pg/mL or at least 100 pg/mL, and up to 500 pg/mL, up to 600 pg/mL, up to 1 mg/mL, up to 2.5 mg/mL, up to 5 mg/mL or up to 10 mg/mL. Preferably the concentration is between 10 pg/mL and 5 mg/mL, more preferably between 50 pg/mL and 2.5 mg/mL, more preferably 100 pg/mL and 1 mg/mL, and still more preferably between 100 pg/mL and 600 pg/ml (such as about 250 pg/ml).

In one embodiment, the deflamin composition comprises less than 20%, less than 10%, or less than 1% by weight or is completely free of lunasin or Blad protein, for example, as defined by the specific sequences provided herein. In another embodiment, none of the deflamin polypeptides comprise any lunasin or Blad sequence, i.e., they do not comprise any portion of lunasin and/or Blad sequence.

Therapeutic uses

When used in therapy to prevent or treat a condition, deflamin is preferably used in a therapeutically effective amount. Preferably, the therapeutically effective amount is nontoxic to the human or animal subject.

The invention provides a deflamin polypeptide composition for use in a method of treating the human or animal body by therapy, wherein said therapy preferably prevents or treats inflammation or cancer. To this end, the invention also provides a method of treating a human or animal comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of an antimicrobial polypeptide comprising deflamin or containing deflamin in addition to antimicrobial polypeptide(s). The invention also provides the use of deflamin in the manufacture of a medicament for treating or preventing inflammation or cancer.

The individual polypeptides that make up deflamin can be administered separately and, accordingly, the invention provides a product comprising a multiplicity of different polypeptides that together form a deflamin composition for simultaneous, separate or sequential use in a method of treating the human or animal body by therapy, wherein said therapy preferably prevents or treats inflammation or cancer.

Deflamin may be administered by any suitable route, for example, intradermally, subcutaneously, intramuscularly, intravenously, intraosseously, intraperitoneally, topically, orally, or transmucosally (such as nasally, sublingually, vaginally, or rectally).

Deflamin is preferably administered together with carriers, diluents and auxiliary substances. Pharmaceutically acceptable carriers include, but are not limited to, liquids such as water, saline, polyethylene glycol, hyaluronic acid, glycerol and ethanol. Pharmaceutically acceptable salts may also be included therein, for example, salts of mineral acids such as hydrochlorides, hydrobromides, phosphates, sulfates and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates and the like. It is also preferred, although not necessary, that the preparation contain a pharmaceutically acceptable carrier which serves as a stabilizer. Examples of suitable carriers which also act as stabilizers for polypeptides include, but are not limited to, pharmaceutical grades of dextrose, sucrose, lactose, trehalose, mannitol, sorbitol, inositol, dextran and the like. Other suitable carriers include, again without limitation, starch, cellulose, sodium or calcium phosphates, citric acid, tartaric acid, glycine, high molecular weight polyethylene glycols (PEGs), and combinations thereof.

Once formulated, the composition can be administered to a subject in vivo using a variety of known routes and techniques. For example, liquid preparations can be provided as an injectable solution, suspension, or emulsion and administered by parenteral, subcutaneous, intradermal, intramuscular, intravenous, intraosseous, or intraperitoneal injection using a conventional needle and syringe, or using a liquid jet injection system. Liquid preparations can also be administered topically to the eyes, skin, hair, or mucosal tissue (e.g., nasal, sublingual, vaginal, or rectal), or provided as a finely divided aerosol suitable for respiratory or pulmonary administration. Other modes of administration include oral administration, suppositories, and active or passive transdermal delivery techniques.

The subject in need of therapy may be any human or animal individual. The subject is typically a chordate, a mammal, an agricultural animal, or a rodent. In preferred embodiments, deflamin may be used in the therapy of subjects at particular risk for inflammation or cancer.

Deflamin may be administered by the use of nucleic acid expression vectors that express deflamin in vivo. The invention provides one or more nucleic acid vectors that together or individually express a deflamin composition for use in a method of treating the human or animal body by therapy, wherein said therapy preferably prevents or treats inflammation or cancer. The nucleic acid vector may be a viral vector or any other type of vector that allows for the administration of the nucleic acid.

The invention also provides a product comprising a multiplicity of nucleic acid vectors co-expressing a deflamin composition for simultaneous, separate or sequential use in a method of treating the human or animal body by therapy, wherein said therapy is preferably to prevent or treat inflammation or cancer.

Antibodies

The invention provides one or more antibodies or fragments thereof that bind to any of the polypeptides corresponding to the sequences SEQ ID NO: 8 to 190 in a specific manner.

Brief description of the drawings

The invention is described below with reference to the accompanying drawings, in which:

Figure 1 shows the internal fragment of the coding sequence of the B-conglutin precursor that corresponds to Blad;

Figure 2 shows the secondary polypeptide structure of Blad;

Figure 3 shows the structure of the Blad tertiary polypeptide;

Figure 4 shows a schematic representation of the methodology used to extract and purify deflamin from Lupinus albus seeds;

Figure 5 shows a comparison between the albumin and globulin polypeptide profiles for each of the eight legume seeds initially analyzed;

Figure 6 is a graph showing the MMP-9 inhibitory activity of the eight legume seeds initially analyzed;

Figure 7 is a graph comparing a cell proliferation assay between the albumin and globulin fractions of each of the eight legume seeds initially analyzed;

Figure 8 shows images of a cell migration wound assay for three legume seeds: Lupinus albus, Cicer arietinum and Glycine max;

Figure 9 is a graph showing the results of the cell migration wound assay comparing the albumin and globulin fractions of each of the eight legume seeds initially analyzed (performed as shown in Figure 8);

Figure 10 is a graph showing gelatinolytic activity by comparing the albumin and globulin fractions of each of the eight legume seeds initially analyzed;

Figure 11 shows zymographic profiles of MMP-9 and MMP-2 activities comparing the albumin and globulin fractions of three legume seeds: Lupinus albus, Cicer arietinum and Glycine max; Figure 12 is a graph showing phytin concentration comparing cooked and raw fractions of six legume seeds analyzed;

Figure 13 is a graph showing saponin concentration comparing cooked and raw fractions of six legume seeds analyzed;

Figure 14 is a graph showing the concentration of phenolic compounds comparing the raw and cooked fractions of six legume seeds analyzed;

Figure 15 is a graph showing soluble protein concentration comparing raw and cooked fractions of six legume seeds analyzed;

Figure 16 shows polypeptide profiles obtained by R-SDS-PAGE comparing the cooked and raw fractions of six legume seeds analyzed;

Figure 17 shows images of cell migration assessed by a wound healing assay comparing various cooked and raw fractions of three legume seeds: Lupinus albus, Cicer. arietino and Glycine max;

Figure 18 is a graph showing the relative migration rates of the wound healing assay comparing various cooked and raw fractions of three legume seeds: Lupinus albus, Cicer arietinum and Glycine max (Figure 17);

Figure 19 is a graph showing cell proliferation comparing various cooked and raw fractions of three legume seeds: Lupinus albus, Cicer arietinum and Glycine max;

Figure 20 is a graph showing gelatinolytic activity comparing various cooked and raw fractions of three legume seeds: Lupinus albus, Cicer arietinum and Glycine max;

Figure 21 is a graph showing the inhibitory effect on MMP proteolytic activity by comparing various cooked and raw fractions of three legume seeds: Lupinus albus, Cicer arietinum and Glycine max;

Figure 22 is a graph showing protein peaks from a partially purified deflamin extract from L. albus seeds fractionated by FPLC gel filtration;

Figure 23 shows the separation of the polypeptide peaks of Figure 22, separated by Tricine SDS-PAGE; Figure 24 is a graph showing the MMP-9 inhibitory activity of each of the protein fractions of Figure 22;

Figure 25 is a graph showing HPLC reverse phase chromatography profiles of the MMP inhibitory fraction isolated from L. albus;

Figure 26 shows electrophoretic profiles under reducing conditions of the MMP inhibitory fractions isolated from L. albus (Figure 25);

Figure 27 is a graph showing the effects of different peak fractions, shown in Figure 25, on MMP-9 activity.

Figure 28 shows the polypeptide composition of L. albus peak 2 collected from the HPLC analysis depicted in Figure 25.

Figure 29 compares the percentage of wound closure for the L. albus sample from Figure 28 with various L. albus protein fractions;

Figure 30 shows images of the wound closure tests corresponding to Figure 29;

Figure 31 is a graph showing the gelatinolytic activity profile corresponding to Figure 29; Figure 32 is a graph showing the quantified activities of MMP-9 and MMP-2 corresponding to Figure 29;

Figure 33 shows the zymographic profiles of MMP-9, MMP-2 and their zymogen enzymatic activities in HT29 extracellular media after exposure of cells to deflamin for 48 h;

Figure 34 shows a representative image of the polypeptide distribution between Lupinus albus seeds simply extracted with buffer (extraction buffer; BE) or after heat treatment (Ht ), and visualized by SDS-PAGE (left) or reverse gelatin zymography (right);

Figure 35 shows representative images of the polypeptide profiles obtained after each step of the deflamin purification protocol as specified at the top of the gels;

Figure 36 is a graph showing the total gelatinolytic activity versus MMP-9 proteolytic activity in the presence of extracts collected at various stages throughout the deflamin purification protocol; Figure 37 is a graph showing the migration of HT29 cells in the presence of extracts collected at various stages throughout the deflamin purification protocol;

Figure 38 shows examples of cell migration now obtained from extracts collected at various stages throughout the deflamin purification protocol (Figure 37);

Figure 39 is a graph showing the effect of different concentrations of deflamin on gelatinolytic activity;

Figure 40 is a graph showing the effect of different concentrations of deflamin on cell migration (Figure 41);

Figure 41 shows examples of cell migration obtained for different concentrations of deflamin; Figure 42 shows the effects of different concentrations of deflamin on cell proliferation; Figure 43 shows a polypeptide profile of deflamin under reducing and non-reducing conditions. The molecular masses of the standards are indicated in kDa;

Figure 44 shows a representative image of the fractionation of deflamin into its constituent polypeptides monitored at 214 nm (reverse phase HPLC chromatography);

Figure 45 shows a representative image of the fractionation of deflamin into its constituent polypeptides monitored at 280 nm (reverse phase HPLC chromatography);

Figure 46 shows polypeptide profiles of each peak collected from the fractionation in Figures 44 and 45, as visualized by SDS-PAGE;

Figure 47 is a graph showing the proteolytic activity of MMP-9 in the presence of fractions 1 to 4 obtained by deflamin fractionation in Figures 44 and 45;

Figure 48 is a graph showing the effect of selected deflamin peaks (fractions 1 to 4 obtained by deflamin fractionation in Figures 44 and 45) on cell migration;

Figure 49 shows the electrophoretic profile of the deflamin fractions that are soluble and those that are precipitated with Ca and Mg after fractionation of deflamin from L. albus into two fractions by Ca2+ and Mg2+;

Figure 50 shows the inhibition of cell invasion in HT29 cells by deflamin and its two subfractions, precipitated or not with Ca and Mg, that is, it shows that the separation of deflamin into two fractions with Ca2+ and Mg2+ influences its antitumor activity;

Figure 51 shows the influence of deflamin from L. albus on the transcription of specific genes in HT29 cells related to inflammation and tumor invasion;

Figure 52 shows the bioactivity (at the level of inhibition of HT29 cell invasion) of deflamin from L. albus in food products, i.e. when used in the manufacture of cooked crackers; Figure 53 shows the analysis of deflamin from L. albus by HPLC and electrophoresis;

Figure 54 shows the mass spectrometric analysis of the two deflamin fragments from L. albus using MALDI-TOF;

Figure 55 shows preliminary results on the anticolitis effects of deflamin in mice with induced colitis;

Figure 56 is a graph showing the effect of various routes of administration of deflamin (and corresponding controls) on colon length of mice with induced colitis;

Figure 57 is a graph showing the effect of various routes of deflamin administration (and corresponding controls) on the degree of intestinal injury in mice with induced colitis;

Figure 58 shows macroscopic observations of colons isolated from the different treatment groups (deflamin and controls) of mice with induced colitis;

Figure 59 shows macroscopic observations of colons isolated from the different treatment groups (deflamin and controls) of mice with induced colitis;

Figure 60 shows the effect of deflamin administration on the histological features of colon inflammation in mice with induced colitis;

Figure 61 shows the effect of deflamin administration on the expression of COX-2 and iNOS in colon tissue in mice with induced colitis;

Figure 62 is a graph showing the effect of deflamin administration on the gelatinase activities of MMP-2 and MMP-9 colon tissue from mice with induced colitis;

Figure 63 shows zymographic profiles showing the effect of deflamin administration on the gelatinase activities of MMP-2 and MMP-9 colon tissue from mice with induced colitis.

Figure 64 is a graph showing the effect of deflamin administration on the development of edema in the rat paw;

Figure 65 is a graph showing the effect of topical administration of deflamin on paw edema in rats;

Figure 66 shows a reverse zymography of blood and feces from mice with induced colitis and treated with deflamin;

Figure 67 shows representative images of wound closure assays showing the cell antimigratory effect of deflamin (purified, cooked and uncooked seeds);

Figure 68 shows representative images of wound healing assays assessing cell migration in the presence of different concentrations of seed extract from Lupinus albus, Cicer arietinum and Glycine max;

Figure 69 shows an SDS-PAGE of deflamin isolated using the diagram depicted in Figure 4 from seeds of Lupinus albus, Glycine max and Cicer arietinum;

Figure 70 is a graph showing a comparison of the anti-gelatinase activity (MMP-9 and MMP-2) of deflamin from seeds of Lupinus albus, Glycine max and Cicer arietinum;

Figure 71 is a graph showing a comparison of the anti-invasion activity of different concentrations of deflamin from seeds of Lupinus albus, Glycine max and Cicer arietinum;

Figure 72 is a graph showing a comparison of cell growth in the presence of deflamin from seeds of Lupinus albus, Glycine max and Cicer arietinum.

Figure 73 shows a reverse zymography performed on a polyacrylamide gel containing gelatin and HT-29 medium with MMP-9 and MMP-2 to detect the presence of deflamin in seeds of other Lupinus species, other legume genera and other non-legume species, including cereals and others;

Figure 74 shows a reverse zymography performed on a polyacrylamide gel containing gelatin and HT-29 medium with MMP-9 and MMP-2 to detect the presence of deflamin in seeds of other species of the Lupinus genus;

Figure 75 shows a representative polypeptide profile of deflamin from Lupinus mutabilis by SDS-PAGE performed under reducing and non-reducing conditions;

Figure 76 shows the representative polypeptide profile of Vigna mungo deflamin by SDS-PAGE performed under reducing and non-reducing conditions;

Figure 77 shows a reverse zymography performed on a polyacrylamide gel containing gelatin and HT-29 medium with MMP-9 and MMP-2 to detect the presence of deflamin in seeds of species of the Triticum genus;

Figure 78 shows the representative SDS-PAGE polypeptide profile of deflamin isolated from various species of the Triticum genus.

Figure 79 shows a 2D IPG gel electrophoresis pH 3-6, 7 cm and SDS-PAGE 17.5% (w/v) acrylamide/bisacrylamide. IEF was performed at 4,000 V, current limit 50 pA/strip, 10,000 V-h. SDS-PAGE separation 65 min at 200 V.

Examples

Technical information

With the increasing incidence of inflammatory diseases, an inevitable increase in medical and pharmaceutical costs is occurring. It seems increasingly likely that the future of human health will depend on two basic areas: traditional clinical therapies for treatment and appropriate diets (including both nutraceuticals and functional foods) for both treatment and prevention. It has been predicted that the continued intake of functional foods and/or nutraceuticals that reduce inflammation will constitute the most effective human tool against most of the ailments that afflict modern societies today. MMP inhibitors (MMPIs) are considered anti-angiogenic agents for primary tumors and deterrents to metastasis, and have also been shown to effectively inhibit precancerous conditions such as colitis and other inflammatory bowel diseases. Over the past decade, a substantial amount of research has focused on the discovery of novel plant foods and compounds that exhibit MMPI activity, however, targeting individual MMPs in a specific manner has proven difficult.

Embodiments of the invention describe a new type of MMPIs that are proteinaceous in nature, survive the digestion process and can be administered orally, intraperitoneally, intravenously or applied topically, and that can be used as a functional food in the prevention/treatment of inflammation, tumorigenesis and cell proliferation, as well as any disease derived therefrom. These MMPIs have been shown to be potent inhibitors of matrix metalloproteinases MMP-9 and MMP-2, thus exhibiting potent anti-inflammatory, anti-tumor and anti-proliferative activities. Embodiments of the invention show deflamin as a useful nutraceutical or in the composition of functional foods in the prevention or treatment of a wide range of diseases.

Brief note on the discovery of deflamina

Embodiments of deflamin comprise novel types of MMP-9 and/or MMP-2 inhibitors extracted from seeds of Lupinus albus and also present in other seeds, both legumes (such as Cicer arietinum and Glycine max) and non-legumes. These findings led to the promising discovery of deflamin, a group of water-soluble polypeptides/small proteins isolated from the edible seeds of a commonly consumed legume species, Lupinus albus, which exhibits highly potent inhibitory activity toward MMP-9 and/or MMP-2 in cultured colon cancer cells and reduces colitis in animal models when administered orally, intraperitoneally, intravenously or topically, without exerting any apparent significant cytotoxicity. Deflamin was also found to be obtained from other seeds, both legumes (e.g., Cicer arietinum and Glycine max) and non-legumes. In the case of Lupinus albus, deflamin comprises in certain embodiments fragments of both large chain B-conglutin and 8-conglutin polypeptides. In addition to high stability at extremes of pH and temperature, preliminary evidence indicates that they also resist digestion, making these polypeptides/small proteins excellent candidates for development into valuable anti-inflammatory agents. These novel polypeptides/small proteins can be produced in certain embodiments as an anti-cancer drug. Embodiments of the invention also include efficient methods for isolating deflamin, suitable for industrial scale-up. A search for homologs in the seeds of other species was undertaken and will continue in the future.

MMP and cancer

Intensive research has been conducted over the past decades to develop new anticancer drugs, both prophylactic and therapeutic. However, despite significant advances in diagnosis, detection and treatment, the overall long-term outcome in patients has not changed significantly over the past decades (Herszényi et al., 2012). In this context, there has been an intensive search for various biological sources to develop novel anticancer drugs to combat this disease, which can be used in prevention, as an aid to chemotherapy or to prevent recurrence, especially in the case of plants, food products or bioactive plant compounds, which can be used as nutraceuticals for prevention and as an aid to chemotherapy (Su et al., 2006). Plants have been shown for several years to be an important natural source of compounds for medical therapies (including anticancer). It has been estimated that over the past 20 years, 25-30% of new drugs entering the US market were discovered in plants. Worldwide, the value of these over-the-counter drugs is estimated at over $40 billion per year. Therefore, pharmaceutical companies and institutions around the world are implementing plant screening programs as a primary means of identifying new drugs.

Colorectal cancer (CRC) is the second most common cause of cancer-related death in Europe, with a huge health and economic burden. Approximately 436,000 new cases and 212,000 deaths occur in Europe each year. Deaths in CRC patients are often caused by metastatic disease rather than the primary tumour itself.

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases involved in connective tissue remodeling (Markle et al., 2010). A subgroup of MMPs, also called gelatinases (MMP-2 and MMP-9), has been shown to be heavily involved in CRC in animal models and patients. Their inhibitors (MMPIs) were shown to be effective in reducing cancer progression/metastasis in in vitro assays and animal models and appear to be most effective in early stages of cancer or in preventing the development of undetected micrometastases after surgery (Coussens et al., 2002; Mook et al., 2004; Zucker and Vacirca, 2004; Sang et al., 2006; Herszényi et al., 2012). In the last decade, the development of synthetic MMPIs became an important branch of research in both academic and industrial settings, and numerous MMP inhibitors have been tested in different clinical trials, especially MMP-2 inhibitors (Hidalgo and Eckhardt, 2001). Because of this, and in view of the involvement of MMP-2 and MMP-9 in various diseases, it is believed that inhibiting the upregulation of specific MMPs may improve the clinical symptoms of patients. However, targeting MMPs in the treatment of diseases has proven to be difficult due to the fact that MMPs are ubiquitously indispensable for normal development and physiology, and previous efforts to inhibit MMP activity in the treatment of cancer patients yielded very unsatisfactory results with severe adverse side effects (Coussens et al., 2002; Ndinguri et al., 2012). Because of this, synthetic peptide inhibitors based on the MMP structure quickly became a hot spot for study of specific inhibitors.

Because MMPs are initially synthesized as zymogens (and are therefore inactive), with propeptides having to be removed from a propeptide domain before the enzyme is activated (Lu et al., 2012; Ndinguri et al., 2012), peptide drugs can inhibit the activation of extracellular MMPs directly, without affecting the expression of intracellular MMPs and thus avoid widespread deleterious side effects (Lu et al., 2012).

Currently, peptide research in drug design and discovery is one of the most promising fields in the development of new drugs. Compared with small molecule compounds, peptide drugs offer several advantages, such as high specificity and low toxicity (Lu et al., 2012). In the last decade, a substantial amount of research has focused on new plant MMPIs that are clinically active against various types of cancer cells. However, for reasons that are not understood, such studies often neglected peptides and small proteins. It is known that several plant species present specific bioactive peptides and small proteins, with functions such as defense against pathogen attack or proteolytic inhibition. This is the case, for example, of legume seeds (Park et al., 2007). The fact that many of these inhibitors are derived from plant foods makes them perfect candidates for use as nutraceuticals and in cancer-preventive diets, especially in the case of colon cancer. Compared with traditional cancer treatments such as chemotherapy or radioactive treatment, peptides and small proteins with high specificity against cancer cells or tumor promoters may present the way to kill cancer cells while protecting normal cells and helping patients recover quickly (Park et al., 2007).

Current findings have led to the discovery that peptides and small proteins from some edible seeds exhibit strong inhibitory activity against MMP enzymes. Advantageously, in certain embodiments, they can pass unchanged through the human digestive tract and can therefore be used for the treatment of colon cancer.

