[go: up one dir, main page]

WO2019116391A1 - Pharmaceutical formulations for management of cancers - Google Patents

Pharmaceutical formulations for management of cancers Download PDF

Info

Publication number
WO2019116391A1
WO2019116391A1 PCT/IN2018/050835 IN2018050835W WO2019116391A1 WO 2019116391 A1 WO2019116391 A1 WO 2019116391A1 IN 2018050835 W IN2018050835 W IN 2018050835W WO 2019116391 A1 WO2019116391 A1 WO 2019116391A1
Authority
WO
WIPO (PCT)
Prior art keywords
formulation
cancer
extract
extracts
combination
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/IN2018/050835
Other languages
French (fr)
Inventor
Chitra Mandal
Eswara Murali SATYAVARAPU
Bikas Pal CHANDRA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Council of Scientific and Industrial Research CSIR
Original Assignee
Council of Scientific and Industrial Research CSIR
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Council of Scientific and Industrial Research CSIR filed Critical Council of Scientific and Industrial Research CSIR
Publication of WO2019116391A1 publication Critical patent/WO2019116391A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/75—Rutaceae (Rue family)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/21—Amaranthaceae (Amaranth family), e.g. pigweed, rockwort or globe amaranth
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/39—Convolvulaceae (Morning-glory family), e.g. bindweed
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/54—Lauraceae (Laurel family), e.g. cinnamon or sassafras
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/74—Rubiaceae (Madder family)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/82—Theaceae (Tea family), e.g. camellia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/906—Zingiberaceae (Ginger family)
    • A61K36/9062—Alpinia, e.g. red ginger or galangal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine

Definitions

  • Natural products having relatively non-toxic nature and abundant availability are the potential source of chemotherapeutic s including anti-cancer molecules like vincristine, vinblastine, etoposide, paclitaxel, camptothecin, topotecan and irinotecan.
  • This natural product combination also has major advantages over the synthetic drug combination by showing higher efficacy and lower side effects.
  • These naturally derived drugs or combinations or extracts are less in cost, which can be easily available for the poor compared to synthetic drugs. Extracts which contain a combination of few medicinal herbs can work together for management of cancer and thereby expected to be less toxic.
  • Yet another objective of the present invention is to provide a formulation which can exhibit the growth-inhibitory activity in different cancer cell lines selected from the group consisting of ovarian, glioma, cervical, lung, pancreas, colon, breast, skin, oral, prostate, head and neck cancers, as well as different types of blood cancer including myeloid leukemia, T-cell/B- cell/Mixed T-cell and B-cell leukemia and cancer stem cells through apoptosis, detachment- induced cell death (anoikis) and inhibition of autophagy.
  • Yet another objective of the present invention is to provide a formulation which can exhibit inhibition of cancer cells migration and regulation of epithelial to mesenchymal transition (EMT), helpful for the management of tumor metastasis.
  • EMT epithelial to mesenchymal transition
  • Yet another objective of the present invention is to provide a formulation which can lead to in vivo efficacy at very low effective dose (ED50) in rodents through oral administration.
  • ED50 effective dose
  • Figure 2 Formulation reduced the growth of tumor in syngenic mice model of breast cancer given a representative image. Similar result was obtained with other types cancer models.
  • Figure 3 Formulation reduced the growth of tumor in mice xenograft model of ovarian cancer (using PA1 cell line) given a representative image.
  • Figure 4 Formulation reduced the growth of tumor in syngenic mice model ovarian cancer (using ID 8 cell line) given a representative image.
  • Figure 5 Reduced growth of melanoma tumor in C57BF6 mice model of melanoma (B16F10 cell line used)
  • Figure 6 Formulation induced apoptosis indicating by Annexin/PI positivity in representative ovarian cancer cell lines. Similar result was obtained with other types cancer cell lines
  • Figure 7 Formulation induces the nuclear fragmentation and condensation depicted by DAPI staining
  • Figure 8 Inhibition of autophagosome formation by formulation analysed by FACS in representative ovarian cancer cell line. Similar result was obtained with other types cancer models
  • Figure 9 Formulation inhibits migration in representative ovarian cancer cells. Similar result was obtained with other types cancer cell lines.