The use of digestion-resistant peptide and small protein MMPIs in studies on CRC control and treatment seems particularly suitable, because the inside of the colon can truly be considered “outside the body,” a situation in which MMPIs will not show side effects unless they are absorbed into the bloodstream.

MMP and inflammation

The worldwide incidence and prevalence of inflammatory bowel diseases (IBD) have increased dramatically over time, evidencing its emergence as a global disease (Molodecky et al., 2012; Burisch et al., 2014). Because mortality in IBD is low (Duricova et al., 2010; Burisch et al., 2014) and the disease is most frequently diagnosed in young people (Loftus et al., 2002; Burisch et al., 2014), the global prevalence of IBD is predicted to continue to increase substantially in the coming years (Abraham & Cho, 2009; Molodecky et al., 2012). Although the etiology of IBD has been widely studied in recent decades (Podolsky, 2002), the pathogenesis of the disease is still not fully understood (Jones et al., 2008; Mikhailov & Furner, 2009).

IBD encompasses three types of diseases: Crohn's disease (CD), ulcerative colitis (UC) and inflammatory bowel diseases not otherwise defined (IBDU). All of them are mainly characterized by chronic mucosal inflammation on pathological histology of the gastrointestinal tract in susceptible individuals (Podolsky, 2002; Danese et al., 2004; Abraham & Cho, 2009; Mikhailov & Furner, 2009). Although IBD significantly reduces the patient's quality of life and is likely to develop precancerous states (Xie & Itzkowitz, 2008; Triantafillidis et al., 2009), general clinical treatments for IBD are prone to induce side effects and present non-specific targets; they are extremely expensive and their curative effects are unsatisfactory (Dyson et al., 2012). According to Xie and Itzkowitz (2008), patients with long-standing IBD are at increased risk of developing CRC. Indeed, many of the molecular alterations responsible for sporadic CRC also play a role in colitis-associated colon carcinogenesis (Xie and Itzkowitz, 2008).

Numerous studies have documented the involvement of MMPs in inflammatory processes in animal models, cell lines, altered tissue cultures and patient biopsies (Baugh et al., 1999; Parks et al., 2004; Murphy & Nagase, 2008; Lee et al., 2013). It has long been recognized that the gelatinases MMP-9 and MMP-2 play important roles in the turnover and degradation of extracellular matrix proteins during cell recruitment in inflammation (Malla et al., 2008) and in other oncologically associated pathological processes, such as tumorigenesis, cell adhesion and metastasis (Herszényi et al., 2012). Although similar in their substrate selectivity, MMP-2 is constitutively expressed in fibroblasts, endothelial cells and epithelial cells and is only moderately implicated in inflammatory diseases (Huhtala et al., 1991), whereas MMP-9 expression is mainly observed in leukocytes (Van den Steen et al., 2002), being highly induced in response to a variety of inflammatory pathologies (Van den Steen et al., 2002) and is the main gelatinase induced during ulcerative colitis and other DII (Garg et al., 2009; Moore et al., 2011).

These discoveries have made MMP-9 a desirable therapeutic target in the prevention and treatment of IBD, as well as in the prevention of early stages of cancer and metastatic migration. However, studies linking MMP-9 inhibition to preclinical and clinical reduction of IBD are very few and it has been difficult to focus on MMPs. This could be achieved, at least partially, by long-term intake of specific, naturally occurring MMP-9 inhibitors that are available in the colon rather than bioavailable in serum. In recent years, it has been highlighted that some foods with nutritional function provide beneficial health effects in the prevention and treatment of certain diseases (Ortega, 2006; Sirtori et al., 2009) and in the last decade a significant amount of research has focused on new plant foods that present MMPIs.

MMPI of plant seeds

For more than 30 years, researchers around the world have considered MMPs as attractive targets for cancer. As a result, many chemical MMPIs have been developed as potential anticancer drugs. Well-known examples are tetracyclines, zoledronate, ethylenediaminetetraacetic acid (EDTA), 1,10-phenanthroline, 25,3R-3-amino-2-hydroxy-4-(4-nitrophenyl)butanol-L-leucine, and Neovastat (trademark) (isolated from shark cartilage). To date, countless MMPIs have been synthesized, some of which have been used as potential therapeutic agents to limit tumor progression (Bourguet et al., 2012). However, only a few small MMPIs have entered the clinical trial phase, most of which (e.g. batimastat®, marimastat®, solimastat®, galardin®, trocade®, prinomastat®, tanomastat®, rebimastat®) were terminated prematurely due to lack of benefit or strong adverse effects (Wang et al., 2012). So far, most cancer clinical trials have been rather disappointing. Furthermore, small non-natural peptides have been synthesized in an attempt to find new MMPIs. Thus, for example, two non-natural dodecapeptides were identified as MMP-2 inhibitors by Lu et al. (2012), while an octadecapeptide was found to be an MMP-9 inhibitor (Qiu et al., 2013).

A different and more recent strategy is the search for MMPI among the multitude of natural products that nature has made available to us.

It has long been known that plant organs and tissues, including seeds, contain metabolites, peptides and proteins with a variety of potentially useful bioactivities, many of which await discovery. One of these bioactivities is related to their ability to inhibit MMPs. In this regard, Tables 2 and 4 of the work reported by Cyr (2001) provide a huge list of plants (stressed or not) whose aqueous, ethanolic and organic extracts exhibit inhibitory activity on human enzymes MMP-2 and M<m>P-2.9, respectively. These extracts surely encompass secondary metabolites, as illustrated by the following additional examples. Withaferin A is a steroidal lactone, derived from Acnistus arborescens, Withania somnifera, and other members of the Solanaceae family, as well as some of its stable derivatives (e.g., 3-azido withaferin A; Rah et al., 2012), abolished secretory MMP-2. The flavonoids chrysin, apigenin, genistein, and their homoleptic copper(II) complexes have also been reported to attenuate the expression and secretion of the metastasis-relevant matrix metalloproteinases MMP-2 and Mm P-9 (Spoerlein et al., 2013).

Many seeds have been reported to contain MMPIs, such as grape (La et al., 2009), soybean, sunflower (Ceccoli et al., 2010) and dried longan (Euphoria longana Lam.) (Panyathep et al., 2013). In grapevine, proanthocyanidins are the MMP inhibitors (Vayalil and Mittal, 2004), while in soybean, the flavonoid genistein and the peptide lunasin (see below) seem to be the active ingredients.

Furthermore, it has long been known that seeds in general, and legume seeds in particular, contain a variety of protein enzyme inhibitors, such as trypsin inhibitors and Bowman-Birk inhibitors. Those from soybean and chickpea show activity against insects. On the other hand, Bowman-Birk inhibitors have been reported to prevent tumor genesis and possess antimicrobial activity. As a result, Birk (1996) concluded that the in vitro effects of proteinase inhibitors in animals should be interpreted with caution when related to humans. However, it should be noted that all of these protein enzyme inhibitors are inactivated by heat treatment (including cooking). In this regard, proteins or glycoproteins from soybean, rice, pea or lupin, and other plant extracts, are known to inhibit M<m>P (Stuhlmann & Joppe, 2013).

A specific mention of lunasin

Lunasin from soybean deserves special mention. Lunasin is a 43-amino acid residue chemopreventive peptide initially identified in soybean and subsequently confirmed to also be present in barley, wheat, rye, Solanum nigrum seeds and Amaranthus (Jeong et al., 2007). Using monoclonal antibodies prepared against the 43-amino acid residue of soybean lunasin, Herrera (2009) conducted a detailed study to detect this peptide in total protein extracts as well as in soluble fractions of seeds from cultivated and wild species of Lupinus. Lunasin was not detected in the albumin or globulin fractions of L. albus. In the analyzed species of the Lupinus genus, a positive immunological signal for lunasin was obtained in the prolamin fraction of seeds of L. albus with testa and in albumin and glutelin. In fractions from seeds without testa of L. montanus and L. stipula, respectively, no lunasin was detected in protein extracts from testa-less seeds of L. albus and from testa-bearing and testa-less seeds of L. mutabilis. An immunological reaction was obtained for polypeptide bands larger than 25 kDa, a result probably derived from the presence of lectins or storage proteins exhibiting lectin activity in lupin cotyledons, which are known to recognize and bind glycosylated IgG after SDS-PAGE fractionation and membrane blotting. Mitchell et al. (2013) could not find a gene (or gene fragment) encoding lunasin in cereals and confirmed its presence in soybean and peanut.

Lunasin is a small subunit peptide (SEQ ID NO: 191) derived from the larger cotyledon-specific 2S seed albumin complex (Gm2S-1) that exhibits anticancer and anti-inflammatory activities. Large-scale animal studies and human clinical trials to determine the efficacy of lunasin in vivo have been hampered by the expense of synthetic lunasin and the lack of a method to obtain gram quantities of highly purified lunasin from plant sources (Seber et al., 2012). A scalable method was developed utilizing the sequential application of anion exchange chromatography, ultrafiltration, and reversed-phase chromatography. This method generates lunasin preparations with a purity of 0.99% with a yield of 442 mg/kg defatted soybean meal. The proposed mode of action of lunasin, presented by the authors in Figure 4 of Kyle et al. (2012), does not include the role of MMP inhibition, i.e. lunasin does not appear to physically interact with MMPs. Rather, physical interactions appear to take place between lunasin, chromatin and histones (Jiang et al., 2016).

A brief reference to Lupinus seed storage proteins

The major seed storage proteins of lupins, termed conglutins, have been classified into four families: a-, p-, y-, and 5-conglutin conglutins. 5-conglutin, the major seed globulin of lupins, is vicilin or the 7S member of the seed storage proteins, whereas a-conglutin is legumin or the 11S member of the seed storage proteins. A total of three a-conglutin, seven p-conglutin, two Y-conglutin, and four 5-conglutin encoding genes were previously identified in narrow-leaf lupin (Lupinus angustifolius) (Foley et al., 2011, 2015). These genes have been named conglutin alpha 1, 2, and 3, conglutin beta 1, 2, 3, 4, 5, 6, and 7, conglutin gamma 1 and 2, and conglutin delta 1, 2, 3, and 4, respectively. Furthermore, the resulting polypeptides undergo extensive and complex processing and assembly processes, resulting in the high degree of microheterogeneity that characterizes these proteins.

A special reference to the protein Lupinus2S, in this genus specifically called 5-conglutin. 5-conglutin belongs to the family of sulfur-rich 2S albumins. The albumin from Lupinus2S seeds, also called 5-conglutin (Sironi et al., 2005), is a monomeric protein comprising two small polypeptide chains linked by two interchain disulfide bonds: a smaller polypeptide chain, consisting of 37 amino acid residues resulting in a molecular mass of 4.4 kDa and a larger polypeptide chain containing 75 amino acid residues with a molecular mass of 8.8 kDa (Salmanowich & Weder, 1997). The unique amino acid sequence of L. albus 5-conglutin has been inferred from the gene sequence. The largest polypeptide chain contains two intrachain disulfide bridges and a free sulfhydryl group (Salmanowich & Weder, 1997). This protein presents specific characteristics unique among L. albus proteins: in addition to its high cysteine content, it presents a low absorbance at 280 nm.

Regarding the physiological role of 5-conglutin, a storage function has been proposed for this class of proteins. The structural similarity to the family of plant cereal inhibitors, which includes bifunctional trypsin/alpha-amylase inhibitors, may suggest a defence function for this protein in addition to its storage function. Its presence in L. albus seeds was assessed to range between 10 and 12%, but more recent data suggest a lower content of approximately 3 to 4%. Lupinus 2S seed albumin is usually present in both the albumin and globulin fractions (Salmanowich & Przybylska, 1994), which explains the authors’ results obtained previously for M<p>inhibitory activity in both protein fractions (Lima et al., 2016).

A concise definition of Blad

Blad is a 20,408.95 Da polypeptide of 173 amino acid residues comprising residues 109 to 281 of the p-conglutin precursor (i.e. pro-G-conglutin). P-conglutin is a globulin and the major storage protein of Lupinus seeds (Figures 1, 2 and 3; Monteiro et al., 2003, 2006). Under natural conditions, Blad accumulates in the cotyledons of Lupinus seedlings between day 4 and 14 after the onset of germination.

Anticancer activities present in food products, with special reference to soybeans

There is abundant evidence in the published literature on the anticancer activities of most edible foods. Legume seeds are no exception and these studies have focused mainly on soybean. Thus, there is much evidence to suggest that compounds present in soybean can prevent cancer in many different organ systems. These include Bowman-Birk inhibitor, trypsin inhibitor, phytic acid, p-sitosterol, isoflavones (e.g. genistein and daidzein) and saponins (Kennedy, 1995). Legume seed proteins and soybean proteins in particular (including BBIS) have been reported to play a role in the levels of anticancer and antimetastasis properties in various animal models (Roy et al., 2010). Champ (2002) reported that soy-derived BBI inhibited or prevented the development of chemically induced liver, lung, colon, mouth, and esophageal cancers in mice, rats, and hamsters. Kennedy and Wan (2002) observed in vitro that 50 to 100 µg soy beta-glucans/mL decreased prostate cancer cell migration. BBI inhibits MMPs and demonstrates efficacy against tumor cells in vitro, animal models, and human clinical trials (Losso, 2008).

Currently, there are no prescription medications that specifically target chronic inflammation. (Of course, there are over-the-counter medications that treat minor, temporary inflammation and the accompanying pain caused by injuries or procedures, such as surgery. However, these are not intended to treat chronic inflammation.) Some medications, such as hydroxychloroquine, which was once used to fight malaria, are helpful in treating some lupus patients, but they do not cure the disease. Aspirin and statins have shown promise in reducing inflammation in some people, but researchers are not sure how helpful those drugs are in that role. With the exception of anti-inflammatory drugs that are far from perfect, such as prednisone, a corticosteroid that carries with it a host of side effects, scientists are still researching how best to contain inflammation.

Cell invasion and integrins

It is generally established that death in patients with colorectal cancer is mainly caused by the metastatic disease rather than the primary tumor itself. Metastasis involves the release of cancer cells from the primary tumor and their adhesion to another tissue or organ. Therefore, cancer cell invasion is a key element in metastasis and requires a) integrins for adhesion/de-adhesion and b) matrix metalloproteinases (MMPs) for targeted proteolysis.

Targeted proteolysis is necessary to open the way in the extracellular matrix for cancer cells to travel through. Wound healing assays provide an estimate of the capacity of cells for cell invasion. Usually, MMP-9 activities are highly correlated with cancer cell invasion, hence reduction in MMP-9 activity inhibits cell invasion and the two activities are usually paired. This is why MMP-9 inhibition is so desired, because it directly inhibits cell invasion, thus inhibiting metastatic death.

On the other hand, cells adhere to a substrate through specific proteins called integrins, transmembrane receptors that bridge cell-cell and cell-extracellular matrix (ECM) interactions. An important function of integrins in cells in tissue culture is their role in cell migration. Recent studies demonstrate that integrins are modulated by tumor progression and metastasis and are closely related to both MMP-9 and MMP-2 activities (Hood & Cheresh, 2002). Targeting and disabling integrins on cancer cell membranes may also be desirable because when cells are released from the tumor, they lack the ability to adhere to other tissue. This will also inhibit metastasis and may even help disaggregate the primary tumor.

Cell proliferation and metabolism

Another target for many studies is specific cytotoxicity against cancer cells. Many bioactive compounds, such as phenolic compounds, strongly reduce cell growth and affect metabolism, reaching toxic levels at high doses. Measuring cell growth and cell metabolism in the presence of a bioactive compound is a direct measure of its toxicity to the cell. The MTT assay uses a specific staining agent that needs to be taken up by living cells, and then metabolized by them to produce a purple color, which is then quantified by spectrophotometry. If the cell is dead or has an altered metabolism, it will not produce the staining agent. Therefore, higher levels of color are indicative of a greater number of living and metabolically active cells. If a compound reduces cell growth or kills cells, there will be a lower level of color.

Dianizing cancer cells to kill them is, in theory, a good approach. However, it can only work if there is a high specificity towards cancer cells and not towards normal (i.e., non-cancerous) healthy cells. Most compounds that kill cancer cells will also kill normal healthy cells at a given dose, and while many studies focus only on the ability of a metabolite (e.g., phenolic compounds) to reduce cancer cell growth, they often do not take into account their effects on controlling healthy cells. One reason this is quite common relates to the fact that, unlike normal, healthy cells, it is relatively easy to grow cancer cells in laboratory conditions. Consequently, later on, at the level of preclinical or even clinical trials, the use of these compounds is hampered by dose-limiting toxicity, insufficient clinical benefits, and extremely adverse side effects.

Therefore, a bioactive agent that reduces cell invasion but does not affect normal cell metabolism (as appears to be the case with deflamin) will likely produce fewer side effects and be safer to use in long-term preventive administrations, because it will not exert cytotoxicity toward normal colon cells.

It was not until about twenty years ago that the biological sciences "woke up" to secondary metabolism. This encompasses the most basic biochemistry and molecular biology, as well as those areas closer to application, such as agriculture and health, and human nutrition. In the past 20 years, 25-30% of new drugs entering the US market were discovered in plants. Globally, the over-the-counter value of these drugs is estimated at over $40 billion annually. However, there is a major difference in dose-dependent bioactivity between bioactive secondary metabolites and small proteins/polypeptides such as those discussed herein.

Beneficial bioactive secondary metabolites (e.g., antioxidant polyphenols) are typically effective at low concentrations. However, above a specific threshold they become highly toxic. In contrast, small proteins/polypeptides are either naturally beneficial (e.g., deflamin) or toxic (e.g., ricin and proteins present in snake and scorpion venoms).

Materials and methods

Materials, solvents and reagents

The 5% (w/v) aqueous solution of 2,4,6-trinitrobenzenesulfonic acid (TNBS) was purchased from Sigma Chemical Co. Ketamine (Imalgene® 1000) and xylazine (Rompun® 2%) were purchased from Bio2 Veterinary Products (Lisbon, Portugal). All other reagents were purchased from Sigma-Aldrich (St. Louis, USA). Dye-labeled gelatin (DQ) was purchased from Invitrogen (Carlsbad, CA, USA).

Measurement of antibacterial activity

The antibacterial activities of deflamin were evaluated in sterile 96-well plates (Greiner Bio-one, Germany), using the microdilution method as described by Bouhdid et al. (2010). Briefly, 50 pL of Müller-Hinton medium (Biokar, France) and 50 pL of deflamin solution (to obtain a deflamin concentration of 100 pg/mL) were added to the first well, and serially diluted 1:2 to each adjacent well, up to 10-fold dilutions. Subsequently, 50 pL of the bacterial suspension with a concentration of 2 x 105 CFU/mL was added to the wells. A positive control (50 pL of Müller-Hinton medium + 50 pL of bacterial suspension) and a negative control (100 pL of Müller-Hinton medium) were performed. The plates were incubated for 24 h at 37 °C, and the absorbance was read at 546 nm (Synergy HT, Biotek, USA) at the beginning of inoculation and at the end of the assay.

Measurement of antifungal activity

Spore suspension was prepared by adding 20 mL of sterile water to 1-week-old fungal cultures grown on Petri dishes containing potato dextrose agar (PDA) as the growth medium. Growth conditions were 25 °C ± 1 °C, in the dark. The spore suspension was filtered and adjusted to a concentration of 105 spores/mL using a hemocytometer. 100 pL of spore suspension was added to Petri dishes containing PDA medium and evenly distributed over the surface of the plate with a sterile rake. Sterile filter paper discs (diameter: 6 mm) were soaked in 6 pL of a deflamin solution (100 pg/mL) and placed on the surface of the medium. Controls were performed by soaking sterile filter paper discs with 6 pL of sterile water. Petri dishes were stored in an incubator (25 °C, dark) and fungal growth was monitored over a period of 2 weeks.

Minimum inhibitory concentrations (MIC)

Minimum inhibitory concentrations (MICs) were assessed in sterile 96-well plates (Greiner Bioone, Germany) using the microdilution method as described previously (Bouhdid et al., 2010). Briefly, 50 pL of RPMI medium was added to each well. Then, 50 pL of each sample was added to the first well and serially diluted 1:2 to each adjacent well, up to 10-fold dilutions. Subsequently, 50 pL of HT29 cell suspension at a concentration of 2 x 105 cells/mL was added to the wells. A positive control (50 pL RPMI medium + 50 pL cell suspension) and a negative control (100 pL RPMI medium) were performed. Plates were incubated for 24 h at 37 °C, and cell growth was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Carmichael et al., 1987). For determination of MMP-9 MIC, medium was collected from each well and gelatinolytic activities were determined with DQ gelatin as described below.

Seeds

In certain embodiments, dried seeds of the following legume species were used: white lupin (Lupinus albus L.), chickpea (Cicer arietinum L.) and soybean (Glycine max L.). Where required, other legume seeds were also used: lentil (Lens culinaris M.), common bean (Phaseolus vulgaris L.), pea (Pisum sativum L.), broad bean (Vicia faba L.) and black-eyed bean (Vigna unguiculata L.).

Cooked seeds

Legume seeds, like many other seeds, are known to contain antinutritional factors, such as digestive enzyme inhibitors, lectins, high concentrations of phytate, non-protein amino acids, etc. Therefore, they should be ingested after cooking to ensure denaturation of protein antinutritional factors. For these reasons, and because deflamin was found to resist boiling, a series of initial experiments were conducted using cooked seeds. To simulate cooking conditions, dried seeds were boiled in distilled water (w/v) until they acquired a soft texture suitable for eating (Xu & Chang, 2008).

Extraction of proteins and non-protein compounds

Extraction of total soluble proteins

In certain embodiments, total soluble proteins from seeds were extracted by shaking for 2 to 3 h at room temperature in 100 mM Tris-HCl buffer, pH 7.5, in a ratio of 1:5 (w/v), containing polyvinylpolypyrrolidone (0.5 g PVPP per 0.5 g fresh weight) and shaking for 4 h at 4 degrees Celsius. The suspension was then centrifuged at 12,000 g for 60 min at 4 degrees Celsius (Beckman J2-21M/E, JA 20,000 rotor). The supernatant was kept and stored in a -20 °C freezer.

Extraction of non-protein compounds

For total phenolic extractions, seeds were ground to flour, defatted with n-hexane, and 10 mL of acetone in water (50%, v/v) was added per 1 g of fresh weight. Samples were shaken for 4 h at room temperature and centrifuged at 12,000 g for 10 min at 4 degrees Celsius (Beckman J2 - 21M/E, rotor JA 20,000). The procedure was repeated twice, and frozen supernatants were collected, pooled, and stored for later analysis. The supernatants were evaporated at 60 °C to dryness and the resulting extract was resuspended in reaction buffer (50 mM Tris-HCl buffer, pH 7.6, containing 150 mM NaCl, 5 mM CaCh and 0.01% v/v Tween 20 with 12% v/v ethanol).

Extraction and isolation of deflamin from seeds

Dried and mature seeds of Lupinus albus L. (lupin) were used in this part of the work. In certain embodiments, identical procedures were followed for the seed of other species. The protein extract MMPI was isolated using its ability to resist boiling and acid denaturation. A method for isolating deflamin from the seeds was developed which is suitable for scale-up to industrial scale (Figure 4).

In one embodiment, the method for purifying deflamine is a clean procedure that follows a sequential precipitation scheme. It is based on the resistance of deflamine to high temperatures, low pH and high ethanol concentrations, and involves the following steps:

- Approximately 100 g ± 0.1 g of dried lupin seed (without embryo and tegument) were ground into flour.

- The flour is degreased with n-hexane and then suspended in water (1:20 w/v; pH adjusted to pH 8.0-8.5), under stirring for 2-3 h at room temperature or

- the fat is simply skimmed off the top of the suspension after suspending the flour in water (1:20 w/v; pH adjusted to pH 8.0-8.5), under stirring for 2-3 h at room temperature. As an alternative to water, on a laboratory scale, the flour can be suspended in 50 mM Tris-HCl buffer, pH 7.5. After stirring for 4 h (or overnight) at 4 degrees C, soluble proteins are obtained by centrifugation at 13,500 g for 30 min at 4 degrees C. The pellet is discarded.

- The protein solution is boiled at 100 degrees C for 10 min and centrifuged at 13,500 g for 20 min at 4 degrees C. The pellet is discarded. The supernatant is collected and the heat-treated (HT) extract is provided.

- Polypeptides/proteins present in the supernatant are precipitated by the addition of diluted HCl to pH 4.0. After centrifugation at 13,500 g for 20 minutes at 4 degrees C, the supernatant is discarded.

- The pellet is resuspended in 40% (v/v) ethanol containing 0.4 M NaCl, shaken for 1 h at room temperature, and centrifuged at 13,500 g for 30 min at 4 degrees C. This treatment brings deflamin into solution, unlike the vast majority of seed storage proteins. The pellet is discarded.

- The supernatant is brought to 90% (v/v) ethanol and stored at -20 degrees C overnight, precipitating deflamin, followed by centrifugation at 13,500 g for 30 min at 4 degrees C. The supernatant is discarded.

For better results and to obtain a purer and cleaner fraction of deflamin, the differential precipitation with 40 to 90% (v/v) ethanol should be repeated. Thus,

- The deflamine present in the sediment is dissolved again in 40% (v/v) ethanol containing 0.4 M NaCl, shaken for 1 h at room temperature and centrifuged at 13,500 g for 30 min at 4 degrees C.

This second wash cleans the deflamine of final contaminants. The sediment is discarded again.

- The supernatant is once again taken up in 90% (v/v) ethanol and stored at -20 degrees C overnight, precipitating the deflamin, followed by centrifugation at 13,500 g for 30 min at 4 degrees C. The supernatant is discarded.

- The final sediment contains pure deflamin, which is subsequently dissolved in the smallest possible volume of Milli-Q water. On a laboratory scale, the deflamin solution is finally desalted in water on Sephadex G-25 columns (NAP-10 columns, GE Healthcare Life Sciences) to remove low molecular mass contaminants.

- The obtained extract was stored in falcon tubes at -20 degrees C.

This methodology was successful in isolating deflamina from L. albus, Cicerarietinum and Glycine max.

Fractionation of deflamine by high performance liquid chromatography

In certain embodiments, the constituent polypeptides of deflamin were fractionated on a high performance liquid chromatography (HPLC) device (Waters 2695 Separations Module) equipped with a Waters 2998 photodiode array detector. Protein samples were separated on a Zorbax 300SB 5 mm, 250 mm x 4.6 mm, C18 reversed phase column. Elution was performed with eluent A (0.1% v/v trifluoroacetic acid, TFA) and solvent B (0.1% v/v acetonitrile in TFA). Peak detection was performed at both 214 nm and 280 nm.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

In certain embodiments, samples were treated with 100 mM Tris-HCl buffer, pH 6.8, containing 100 mM mercaptoethanol, 2% (w/v) SDS, 15% (v/v) glycerol, and 0.006% (w/v) m-cresol purple, and heated to 100 degrees C for 5 min. One-dimensional electrophoresis was performed, following the method described by Laemmli (1970) on a 12.5% (w/v) acrylamide resolving gel and a 5% (w/v) acrylamide stacking gel, and was run on a vertical electrophoresis unit at 100 V and 20 mA per gel. Gels were fixed for 20 min in 10% (w/v) TCA and stained with 0.25% (w/v) Coomassie Brilliant Blue R-250 (CBB R-250), 25% (v/v) 2-propanol, and 10% (v/v) acetic acid. Destaining was performed in a solution of 25% (v/v) 2-propanol and 10% (v/v) acetic acid.

Mass spectrometry (MS) analysis

In certain embodiments, selected isolated peaks were analyzed by LC/MS on a 5600 TripleTOF (ABSciex®) in information dependent acquisition (IDA) mode. Peptides were resolved by liquid chromatography (nanoLC Ultra 2D, Eksigent®) on a reversed phase MicroLC ChromXPTM C18CL column (300 µm ID x 15 cm length, 3 mm particles, 120 A pore size, Eksigent®) at 5 µL/min. Peptides were eluted into the mass spectrometer with a multi-step gradient: 0-2 min linear gradient from 5 to 10%, 2-45 min linear gradient from 10% to 30%, and 45-46 min at 35% acetonitrile in 0.1% FA. Peptides were eluted into the mass spectrometer using an electrospray ionization source (DuoSpray™ Source, AB Sciex) with a 50 mm internal diameter (ID) stainless steel emitter (New Objective).

For information-dependent acquisition (IDA) experiments, the mass spectrometer was set to scan full spectra (350–1250 m/z) for 250 ms followed by up to 100 MS/MS scans (100–1500 m/z starting with a dynamic accumulation time – minimum 30 ms for precursor above intensity threshold of 1000 – in order to maintain a cycle time of 3.3 s). Candidate ions with a charge state between 2 and 5 and counts above a minimum threshold of 10 counts per second were isolated for fragmentation and an MS/MS spectrum was collected before those ions were added to the exclusion list for 25 s (Analyst® TF 1.6 mass spectrometer, ABSciex®). A rolling collision with a collision energy spread of 5 was used. Two IDA experiments were performed for each sample with the second analysis carried out with a holdout list of previously identified peptides.

Protein identification was obtained using Protein Pilot™ software (v 5.0, ABSciex®) with the following search parameters: identification from the uniprot database March 2016, no alkylation or digestion for peptide samples. An unused score value of 1.3 and a peptide confidence filtering of 95% and >0 contribution are used as criteria for protein filtering.

Cell cultures

Biological material

Throughout this work, the human colon adenocarcinoma cell line, HT29 (ECACC 85061109), obtained from an adenocarcinoma of a 44-year-old Caucasian woman, was used. HT29 cell lines were maintained in RPMI medium supplemented with 10% (w/v) heat-inactivated fetal bovine serum (FBS) and 200 mM glutamine, 2x104 IU.mL-1 penicillin and 20 mg.mL-1 streptomycin at 37 °C in a humidified atmosphere of 5% (v/v) CO2. Routine observation of cell viability was performed by inverted microscopy.

Cell proliferation, adhesion and viability assays

HT29 cells cultured in 96-well plates were seeded (2X10/well), samples were added to the growth media at the required concentrations (e.g. 100 pg/mL) and incubated for 24 h. After each treatment, extracellular media was collected and cells were washed with phosphate buffered saline (PBS) to remove non-adherent cells. Cells that were attached to the bottom were harvested with 0.15% (w/v) trypsin in phosphate buffer. Cell proliferation and viability were determined by the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay described by Carmichael et al. (1987). Cell number was determined using a hemocytometer, with trypan blue staining, which allowed quantification of a) cell adhesion, b) cytotoxicity and c) cell growth. All treatments were performed in duplicate in at least 3 independent experiments.

Cell migration assays: in vitro wounding assay

In certain embodiments, for analysis of cell migration, wound healing assay was performed. HT29 cells (5x105 cells/well) were seeded in 6-well plates and allowed to reach 80% confluency. "Wounds" were made by scratching along the cell monolayer to create an open space, thus mimicking a wound. Cells were then washed twice with PBS to remove floating debris. Each well was then filled with fresh medium with or without the presence of different concentrations of potential inhibitors, at different concentrations (e.g., 100 pg/mL), and allowed to grow for up to 48 h. At the end of the treatments, the invaded area or the number of cells in the scratch area of each well was determined and compared to the initial area at 0 h, and the cells were photographed under a phase contrast microscope. The area covered by cells migrating to the peeled area at the start of treatment was calculated. This comparison allowed the evaluation of the inhibitory effect (if any) exerted by each protein fraction on the migratory capacity of HT29 cells. The area of cell migration was counted in three to five random fields of each treatment in triplicate and was expressed as a percentage relative to time 0 (area covered by cells migrated to the peeled area at the start of treatment).

Enzyme assays: effect of deflamin on the catalytic activities of MMP-9 and MMP-2

Quantification of gelatinolytic activity with MMP-9

For the fluorogenic gelatin assay, DQ gelatin was purchased from Invitrogen (Carlsbad, CA, USA) and dissolved in water at 1 mg/mL. All solutions and dilutions were prepared in assay buffer (50 mM Tris-HCl buffer, pH 7.6, containing 150 mM NaCl, 5 mM CaCl2, and 0.01% v/v Tween 20). DQ gelatin was used at a concentration of 2.5 pg/mL in all experiments.

In a 96-well macroassay plate (chimney, 96 wells, black), 0.1 nM (for a final volume of 200 pL) of the enzyme (MMP-9, Sigma) was added. For inhibitor assays, the required amount of inhibitor (e.g. 100 pg/mL) was added and the plate was incubated for 1 h at 37 degrees C. Subsequently, DQ-gelatin was added to a final concentration of 2.5 pg/mL and allowed to incubate again for 1 h. Fluorescence levels were then measured (ex. 485 nm/em. 530 nm). Positive (no inhibitor) and negative (no enzyme) controls were included in each experiment to correct for possible proteolytic activities present in the protein samples. All data were corrected by subtraction of their respective negative controls.

Quantification of gelatinolytic activity in cellular media

Extracellular HT29 medium was collected to quantify the gelatinolytic activities present after exposure to the different inhibitors. The detected activity is due to the presence of both enzymes, MMP-9 and MM-2. The assay was carried out in the same way as described above, with the exception that HT29 medium from each treatment was added to each well (150 pL).

Total gelatinolytic activity of colonic tissues

Combined MMP-9 and MMP-2 activities were determined in colons of mice exposed to deflamin extract treatments as described by Medina et al. (2006) with few alterations. Briefly, colonic tissue was homogenized at a 1/100 (w/v) ratio in 50 mM Tris-HCl buffer, pH 7.6, containing 150 mM NaCl. Samples were sonicated three times for 10 s each at 1 min intervals. After 10 min on ice, protein extracts were centrifuged for 10 min at 13,000 g and 4 °C, supernatants were stored, and protein concentrations were determined by a modification of the Lowry method (Lowry et al.,1951). Samples were stored at -80 °C until analysis. Protein extraction from each colon, as described above, was used to quantify the respective gelatinolytic activities. The fluorogenic substrate dye-tempered gelatin (DQ) purchased from Invitrogen (Carlsbad, CA, USA) was used to quantify MMP-9 and MMP-2 activities. DQ gelatin was dissolved in water at 1 mg/mL according to the manufacturer's instructions. All solutions and dilutions were prepared in assay buffer (50 mM Tris-HCl buffer, pH 7.6, containing 150 mM NaCl, 5 mM CaCl2, and 0.01% v/v Tween 20). A 96-well microassay plate (chimney, 96 wells, black) was used. Colonic tissue supernatant from each treatment (100<j>L) was loaded into each well. Subsequently, DQ gelatin (at a final concentration of 2.5 µg/mL) was added to each well and the plate was allowed to incubate for 1 h. Fluorescence levels (ex. 485 nm/em. 530 nm) were measured. All data were corrected by subtracting their corresponding negative controls.

Zymography

In zymographic assays, two enzymes (MMP-9 and MMP-2, commonly known as gelatinases) as well as their corresponding zymogens or proenzymes (pro-MMP-9 and pro-MMP-2) were detected. By definition, a zymogen or proenzyme is inactive due to the presence of a short sequence of amino acids (called a prosequence) that typically blocks access to the active site. In the current situation, MMP enzymes are synthesized in this form to prevent, for example, them from starting to degrade the ribosome while they are still bound to it during protein synthesis. These proteins are thus synthesized in an inactive form and converted to their mature, natural form at their site of action. In the zymographic assays described herein, progelatinases also become active because they are denatured by SDS, thus exposing the catalytic site - Hence the slightly higher mass of the proenzymes in zymographic gels because they still retain the short cysteine switch amino acid sequence.

Gelatin zymography

Gelatin zymography was performed according to standard methods with the following modifications: To determine metalloproteinase activity in culture supernatants of HT29 cancer cell lines, SDS-polyacrylamide gels (12.5% w/v acrylamide) copolymerized with 1% (w/v) gelatin were prepared. Cell culture supernatants were treated with non-reducing buffer containing 62.6 mM Tris-HCl pH 6.8, 2% (w/v) SDS, 10% (v/v) glycerol and 0.01% (w/v) bromophenol blue and loaded into each well. Electrophoresis was carried out at 100 V for 2 h. After electrophoresis, the gels were washed three times in renaturation buffer (2.5% v/v Triton X-100) for 60 min each, to remove SDS. The gels were then incubated overnight with developing buffer (50 mM Tris-HCl buffer, pH 7.4, containing 5 mM CaCl2, 1 mM ZnCl2, and 0.01% w/v sodium azide). The gels were stained with 0.5% (w/v) Coomassie Brilliant Blue G-250 for 30 min and destained with a methanol:acetic acid:water solution (50 : 10 : 40). The intensities of the protein bands were determined by densitometry by measuring the peak areas using image processing software.

Reverse gelatin zymography

Reverse gelatin zymography, used to detect and quantify MMPI proteins in different samples, was performed as described by Hawkes et al. (2001,2010), with some modifications. Protein samples were treated with zymographic buffer (313 mM Tris-HCl buffer, pH 6.8, containing 10% (w/v) SDS, 50% (v/v) glycerol and 0.05% (w/v) bromophenol blue) and loaded onto SDS-polyacrylamide bulk gels (12.5% w/v acrylamide) copolymerized with gelatin (1% w/v) and conditioned medium (1.0 mL) from a cell line expressing MMP-2 and mMP-9 or with different concentrations of MMP-9 (e.g., 1 jmol/mL). Electrophoresis was performed as described above and gels were washed three times (60 min each) in 2.5% (v/v) Triton X-100 to remove SDS and incubated overnight as described above for substrate zymography. Gels were stained with 0.5% (w/v) Coomassie Brilliant Blue G-250. Dark areas marked MMPI-mediated inhibition of gelatin degradation. Dark bands visible against a white background marked MMPI-mediated inhibition of gelatin degradation (Hawkes, 2001).

Gelatin zymography of colon extracts

To determine specific metalloproteinase activities in colon extraction supernatants, gelatin zymography was performed according to standard methods (Tothet al., 2012), with the following modifications: SDS-polyacrylamide gels (12.5% w/v acrylamide) were copolymerized with 1% (w/v) gelatin. Colon extraction supernatants, previously treated with a non-reducing buffer containing 62.6 mM Tris-HCl buffer, pH 6.8, 2% (w/v) SDS, 10% (v/v) glycerol and 0.01% (w/v) bromophenol blue, were loaded into each SDS-gel well. Electrophoresis was carried out as described above, on a 12.5% (w/v) acrylamide resolving gel and a 5% (w/v) acrylamide stacking gel, run on a vertical electrophoresis unit at 200 V and 20 mA per gel. After electrophoresis, the gels were washed three times in 2.5% (v/v) Triton X-100 for 60 min each, to remove SDS. The gels were then incubated for two days with developing buffer (50 mM Tris-HCl buffer, pH 7.4, containing 5 mM CaCl2, 1 µM ZnCl2, and 0.01% w/v sodium azide), stained for 30 min with 0.5% (w/v) Coomassie Brilliant Blue G-250 in 50% (v/v) methanol and 10% (v/v) acetic acid, and destained with a solution of 50% (v/v) methanol and 10% (v/v) acetic acid. The white bands visible against a blue background marked the gelatinase activity of each of the proteinases (Tothet al., 2012).

Animal models of experimental inflammatory diseases

Colitis inflammation model

Animals

Male CD-1 mice, weighing 25–30 g and 5–6 weeks old (Harlan Iberica, Barcelona, Spain) were housed in standard polypropylene cages with free access to food and water, under a controlled environment in a room maintained at 22 degrees C ± 1 degrees C with a 12-h light/12-h dark cycle at the Faculty of Pharmacy, Central Animalarium, University of Lisbon.

Care and maintenance of animals for in vivo experiments

The experiments were carried out in accordance with the Home Office Guidance in the Operation of Animals (Scientific Procedures) Act 1986, published by Her Majesty's Stationary Office, London, UK, and the Institutional Animal Research Committee Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication no. 85-23, revised in 1996), as well as with the currently adopted EC regulations (Directive 2010/63/EU). Finally, the studies performed are in accordance with the ARRIVE Guidelines for Reporting Animal Research' summarized at http://www.nc3rs.org.uk. This experimental protocol was also approved by the Ethics Committee of the Faculty of Pharmacy, University of Lisbon. Furthermore, colleagues from the Faculty of Pharmacy, University of Lisbon, are authorized by the Directorate General of Veterinary Medicine of Portugal to coordinate and carry out independent animal research. All studies were conducted using 5-week-old male Wistar rats weighing 100–150 g (Harlan Iberica, Barcelona, Spain). All animals received a standard diet and water ad libitum.

Colitis model using oral (p.o.) administration of deflamin

Induction of experimental colitis

2,4,6-Trinitrobenzene sulfonic acid (TNBS) was instilled as a single intracolonic dose as described previously (Impellizzerie et al., 2015). Briefly, mice were starved for 24 h. On the day of induction (day 0), mice were anesthetized with 100 mg/kg ketamine and 10 mg/kg xylazine. Then, 100<j>L of TNBS solution was administered through a catheter carefully introduced up to 4.5 cm into the colon. Mice were kept for 1 min in the Tredelenburg position to prevent reflux. Four days after induction, mice were anesthetized and blood samples were collected by cardiac puncture. Mice were euthanized by cervical dislocation and necropsied. The abdomen was opened by a midline incision. The colon was removed, freed from surrounding tissues, and opened longitudinally for observation and grading of diarrhea severity. The colon was then lavaged with phosphate-buffered saline for macroscopic tissue observation and subjected to biochemical analysis or postfixed in paraformaldehyde for further processing.

Experimental groups

The animals were randomly distributed into four experimental groups as described:

1. Sham group (n = 6): Animals were subjected to the procedures described above with the exception that intracolonic administration was with 100 µL of saline. During the 4 days of the protocol, animals were orally administered 10 mL/kg of distilled water.

2. Ethanol group (n = 6): Animals were subjected to the procedures described above with the exception that intracolonic administration was with 100 µL of a 50% (v/v) ethanol/water solution. During the 4 days of the protocol, animals were orally administered 10 mL/kg of distilled water.

3. TNBS group (n = 10): animals were administered 100 pL of 2.5% (w/v) TNBS in 50% (v/v) ethanol. During the 4 days of the protocol, animals were administered 10 mL/kg of distilled water orally.

4. TNBS extract (or deflamin) group (n = 10): animals were administered 100 pL of 2.5% (w/v) TNBS in 50% (v/v) ethanol. During the 4 days of the protocol, the animals were administered orally (p.o.) the extract (or deflamin; 15 mg/kg).

Oral administrations were performed daily, starting 3 h after the initial administration of TNBS, via gastric tube.

Macroscopic assessment of colitis severity

Following removal of the colon, a longitudinal incision was made for observation of the contents and grading of diarrhea severity by an observer blinded to the experimental groups. The colon was then rinsed with saline and viewed macroscopically through a surgical microscope for closer observation of the tissue and imaging of the lesion. The colon was then measured, as was the extent of the lesion (if present).

Hemorrhagic injury assessment

Fecal hemoglobin, as an index of hemorrhagic injury, was measured by a quantitative method by immunoturbidimetry (Kroma Systems).

Histology and immunohistochemistry procedures

Colons were removed, fixed in 4% (w/v) paraformaldehyde in PBS for 72 h at room temperature, dehydrated through a graded ethanol series, and embedded in paraffin (n = 3 per group). Hematoxylin and eosin (H&E) staining was performed as described previously (Rocha et al., 2015) and images were acquired using an Axioscop brightfield microscope (Zeiss, Gottingen, Germany).