  • EMT epithelial to mesenchymal transition
  • the formulation as described above is having anti-cancer and immunomodulatory activity.
  • the combination of formulation with clinically approved anti-cancer drugs showed good synergistic effect and thereby expected to reduces the side -effect and toxicity arising due to the high dosage of known anti-cancer drugs.
  • step (b) mixing this extract of step (b) with the hydro-alcoholic (1:1) extract of one or more than one plant selected from the group consisting of Alpinia galangal, Rubia cordifolia, Camellia sinensis, Aegle marmelos, Cinnamomum tamala, Spinacia oleracea, Ocimum tenuiflorum and Ipomoea aquatic
  • the formulation or combination of formulation of extracts and clinically approved anti-cancer drug can be administered by oral, mucosal or intra peritoneal administration.
  • Another purpose of the present invention is to provide a kit for the treatment and management of cancer, wherein the kit comprising
  • the pamphlet contains instructions that administration of said combination provides synergistic effect in comparison with the administration of either formulation of extracts or clinically approved anti -cancer drug alone.
  • Natural products known to be the best source to treat many diseases like cancer We have screened many plants sources for their anti-cancer activity and from that finally selected nine different herbs which having a potent anti-cancer activity.
  • the selected plants include M. Koenigii, Alpinia galangal, Rubia cordifolia, Camellia sinensis, Aegle marmelos, Cinnamomum tamala, Spinacia oleracea, Ocimum tenuiflorum and Ipomoea aquatic.
  • the present invention provides a synergistic formulation comprising extract of Murraya koenigii and extract of one or more than one plant selected from the group consisting of Alpinia galangal, Rubia cordifolia, Camellia sinensis, Aegle marmelos, Cinnamomum tamala, Spinacia oleracea, Ocimum tenuiflorum and Ipomoea aquatic, optionally along with one or more pharmaceutically acceptable preservative, lubricant and diluent for the management of cancers through growth inhibitory activity and immunomodulation.
  • the ratio of extract of Murraya koenigii extract of one or more than one plant selected from the group consisting of Alpinia galangal, Rubia cordifolia, Camellia sinensis, Aegle marmelos, Cinnamomum tamala, Spinacia oleracea, Ocimum tenuiflorum, and Ipomoea aquatic in the formulation is in the range of 8-65:35-92.
  • the formulation comprising Murraya koenigii and Aegle Marmelos, wherein the ratio of extract of Murraya koenigii: Aegle Marmelos is in the range of 23-33:67-77 (Table-3A, Example-6).
  • the ratio of extract of Murraya koenigii: Alpinia galangal in the present formulation comprising Murraya koenigii and Alpinia galangal is in the range of 8-27:73-92 (Table-3C, Example-6).
  • the ratio of extract of Murraya koenigii: Rubia cordifolia in the present formulation comprising Murraya koenigii and Rubia cordifolia is in the range of 27-60:40-73 (Table-3D, Example-6).
  • the present invention also describes about the synergistic formulation comprising 4 herbs i.e Murraya koenigii, Alpinia galangal, Rubia cordifolia and Camellia sinensis, wherein ratio of extracts of Murraya koenigii, Alpinia galangal, Rubia cordifolia and Camellia sinensis is in the range of 10-40:10-60:18-25:12-25 (Formulation 1, Example 7A , Table 3E).
  • the present invention further provides a synergistic formulation comprising 5 herbs i.e. Murraya koenigii, Alpinia galangal, Rubia cordifolia, Camellia sinensis and Aegle marmelos, wherein ratio of extracts of Murraya koenigii, Alpinia galangal, Rubia cordifolia, Camellia sinensis and Aegle marmelos is in the range of 8-30:10-54:16-30:11-15:11-15 (Formulation 2, Example 7B, Table 3F).
  • a synergistic formulation comprising 5 herbs i.e. Murraya koenigii, Alpinia galangal, Rubia cordifolia, Camellia sinensis and Aegle marmelos, wherein ratio of extracts of Murraya koenigii, Alpinia galangal, Rubia cordifolia, Camellia sinensis and Aegle marmelos is in the range of 8-30:10-
  • synergistic formulation 1 and formulation 2 another synergistic formulation comprising all 9 herbs is also prepared by mixing extracts of Murraya koenigii, Alpinia galangal, Rubia cordifolia, Camellia sinensis, Aegle marmelos, Cinnamomum tamala, Spinacia oleracea, Ocimum tenuiflorum and Ipomoea aquatic in the ratio of 10:30:20:10:10:7.5:7.5:2.5:2.5 (Example-7B, Table 3G).
  • the formulation may be in different dosage forms selected from the group consisting of tablet, capsule, gel, ointment, lotion and suspension suitable for oral, mucosal or intra peritoneal administration.
  • the preservative used in the formulation is selected from the group consisting of methyl paraben or propyl paraben and it is preferably in 0.l-0.5%.
  • the lubricant used in the formulation is selected from the group consisting of magnesium stearate or stearic acid and it is preferably in 0.5 to 10%.
  • the binder used in the formulation is selected from the group consisting of sucrose or lactose and it is preferably in 0.5 to 10%.
  • the growth inhibitory effect of the present formulation was studied in various cancer cells including ovarian, glioma, cervical, lung, pancreas, colon, breast, skin, oral, head and neck cancers, myeloid leukemia, T-cell/B -cell/Mixed T-cell and B-cell leukemia through killing of cancer cells as determined by MTT and tryphan blue dye exclusion method by incubating cells with formulation by varying doses (0-30 pg/ml) and time (24-72 hours).
  • the IC50 for values for all these cancer cells varied within the range of 10-20 pg/ml (Table 2) after exposure of formulation. However, no viable cells were observed at higher concentrations ( ⁇ >30 pg/ml).
  • formulation showed no toxicity towards normal cells (WI-38, Figure 1), even at >200 pg/ml dose. No inhibition in the cell proliferation was observed in solvent control. So, formulation was found to be a non-toxic agent under in vitro condition. Under indentical conditions, the IC 50 for values for all these individuals plant extracts from nine different plants was also initially tested against cancer cells (Table 2).
  • in vivo growth inhibitory effect of the formulation was studied in three representative cancer models namely, breast cancer ( Figure 2), ovarian cancer ( Figure 3-4) and melanoma ( Figure 5) through oral administration of formulation.
  • Reduced rate of tumor growth was observed after treatment with formulation (200- 400 mg/kg body weight for 10-20 days).
  • Treated animal shows enhanced survivability through immunomodulation.
  • Body weight ( Figure 10) histopathology data of liver, heart, spleen and kidney (Figure 11) suggesting no adverse effect on these animals.
  • the present formulation controls the cancer cells in a dual mode like both by inhibiting autophagy ( Figure 8) as well as by inducing detachment induced cell death (anoikis) which are crucial for metastasis (secondary tumor formation which is incurable). Therefore, the present formulation not only kills the cancer cells (apoptosis), it could also manage the cancer metastasis and controls the metastasis through regulating anoikis (detachment induced cell death) and autophagy (self eating process; Figure 8). It has been observed that inhibition of migration and regulation of the epithelial to mesenchymal transition (EMT) is helpful to control tumour metastasis (Figure 9).
  • EMT epithelial to mesenchymal transition
  • Inhibition of autophagy, migration, EMT and inducing anoikis cell death in cancer cells after treatment with formulation indicates that the formulation could be used to manage tumor metastasis in patients and can prevent the secondary tumor formation.
  • the present invention further elaborates the immunomodulatory activity of formulation (Table-8A).
  • the levels intra cellular cytokines IL-2 and IL-4 decreased and IL-10 and IL-12 increased in treated mice indicating enhanced immunomodulation and suggesting better therapeutic efficacy of this formulation.
  • the present formulation showed synergistic effect with clinically approved drugs like cis- platin, paclitaxel and 5-fluorouracil in cancer cell lines (Table 4). Because of the synergistic effect, combination of formulation and clinically approved drug which reduced the dose of anti cancer drug.