The degree of colonic inflammation and damage on microscopic cross sections was semiquantitatively graded from 0 to 3: 0, normal colon without lesions, mucosa of uniform thickness, straight crypts, normal crypt architecture, no cellular infiltration, edema, or exudate meaning no signs of inflammation; 1, colon with mild lesions, mucosal erosion, and small superficial ulcers scattered along the length of the colon, with slight crypt loss and mononuclear cell infiltration; 2, colon with moderate lesions, bowels with extensive erosion and ulceration, with moderate crypt loss and neutrophil infiltration; 3, colon with very severe ulceration, thin mucosa with crypt loss and markedly increased neutrophil infiltration and acute inflammatory exudate.

For immunostaining, 6 pm thick sections were subjected to antigen retrieval in 20 mM citrate buffer with 1.5% (v/v) H2O2 for 15 min at room temperature in the dark, incubated for 10 min in Tris/EDTA buffer at 84 °C, and blocked for 1 h at room temperature in 1% (w/v) bovine serum albumin (BSA) in PBS. Primary antibodies [rabbit anti-COX2 (Cell Signaling #4842, 1:100) and mouse anti-iNOS (BD Transduction Laboratories #610328, 1:100)] in 0.5% (w/v) BSA in PBS were used overnight at 4 degrees C. After washing in PBS, sections were incubated for 1 h at room temperature with horseradish peroxidase-coupled anti-rabbit antibodies (Santa Cruz Biotechnology, 1:5000) in 0.5% (w/v) BSA in PbS, incubated for 10 min in SIGMAFa St DAB with metal enhancer (Sigma, USA), and mounted with Entellan (Merck, Germany). Tissue sections were visualized with an AxioScope brightfield microscope (Zeiss, Gottingen, Germany).

Colitis model using intraperitoneal (i.p.) injection administration of deflamin

The anti-inflammatory effect of deflamin against colitis administered intraperitoneally was also tested. Mice were treated exactly as described above, except that deflamin extract was administered by intraperitoneal injection. The following experimental groups were tested:

1. Sham group (n = 6): Animals underwent the procedures described above with the exception that intracolonic administration was with 100 pL of saline. During the 4 days of the protocol, animals were orally administered 10 mL/kg of distilled water or were injected intraperitoneally with the same amount of saline.

2. Ethanol group (n = 6): animals were subjected to the procedures described above with the exception that intracolonic administration was with 100 pL of a 50% (v/v) ethanol/water solution.

During the 4 days of the protocol, the animals were orally administered 10 mL/kg of distilled water injected intraperitoneally with the same amount of saline solution.

3. TNBS group (n = 10): Animals were administered 100 pL of 2.5% (w/v) TNBS in 50% (v/v) ethanol. During the 4 days of the protocol, animals were administered orally 10 mL/kg of distilled water injected intraperitoneally with the same amount of saline.

4. TNBS extract (or deflamin) group (n = 10): animals were administered 100 pL of 2.5% (w/v) TNBS in 50% (v/v) ethanol. During the 4 days of the protocol, animals were injected intraperitoneally (i.p.) with extract (or deflamin; 10 mg/kg).

Intraperitoneal injection administrations were performed daily, starting 3 h after the initial administration of TNBS, by gastric tube, as described above, and the same assessments were performed, as described for oral administrations.

Preventive effects versus curative effects of deflamina

To compare the preventive effect of deflamin with its curative effect, mice were maintained and treated as described above and randomly assigned into four experimental groups:

1. Sham group (n = 6): Animals underwent the procedures described above with the exception that intracolonic administration was with 100 pL of saline. During the 4 days of the protocol, animals were orally administered 10 mL/kg of distilled water.

2. TNBS group (n = 10): Animals were administered 100 pL of 2.5% (w/v) TNBS in 50% (v/v) ethanol. During the 4 days of the protocol, animals were administered 10 mL/kg of distilled water orally.

3. TNBS deflamin p.o. (n = 9): Animals were administered 100 pL of 2.5% (w/v) TNBS in 50% (v/v) ethanol. Animals were administered deflamin orally (15 mg/kg) during the 4 days of the protocol.

4. Preventive treatments with TNBS deflamin (n = 10): Three days before TNBS induction, deflamin (15 mg/kg) was administered orally to the animals. The animals were then given 100 pL of 2.5% (w/v) TNBS in 50% (v/v) ethanol.

During the 4 days of the protocol, the animals were administered deflamin orally (15 mg/kg).

Following the 4-day experiment, macroscopic assessment of colitis severity was performed as described above.

Carrageenan-induced paw edema inflammation model

Care and maintenance of animals for in vivo experiments

The experiments were carried out in accordance with the Home Office Guidance in the Operation of Animals (Scientific Procedures) Act 1986 published by Her Majesty's Stationary Office, London, UK and the Institutional Animal Research Committee Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication no. 85-23, revised in 1996) as well as with the currently adopted EC regulations. Finally, the studies conform to the ARRIVE Guidelines for Reporting Animal Research' summarized at http://www.nc3rs.org.uk. Therefore, the Ethics Committee of the Research Institute endorsed the animal study protocol, also considering that the authors Sepodes and Rocha are authorized by the Portuguese General Veterinary Directorate to coordinate and carry out independent animal research. All studies were conducted using 5-week-old male Wistar rats weighing 100 to 150 g (Harlan Iberica, Barcelona, Spain). All animals received a standard diet and water ad libitum.

Paw edema inflammation model using oral administration of deflamin

Induction of experimental paw edema and assessment of edema severity. Carrageenan-induced paw edema of the rat hind paw is a suitable model to study acute local inflammation and is widely considered one of the most useful models in the assessment of anti-inflammatory activity. This model was used to test the anti-inflammatory activity of deflamin administered orally or topically.

The paw edema model

Paw edema was induced by a single subplantar injection into the left hind paw of the rat of 0.1 mL of a sterile 1% (w/v) carrageenan A saline solution. Paw volume was measured by a volume displacement method using a plethysmometer (LE7500 digital plethysmometer; Letica Scientific Instruments, Letica, Spain). Paw volume was measured immediately after carrageenan injection (VO or basal volume) and 6 h later (V6 h). The increase in paw volume was taken as the edema volume.

Experimental groups

Animals were randomly assigned into the following groups as described:

1. Control group (n = 6): Animals were subjected to a subplantar injection in the left hind paw of the rat with 0.1 mL of sterile saline and were administered saline (1 mL/kg, i.p.).

2. Carrageenan group (n = 6): Animals were subjected to paw edema induction and administered saline (1 mL/kg, i.p.).

3. Deflamin group (n = 5): Animals were subjected to paw edema induction and pretreated with deflamin extract (15 mg of crude extract per kg, administered orally and i.p.) 30 minutes before carrageenan A injection.

4. Indomethacin group (n = 6): Animals were subjected to paw edema induction and pretreated with indomethacin (10 mg/kg, i.p.) 30 minutes before carrageenan A injection.

5. Trolox group (n = 6): Animals were subjected to paw edema induction and pretreated with Trolox (30 mg/kg p.o.) 30 minutes prior to carrageenan A injection.

Colitis model using topical administration of deflamin

Animals were treated essentially as described above and assigned into groups as described below:

1. Control group (n = 6): Animals were subjected to a subplantar injection in the left hind paw of the rat with 0.1 mL of sterile saline and were administered saline (1 mL/kg, i.p.).

2. Carrageenan group (n = 6): Animals were subjected to paw edema induction and administered saline (1 mL/kg, i.p.).

3. Deflamin group (n = 5): Animals were subjected to paw edema induction and topically treated with deflamin extract (dissolved in water and glycerol 30:70, v/v) after carrageenan A injection.

4. Control group (n = 6): animals were subjected to paw edema induction and treated topically with glycerol solution (water and glycerol 30:70, v/v) after carrageenan A injection.

Statistical analysis

For the animal colitis model, all results were expressed as the mean ± SEM of observations, where n represents the number of animals studied. Results were compared using a one-way ANOVA test, followed by a Bonferroni post hoc test using GraphPad Prism 5.0 software (GraphPad, San Diego, CA, USA). For gelatinolytic activities, all experiments were performed in triplicate, at least on three independent occasions and data were expressed as the mean ± standard deviation (SD). SigmaPlot software (version 12.5) was used to compare different treatments, using one-way and two-way analysis of variance (ANOVA). Tukey's test was used to compare differences between groups and statistical differences with a P value less than 0.05 were considered statistically significant. Results

MMPI activities in total seed extracts of legume species

In a preliminary study recently published in Food Chemistry (Lima et al., 2016) and prior to the discovery of deflamin, a series of total seed extracts were examined for the presence of MMPI inhibitory activities. The seeds under study were: Cicerarietinum L. (chickpea), Glycine max L., (soybean), Lens culinaris M. (lentil), Lupinus albus L. (lupin), Phaseolus vulgaris L. (common bean), Pisum sativum L. (pea), Vicia faba L. (broad bean) and Vigna unguiculata L. (black-eye bean). The most promising, which were further studied and will be discussed in this paper, were chickpea, lupin and soybean (Figures 5 and 6-11; Lima et al., 2016). Figure 5 compares the albumin and globulin polypeptide profiles for each of the eight legume seeds initially analyzed. Figure 5 shows representative images of the polypeptide distribution between albumins and globulins from eight legume seed species separated by SDS-PAGE. G - globulins, A - albumins. Protein extracts (40 µg per lane) were loaded onto 12.5% (w/v) polyacrylamide gels under reducing conditions (Lima et al., 2016).

It should be mentioned that the extractions of total seed proteins, total albumins and total globulins were performed under natural conditions. Therefore, Figure 6-11 addresses the effects of a series of seed subfractions on the MMPI activities present in the seeds of the eight species:

- Total soluble extracts (including thus low molecular weight metabolites such as flavonoids and other polyphenols, and peptides and proteins, presumably including lunasin) in Figure 6; Figure 6 refers to MMP-9 inhibitory activity. The effect of total soluble extracts of eight different legume seeds (Lupinus albus, Cicer arietinum, Vicia faba, Phaseolus vulgaris, Pisum sativum, Vigna unguiculata, Lens culinaris and Glycine max) on MMP-9 proteolytic activity. Total soluble extracts were added to a reaction mixture containing MMP-9 and gelatinolytic activity was determined by fluorogenic DQ assay.

- Albumins and globulins (a 24 h exposure) on HT29 cell proliferation in Figure 7; Figure 7 refers to the HT29 cell proliferation assay. HT29 cells were cultured for 24 h in the presence of albumin or globulin fractions previously extracted from the eight seed species.

- Albumins and globulins (24 h exposure) on HT29 cell migration are shown in Figures 8 and 9; Figure 8 refers to the wound assay of HT29 cell migration. Cells were grown until 80% confluent and the monolayer was scratched with a pipette tip (day 0). Cell migration was determined after 48 h exposure of HT29 cells to albumin or globulin fractions from the eight seed species. Examples of cell migration obtained for the most inhibitory seed extracts: L. albus, C. arietinum and G. max.

Figure 9 refers to the wounding assay of HT29 cell migration. Cells were grown to 80% confluence and the monolayer was scratched with a pipette tip (day 0). Cell migration was determined after 48 h exposure of HT29 cells to albumin or globulin fractions from the eight seed species. Relative migration rates (D),

- albumins and globulins (a 48 h exposure) on the proteolytic activity of gelatinases present in the extracellular media of HT29 in Figure 10;

Figure 10 refers to the proteolytic activity of gelatinases present in the extracellular media of HT29 after a 48 h exposure to albumin or globulin fractions isolated from the eight seed species, quantified by the fluorogenic DQ method.

- Zymographic profiles of MMP-9 and MMP-2 activities present in the extracellular media of HT29 cells after 48 h exposure to albumins or globulins in Figure 11.

Figure 11 depicts the zymographic profiles of MMP-9 and MMP-2 activities present in the extracellular media of HT29 after a 48 h exposure of the cells to albumin or globulin protein fractions. Only the seed extracts producing the most marked inhibitions are shown (i.e., L. albus, C. arietinum, and G. max). Polyacrylamide gels (12.5% w/v acrylamide) were copolymerized with 1% (w/v) gelatin. The relative activities of the MMP-9 and MMP-2 bands were calculated as a % of the controls. G, Glob – total globulin fraction containing 100 µg protein/mL; A, Alb – total albumin fraction containing 100 µg protein/mL. All values represented are the mean of at least three replicate experiments ± SD, and are expressed as a percentage of the corresponding control. Vertical bars represent SD. *P < 0.05, **P < 0.001.

It is important to mention that none of these studies/extracts involved cooked seeds, boiled extracts, extracts exposed to low pH values and/or extracts subjected to the digestive process. The results obtained clearly indicate the presence of MMP inhibitory activity(ies) in the seeds of the eight seeds studied, with the greatest prominence, in decreasing order of magnitude, in lupin, soybean and chickpea. Such MMP inhibitory activities were attributed to both secondary metabolites, which are widely reported in the literature (e.g. soybean genistein), and to a low molecular mass protein (i.e. lunasin).

Although consumption of sweet lupin seeds is increasing worldwide, with a multitude of applications by the food industry, wild seeds (i.e. those containing bitter or alkaloid) have been used as human food since ancient times and continue to be used as a snack in Mediterranean countries, provided they are boiled and the alkaloids are cleaned by soaking in running water. Interestingly, the same alkaloids that make wild-type seeds toxic appear to exert beneficial effects on diabetes mellitus. However, in the everyday Western lifestyle, other legume seeds are consumed more often and in greater quantities than lupins, such as chickpea and soybean. Embodiments of the invention focus on these last three species, with some additional results also focusing on some non-legume seeds.

Cooking and the richness of seeds in bioactive metabolites

As mentioned above in the Materials and Methods section, dried seeds, including those of cereals and legumes, are not ingested as such for a couple of reasons. First, they are quite hard to chew. Second, and more importantly, legume seeds, like many other seeds, are well known to contain antinutritional factors, such as digestive enzyme inhibitors, lectins, high concentrations of phytate, non-protein amino acids, etc. Therefore, they must be ingested after cooking to ensure denaturation of proteinaceous antinutritional factors as well as destruction or leaching of some non-protein constituents. For these reasons and because deflamin was found to resist boiling, several initial experiments were conducted using cooked seeds.

In addition to proteins, plant seeds contain a wide variety of bioactive secondary metabolites, most of which retain much of their biological activity after cooking.

The following examples of phytin, saponins, phenolic compounds and protein exemplify their concentration in both raw and cooked seeds of various legume species. It should be noted, as referred to above, that the reduction (expressed per unit of dry weight) of a specific compound induced by cooking, from phytin to proteins, is typically due to both molecular destruction and leaching into the surrounding water. Hence the popular knowledge that the water in which vegetables are cooked should be used to prepare soup.

In the following block of experiments, Pisum sativum and Vigna unguiculata were abandoned due to their poor results regarding MMP inhibition (see above). Therefore, the experiments concern only six species, namely Cicerarietinum, Glycine max, Lens culinaris, Lupinus albus, Phaseolus vulgaris and Vicia faba.

Fitina

Phytin (also referred to as phytate, PA, inositol hexaphosphate and IP6), for example, is considered an antinutrient, since when present in excess it inhibits digestive enzymes (e.g. trypsin, pepsin, a-amylase and p-glucosidase) and binds to certain minerals (most particularly zinc and, to a lesser extent, calcium and chromium), thus interfering with their bioavailability (website 1).

Figure 12 represents the seed phytin content of several species under study. The phytin content of the seeds appears to be fairly constant among the species studied and is not affected by cooking in any significant way.

Figure 12 represents the phytin concentration in the seeds of several legumes, as quantified by the method described by Gao et al. (2007). The vertical bars represent the mean ± SD of at least three biological replicates.

Saponins

Saponins have considerable potential as pharmaceutical and/or nutraceutical agents in natural or synthetic form. They have been shown to have anticarcinogenic, neuroprotective, anti-inflammatory and antioxidant activities, among others (Rao & Gurfinkel, 2000).

Figure 13 represents the seed saponin content of several species under study. Unlike phytin, saponin content varies significantly among the species analyzed, with the highest values obtained for soybean and chickpea and the lowest for lupin. Boiling consistently reduced the amount of saponins present, with losses ranging from approximately 7% (lentil) to over 90% (chickpea). Figure 13 represents the saponin concentration in the seeds of several legumes, as quantified by the method described by Hiaie et al. (1976). The vertical bars represent the mean ± SD of at least three biological replicates.

Phenolic compounds

Phenolic compounds are universally found in plants and are known to exhibit high antioxidant capacity and free radical scavenging ability. Therefore, they are generally considered as potential agents to prevent and treat many oxidative stress-related diseases, such as cardiovascular diseases, cancer, aging, diabetes mellitus, and neurodegenerative diseases, mainly due to their cardioprotective, anticancer, anti-inflammatory, and antimicrobial bioactivities (Li et al., 2014). However, major concerns involve their bioavailability and potential toxicity, as the vast majority of studies do not consider their resistance to cooking and digestive process. Moreover, unlike bioactive proteins, their beneficial/harmful bioactive effects are usually dose-dependent.

Seed phenolic concentration was highest in lupin, followed by soybean, common bean and lentil (Figure 14). Supposedly, the higher the level of polyphenols in the seed, the higher its anticancer bioactivity. Cooking drastically reduced (between 60 and 90 % depending on the species) the amount of seed phenols. Figure 14 shows the concentration of phenolic compounds in the seeds of several legumes, as quantified by the Folin-Ciocalteau method (Attard, 2013). The vertical bars represent the mean ± SD of at least three biological replicates.

Total soluble proteins

Proteins comprise an incredible class of biomolecules that perform an enormous variety of bioactivities unmatched by any other class of molecules. Selecting a previously unknown protein and determining its biological function is not only difficult but also one of the most challenging and interesting tasks for biological and chemical researchers. In addition to performing the biological role for which they evolved, proteins can also be used to benefit humanity due to a wide range of beneficial activities. Thus, for example, the Food and Drug Administration (FDA) authorized the use, on food labels and in food labeling, of health claims about the association between soy protein and reduced risk of coronary heart disease (FDA, 1999).

Soybeans contain, as expected, the highest amount of total soluble protein (297.4 mg/g dry weight), followed, in decreasing protein concentration, by lupin (190.4 mg/g dry weight), chickpea (138.2 mg/g dry weight), broad bean (114.5 mg/g dry weight), lentil (79.0 mg/g dry weight) and common bean (720.0 mg/g dry weight); (Figure 15). After cooking, these values were reduced in all cases to values below 20 mg/g dry weight, with the highest protein concentrations obtained for lentil, lupin, chickpea and soybean. It is important to note that Figure 15 refers to soluble proteins and that cooked seeds certainly contain most of their protein in an insoluble and denatured form.

Figure 15 shows the concentration of total soluble proteins in seeds of various legumes, quantified by the Bradford method (Nobel, 2000). The vertical bars represent the mean ± SD of at least three biological replicates.

The data presented in Figure 16 allow a comparison for each species between the polypeptide profiles of the soluble proteins of intact seeds and those of the corresponding cooked seeds. As expected, the profiles are completely different with the polypeptides that survived cooking in each case being present in the C lines. It should be noted that the lines in Figure 16 do not contain the same amount of protein. Rather, they correspond to a fixed amount of seed dry weight. Therefore, direct quantitative and qualitative comparisons can be made for each species.

Figure 16 shows representative polypeptide profiles obtained by SDS-PAGE (17.5% w/v acrylamide supplemented with 10% v/v glycerol; under reducing conditions) of soluble protein fractions extracted from raw (NC) and cooked (C) seeds. To allow both quantitative and qualitative comparative analyses, NC and C fractions were suspended and loaded onto the gel in equal volumes.

Embodiments of the invention relate primarily to Cicerarietinum, Glycine max and Lupinus albus.

Cell migration

The data presented in Figure 17 and Figure 18 show that soluble extracts prepared from C. arietinum, G. max, and L. albus inhibit HT29 cell migration. However, considerable differences were found between extracts, species, and condition (i.e. cooked or not) of the seeds. Thus, all non-protein extracts examined showed a marked inhibition on cell migration. Among soluble protein extracts prepared from raw seeds, cell migration inhibitory activity was almost negligible for soybean, intermediate for lupin, and slightly higher for chickpea. However, it was among soluble protein extracts prepared from cooked seeds that a striking and surprising result was achieved: unlike chickpea and soybean, cooking lupin seeds exhibits a dramatic effect in enhancing the cell migration inhibitory activity of the resulting soluble protein extract. Surprisingly, and again unlike chickpea and soybean, exactly the same effect is achieved with regard to the non-protein extract prepared from cooked lupin seeds. These data suggest that lupin seeds contain both proteins and low molecular weight non-protein compounds that exhibit HT29 cell migratory activity whose effect is significantly enhanced by pre-cooking the seeds, making them particularly suitable with regard to human nutrition.

This study may be the first to consider the effect of cooking (a topic of utmost importance in relation to human health and nutrition) legume seeds on the migratory potential of cancer cells.

Several hypotheses can be put forward to try to explain the increase in cell migration inhibitory activity of soluble protein and non-protein extracts prepared from cooked lupine seeds versus raw seeds.

1. A concentration effect. It is known that most proteins in lupin seeds are denatured and leach into the surrounding water during cooking. It is also known that many (if not most) of the low molecular weight metabolites are destroyed or leached into the boiling water. In the wound healing assays depicted in Figure 17 and 18, 100 µg soluble protein/mL or 10 mg soluble non-protein biomolecules/mL were used. Compared to extracts prepared from raw seeds, this means a large concentration effect for those metabolites and proteins that survived the cooking process.

2. A heat-induced dissociation effect is irreversible in the case of oligomeric proteins, unless the individual subunits or a remaining part of the original oligomer are soluble and exhibit increased bioactivity.

3. Deflamination may also occur in both the soluble protein fraction and the low molecular weight soluble non-protein metabolite fraction.

However, the same effect was not observed for chickpeas or soybeans, which is what makes lupins so 'special'.

Figure 17 shows representative images of HT29 cell migration assessed by wound healing assay (A). Cells were grown until 80% confluent. Cell migration was determined after 48 hours exposure of HT29 cells to buffer (control), non-protein extracts (10 mg/mL) or protein extracts (100 pg/mL) of the three seed species under study. Figure 18 shows the relative migration rates represented in (B). NP: soluble non-protein extract prepared from raw seeds; NPC: soluble non-protein extract prepared from cooked seeds; P: soluble protein extract prepared from raw seeds; PC: soluble protein extract prepared from cooked seeds. Vertical bars represent the mean ± SD of at least three biological replicates *P < 0.05.

Unlike soluble low molecular weight non-protein compounds, the soluble soy protein fraction prepared from raw seeds did not show significant inhibitory activity on HT29 cell migration. This may be surprising given the known presence of Bowman-Birk inhibitors (BBI) in G. max seeds. A possible explanation may lie in the fact that Fereidunian et al. (Fereidunian et al., 2014) purified 12 mg BBI/g soybean seed, a value corresponding to approximately 9% of the total soluble soy protein. A simple extrapolation reports that approximately 9 ug of soy BBI was included in the soluble protein extract prepared from raw soybeans used in the assay (Figures 17 and 18), an amount well below that used by Fereidunian et al.

Cell viability and proliferation

The results presented in Figure 19 show the effect of the different extracts, species and condition (i.e. cooked or not) of the seeds on HT29 cell proliferation. With the possible exception of the soluble non-protein extract prepared from chickpea seeds, it seems reasonable to conclude that all extracts exhibit low toxicity to HT29 cells, as these cells remain viable after a 24-h exposure. Furthermore, they do not appear to inhibit cell proliferation. Giron et al. (Giron-Calle et al., 2004) detected a potent inhibition of Caco-2 cell proliferation in the presence of soluble acetone metabolites extracted from chickpea seeds. Figure 19 represents a cell proliferation assay. HT29 cells were cultured for 24 h in the presence of buffer (control), non-protein extracts (10 mg/mL) or protein extracts (100 µg/mL) of the three seed species under study. NP: soluble non-protein extract prepared from raw seeds; NPC: soluble non-protein extract prepared from cooked seeds; P: soluble protein extract prepared from raw seeds; PC: soluble protein extract prepared from cooked seeds. Vertical bars represent the mean ± SD of at least three biological replicates. *P < 0.05. In contrast to the results shown in Figure 19, published data on legume seed proteins point to an inhibitory role at the level of cell proliferation. Fereidunian et al. (Fereidunian et al., 2014) observed a reduction greater than 50 % in HT29 cell proliferation in the presence of 200 pg BBI/mL. This value increases to over 80 % for a BBI concentration of 400 pg/mL. Chickpea BBI has been reported to inhibit breast cancer cell proliferation in vitro, while common bean, soybean and chickpea BBIs reduced prostate cancer cell proliferation in vitro as well (Magee et al., 2012).

Badawi and colleagues (Badawiet al.,2005) reported inhibition of prostate cancer cell proliferation and cell migration as well as secretion of MMP-9 and MMP-2 by water-soluble condensed black bean tannins. Parket al. (2013) reported that fisetin obtained from methanolic extract of Dalbergia odorifera inhibits MMP, proliferation and invasiveness of fibrosarcoma HT-1080 cells, and Aparicio-Fernandezet al. (2006) using human adenocarcinoma HeLa cells and human precancerous keratinocytes (HaCaT) found that 100% crude methanol extract of black Jamapa bean teguments shows an inhibitory effect on the proliferation of HeLa cancer cells but is less aggressive on HaCaT precancerous cells. Zhou et al. (1999) showed that soy isoflavones (genistein and daidzein) or soy phytochemical concentrate inhibited the growth of LNCaP, DU 145 and PC-3 prostate cancer cells in vitro.

Gelatinolytic activity in extracellular media of HT29

Under normal conditions (e.g., in the absence of inhibitors), cancer cells secrete MMP-9 and MP-2 into the external environment to degrade matrix proteins, thereby allowing the cells to migrate. Any condition that inhibits gelatinases will inhibit metastasis formation. The discovery of natural MMP inhibitors is therefore of potential interest.

As demonstrated in the previous section, some soluble extracts containing proteins and low molecular mass metabolites prepared from lupin, chickpea and soybean show a strong inhibitory effect on HT29 cell migration, but not on HT29 cell proliferation. This difference can be explained by their mechanism of action. One of the main factors associated with cancer cell invasion is the activity of MMP-9 and MMP-2 enzymes. Therefore, these activities were determined in the extracellular media of HT29 after a 48-hour exposure to various extracts (Figure 20), quantified by the fluorogenic DQ method.

Figure 20 shows the proteolytic activity of total MMP activity in HT29 extracellular media quantified by the fluorogenic DQ method. HT29 cells were cultured for 48 hours in the presence of buffer (control), non-protein extracts (10 mg/mL) or protein extracts (100 pg/mL) of the three seed species under study. NP: soluble non-protein extract prepared from raw seeds; NPC: soluble non-protein extract prepared from cooked seeds; P: soluble protein extract prepared from raw seeds; PC: soluble protein extract prepared from cooked seeds. Vertical bars represent the mean ± SD of at least three biological replicates *P < 0.05.

The results presented in Figure 20 show that all tested extracts exhibited some degree of inhibition on the level of gelatinase activities present in the extracellular media of HT29. However, compared to the control, extremely strong inhibition (between 80 and 90%) was found for all soluble protein extracts (whether prepared from raw or cooked seeds) and for the soluble protein mp extract prepared from raw soybeans.

Overall, it is interesting to note that HT29 cell migration was mainly affected by soluble non-protein metabolites (Figures 17 and 18), whereas the total MMP inhibition capacity in HT29 extracellular media was essentially conditioned by soluble proteins (Figure 20). These results suggest that legume proteins are more efficient in inhibiting MMP activity, whereas soluble non-protein metabolites seem to inhibit cell migration through other mechanism(s) of action.

The assays illustrated in Figure 20 measure total gelatinolytic activity, i.e. that of MMP-9 (92 kDa) and MMP-2 (72 kDa) combined. To separate these two activities, a different approach must be used, such as zymography or reverse zymography. The results of such experiments (data not shown) indicate that MMP-9 is the main target. Indeed, zymography of extracellular media collected after a 48-hour incubation of HT29 cells with each of the four extracts used in Figure 20 showed inhibitory activity affecting essentially MMP-9 and pro-MMP-9 (83 kDa).

This set of experiments seems to indicate that soluble proteins from lupin, chickpea and soybean inhibit HT29 cell migration essentially through the inhibition of MMP-9. However, it should be noted that different reports on the inhibitory activities of gelatinases produce contradictory results even within this same study. Thus, the experiments described immediately above indicate that both the protein fraction and the metabolite fraction from lupin, chickpea and soybean strongly inhibit MMP-9 but not MMP-2. In contrast, the experiment illustrated in Figure 32 shows that the total protein fraction from lupin strongly inhibits both gelatinases. Identical results are presented in the available literature. Soybean BBI inhibits both MMP-9 and MMP-2 at concentrations of 200 and 400 pg/mL (much higher concentrations than those used in the present study; Fereidunian et al., 2014). Bawadi et al. (2005) reported that a 24-hour incubation of Caco-2 colon cancer cells, MCF-7 and Hs578T breast cancer cells, and DU 145 prostate cancer cells with water-soluble black bean tannins resulted in a sharp decrease in the levels of active MMP-2 and MMP-9 secreted into the culture medium at tannin concentrations above 12<j>M. At 15<j>M, fisetin inhibited 50% of MMP-9 and other MMPs, but apparently not MMP-2 (Park et al., 2013). Phytin at 2.5 mM inhibited the expression of MMP-9, MMP-2, and other MMPs in phorbol-12-myristate 13-acetate (PMA)-stimulated Caco-2 colon cancer cells; (Kapralet al.,2012). Treatment of HT-1080 fibrosarcoma cells with soybean saponins inhibited mRNA expression and reduced the amounts of secreted MMP-2 and MMP-9 (Kang et al., 2008).

A striking observation is evident: with the exception of protease inhibitors (e.g., trypsin inhibitor and BBI), few experiments have been performed to assess the possible inhibitory effect of proteins on MMP activities.

Figure 21 illustrates another experiment in which the inhibitory activity of chickpea, soybean and lupin seed extracts on commercial MMP-9 was evaluated using the same seed mass in all cases, allowing a direct comparison and emulating the ingestion of these seeds. Figure 21 shows the inhibitory effect of soluble seed extracts on the proteolytic activity of MMP-9. The extracts were added to a reaction mixture containing commercial MMP-9 and the gelatinolytic activity was determined by the fluorogenic DQ assay. The volume of extract (100<j>L) added to each reaction mixture corresponded to the same seed mass. NP: soluble non-protein extract prepared from raw seeds; NPC: soluble non-protein extract prepared from cooked seeds; P: soluble protein extract prepared from raw seeds; PC: soluble protein extract prepared from cooked seeds. Vertical bars represent the mean ± SD of at least three biological replicates *P < 0.05.

Overall, this experiment shows that both the protein fraction and the non-protein fraction inhibit MMP-9 activity. This inhibitory action appears to be greater for the non-protein fraction and stronger in lupins than in chickpeas or soybeans. As expected, the effect of cooking the seeds decreased, but only marginally, the inhibitory power of all fractions. These results explain the effect observed in Figures 17 and 18, where the higher HT29 cell migration (as assessed by the wound healing assay) obtained for the non-protein and protein fractions of cooked lupin seeds, compared to raw seeds can be attributed to a concentration effect of the cooking-resistant active ingredients.

Isolation of potentially novel inhibitors of MMP-9 and MMP-2 from Lupinus albus seeds

Discovery of deflamin

To isolate the protein fractions found to be responsible for MMP-9 inhibition, L. albus proteins were separated according to their natural size and their inhibitory activity against MMP-9 was analyzed. Figures 22 to 24 depict the size exclusion chromatography (SEC) obtained for the extraction of total protein from L. albus, the electrophoretic protein profiles of the collected fractions F1 to F6, and the MMP-9 inhibitory activity for each fraction. Peptides, polypeptides, and proteins were extracted from L. albus seeds as described in the Materials and Methods section. The desalted extract, containing the total protein extract, was fractionated using the Akta system by size exclusion chromatography on a Superdex 75 column. Protein peaks were collected as fractions F1 to F6 (as depicted in Figure 22). Proteins/polypeptides were in certain embodiments separated according to their molecular size using a buffer containing urea and dithiothreitol (DTT), which allowed the separation of different low molecular mass fractions. Other buffers were tested which failed to allow efficient separation of proteins/polypeptides from L. albus in this size range, in agreement with previous results obtained in the laboratory. Each fraction was then tested for MMP-9 inhibitory activity using a DQ gelatin assay. The figure depicts the effect on MMP-9 activity of each fraction (F1-F6).

Figure 24 clearly shows that fraction 4 causes 100% inhibition of MMP catalytic activities. This fraction was later confirmed to contain deflamin.

Figures 22-24 show that peptides, polypeptides and proteins were extracted from L. albus seeds as described in the Materials and Methods section. The desalted extract, containing the total protein extract, was fractionated using the Akta system by size exclusion chromatography on a Superdex 75. Protein peaks were collected as fractions F1 to F6 (22) and separated by Tricine SDS-PAGE under reducing conditions (23). (24) Each fraction was tested for MMP-9 inhibitory activity using the DQ gelatin assay. Results are expressed in arbitrary fluorescence units and represent an average of three replicates ± SD. As seen in Figures 22 to 24, only fraction 4 showed a very high level of MMP-9 inhibition. This sample was subsequently fractionated by reverse phase (RP) HPLC to analyze the specific peptides responsible for this activity. Figures 25 to 27 show the HPLC profiles obtained for fraction 4. Essentially four peaks were obtained, each of which was analysed by SDS-PAGE and its MMP-9 inhibitory activity assessed by the DQ gelatin assay. Peak 2 exhibited the highest MMP-9 inhibitory activity and, to a lesser extent, so did peak 3 (Figure 27). Interestingly, at this stage, it is concluded that peak 2 is composed of at least two, probably more polypeptides (box 2 in Figure 25).

It should be noted that all protein fractions used in Figures 28 to 32 were previously subjected to boiling and low pH values, to determine their resistance to denaturation while simulating the digestive process, as well as part of the isolation procedure, and were subsequently used for HT29 cell wound closure assays and gelatinolytic assays, as well as zymographic analysis. This clearly indicates the resistance of deflamin to boiling and low pH values.

Figure 31 shows the gelatinolytic activities of HT29 cell media in the presence of the same protein fractions, while Figure 32 evaluates the activities of both MMP-9 and MMP-2 in zymographic separations. The results show that the polypeptides composing peak 2 collected from the HPLC chromatogram shown in Figure 25 are indeed potent MMP inhibitors, especially after heat treatments, and that they can inhibit both the catalytic activities of MMP-9 and MMP-2.

Analysis of HT29 extracellular media after a 48-hour incubation of the cells with spike 2 protein (100 µg protein/mL) revealed that deflamin inhibits both MMP-9 and MMP-2 activity, as shown by the fluorogenic gelatin DQ method (Figure 32) and by zymography (Figure 33). The black background (blue when viewed in color) visible in Figure 33 is due to intense staining of gelatin by Coomassie brilliant blue, which copolymerizes with acrylamide to form the gel matrix. The presence of active MMP enzymes or proenzymes, banded pro-MMPs, locally degrades gelatin embedded in the matrix, resulting in a white band. The presence of an inhibitor (e.g. deflamin) blocks the proteolytic action of MMPs, allowing staining of unaltered gelatin. Therefore, the absence of white bands in the 'deflamin' line in Figure 33 reveals that 'deflamin' at 100 µg/m L completely inhibited the activity of all forms of MMP present in the HT29 extracellular media and indicates that this inhibition was not reversed during the zymographic assay.

It should be noted that the fraction loaded onto the gels depicted in Figure 33 is not exactly deflamin. In fact, in certain aspects, deflamin is a protein fraction obtained from seeds following the isolation methodology detailed in Figure 4; in certain embodiments this is part of its definition. The bioactive fraction used in Figure 33 was purified using a different procedure.

Figure 33 shows representative images of the zymographic profiles of MMP-9 and MMP-2 enzymatic activity in HT29 extracellular media after a 48-hour exposure of the cells to 'deflamin' (100 µg protein/mL). Control: HT29 cells incubated for 48 hours in the absence of 'deflamin'.

Although capable of inhibiting cell invasion (Figure 30), the same concentration of 'deflamin' did not affect cell multiplication, suggesting absence of cytotoxicity.

Purification and characterization of deflamin from Lupinus albus.

An embodiment of the inventive methodology (see Figure 4) was developed to extract and purify deflamine from seeds, which is suitable for scale-up, allowing its mass production in industrial facilities. The procedure is described in detail in the Methods section.

It should be noted that the HPLC step referred to in the Methods section is used to fractionate the constituent polypeptides of deflamin and not to isolate deflamin.

Sequential extractions allow the isolation of deflamin from L. albus, which presents a higher MMPI activity than the total extracts.

Figure 34 shows a representative image of the polypeptide distribution between Lupinus albus seeds simply extracted with buffer (buffer extraction; BE) or after heat treatment (HT), and visualized by SDS-PAGE (left) and reverse gelatin zymography (right).

Protein extracts (50 µg/mL) were loaded onto 17.5% (w/v acrylamide) polyacrylamide gels, copolymerized with gelatin and MMP-9 in the case of reverse zymography.

The polypeptide band visible in both BE and HT lines with a molecular mass less than 20 kDa corresponds to deflamin. As shown in Figure 34, deflamin maintains its biological activity after heat treatment.

Preliminary results suggested that the MMPI protein fraction of L. albus (i.e. deflamin) was highly water-soluble and showed resistance to heat denaturation. Therefore, an isolation method using sequential precipitations (suitable for future scale-up to an industrial scale) was established.

Representative images of the electrophoretic profiles obtained after several sequential extractions to isolate the active protein fraction of MMPI are shown in Figure 35.

Figure 35 shows representative images of the polypeptide profiles obtained after each step of the purification method specified on the top of the gels. Protein samples (25 µg) were loaded onto 17.5% (w/v acrylamide) polyacrylamide gels. MW- molecular weight markers; BE-buffer extraction; HT s - heat treatment, supernatant; HT p - heat treatment, pellet; pH4 s - acid precipitation, supernatant; pH4 p - acid precipitation, pellet; 40% - 40% v/v ethanol containing 0.4 M NaCl, supernatant; 40% w - 40% v/v ethanol containing 0.4 M NaCl, pellet; 90% - 90% v/v ethanol overnight at -20 °C, pellet; and D - deflamin.

Analysis of the polypeptide profiles after each step of the isolation method depicted in Figure 4 revealed the gradual purification of a protein fraction with a molecular mass below 20 kDa, which was named deflamin. The main protein fractions obtained, i.e., total protein extract/buffer extraction (BE), heat-treated extract (HT) and isolated deflamin fraction, were compared for their MMPI activities using the DQ gelatin assay. The results obtained are shown in Figure 36.

At the tested protein concentration (50 µg/mL), Figure 36 shows that all samples were able to significantly inhibit MMP-9 proteolytic activity. However, significant differences (P<0.05) were observed between the samples tested, with the highest inhibition (P<0.05) detected for deflamin, which induced a greater than 80% reduction of MMP-9 activity.

That is, in Figure 36, buffer extraction (BE), heat treatment (HT) and deflamin (D) protein fractions were obtained from L. albus seeds and were used to evaluate their inhibitory activity on MMP-9 proteolytic activity in DQ-gelatin. The negative control (C) does not inhibit MMP-9, resulting in 100% proteolytic activity for this protease. Protein samples were added at a concentration of 50 µg/ml and gelatinolytic activity was measured by the DQ fluorogenic assay. MMP activities are expressed as relative fluorescence in % of controls, and represent the averages of at least three replicate experiments (n=3)±SD. *P<0.05, **P<0.001.

In summary, as deflamin is gradually purified, its inhibitory effect as MMPI increases.

Deflamina from L. albus is more effective in inhibiting colon cancer cell invasion and proliferation

Note: The word 'more' in this title was used with a double meaning.

1- As the isolation methodology proceeds from the initial total protein extract to purified deflamin, its biological activity gradually increases, reaching a maximum with isolated deflamin. Since the comparative tests performed after each purification step use identical amounts of proteins from each sample, as the degree of purification of deflamin increases, the amount of deflamin relative to the total protein in each fraction also increases, justifying the increase in deflamin bioactivity when moving from less pure to purer fractions of deflamin.

2- Deflamin is a poor inhibitor of cell multiplication (meaning low cytotoxicity; see below; compare figures 39 to 42), but a potent inhibitor of colon cancer cell invasion and proliferation.

The activities of isolated deflamin in HT29 cells were characterized while comparing with the total extract and the heat-treated extract of L. albus. Figures 37 to 38 show the effect of each of these protein fractions on HT29 cell migration after 48 hours of exposure to the total extract, the heat-treated extract, and isolated deflamin (50 µg protein/mL).

Figures 37 and 38 show HT29 cell migration after exposure to buffer extraction (BE), heat treatment (HT) and isolated deflamin (D), as determined by wound healing assays. (Figure 37) - Relative migration rates. Values are the averages of at least three replicate experiments SD, and are expressed as % wound closure relative to day 0. (Figure 38) - Examples of cell migration obtained for the highest inhibitory protein fraction, i.e. deflamin. Cells were grown to 80% confluence and the monolayer was scratched with a pipette tip (day 0). Cells were then exposed to 50 µg protein/ml extract and cell migration was recorded after 48 h. *P<0.05, **P<0.001.

These results show that deflamin exhibited the highest inhibition on migration rates compared to the other protein samples studied (P<0.05), inducing a 60% reduction in cell migration rates. Furthermore, at the concentration used, HT and deflamin were statistically different from the controls (P<0.05), while the BE sample remained statistically similar to the controls (P>0.05). This result may explain, at least to some extent, why deflamin was not discovered earlier.

The deflamin activities of L. albus are dose-dependent.

The methodology developed to isolate deflamin (depicted in Figure 4 and described in detail in the Methods section) proved to be highly efficient in isolating the MMPI fraction responsible for the MMPI activities of L. albus. The effect of this fraction (i.e. deflamin) was further tested to see if it was dose-dependent. Four different concentrations of deflamin (100, 50, 10 and 5 µg/mL) were tested using the DQ gelatin method and the wound invasion assay in HT29 colon cancer cells and the results are expressed in Figures 39, 40 and 41, respectively.

Figure 39 shows the effect of different concentrations of deflamin (100, 50, 10 and 5 µg/mL) on gelatinase activities. Four different concentrations of deflamin were obtained from L. albus seeds and were used to evaluate its inhibitory activity on the proteolytic activity of MMP-9 in DQ-gelatin. The negative control (C) does not inhibit MMP-9, resulting in 100% proteolytic activity for this protease. Deflamin was added at concentrations of 100, 50, 10 and 5 µg/mL and gelatinolytic activity was measured by the DQ fluorogenic assay. Gelatinase activities are expressed as relative fluorescence in % of controls, and represent the averages of at least three replicate experiments (n = 3) ± SD.

Figures 40-41 show HT29 cell migration after exposure to different concentrations of deflamin, as determined by wound healing assays. (Figure 40) - Relative migration rates. Values are the averages of at least three replicate experiments ± SD, and are expressed as % wound closure relative to time 0. (Figure 41) - Examples of cell migration obtained for the four deflamin concentrations tested plus the control. Cells were grown to 80% confluence and the monolayer was scratched with a pipette tip (day 0). Cells were then exposed to deflamin at 100, 50, 10 and 5 µg/m L and cell migration was recorded after 48 h. ** represents P<0.001 and represents P<0.05 compared to controls.

Figure 40 shows that all tested concentrations (100, 50, 10 and 5 µg/mL) were able to significantly inhibit the proteolytic activity of gelatinase (P<0.001), compared to controls. However, the level of inhibition at each concentration differed, in a dose-dependent manner, with the highest inhibition detected for 100 µg/m L deflamin, which induced a greater than 90% reduction of gelatinolytic activity. Figures 40 and 41 show the ability of deflamin to inhibit colon cancer cell invasion.