  • Dose reduction index (DRI) in Table-4 represents the fold reduction in dose of these clinically used anti-cancer drugs and thereby to reduce side effects/toxicity. Paclitaxol dose reduced to 1.5-2.0 fold, cisplatin dose reduced by 2.4-4.0 fold and 5-fluoruracil dose reduced by 1.5-2.5 fold.
  • the present invention further provides a process for the preparation of formulation, wherein the fresh plant parts of M.koenigii were washed with water, dried, ground into powder. The powder was then extracted with water for 2-3 hour at 40-60 °C. The dust so obtained was subjected to maceration with ethanol for 24-96 hour at 25 to 35 °C, filtered and dried at 40-60 °C.
  • the extract was finally mixed with the hydro-alcoholic (water: ethanol 1:1) extract of one or more than one plant selected from the group consisting of Alpinia galangal, Rubia cordifolia, Camellia sinensis, Aegle marmelos, Cinnamomum tamala, Spinacia oleracea, Ocimum tenuiflorum and Ipomoea aquatic.
  • the fresh plant parts used are selected from the group consisting of leaves, stem, root and flowers.
  • the present invention provides a kit for the treatment and management of cancer, wherein the kit comprising (i) formulation of extracts optionally with one or more pharmaceutically acceptable preservative, lubricant, diluent and any other excipients
  • the pamphlet contains instructions that administration of said combination provides synergistic effect in comparison with the administration of either formulation of extracts or clinically approved anti -cancer drug alone.
  • PA1 ovarian cancer, MIAPaCa-2 pancreatic cancer, A549 (lung carcinoma), B16F10 (mouse melanoma), HeLa (cervical), HCT116 (colon), WI-38 (human lung fibroblasts), U87MG (glioma), K562 (leukemia) and many other cell lines collected from American Type Culture Collection, Manassas, VA, USA, were cultured in MEM or RPMI-1640 or IMDM with 10% FCS with antibiotic and antimycotic. These cells were cultured till 80% confluence under humidified atmosphere of 95% air, 5% Carbon dioxide at 37°C. Cells were washed once with IX PBS and harvested with Trypsin-EDTA (0.05%) for future purpose. Additionally, U87MG stems like cells was also used.
  • Example 5A Biological activity of individual extracts by MTT assay
  • Example 7B More snergistic enhancement of cytotoxicity of five different extracts after combination
  • Example 12B Sub-acute toxicity studies
  • mice Female BALB/c mice were injected subcutaneously with mouse breast cancer cell lines 4T1. Tumors (450-200 mm 3 ) were generated within 7 days. Mice were administered orally with formulation for 36 days. Mice weight/tumor volume/weight was calculated ( Figure 2). Similar results were obtained with other formulations.
  • Example 14 Effect of formulation 1 on ovarian cancer xenograft model
  • Example 15 Effect of formulation on ovarian cancer syngenic mice model
  • mice Female BALB/c mice were injected subcutaneously with mouse ovarian cancer cell (ID8) for syngenic model. Tumors (450-200 mm 3 ) were generated within 7 days. Mice were administered orally with formulation for 14 days. Mice weight/tumor volume/weight was calculated ( Figure 4). Similar results were obtained with other formulations.
  • Example 16 Effect of formulation on melanoma syngenic mice model
  • Example 17 Formulation 1 induced homeostasis of immune system in ovarian cancer mice model
  • IL- 2/IL-4/IIL-10/IL-12 Intracellular cytokines
  • CD4/CD8 percent positive cells indicated the formulation 1 maintains the homeostasis of immune system in cancer mice after treatment (Table -8A). Similar results were obtained with other formulations.
  • Example 18A Flow cytometric analysis of apoptosis
  • Human ovarian cancer cell line was plated on cover slip and then treated with formulation 1 (10-30 pg/ml) for 24 hours. These adhered cells were washed with PBS and stained with 1 pg/ml DAPI in methanol for 5 minutes at room temperature (-25° C) and washed thrice with PBS. These slides were mounted on glass slide and images were capture with microscope ( Figure 7). Other formulations also induced apoptosis. The experiments were performed using different types of human cancer cells which exhibited similar result.