Figures 40 and 41 show that the ability of deflamin to inhibit HT29 cell migration gradually increases with deflamin concentration, from 5 to 50 µg deflamin/mL. However, for the highest deflamin concentration studied (100 µg/mL), an interesting and different result was obtained: HT29 cells were completely detached at 100 µg deflamin/mL (see Figures 40 and 41), justifying the absence of this concentration in the graph in Figure 40. Deflamin from L. albus does not reduce cell growth or metabolism in colon cancer cells.

To analyze whether deflamin was cytotoxic to HT29 cells and whether it influenced cell growth, the same concentrations were analyzed using a standard cell proliferation assay.

Figure 42 illustrates the number of live HT29 cells after growth in the presence of different concentrations of deflamin (100, 50, 10 and 5 µg/mL), determined after staining with MTT (which can only be metabolized by living cells). The results show that a 2-day exposure to deflamin did not induce a significant reduction (P>0.001) in cell growth or in the number of live cells, when compared to controls. Furthermore, there were no visible cytotoxic effects. This result indicates that deflamin is relatively non-cytotoxic to HT29 cells even at 100 µg deflamin/mL.

Figure 42 shows cell proliferation after 24 h exposure to different concentrations of deflamin. Cells were cultured for 24 h in the presence of 100, 50, 10 and 5 µg of protein/ml of extract and stained with MTT. Values represented are the means of at least three replicate experiments (n=3) ± SD and are expressed as a percentage of the control.

Since cell growth was not affected by deflamin, minimum inhibitory concentrations (MICs) for cell invasion, cell detachment, MMP inhibition, and cell growth were determined.

The results presented in Figure 42 show that a 2-day exposure to deflamin did not induce a significant reduction (P>0.001) in cell growth or in the number of live cells, when compared to controls. Furthermore, there are no visible cytotoxic effects (data not shown). This result indicates that deflamin is relatively nontoxic to HT29 cells even at 100 µg deflamin/mL and does not interfere with normal cellular metabolism.

However, for the highest dose of deflamin, HT29 cells were detached (Figures 40-41) which if not related to any degree of cytotoxicity, could possibly be related to cell adhesion. It is known that cells adhere to a substrate by their integrins, i.e., transmembrane receptors that bridge cell-cell and cell-extracellular matrix (ECM) interactions. An important function of integrins on cells in tissue culture is their role in cell migration. Recent studies have shown that integrins are modulated by tumor progression and metastasis and are strongly linked to both MMP-9 and MMP-2 activities. However, few studies have shown a cell detachment effect in the presence of MMPI. These results suggest that the mode of action of deflamin involves a broader mechanism that induces more than just gelatinase inhibition. The observation that the highest dose of deflamin tested (i.e., 100 µg/mL) did not cause apparent cytotoxic effects suggests that it is not harmful to the digestive system and can therefore be used in preventive diets without any side effects.

Since cell growth was not affected by deflamin, the minimum inhibitory concentrations (MIC) and the concentration necessary to induce a 50% effect (EC50) of deflamin were determined in different tests: cell growth, cell invasion, cell separation and MMP inhibition. The results are presented in Table 1.

Table 1. Determination of minimum inhibitory concentrations (MIC) and concentrations inducing a 50% effect (EC50) for deflamin bioactivities on cell growth, cell invasion, cell detachment, and MMP inhibition. Results are expressed in µg/mL. ND: not determined.

Under the conditions tested, the MIC values for cell invasion and MMP inhibition were lower than the MICs found for the other parameters studied. A deflamin concentration of 10 µg/mL-1 was found to be sufficient to significantly inhibit 50% of cell invasion (P<0.05), making it the EC50 value for cell invasion. For MMP inhibition, the EC50 is 10 µg deflamin/mL as well. This is in agreement with the high relationship between MMP-9 activities and cell invasion, and corroborates that MMP inhibition is at least one of the main modes of action of deflamin. However, the MIC levels determined for cell invasion were lower than 10 µg/mL, but were not statistically significant (P<0.05) at 5 µg/mL, whereas MMPs were already significantly inhibited in the presence of 5 µg/mL, which is why the MIC values are lower at this concentration. With lower MIC values for MMP inhibition than for cell invasion, MMP inhibition is expected to only induce a notable reduction in cell invasion after a certain degree of inhibition. On the other hand, the MIC for cell detachment was only achieved for >100 µg/mL, at the highest deflamin concentrations tested, at which no significant cellular toxicity was detected.

Clearly, MMP inhibition and reduction in cell invasion are the strongest activities of deflamin, compared to impairment of cell growth or cytotoxicity which were only affected to a very low extent. This could be of significant importance. MMPIs with high specificity and low side effects have been very difficult to find, and most clinical trials yielded unsatisfactory results. On the other hand, in diets to prevent cancer, reducing MMP-9 and -2 activities in low amounts is desirable, but low levels of toxicity against colon cells even at higher doses are a very important requirement. Compared to low molecular mass compounds such as polyphenols, polypeptide MMPIs offer several advantages such as high specificity and low toxicity. Compared to traditional cancer treatments such as chemotherapy or radioactive treatment, peptides and small proteins with high specificity against tumor promoters such as MMPs that simultaneously exhibit low toxicity may represent the future in cancer treatment/prevention.

Deflamin from L. albus is a low molecular mass complex of B-conglutin and 5-conglutin fragments.

Since the deflamin fraction analyzed in Figure 35 appeared to be composed of more than one polypeptide band, the same sample (i.e., isolated deflamin) was fractionated by electrophoresis, under reducing and non-reducing conditions to detect its polypeptide composition, as well as to determine the potential presence of disulfide bonds. The results obtained are presented in Figure 43.

Figure 43 shows the polypeptide profile of deflamin under reducing and non-reducing conditions. The image is representative of the polypeptide distribution of deflamin isolated from Lupinus albus seeds separated by SDS-PAGE under reducing (R) and non-reducing (NR) conditions. Deflamin (50 pg/mL) was loaded onto a 17.5% (w/v) polyacrylamide gel with reducing buffer (100 mM Tris-HCl, pH 6.8, containing 100 mM mercaptoethanol, 2% (w/v) SDS, 15% (v/v) glycerol, and 0.006% (w/v) m-cresol purple) and non-reducing buffer (100 mM Tris-HCl, pH 6.8, containing 2% (w/v) SdS, 15% (v/v) glycerol, and 0.006% (w/v) m-cresol purple).

Deflamin was further analyzed by reverse phase HPLC, to separate its different polypeptide constituents. Figures 44 to 46 show the chromatographic profiles obtained at 280 and 214 nm, and the respective electrophoretic patterns. The results show the presence of the standard bands of deflamin, dispersed along peaks 2 to 4.

The peak at 280 nm from the reversed-phase HPLC column at 45 to 50 minutes contains neither protein nor bioactivity. For this reason, its study was discontinued.

To determine the peak fraction with the highest activity, the MMPI activities of the 4 peaks were further determined (Figures 47 and 48), using the gelatin DQ assay and cell invasion assays. The results are shown in Figures 47 and 48, respectively.

The results seem to suggest that all the selected peaks showed some degree of MMP inhibition and also inhibited invasion, but peak 3 was the one that showed the highest observed activities. The 4 selected peaks were further analyzed by mass spectrometry, for their identification.

Deflamin is the first protein MMPI that can be purified by a cost-effective and scalable procedure.

Legume seeds have long been recognized to contain a variety of proteinaceous enzyme inhibitors, such as trypsin inhibitors and BBIs. However, although the presence of naturally occurring MMPIs can be considered ubiquitous in plant tissues, they all present several disadvantages when considering their production for clinical purposes: toxicity at high concentrations or prolonged exposures, chemical inactivation (e.g., denaturation) or destruction (e.g., proteolysis) upon cooking and/or by the digestive process; the lack of a specific target; and a high-cost and inefficient isolation method, which prevents MMPIs in general from being subjected to efficient scale-up at an industrial level. The isolated deflamin reported in this work overcomes all these limitations, as it is resistant to boiling and is an enzyme inhibitor; on the other hand, the developed sequential precipitation method is simple, cost-effective, and easily applicable in an industrial context. As a mixture of edible polypeptides naturally occurring in lupin seeds, it does not pose the problem of toxicity at higher doses than most phenolic compounds and other bioactive secondary metabolites, and the use of acid and ethanol precipitations ensures the removal of potential toxic contaminants as well as higher molecular mass proteins.

The yield of the extraction procedure is presented in Table 2. The results show that for every 100 g of dry seed, 100 mg of deflamin are obtained, which corresponds to approximately 0.5% of the total protein content of the seed.

Table 2. Yield (expressed in %) of dry seeds of L. albus in deflamina.

These results corroborate that deflamin is indeed present in very low concentrations in the seed, with the lowest activities being observed in the BE fractions. It is also suggested that consumption of lupin alone may not provide enough deflamin to induce the same effects that its isolated form can provide.

It is important to mention that the low yields of the extraction procedure are not due to the method itself, but rather to the low amount of deflamin in the seed. Still, the relative ease of the procedure and the possibility of scaling up to larger quantities, in a cost-effective and simple manner, using flow filtration and centrifugation, as well as low-cost reagents such as ethanol, suggest a high potential for industrial production.

Furthermore, given the potential of deflamin, the method developed in the context of the present invention is also of particular importance to pursue a more thorough characterization of this protein fraction, such as its identity, dose-response effects and EC50, as well as clinical and preclinical studies. Research on other lupin varieties and species, on seeds of other legume species and on seeds of other plant families, as well as changes in their growth conditions, may provide an increase in the amount of deflamin in the seed. The fact that isolated deflamin is efficient in inhibiting MMP-9 and reducing cancer cell invasion suggests its high potential for a wide range of clinical uses. Since MMP-9 is closely involved in inflammation as well as in oncological processes, deflamin MMPI could possibly be used both in anticancer approaches and in anti-inflammatory treatments, especially those related to the digestive tract, such as colorectal cancer (CRC) and inflammatory bowel diseases (IBD).

Identification of the polypeptides comprising deflamin from L. albus by mass spectrometry - I

Deflamin was isolated from Lupinus albus seeds following the methodology detailed in the example in Figure 4. Deflamin was subsequently fractionated by RP-HPLC into the four peaks depicted below. The polypeptides comprising each of these peaks as indicated in Figure 46 were identified by mass spectrometry. The results obtained are presented in (peak 1), (peak 2), (peak 3) and (peak 4) below and indicate the presence of the following peaks:

Peak 1: Conglutin beta fragments 1,2, 3, 4, 5 and 7. Note that p-conglutin fragments spanning the entire molecule were detected. No 5-conglutin fragments were detected in peak 1.

Peak 2: fragments of conglutin beta 1, 2 and 6, and large chain of conglutin delta-2. Note that fragments of p-conglutin and 5-conglutin were detected that encompass the entire molecule of their precursors.

Peak 3: fragments of conglutin beta 1 and large chain of conglutin delta-2. Note that fragments of p-conglutin and 5-conglutin were detected that encompass the entire molecule of their precursors.

Peak 4: Conglutin beta fragments 1, 2, 3, 6 and 7. Note that p-conglutin fragments spanning the entire molecule were detected. No 5-conglutin fragments were detected in peak 1.

That is, Figures 44 to 46 show representative images of the fractionation of deflamin by RP-HPLC and SDS-PAGE into its constituent polypeptides. Deflamin was extracted and purified from Lupinus albus seeds by the methodology developed and illustrated in Figure 4. (Figures 44) and (45) - reverse phase (RP)-HPLC chromatography of deflamin monitored at 214 nm (Figure 44) and at 280 nm (Figure 45). (Figure 46) Polypeptide profile of each peak collected from the HPLC run as visualized by SDS-PAGE (17.5% w/v acrylamide) performed under reducing conditions (R-SDS-PAGE). Protein peaks (50 µg) were loaded onto 17.5% (w/v) polyacrylamide gels. Total polypeptides were stained with Coomassie brilliant blue.

Figure 47 shows the proteolytic activity of MMP-9 from fractions 1 to 4 obtained by HPLC fractionation of deflamin. Protein samples were added at a concentration of 25 µg/ml and gelatinolytic activity was measured by the fluorogenic DQ assay. Results are expressed in arbitrary fluorescence units and represent the averages of at least three replication experiments (n=3) ± SD. ** represents P<0.001 and * represents P<0.05 when compared to controls.

Figure 48 shows HT29 cell migration after exposure to each of the selected deflamin peaks collected after RP-HPLC fractionation - relative migration rates. Values are the averages of at least three replication experiments ± SD, and are expressed as % wound closure relative to day 0. Cells were grown to 80% confluence and the monolayer was scraped with a pipette tip (day 0). Cells were then exposed to 25 µg protein/ml extract and cell migration was recorded after 48 h. ** represents P<0.05.

Two main conclusions can be drawn from all these results:

- L. albus deflamin is composed of a complex mixture of p-conglutin and 5-conglutin fragments;

- p-conglutin and 5-conglutin are both precursors of deflamin from L. albus.

Mass spectrometry analysis of peak components (see Figures 44-46 and SEQ ID NO: 194-197) of deflamin from L. albus.Fractionation of deflamin from L. albus by RP-HPLC resulted in 4 peaks, each containing polypeptides that were identified by mass spectrometry.

The color code indicates the confidence of the peptides:

• green residues correspond to peptides with 95% confidence;

• yellow for peptides with confidence between 50 and 95%;

• red for peptides with confidence below 50%;

• and grey corresponds to unidentified waste.

Division of deflamina from L. albus into two fractions with Ca2+ and Mg2+

Deflamin isolated from L. albus comprises two fractions (one derived from p-conglutin, the other from 8-conglutin) that are not separated by HPLC, but can in fact be separated by the addition of Ca2+ and Mg2+, since p-conglutin binds to these cations, self-aggregates and becomes insoluble.

Methodology

The deflamin fraction isolated by HPLC was lyophilized and dissolved in water and shaken. CaCl2 and MgCl2 from a stock solution were added to the deflamin solution to reach 10 mM CaCl2 and 10 mM MgCl2, followed by shaking for 4 h or overnight. The suspension was centrifuged for 1 h at 30,000 g. The supernatant and pellet were desalted on NAP-10 columns previously equilibrated in water and lyophilized for future analysis. All operations were performed at 4 °C.

Both lyophilized fractions were used for electrophoretic separation, and their MMP-9 inhibitory activity was determined using the fluorogenic DQ-gelatin assay, the wound healing assay in HT29 cells, and substrate zymography, as described previously.

Results

Figure 49 shows the electrophoretic profiles of both Ca/Mg-soluble and insoluble deflamin fractions. D- deflamin isolated by RP-HPLC; Ds- fraction of deflamin that did not precipitate in the presence of cations (supernatant); Dp- fraction of deflamin that precipitated in the presence of cations (sediment).

Splitting L. albus deflamin into two fractions influences its anticancer activity

When split into two fractions, deflamin from L. albus loses its cell invasion inhibition activity. But when these fractions are combined again, it regains some of the initial activity. These results, presented in Figure 50, suggest that the two fractions are essential for the expression of deflamin bioactivity.

Figure 50 shows the inhibition of cell invasion in HT29 cells by deflamin and deflamin subfractions, precipitated or not with a solution of 10 mM CaCh and 10 mM MgCl2. C - control; D - deflamin; Ds - fraction of deflamin that did not precipitate in the presence of cations (supernatant); Dp - fraction of deflamin that precipitated in the presence of cations (pellet); and Ds+p - both fractions, Dp and Ds, combined in equal parts.

Influence of L. albus deflamin on the expression of genes related to inflammation and tumor invasion

Based on the natural history of certain diseases and epidemiological studies, a strong association has been established between certain chronic inflammatory conditions and the eventual development of tumors. Certain genes are expressed to an increased extent during these pathologies and the proteins they express are often recognized as biomarkers of inflammation and tumor genesis. Examples include several types of MMPs, such as MMP-1, MMP-7, MMP-9 and MMP-2, as well as tumor necrosis factor alpha (TNFa), a cell signaling protein (cytokine) involved in systemic inflammation, nuclear factor kappa B (NF-kB), a protein complex that controls DNA transcription, and TIMP1, an endogenous tissue inhibitor of metalloproteinases, as well as the inflammatory mediators iNOS and COX2. Some of these were tested in Figure 51.

Methods

HT29 cells were exposed to 50 pL/g deflamin and allowed to grow for 24 h. Total RNA was extracted from HT29 cells using the NZY Total RNA Extraction Kit (Nzytech) with some modifications, and quantification was performed on a Synergy HT Multiplate reader, with Gene5 software, using a Take 3 multi-volume plate (Bio-Tek Instruments Inc. Winooski, USA). For reverse transcription, the RevertAid Reverse Transcriptase oligod(T) kit (Thermo Scientific) was used according to the manufacturer's recommendations.

A set of primers for specific genes related to inflammation and cancer invasion was used. When amplification confirmed expression, transcripts were quantified by real-time PCR (qPCR), performed in 20 µl reaction volumes composed of cDNA derived from 2 pg of RNA, 0.5 pM of gene-specific primers (Table 3), and SsoFast EvaGreen Supermixes (Bio-Rad, Hercules, CA) using an iQ5 real-time thermal cycler (BioRad, Hercules, CA). Reaction conditions for cycling were 95 °C for 3 min followed by 40 cycles at 95 °C for 10 s, 61 °C for 25 s, and 72 °C for 30 s. Melting curves were generated in each case to confirm the amplification of single products and the absence of primer dimerization. Each analysis was performed in triplicate reactions, each in three biological replicates (n=9, where each replicate is the average of three technical measurements). The corresponding quantification cycles (Cq) obtained by the iQ5 optical system software (Bio-Rad, Hercules, CA) were exported to an MS Excel spreadsheet (Microsoft Inc.) for quantification. The Cq values of each gene of interest were normalized to actin (ct). Relative gene expression values under deflamin exposures are presented in Figure 51 below, as fold change values relative to control conditions.

Table 3 - Specific primer pairs used to assess transcription of the genes under study.

NM_001145938 HsMMPl TTCGGGGAGAAGTGATGTTC TTGTGGCCAGAAAACAGAAA NM.002423 HsMMP7 GTATGGGACATTCCTCTGATCC CCAATGAATGAATGAATGGATG NM.004994 HsMMP9 GCACGACGTCTTCCAGTACC CAGGATGTCATAGGTCACGTAGC NM.000963 HsCOX-2 TGAGCATCTACGGTTTGCTG AACT G CTCAT CAC CCCATTC NM_001165412 HsNFKpi TGGAGTCTGGGAAGGATTTG CGAAGCTGGACAAACACAGA NM_003254 HsTIMPl AGGCTCTGAAAAGGGCTTCC GGACACTGTGCAGGCTTCAG

Results

The results presented in Figure 51 suggest that deflamin does not significantly alter the expression of specific genes related to inflammation and tumor genesis, nor to MMP-9, corroborating the hypothesis that deflamin does not have a significant direct activity on gene expression, but rather acts through direct interaction with MMP-9. Of note is a small inhibition of the expression of genes associated with MMP-1 and MMP-7, which generally show increased expression during advanced metastatic disease.

Figure 51 shows in other words the transcriptional responses to deflamin in HT29 cells.

Deflamin activity in food products

Deflamin from L. albus was used in the manufacture of baked crackers. It is important to note that during this process, temperatures are raised up to 180 °C. However, the results obtained in Figure 52 show that deflamin maintained its cancer cell invasion inhibitory activity in baked crackers.

Figure 52 shows the inhibition of cell invasion in HT29 cells by protein extracts prepared from biscuits containing (D) or not (C) deflamin. C- control; CF- uncooked control biscuits; CS - cooked biscuits; DF - uncooked biscuits containing deflamin; DS- cooked biscuits containing deflamin.

Isolation and characterization of deflamin from L. albus-II

Analysis of deflamin by RP-HPLC and electrophoresis

Figure 53 shows representative images of the fractionation of deflamin from L. albus by RP-HPLC (using acetonitrile and TFA) and the corresponding SDS-PAGE. Deflamin was extracted and purified from Lupinus albus seeds.

Figure 53 shows HPLC and electrophoretic profiles. A - reverse phase (RP)-HPLC chromatography monitored at 280 nm. The deflamin peak is identified by the arrow. B - polypeptide profile of the deflamin peak collected from the HPLC run visualized by SDS-PAGE (17.5% w/v acrylamide) performed under reducing conditions. The protein peak eluting at 30 min (50 |µg) was loaded on a 17.5% (w/v) polyacrylamide gel (acrylamide) and stained with Coomassie brilliant blue.

Mass spectrometry analysis of the two deflamin fragments by MALDI-TOF - II

Methods

The instrumentation comprised Bruker-Daltonics Ultraflex II MALDI-TOF TOF, equipped with a LIFT cell and N2 laser.

Ionization: MALDI

Mode of operation

The mass spectrometer was operated with positive polarity in linear mode and spectra were acquired in the m/z range 5000–20000. A total of 1000 spectra were acquired at each spot position at a laser frequency of 50 Hz. External calibration: a Bruker protein calibration standard I ([M+H]+ of insulin (5734.51 m/z); ubiquitin I (8565.76 m/z), cytochrome c (12360.97 m/z), myoglobin (16952.30 m/z); [M+2H]2+ of cytochrome c (6180.99 m/z) and myoglobin (8476.65 m/z)).

Results

MALDI-TOF analysis of deflamin from L. albus clearly shows the presence of two blocks of polypeptide fragments with masses of 13 and 17 kDa, composed of several homologous fragments with slightly different lengths (Figure 54).

Figure 54 shows, in other words, deflamin from L. albus analysed by MALDI-TOF MS.

Antimicrobial activity of deflamin

The antimicrobial activity of deflamin was tested against the following microorganisms and found to be nil:

Gram+ bacteria:

•Listeria monocytogenes (NCTC 11994)

•Bacillus cereus (NCTC 7464)

•Staphylococcus aureus (NCTC 10788)

Gram-negative bacteria:

•Escherichia coli (NCTC 9001)

•Salmonella Goldcoast(NCTC 13175).

Fungus:

•Alternaria alternata

• Botrytis cinerea

• Phaeoacremonium aleophilum

• Alternariasp.

•Penicillium sp.

Animal models of colitis

Preliminary results on the anticolitis effects of deflamina

Preliminary results on the bioactivity of deflamin on colitis are shown in Figure 55. Preliminary results on the effects on colitis-induced lesions compared to control and deflamin treatments in mouse models. The trials were performed as a preliminary study with only one concentration of deflamin introduced into their diets after induction of colitis.

Administration of deflamin reduces the macroscopic and functional signs of colitis injury.

To verify the anti-inflammatory effects of deflamin, its effects were tested in mice with TNBS-induced colitis using two types of administration, oral (p.o.) and intraperitoneal (i.p.) injection. Deflamin was administered 3 h after the induction of colitis. Figures 56 and 57 show the effect of deflamin on colonic length (cm) and extent of intestinal injury (cm), respectively.

Figure 56 shows the effect of deflamin administration on colon length (cm). Sham group (n = 6), EtOH group (n = 6), TNBS group (n = 10), TNBS+deflamin p.o. group (n = 9), and TNBS+deflamin i.p. group (n = 10). #P<0.001 vs sham group, *P<0.001 vs TNBS group.

Figure 57 shows the effect of deflamin administration on the extent of intestinal injury (cm). Sham group (n = 6), EtOH group (n = 6), TNBS group (n = 10), TNBS+deflamin p.o. group (n = 9), TNBS+deflamin i.p. group (n = 10). #P<0.001 vs control group, *P<0.01 vs TNBS group.

Figure 58 shows the effect of deflamin administration on macroscopic observation of the colon. (A) Sham group (n=6), (B) EtOH group (n=6), (C) TNBS group (n=8), (D) TNBS+deflamin p.o. group (n=9). #P<0.001 vs sham group, *P<0.01 vs TNBS group.

Figure 59 shows the effect of deflamin administration on macroscopic observation of the colon. (A) Sham group (n = 6), (B) EtOH group (n 6), (C) TNDS group (n 0), (D) TNBS+deflamin i.p. group (n = 10). #P<0.001 vs sham group, *P<0.01 vs TNBS group.

Animals in the sham and ethanol groups showed no macroscopic signs of colonic injury, and no mortality, whereas intracolonic injection of TNBS/EtOH led to a highly significant (P<0.05) decrease in colon length and an increase in the extent of visible injury (ulcer formation). In the deflamin treatment group (both p.o. and i.p.), all macroscopic signs of colonic injury were significantly reduced, compared to the TNBS group (Figures 56 and 57).

The results show that deflamin administration (both i.p. and p.o.) produced an overall reduction in colonic inflammation and, in the case of p.o. administration, a significant attenuation (P<0.05) of the reduction in colon length and a significant reduction (P<0.05) in the extent of visible injury (colitis formation). These differences can be easily perceived by microscopic observation of fresh and rinsed colons immediately after collection at the end of the experiments. Figures 58 and 59 show representative images of these microscopic observations obtained by surgical microscope. Four days after intracolonic administration of TNBS, the colons appeared flaccid and filled with liquid feces. Image observations show a clear attenuation of colon injury in deflamin-treated animals compared to TNBS-induced colitis (Figures 42 and 43). Regarding the type of administration, some differences were observed between the two types of administration when comparing the morphological signs of colitis and its extension. Colonic injury was greater in ip administrations (Table 1 and Figure 59; P<0.05). Macroscopic observations also corroborate this trend when comparing the two treatments (i.e. p.o. vs. i.p.; Figures 58 and 59).

Deflamin attenuates the histological features and inflammatory markers of colitis lesions.

To clarify the mechanisms responsible for the effect of deflamin, the severity of histological lesions is analyzed and the presence of specific markers of inflammation and cancer progression, COX2 and iNOS, is also determined.

Decreased expression of cyclooxygenase-2 (COX-2) and nitric oxide synthase (iNOS) in colon tissue from experimentally induced colitis (D'Acquisto et al, 2002), is associated with reduced severity of colitis and alleviation of macroscopic and microscopic signs of the disease. Specifically in the gut, upregulation of NO production through expression of inducible iNOS represents part of a rapid intestinal antibacterial response; however, NO has also been associated with the initiation and maintenance of inflammation in human IBD (Kolios et al, 2004) and studies have shown that the level of iNOS-derived NO correlates well with disease activity in ulcerative colitis (Cross & Wilson, 2003). Nitric oxide is produced and released locally in much greater amounts in the inflamed gut than in the non-inflamed gut and has in fact been suggested as a new clinical biomarker for the diagnosis and monitoring of IBD patients (Lundberg et al, 2005). Similar to COX-2, immunostaining assays in the experimental colitis study showed that there was indeed increased expression of iNOS in animals undergoing colitis induction.

Histological evaluations are presented in Figure 60.

Figure 60 shows the effect of deflamin administration on the histological features of colon inflammation. (A) Sham group (n = 6), (B) EtOH group (n = 6), (C) TNBS group (n = 8), (D) TNBS deflamin p.o. group (15 mg/kg, n = 9), (E) Tn Bs deflamin i.p. group (10 mg/kg, n = 10).

While control samples showed a normal colon without lesions, a mucosa of uniform thickness, normal crypt architecture and no signs of inflammation, in the TNBS-treated colons exhibited severe ulceration with crypt loss and a thinner mucosa with a marked neutrophil infiltration, equivalent to a score of 3.

In comparison, samples from animals administered deflamin showed more moderate lesions with partially intact crypts and some neutrophil infiltration, indicating a damage score of less than 2, particularly with p.o. administrations.

Figure 61 shows the effect of deflamin administration on COX-2 and iNOS expression in colon tissue. (A) - COX-2 expression: (1) sham group, (2) TNBS group, (3) TNES+deflamin orally group, (4) TNBS deflamin ip. group; (B) - iNOS expression: (1) sham group, (2) TNBS group, (3) TNES deflamin p.o. group, (4) TNBS geflamin i.p. group.

Figure 61 shows the expression of COX-2 and iNOS in colonic tissues. The results show that TNBS treatment induced a marked increase in the expression of COX-2 and iNOS along the remaining crypts, indicated by the brown color compared to control samples. In agreement with histological observations, deflamin-treated animals exhibited reduced staining for both COX-2 and iNOS, indicating a reduction in inflammatory processes in the colon, especially in the p.o. group.

The effects of deflamin are induced in preventive and curative treatments

Since p.o. administrations induced better outcomes, it is further tested whether a preventive approach through oral dietary supplementation with deflamin, rather than a curative approach alone, would present similar effects.

Table 4 shows the morphological and functional signs of colitis in both treatments, curative (D - p.o. administration of deflamin 3 h after TNBS induction) and preventive (Dp - o.p. of deflamin 3 days before TNDS administration).

Animals in the sham and ethanol groups showed no macroscopic signs of colon injury, and no mortality, whereas intracolonic injection of TNBS/EtOH led to a highly significant (P<0.05) decrease in colon length and an increase in the extent of visible injury (ulcer formation) and severity of diarrhea, exhibiting a 50% mortality rate.

Table 4. Morphological and functional observations of the colon, immediately after collection, for both curative (D - p.o. administration of deflamin 3 hours after TNBS induction) and preventive (Dp - p.o. administration of deflamin 3 days before TNBS administration) treatments. *p < 0.001 versus sham group; #p < 0.05 versus TNBS group; and ^p < 0.05 D group versus Dp group.

The results show that deflamin administration, both curative and preventive, led to an overall reduction in colonic inflammation, with a significant attenuation (P<0.05) of colonic length and a significant reduction (P<0.05) in the extent of visible injury (ulcer formation). In addition, a significant decrease (P<0.05) was observed in the severity of diarrhea, mortality rates, and overall histological features of colonic inflammation compared to the TNBS group. Furthermore, there were no significant differences (P>0.05) observed between the preventive and curative approaches and between both deflamin treatments and controls.

Deflamin reduces MMP-9 activity in fresh colon tissues

To test whether the anti-inflammatory effects observed in deflamin treatments were due to the inhibition of MMP-9 and/or MMP-2, the gelatinolytic activities of these enzymes were analyzed in fresh colon tissues from the different groups of experimental animals (curative and preventive), using the DQ gelatin kit and zymographic assays. Figure 62 shows the total gelatinolytic activity present in the colon samples, quantified by the DQ gelatin method.

Figure 62 shows the effect of deflamin administration on the gelatinolytic activities of these enzymes in colon tissue of colitis-induced mice. The proteolytic activity of the enzymes was quantified by the fluorogenic DQ method. Results are expressed as relative fluorescence as a % of controls and represent the average of at least three replicate experiments (n = 3) ± SD. GTNBS group = C+; sham group C-; D= curative treatment with deflamin (15 mg/kg, n = 9, p.o.); and Dp = preventive treatment with deflamin (15 mg/kg, n = 6, p.o.). *p<0.001 versus sham group; #p < 0.05 versus TNBS group; and ^p < 0.05 group D versus Dp.

Figure 62 shows that TNBS induced a highly significant increase in gelatinolytic activities, compared to controls (P<0.001), while both curative and preventive administrations of deflamin significantly reduced (P<0.05) total MMP-9 MMP-2 activity compared to TNBS treatment, but there was no significant difference (P>005) between curative and preventive administrations.

Since the DQ gelatin assay provides evidence of total gelatinolytic activity in colon tissue (i.e., MMP-9 MMP-2) with no possible distinction between the two enzymes, their specificity was further analyzed by substrate zymography, where MMP-9 and MMP-2, in their native and zymogenic forms, can be easily separated by electrophoresis. Figure 63 shows an example of a zymographic profile of the protein extracts obtained from the colonic tissues in the different experimental groups. The white bands show the gelatinolytic activity of the specific bands.

Figure 63 shows the effect of deflamin administration on the gelatinolytic activities of MMP-2 and MMP-9 of colitis-induced mice. Representative image of the zymographic profiles of MMP-9 and MMP-2 activities of the colons. The protein extracts of the colon were loaded on 12.5% (w/v) polyacrylamide gels copolymerized with 1% (w/v) gelatin. TNBS group (n = 10); Sham group (n = 6); D colon of animals treated with deflamin in curative treatments (15 mg/kg, n = 9, p.o.) and Dp = colon of animals treated with deflamin in preventive treatments (15 mg/kg, n = 9, p.o.).

Zymographic profiles show how TNBS increased not only MMP-9 activity but also MMP-2 activity, both in the wild-type and zymogenic forms of the enzymes, compared to controls where there was low activity of the active forms of MMP-2 and MMP-9. However, in the deflamin treatments there was an evident reduction in the activities of MMP-9 and MMP-2 (wild-type and zymogenic forms), compared to the TNBS group.

However, the specificity and intensity of MMP inhibition differed between treatments. While in the curative treatments both MMP-2 and MMP-9 were similarly reduced (and for both enzyme forms), this trend was not the same in the preventive approach, where MMP-9 was clearly and visibly more inhibited than pro-MMP-9, while pro-MMP-2 was inhibited but MMP-2 was not (Figure 63).

Deflamin is also bioactive in other models of acute inflammation

Figure 64 shows the effect of deflamin administration on the development of carrageenan-induced rat paw edema 6 h after edema induction. Effect of a single administration of deflamin extract (15 mg/kg; n = 5; p.o.) compared with the effect of a single administration of carrageenan (1 mL/kg; n = 6; i.p.), indomethacin (10 mg/kg; p.o.; n = 6), tempol (30 mg/kg; n = 8; p.o.), or Trolox (30 mg/kg; n = 8; p.o.). SF: subplantar injection of 0.1 mL of sterile saline and administered with saline (1 mL/kg, i.p., n = 6). Data are presented as means with standard errors. *p < 0.001 versus SF group; #p < 0.001 versus carrageenan group.

Figure 65 shows the effect of topical administration of deflamin on paw edema in rats 6 h after edema induction by carrageenan. Effect of a single administration of deflamin extract (15 mg/kg; n = 3; p.o.) compared with the effect of a single administration of carrageenan (1 mL/kg; n = 6; i.p.). SF: subplantar injection of 0.1 mL of sterile saline and administered with saline (1 mL/kg, i.p., n = 6). Data are presented as means with standard errors. *p < 0.001 versus SF group; #p < 0.05 versus carrageenan group; and p < 0.01 versus SF group. Deflamin was tested to see if it is able to reduce inflammation in another animal model of inflammation: carrageenan-induced paw edema, also using different administrations: p.o., i.p. or topically applied. Figure 64 shows the effect of deflamin on paw edema under p.o. and i.p. administration conditions, and Figure 65 shows the result for topical administration, represented as % increase in paw volume. For the carrageenan group, a highly significant increase (P<0.001) in the % of paw volume was observed in both figures, while treatment with anti-inflammatory controls significantly reduced it (P<0.05).

The results show that deflamin treatments reduced the percentage increase in paw volume after carrageenan administration, but were only significant (P<0.05) for topical applications (Figure 65), while in i.p. and p.o. administrations (Figure 58) the effect was not significantly different from the carrageenan group (P>0.05).

Digestibility of deflamin

Figure 66 shows a reverse zymography of mouse blood and feces (inner and outer fractions, respectively). Deflamin was administered orally to rats for 0 (control), 4 (T4) and 7 days (T7). M: molecular mass markers.

Reverse zymography (Figure 66) shows the presence of a proteinase inhibitor (i.e. deflamin) in mouse feces, but not in mouse blood. This result is corroborated by the data presented in Figure 34. Although not clearly visible in Figure 66, this activity was found to be more intense in animals that ingested deflamin for a longer period of time (i.e. 7 days).

These results suggest that deflamin survives the mouse digestive process because it maintains its biological activity after passing through the colon. No inhibitory activity was detected in mouse blood, suggesting that deflamin may not enter the bloodstream. Alternatively, the methodology followed may not have been sensitive enough to ensure its detection in blood samples.

Ingestion of deflamin by mice led to its detection in the external fraction (i.e. colon). Weak evidence suggests that orally administered deflamin may not enter the bloodstream. This could be interpreted as meaning that after exerting its bioactivity, deflamin is excreted in the mouse faeces. If the absence of deflamin in the bloodstream of fed animals is confirmed, this could be highly advantageous in the sense of avoiding the well-known side effects resulting from a non-specific inhibition of MMPs in the body in general, widely described for synthetic MMPIs. Importantly, it is important to highlight the perspectives opened by the observation that deflamin appears to maintain its biological antigelatinolytic activity even after passing through the entire digestive tract of the mouse, something that is intimately associated with its potential application in human health and nutrition.

Effect of seed cooking on the ability of L. albus to inhibit HT29 cell migration

Many thermolabile bioactive compounds have been described in seeds. However, most seeds are ingested after cooking for various reasons, including, for example, the presence of toxic substances, thermolabile peptides, proteins (e.g., lectins, enzyme inhibitors, and enzymes releasing toxic compounds such as glycosidases present in cyanogenic plants), and other compounds. Under these conditions, bioactive compounds present in functional seeds often need to withstand cooking in addition to surviving the digestive process.

Figure 67 shows the antimigratory effect of deflamin on HT29 cells. Representative images of wound closure assays in HT29 cells after treatments with deflamin or with soluble protein extracts from cooked seeds and raw seeds. Cells were grown to 80% confluence and wounds were made by scratching the cells with a pipette tip (0 h). Cells were then exposed to 100 µg of protein extract/ml and wound healing was monitored after 48 h.

Figure 67 analyses the effect of seed cooking on the ability of L. albus seeds to inhibit HT29 cell migration by the wound healing assay. Isolated deflamin totally inhibited HT29 cell migration at a protein concentration of 100 µg/ml. In fact, this concentration of deflamin not only blocked cell migration, but also detached them from the solid support. The total seed protein extract also inhibited cell migration, an effect that was found to be particularly strong in the case of cooked seeds. This seemingly surprising result can be explained by the concentration effect on deflamin and other heat-resistant proteins exerted by boiling. In other words, the amount of deflamin present in 100 µg of total soluble seed protein is higher in cooked than in raw seeds simply because many seed proteins were denatured (and therefore rendered insoluble) by the heat treatment.

It is possible to conclude that deflamin exhibits a potent ability to inhibit cell invasion and MMP-9 and MMP-2 activities at low concentrations, without significantly affecting colon cell growth. Therefore, deflamin shows high potential as an anti-inflammatory and antitumor agent in CRC. Its high resistance to the digestive process, boiling and low pH values make deflamin an excellent candidate to be used as a nutraceutical in human health and nutrition. Its bioactivity is equally potent in the cooked total seed extract, an observation that makes lupins an excellent functional food, to be implemented worldwide as another great benefit of the well-established Mediterranean diet. Included on November 16, 2010 by the Intergovernmental Committee of UNESCO in the Representative List of Intangible Cultural Heritage of Humanity.

The amino acid residue sequence of lunasin and those of the deflamin precursors

The data presented above indicate the presence of large chain fragments of p-conglutin and 8-conglutin-2 in deflamin preparations. Therefore, p-conglutin and large chain 8-conglutin-2 can be considered as precursors of deflamin.

The NCBI BLAST (Basic Local Alignment Search Tool) tool available at http://blast.ncbi.nlm.nih.gov/Blast.cgi, was used to check for possible similarities in amino acid residue sequences between the soybean 43-amino acid residue lunasin and the proteins whose polypeptides were identified as components of deflamin, namely, p-conglutin fragments and the 8-conglutin heavy chain.

Where appropriate, the ExPASy BLAST tool available at http://web.expasy.org/tmp/1week/blastf29720.html was also used.

Based on the previous observation that deflamin apparently comprises a mixture of G-conglutin and 8-conglutin fragments <10 kDa, the query sequences used in the BLAST analyses were as shown in lunasin (Glycine max) (SEQ ID NO: 191), p-conglutin (Lupinus albus) (SEQ ID NO: 192), 8-conglutin-2 large chain (Lupinus angustifolius) (SEQ ID NO: 193) and 8-conglutin (Lupinus albus) (SEQ ID NO: 194).

Compared to all entries present in protein databases, the amino acid residue sequence of lunasin (Glycine max) shows a very strong homology (more than 95%) with the 2S albumin of G. max. This was clearly illustrated by appropriate BLAST analyses.

A comparison of the amino acid residue sequence of lunasin with that of the deflamin precursors was also made. No homology was found in the amino acid residue sequence between lunasin and the beta-conglutin precursor of L. albus and lunasin versus large-chain delta-2 conglutin. Considerable homology was found between the amino acid sequence of lunasin and the fragment corresponding to the light chain of 8-conglutin of L. albus.

Along the 43 amino acid residues of lunasin, there is a stretch of 25 amino acids that exhibits 48% homology (12 residues out of 25) with the p-conglutin region of Lupinus albus, which comprises residues 21 to 45.

It is possible to conclude that the amino acid sequence of lunasin shows no homology with that of the deflamin precursors, namely the large chain of p-conglutin and 8-conglutin-2.

SEQ ID NO: 195 confirms that lunasin contains a 25-long region whose amino acid sequence shows 48% homology to a corresponding region within the small 8-conglutin chain of Lupinus albus, but not to the large delta-2 conglutin chain of Lupinus angustifolius. However, this observation is contradicted by results reported by Herrera (2009).

SEQ ID NO: 195 shows sequence matches between 8-conglutin from Lupinus albus (accession number Q333K7; black, orange and red) and two polypeptides: lunasin from Glycine max (accession number AF005030; green) and large-chain delta-2 conglutin from Lupinus angustifolius (accession number P09931; blue).

In addition to a different sequence of amino acid residues, another important difference between lunasin and deflamin concerns their susceptibility to digestion proteolysis. While deflamin resists the digestive process, lunasin does not (Cruz-Huerta et al., 2015). Furthermore, unlike deflamin, lunasin is not amenable to scale-up extraction and purification, making this bioactive peptide unsuitable for mass production.

Deflamina from seeds other than L. albus- I

Deflamin was found to be present in seeds other than Lupinus. Figure 68 shows the inhibitory effect exerted on HT29 cell migration by various concentrations of total soluble protein extracts from L. albus, C. arietinum and G. max. The results indicate the presence of cell migration inhibitory activity in the seeds analyzed.

Figure 68 shows representative images of HT29 cell migration as assessed by the wound healing assay. Cells were grown to 80% confluence and the monolayer was scraped with a pipette tip (0 h). Cell migration was determined after a 48 h exposure of HT29 cells to buffer (control), and various concentrations of total soluble proteins were extracted from the seeds of L. albus, C. arietinum, and G. max.

Subsequently, deflamin was purified and isolated from the seeds of L. albus, C. arietinum and G. max following the procedure described in Figure 4 for L. albus. The polypeptide profiles of deflamin from L. albus (designated as deflamin La), G. max (designated as deflamin Gm) and C. arietinum (designated as deflamin Ca) are shown in Figure 69.

The results presented in Figures 70, 71 and 72 compare the antigelatinolytic activity measured in vitro by the DQ gelatin assay, the inhibitory activity upon HT29 cell migration determined by the wound healing assay and the growth of HT29 cells analysed by the MMT method of deflamin isolated from L. albus (deflamin La), G. max (deflamin Gm) and C. arietinum (deflamin Ca), respectively.

The data presented in Figures 70 to 72 reveal that deflamin from soybean and chickpea also inhibit MMP-9 and MMP-2 gelatinases in vitro, as well as migration of hT29 cells, but do not affect HT29 cell growth to any significant extent, in parallel with the results obtained for deflamin from lupins. However, deflamin from lupins appears to be more potent than that from soybean or chickpea. This last observation may not be relevant as regards the Mediterranean diet mentioned above, since the population usually ingests larger amounts of chickpea or soybean at each meal than lupins.