  • PA1 (lxlO 6 ) were treated with formulation 1 for 24 hours. Then green detection reagent was added and incubated at 37 °C for 30 minutes. Cells were washed and analyzed by FACS. The experiment was performed in a representative human cancer cell line PA1 ( Figure 8). Other formulations were also inhibited autophagosomes.
  • PA1 (lxl0 6 /ml/well) were cultured to >90% confluence and scratch-wounds were made and cultured with formulation 1 for 24 hours. Wound size was calculated and expressed in relative percent compared to untreated cells. The experiment was performed using different types of human cancer cells (Figure 9). Similar results were obtained with other formulations.
  • This formulation is a commercially viable product.
  • Maximum tolerated dose is very high, -2000 mg/kg/day.
  • This may be available as tablet/capsule/ointment or any other form.

Landscapes

  • Health & Medical Sciences (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Alternative & Traditional Medicine (AREA)
  • Biotechnology (AREA)
  • Botany (AREA)
  • Medical Informatics (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines Containing Plant Substances (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The present invention relates to a formulation comprising at least one plant extract of Murraya koenigii and extract of one or more than one plant selected from Alpinia galangal, Rubia cordifolia, Camellia sinensis, Aegle marmelos, Cinnamomum tamala, Spinacia oleracea, Ocimum tenuiflorum and Ipomoea aquatic, optionally along with pharmaceutically acceptable preservative, lubricant and diluent for management of various cancers and cancer stem-like cells through growth-inhibitory effect and immunomodulatory activity. A combination of formulation of extracts and anti-cancer agents reduced the dosage of anti-cancer drug and thereby minimizes the side-effect or toxicity. Formulation is non-toxic to normal cell lines and different tissues in normal animals and reduced the tumor growth in different rodent cancer models through oral route. The present invention further provides a method of treatment using the formulation of extracts of plants, kit comprising formulation and anti-cancer drug and a process for the preparation of formulation of extracts of plants.

Description

PHARMACEUTICAL FORMULATIONS FOR MANAGEMENT OF CANCERS
FIELD OF INVENTION:
The present invention relates to a formulation comprising extract of Murraya koenigii and extract of a plant selected from the group consisting of Alpinia galangal, Rubia cordifolia, Camellia sinensis, Aegle marmelos, Cinnamomum tamala, Spinacia oleracea, Ocimum tenuiflorum, Ipomoea aquatic and combination thereof for management of cancer. The formulation is having growth-inhibitory activity against different cancer cells and cancer stem-like cells for the management of cancer. This formulation also reduces the growth of tumor and increases the survivability of the treated animals possibly through immunomodulation. The present invention also relates to the combination of formulation of these extracts and clinically approved known anti-cancer drugs which helps in reducing dosage of these toxic anti-cancer drugs administered normally. The present invention further relates to a process for the preparation of formulation containing above-mentioned plant extracts.
BACKGROUND AND PRIOR ART OF INVENTION:
Cancer is one of the leading causes of morbidity and mortality worldwide. According to world health organization, approximately 14 million new cases in 2012 were reported. The number of new cases is expected to rise by about 70% over the next 2 decades. Cancer is the second leading cause of death globally and was responsible for 8.8 million deaths in 2015.
The standards of treatment of cancer include radiation therapy, chemotherapy and surgery. Chemotherapy uses paclitaxel, platin based drugs like cisplatin, topotecan and PARP inhibitors like rucaparib, tamazolamide, alone or combination of these drugs. Many of the clinically approved targeted drugs quickly acquired drug resistance. Major of cancer drugs especially from synthetic origin shows very severe side effects which include joint and muscle pain, fatigue, physical weakness, general malaise and serious side effects include nausea and vomiting, rash, and liver toxicity and dizziness. Some anti-cancer drugs show severe allergic reaction. Number of anti-cancer preventative vaccines is also limited. Although patient survival is favorable in early- stage cancer, patients in advanced stages suffer greatly resulting in a shorter survival rate. The costs of these drugs are beyond the reach of the poor.
Natural products having relatively non-toxic nature and abundant availability are the potential source of chemotherapeutic s including anti-cancer molecules like vincristine, vinblastine, etoposide, paclitaxel, camptothecin, topotecan and irinotecan. This natural product combination also has major advantages over the synthetic drug combination by showing higher efficacy and lower side effects. These naturally derived drugs or combinations or extracts are less in cost, which can be easily available for the poor compared to synthetic drugs. Extracts which contain a combination of few medicinal herbs can work together for management of cancer and thereby expected to be less toxic.
To summarize major problems in anti-cancer therapy, there is a high proportion of chemo- resistance with standard regimens. High costs of treating cancer and increased length of stay in hospital are additional problems. Moreover these patients are highly immunosuppressed.
Accordingly, the applicants felt the need to provide an inexpensive formulation from easily available natural sources mainly from abundantly available edible plants for successful management of cancer with immunomodulatory activity. More importantly this formulation should be non-toxic to normal cells.
References may be made to publication (J Amer Sci., 2011, 7, 80-83), wherein authors have found a mixture containing 13 different compounds when used only ethanol for extraction. Most of the molecules present in their extract separately are reported to have anti-oxidant and anti microbial property as indicated in database. No experimental evidences were provided.
References may be made to publication ( Asian J Pharm Clin Res, 2014, 7, 83-86), wherein extraction of Murraya koenigii plant was reported with ethanol. The IC50 value of the reported ethanolic extract is -50-60 pg/ml.
References may be made to publication ( BMC Complementary and Alternative Medicine, 2015, 15, 306), wherein authors made water extraction of Murraya koenigii plant at 60 °C for 2 hrs which showed activity against breast cancer cells.
References may be made to publication (. Nutrition Research, 2003, 23, 1297-1444), wherein authors have reported the acetone: water (3:2) extract of Murraya koenigii plant and tested topically only for skin papillomagenesis.
In another prior art, WO 2007026201 Al and EP 1933945 B l, wherein it was disclosed that water extract of Murraya koenigii plant alone showed the activity against prostate cancer at 200 pg/ml and 100 pg/ml concentration.
Further reference may be made to W02008051523, wherein mahanine, a carbazole alkaloid purified from Murraya koenigii plant induces the expression of an epigenetically silenced gene, RASSF1A, in prostate cancer cells. The effect of mahanine on RASSF1A expression in skin, lung, pancreas, colon, breast and ovarian cancer cell lines was also examined. In all cases, mahanine induced epigenetically silenced gene RASSF1A.
References may be made to US8637679, wherein it is reported that mahanine isolated from Murraya koenigii is effective as potential anticancer agent against colon, cervical, glioblastoma multiform (GBM) and pancreatic cancers. References may be made to publication ( Cancer Letters 2014, 351, 81-90 and Apoptosis, 19, 149-164), wherein the synergistic activity of mahanine from Murray a koenigii with cisplatin in cervical cancers was reported. Mahanine with 5-fluorouracil in combination exhibited 4 fold more synergistic effect in colon cancer.
References may be made to publication ( BMC Complementary Alternat. Med., 2015, 15, 306), wherein it was also reported that the aqueous extract from M. koenigii can show immunomodulatory property.
References may be made to publication ( Iran J Cancer Prev. 2014 Summer, 7, 142-14) wherein authors have reported the antiproliferative activity of aqueous extract of Alpinia galangal on human gastric tumor and L929 cell lines at ~> 250 pg/ml.
References may be made to publication ( Journal of Advances in Biology & Biotechnology, 2016, 5, 1-8) wherein it was reported the cytotoxicity against breast cancer cell line, MDA-MB-231 with IC50 = 44 pg/ml by Rubia cordifolia aqueous extract.
Ethanolic extract of root of Rubia cordifolia induced apoptosis in HeLa human cervical cell line ( Science Secure Journal of Biotechnology 2012, 1, 39-42).
Methanolic, dicloromethane, petether fractions of Rubia Cardifolia showed anti-cancer effect in cervical cancer cell one, HeLa and in Larynx carcinoma cell line Hep-2 at 30-50 pg/ml (International Journal of Pharmacy and Pharmaceutical Sciences, 201, 3,70-71).