Brief discussion of selected topics from the results presented

Deflamin is a novel digestion-resistant gelatinase inhibitor that reduces colitis lesions through oral supplementation. MMP-9 inhibitors (MMPIs) are primarily considered as antiangiogenic agents for primary tumors and metastasis deterrents, but they have also been shown to effectively inhibit precancerous conditions such as colitis and other inflammatory bowel diseases. For more than 30 years, researchers worldwide have considered MMPs to be attractive therapeutic targets for both cancer and inflammation. As a result, countless MMPIs have already been synthesized, some of which have been used as potential therapeutic agents (Bourguet et al., 2012). However, only a few small MMPIs entered the clinical trial stage, most of which were terminated prematurely due to lack of benefits or strong adverse side effects (Wang et al., 2012). Ideally, for a specific MMP-9 inhibitor to be successfully used in inflammatory bowel disease (IBD) treatments as a dietary supplement, it should be available in the colon, rather than bioavailable in serum, be resistant to the digestive process and nontoxic to health. The results presented here clearly show that deflamin survives the digestion process and is able to attenuate the lesions caused by TNBS-induced colitis, leading to a reduction in several functional and histological markers of colonic inflammation, namely: attenuation of the decrease in colonic length, reduction in the extent of visible injury (ulcer formation), decrease in the severity of diarrhea, reduction in mortality rate, reduction in mucosal bleeding and reduction in the general histological features of colonic inflammation. Furthermore, this effect was evident in p.o. treatments as well as in i.p. treatments, corroborating its potential use in a dietary approach. Indeed, the overall results obtained from oral administrations suggest that deflamin is not only resistant to digestion, but was more efficient than i.p. administration, treatment in reducing colitis lesions possibly acting more effectively in situ. Interestingly, a preventive approach, with a longer dietary administration of deflamin, was not significantly different from the curative approach, in which deflamin was administered only 3 h after TNBS induction. This suggests that it may act as an inflammatory deterrent effectively and rapidly, suggesting a potential use as a nutraceutical in both acute and chronic inflammation.

Oral administration of deflamin reduces the expression of inflammatory markers involved in the inflammatory signaling cascade.

Histological analysis and expression of some important inflammation markers also corroborated the anti-inflammatory effects of deflamin. Our immunostaining assays performed on the colon of animals from the experimental colitis study showed that induction of colonic inflammation led to increased expression of COX-2 compared to sham animals, which is consistent with clinical and epidemiological studies demonstrating the important role of COX-2 and prostaglandins in the progression of intestinal inflammation in IBD patients (Ogasawara et al., 2007; Chen et al., 2014). Administration of deflamin led to reduced COX-2 staining, indicating that it altered COX-2 expression in injured intestinal tissue.

Furthermore, specifically in the intestine, an upregulation of nitric oxide production was observed. This compound is reported to be produced and released locally in much higher quantities in the inflamed intestine than in the non-inflamed intestine, and is suggested as a new clinical biomarker for the diagnosis and monitoring of IBD patients (Lee et al., 2013). Similar to COX-2, immunostaining assays in the experimental colitis study showed that there was indeed increased iNOS expression in animals subjected to colitis induction. Again, administration of deflamin, particularly orally, was able to reduce iNOS expression and thus contribute to the deterioration of the inflammatory process in the colon.

Deflamin inhibits MMP-9 and MMP-2 in vivo

Deflamin has now been shown to inhibit MMP-9 and MMP-2 in colon cells in in vitro assays, and to be resistant to heat and acid denaturation, making it a good candidate for the treatment of IBD and colon cancer. However, in vivo testing was required to further determine its effectiveness after digestion. In this regard, the overall physiological and morphological results were able to corroborate that deflamin can indeed inhibit the colitis-induced increase in MMP-9 and MMP-2 activities observed in the TNBS group, leveling them to activity intensities closer to those observed in controls.

Interestingly, although no morphological and functional differences were observed between preventive and curative treatments, there were significant differences between the specific inhibitions of both enzymes in the zymographic assays. While in curative treatments deflamin seems to act directly on the two gelatinases strongly induced by TNBS, in preventive treatments a specific inhibition of the active form of MMP-9 and of pro-MMP-2 was observed, but not of pro-MMP-9, nor the active form of MMP-2. MMPs are generally synthesized as zymogens (pro-MMPs), with their catalytic activity blocked by a cysteine switch and are only activated by its elimination, through limited proteolysis. In zymographic assays, progelatinases also become active because they are denatured by SDS, thus exposing the catalytic site (hence the slightly higher mass of the proenzymes in zymographic gels because they still retain the short amino acid sequence of the cysteine switch). The fact that deflamin only inhibits the active form of MMP-9 suggests that it displays a certain degree of specificity towards this form, perhaps towards the catalytic site, only exposed in the active form.

On the other hand, pro-MMP2 appears to be inhibited but not MMP2. Because MMP-2 is one of the proteases that activate MMP-9, it seems plausible that, with longer exposure to deflamin, high inhibition of MMP-9 would induce, through feedback, increased activation of MMP-2 to activate MMP-9.

Although longer exposure to deflamin seems to suggest more profound effects on gelatinase synthesis and activation, the results suggest that its administration with a curative approach is as effective in reducing colitis lesions as the preventive mode of administration. Nevertheless, the higher specificity towards MMP-9 is important because it ensures low side effects, unlike most broad-range MMPIs used in the clinic.

Deflamin also reduces inflammation in other models

To elucidate its scope as an anti-inflammatory agent, it was further analyzed whether deflamin could reduce inflammation in paw edema. Although there was a reduction in % of paw volume in both i.p. and p.o. administrations, they were not statistically significant, suggesting that absorption and distribution of deflamin through the bloodstream is limited in both administrations. However, the significant efficacy observed in topical administrations of deflamin corroborates that its effect is greater when applied in situ. It also suggests that deflamin can be absorbed through the skin. Considering the relationship between MMP-9 and skin cancer diseases (Philips et al., 2011), the present results open new possibilities for clinical applications of deflamin.

Conclusion -1

As a potent inhibitor of matrix metalloproteinases MMP-9 and MMP-2 and exhibiting powerful anti-inflammatory activities, deflamin represents a new type of edible proteinaceous MMPI that survives the digestion process and can be used as a functional agent in foods and in the prevention/treatment of inflammation as well as any diseases derived from it. Being effective in oral, intravenous or topical applications, deflamin may prove useful as a functional food in the prevention or treatment of a wide variety of diseases.

Deflamin from L. albus is a mixture of large chain fragments of p-conglutin and 5-conglutin.

It is quite interesting the presence of p-conglutin and 5-conglutin fragments in deflamin. P-conglutin is a trimeric protein devoid of disulfide bridges in which the monomers are formed by a large number of polypeptides, glycosylated or not, ranging from 16 to more than 70 kDa, but a large number of proteolytic processing sites give rise to the abundance of 7S protein subunits were observed. Its complete post-germination degradation strongly supports the storage function of p-conglutin. Interestingly, another fragment of this protein is known for its potent bioactivities against fungi: Blad, an abundant 20 kDa transient p-conglutin-derived polypeptide chain that displays lectin-like activity. Being highly reactive and with the presence of a bioactive cupin domain, it is possible that other p-conglutin fragments exist with specific activities yet to be discovered. However, previous work revealed that deflamin exhibits neither antifungal nor bactericidal activity (results not shown), and the sequences of deflamin fragments do not match those of Blad.

5-Conglutin belongs to the family of 2S sulfur-rich albumins that might also have unknown specific bioactivities in lupin. Lupinus 2<s> seed albumin, also called 5-conglutin, is a monomeric protein comprising two small polypeptide chains linked by two interchain disulfide bonds: a smaller polypeptide chain, consisting of 37 amino acid residues resulting in a molecular mass of 4.4 kDa, and a larger polypeptide chain containing 75 amino acid residues with a molecular mass of 8.8 kDa. This latter larger polypeptide chain is somewhat similar to some of the polypeptide profiles obtained for deflamin, particularly in peak 3 (see Figures 44 to 46). The larger polypeptide chain contains two intrachain disulfide bridges and a free sulfhydryl group. This could provisionally explain the slight difference in apparent molecular mass detected between R- and NR-SDS-PAGE of deflamin (see Figure 43). This protein presents specific inherent features unique among L. albus proteins: in addition to its high cysteine content, it exhibits a low absorbance at 280 nm.

Regarding the physiological role of 5-conglutin, a storage function has been proposed for this class of proteins. However, the structural similarity with the family of plant cereal inhibitors, which includes bifunctional trypsin/amylase inhibitors, may suggest a defense function for this protein in addition to its storage function and could corroborate its role as MMPI. The presence of free sulfhydryl groups in 5-conglutin could be related to a high degree of affinity towards the Zn2+ active site in MMPs and could explain their mode of inhibition. Indeed, one way to isolate these conglutins is by Zn precipitation. Furthermore, its presence in L. albus seeds was evaluated to be approximately 3 to 4 % of the seed weight, which is in agreement with the yields obtained previously. Furthermore, 2S albumin from Lupinus seed is typically present in both the albumin and globulin fractions, which explains the results previously obtained for MMP inhibitory activity in the two protein fractions (Lima et al., 2016).

HPLC peaks 1 and 4 of deflamin (Figures 44-46) were composed of only p-conglutin fragments and still exhibited MMPI activities, albeit at lower levels. Therefore, the increased activity appears to be attributable to a specific mixture comprising fragments of both p- and 5-conglutin proteins and not exclusively to p-conglutin. The fact that only the large polypeptide chain of 5-conglutin was present in deflamin might suggest that its three sulfhydryl groups might be free to interact with MMPs or G-conglutin fragments (this might explain the presence of a cluster of apparently three minor bands of higher molecular mass comprising deflamin) and that this complex exhibits the highest activity. Alternatively, the smaller polypeptide chain of 5-conglutin may be present in deflamin.

Conclusion - 2

In the last decade, a substantial amount of research has focused on the discovery of novel plant foods containing MMPIs, but few, if any, present the potential of deflamin, as it is easy to isolate and displays high MMP-9 inhibitory activities. Deflamin has been characterized as a complex mixture of soluble fragments of two specific protein precursors: 5- and p-conglutins. Overall, this polypeptide mixture was shown to be highly soluble in water; its bioactivities resist boiling, low pH values, and possibly digestive proteases; it strongly inhibits matrix metalloproteinase (MMP)-9 and/or MMP-2, i.e., it is an inhibitor of MMP (MMPI) at low concentrations and in a dose-dependent manner, and it reduces the invasiveness of the human colon adenocarcinoma cell line HT29 without exerting cytotoxicity. These data were strongly supported by a series of preclinical studies performed with animal models. These features make deflamin a new type of MMPI that can be used as an ingredient in functional foods in the prevention/treatment of tumorigenesis and cell invasion, as well as any diseases derived from them. As a potent inhibitor of matrix metalloproteinases MMP-9 and MMP-2, deflamin may be useful in functional foods in the prevention and treatment of a wide variety of diseases related to MMP-9 activity. Its efficacy when administered orally and its ability to survive the digestive process suggest that it can act efficiently in the colon, without exerting the harmful side effects that characterize synthetic MMPIs, making deflamin an excellent candidate to be used in the prevention and treatment of colorectal cancer.

Conclusion - 3

Deflamina presents:

- Deflamin exhibits potent gelatinolytic activity in a dose-dependent manner;

- Deflamin exhibits potent inhibition of colon cancer cell invasion in a dose-dependent manner;

- high concentrations of deflamin detach colon cancer cells from the solid surface; - deflamin does not appear to induce significant cytotoxic effects on colon cancer cells, even at a concentration of 100 |jg. mL’1;

- Deflamin does not induce a significant reduction in cell growth or a reduction in the number of living cells.

In one or more further embodiments, deflamin may actually be produced or enhanced during its own purification and isolation as a result of the harsh in vitro treatments imposed on lupin seed storage proteins, which disassemble oligomeric structures, cleave polypeptides by limited proteolysis, and remove most of the polypeptides that are no longer water-soluble. Other polypeptides are released and may become part of deflamin. In other words, it is possible that deflamin, apparently composed of a mixture of polypeptides that are fragments derived from different protein precursors, is formed in vitro after extraction of the storage proteins and their partial denaturation/proteolysis.

The presence of deflamin in seeds of other species - II

To identify the presence of deflamin in seeds of other species, reverse zymography of soluble proteins from different edible seeds was performed (Figure 73). Figure 73 shows representative images of reverse zymography performed on a 12.5% (w/v) acrylamide SDS-PAGE gel with gelatin and 1 ml of HT-29 medium containing MMP-9 and MMP-2. Each well was loaded with 50 µg of protein from the total extracts of the different seed species. Legumes: V.a -Vigna angularis(azuki bean); V.r -Vigna radiata(mung bean); V.m -Vigna mungo(urad bean); L.m-Lupinus mutabilis(Andean lupine). Cereals: T.a -Triticum aestivum(soft wheat); A.s -Avena sativa(oats); Page -Pennisetum glaucum(millet); T.t -T. turgidumvar. turanicum (kamut wheat); Other dicotyledons: C.q-Chenopodium quinoa(quinoa), H.a -Helianthus annus(sunflower); P.d -Prunus dulcis(almond); C -Curcubitasp. (pumpkin); F.tFagopyrum tataricum(buckwheat); S.hSalvia hispanica (chia). Arrows indicate the presence of deflamin.

Among the other species analysed, and in addition to those initially evaluated for deflamin (i.e. L. albus, C. arietinum and G. max), only seeds of Vigna mungo (urad bean), Lupinus mutabilis and Triticum turanicum showed deflamin-like bands. Therefore, other legume species, particularly of the genus Lupinus, and also of the genus Triticum, were further analysed.

Presence of deflamina in the seeds of other species of the genus Lupinus

Seeds of the following Lupinus species were analyzed for the presence of deflamin:

-L. albus

- L. mutabilis

- L. hispanicus

- L. nootkatensis

- L. angustifolius

- L. luteus

The reverse zymography gel is shown in Figure 74 below. Although it is difficult to appreciate in Figure H, all Lupinus species analyzed were shown to contain deflamin.

Figure 74 shows the reverse zymography performed on a 12.5% polyacrylamide gel with gelatin and 1 ml of HT-29 medium containing MMP-9 and MMP-2. Each well was loaded with 50 µg of the total extracts of the different seed species: L. albus; L. mutabilis; L. hispanicus; L. nootkatensis; L. angustifolius; and L. luteus.

Isolation of deflamin from seeds of L. mutabilis

Figure 75 shows representative polypeptide profiles of the potential deflamin homolog under reducing (R) and non-reducing (NR) conditions by SDS-PAGE on a 12.5% (w/v) acrylamide gel, using the deflamin isolation method. Wells were loaded with 100 µg of purified protein from L. mutabilis.

The presence of deflamin in the seeds of other legume species

Purification of deflamin from Vigna mungo (urad bean) seeds yielded a polypeptide pattern quite different from that of Lupinus species (Figure 76).

Figure 76 shows representative polypeptide profiles of the potential deflamin homolog under reducing (R) and non-reducing (NR) conditions by SDS-PAGE on 12.5% (w/v) acrylamide gel, using the deflamin isolation method. Wells were loaded with 100 µg of purified Vigna mungo protein.

Isolation of deflamina from seeds of species of the genus Triticum

The following Triticum species were analyzed for the presence of deflamin:

-T. spelta(spelt)

-T. turgidumvar. durum wheat (hard wheat)

-T. aestivum (common wheat)

-T. turgidumvar,turanicum(kamut)

Reverse zymography gels are shown in Figure 77 below. Reverse zymography showed the presence of MMP-9 inhibitory bands in the species:

-T. spelt

-T. turgidumvur,var. durum

- Triticum turgidum var. turanicum*,

and most likely other ancient wheat species and varieties, but apparently not T. aestivum.* Khorasan wheat or Oriental wheat (Triticum turgidums sp. turanicum also called Triticum turanicum), known commercially as kamut, is a tetraploid wheat species. Identifications sometimes seen as T. polonicum appear to be incorrect. Recent genetic evidence from DNA fingerprinting suggests that the variety may be derived from a natural hybrid between T. durum and T. polonicum.

Figure 77 shows the reverse zymography performed on 12.5% (w/v) acrylamide gel with gelatin and 1 ml of HT-29 medium containing MMP-9 and MMP-2. Each well was loaded with 50 µg of protein in the total extracts of different seed species: T. spelta (spelt); Turgidum var. trigo durum (durum wheat); T. aestivum (soft wheat); and Triticum turgidum var. turanicum (kamut).

Deflamin was then isolated from the seeds of these species (i.e. T. spelta, T. turgidum var. durum and Triticum turgidum var. turanicum). The results are expressed in Figure 78 below.

Figure 78 shows representative polypeptide profiles of the putative deflamin homolog in reducing (R) and non-reducing (NR) buffer by D-PAGE on a 12.5% (w/v) acrylamide gel.

Claims (13)

1. Method of extracting deflamine from suitable seeds, comprising: - at least one high temperature stage of at least 80 degrees Celsius or boiling; and - at least one low pH stage of pH 4 or lower; - at least one step of contacting the extract with high ethanol concentrations of at least 30%, 40%, 70% or at least 90% v/v ethanol; wherein preferably said method comprises the following steps in this(a) order/sequence or in a(n) different order/sequence: (a) boiling the intact seeds in water, followed by extraction in water or buffer and removal of fat, or reducing the intact seeds to flour, extraction in water or buffer followed by removal of fat and boiling, or removal of fat from the flour followed by extraction in water or buffer and boiling, (b) exposing the soluble fraction to a sufficiently low pH value of pH 4.0 or below to allow precipitation of most of the remaining proteins/polypeptides, (c) resuspending the precipitated fraction in 30% to 50% (v/v) ethanol, preferably about 40% (v/v) ethanol, with the solution also optionally containing 0.4 M NaCl, to obtain a supernatant containing deflamin, and optionally perform the following steps: (d) completing the soluble fraction with 90% (v/v) ethanol to precipitate deflamin and optionally storing at 5 °C to -30 °C, preferably at -20 °C, or precipitate deflamin by other means, such as lyophilization, (e) clean the precipitated deflamine from contaminants by repeating steps (c) and (d), (f) dissolve the precipitated deflamine, for example, in water, and desalinate. 2. Method of extracting deflamin from suitable seeds depending on claim 1, said method comprising the following steps in the same order/sequence or in a different order/sequence: • provide flour from said seeds; • degrease said flour; • boil the remaining sample from said degreasing stage for a period; • centrifuge said sample for a period; • thereafter discard the resulting pellet and further process a resulting supernatant to precipitate the polypeptides while reducing their pH; • centrifuge further to obtain additional pellet; and discard said supernatant; and • further processing said additional pellet with ethanol and centrifuging to obtain an additional pellet which is then discarded; the remaining supernatant comprises the deflamin, in which optionally: - the method further comprises the step of adding ethanol to said remaining supernatant and storing for a period to allow precipitation of the deflamin and further centrifuging to obtain a pellet of deflamin, and/or - the method further comprises the step of one or more additional ethanol precipitations. 3. Method according to claim 2, wherein said seeds are seeds of Lupinus albus, Ciceraritinum or Glycine max, and/or wherein said seeds are cooked. 4. A deflamin polypeptide composition for use in a method of treating the human or animal body by therapy, wherein said therapy is preferably to prevent or treat inflammation or cancer, wherein said deflamin polypeptide composition comprises deflamin as obtainable by the method according to any one of claims 1 to 3. 5. Deflamin polypeptide composition for use according to claim 4, wherein said composition comprises a mixture of polypeptides obtainable from seed storage proteins by the method according to any one of claims 1 to 3 each comprising a sequence which is a portion of a conglutin protein, - wherein said portion has a length of at least 5, 10, 20, 30 or 50 amino acids, and/or - wherein said conglutin protein is optionally a beta conglutin or delta conglutin; I - wherein said conglutin protein is optionally a beta 1,2, 3, 4, 5, 6 or 7 conglutin protein or a delta 2 conglutin protein. 6. Deflamin composition for use according to claim 4 or 5, comprising at least one mixture of up to 100 polypeptides having a length of 10 to 200 amino acids. 7. Deflamin composition for use according to any one of claims 4 to 6, which is obtained by extraction of plant material or by recombinant expression. 8. Deflamin composition for use according to any one of claims 4 to 7, wherein any of said polypeptides comprises the sequence of a plant seed protein or has at least 70% identity to a plant seed protein, wherein said plant is optionally of the genus Lupinus, Cicero Glycine and said protein is optionally a conglutin protein or a natural homologue of any conglutin. 9. A method of preparing deflamin comprising expressing the deflamin polypeptides from one or more nucleic acids encoding the polypeptides and purifying said polypeptides, wherein said expression is preferably in a cell, and wherein the deflamin polypeptides are obtainable by the method according to any one of claims 1 to 3. 10. One or more nucleic acid vectors expressing together or individually a deflamin composition for use in a method of treating the human or animal body by therapy, wherein said therapy is preferably to prevent or treat inflammation or cancer, wherein said deflamin composition is optionally as defined in any one of claims 5 to 8, and wherein the deflamin is obtainable by the method according to any one of claims 1 to 3. 11. Product comprising a multiplicity of different polypeptides as defined in any one of claims 5 to 8, which together form a deflamin composition for simultaneous, separate or sequential use in a method of treatment of the human or animal body by therapy, wherein said therapy is preferably to prevent or treat inflammation or cancer, and wherein the deflamin is obtainable by the method according to any one of claims 1 to 3. 12. Product comprising a multiplicity of nucleic acid vectors together expressing a deflamin composition for simultaneous, separate or sequential use in a method of treating the human or animal body by therapy, wherein said therapy is preferably to prevent or treat inflammation or cancer and wherein deflamin is obtainable by the method according to any one of claims 1 to 3. 13. Composition that is: (i) a deflamine composition which is obtainable by the method according to any one of claims 1 to 3, and which is optionally as defined in any one of claims 5 to 8; or (ii) a composition comprising one or more nucleic acids encoding individually or together a deflamin polypeptide composition obtainable by the method according to any one of claims 1 to 3, optionally wherein - said nucleic acids are expression vectors capable of expressing a deflamin composition, preferably a viral vector, and/or said composition is in the form of a pharmaceutical composition which optionally also comprises a pharmaceutically acceptable carrier or diluent.