References may be made to publication (Anticancer Agents Med Chem. 2015, 15, 26-36), wherein authors have reported the benefits of Camellia sinensis in killing bladder cancer cells and protecting healthy cells. Copper nanoparticles prepared with aqueous extract of Camellia sinensis which showed anti-cancer effect on HT-29, MCL-7, and MOLT-4 human cancer cell lines ( Asian J Pharm Clin Res. 2017, 10, 71-77). It also inhibited Caco-2 Colorectal Cancer Cell Line (Asian Pac J Cancer Prev. 2018, 19, 1697-1701).
References may be made to publication ( South African Journal of Botany 2016, 104, 6- 14), wherein it was also reported that petroleum ether fraction of Cinnamomum tamala methanol extract induced cell death in human lung carcinoma cell line (A549), human breast adenocarcinoma cell line (MCL7) and human glioblastoma cell line (U-87MG) with no toxicity on normal cells. A compound obtained from Cinnamomum tamala f horny 1 acetate) inhibits ovarian cancer cell line A-2780 (Pakistan journal of pharmaceutical sciences, 2015, 28, 969-72).
Reference may be made to publication (Pharmaceutical Biology, 2008, 37, 285-290), wherein authors have reported cytotoxicity against human fibrosarcoma cells (HLS cells) in culture at 50 pg/ml and above by Ocimum sanctum L. (Labiatae) ethanolic extract. Reference may be made to publication ( Food Research 2018, 2, 154-162), wherein it was also reported that the Ocimum sanctum L. (Labiatae) methanolic extract induced cell death in human breast cancer cell lines.
Reference may be made to publication ( Indian J Pharm Sci. 2013,75,628-634), wherein authors have reported the anti-cancer activity of Aegle marmelos leaf extracts prepared using hexane, petroleum ether, ethanol and chloroform in various cancers including lung (A-549), colon (CoLo-05), ovary (IGR-OV-l), prostrate (PC3), leukaemia (THP-l) and breast (MCF-7) cancer at a dose of 100 pg/ml.
Reference may be made to publication ( Journal of Natural Pharmaceuticals, 2013, 4, 66- 70), wherein authors have reported the anti-cancer activity of Ipomoea aquatica ethanol extract against prostrate cancer cell line, LNCaP at IC50 dose 266 g/mL.
Reference may be made to publication ( Indian J Pharmacol, 200, 37, 397-398), wherein authors have reported the anti-cancer activity of Ipomoea aquatica methanol extract and fractions from this extract against cervical cancer cell line, Hep2 and lung cancer cell line, A549.
Reference may be made to publication (Int. J. Pharm. Sci. Rev. Res. 2015, 33, 225-227), wherein authors have reported the anti-cancer activity of Spinacia oleracea ethanolic extract in Lung (A549), cervix (Hela), leukemia (K562), MDA-MB and liver (HepG2) cancers cell lines respectively.
OBJECTIVE OF THE INVENTION:
The main objective of the present invention is to provide a synergistic formulation comprising extracts of selected plants for the management of cancers and cancer stem like cells via growth-inhibitory activity, inhibition of autophagy and immunomodulation.
Another objective of the present invention is to provide a combination of formulation of plant extracts and any clinically approved anti-cancer drug in order to reduce the dose of highly toxic anti-cancer agents.
Yet another objective of the present invention is to provide a formulation which can exhibit the growth-inhibitory activity in different cancer cell lines selected from the group consisting of ovarian, glioma, cervical, lung, pancreas, colon, breast, skin, oral, prostate, head and neck cancers, as well as different types of blood cancer including myeloid leukemia, T-cell/B- cell/Mixed T-cell and B-cell leukemia and cancer stem cells through apoptosis, detachment- induced cell death (anoikis) and inhibition of autophagy. Yet another objective of the present invention is to provide a formulation which can exhibit inhibition of cancer cells migration and regulation of epithelial to mesenchymal transition (EMT), helpful for the management of tumor metastasis.
Yet another objective of the present work is to provide a formulation in different dosage forms selected from the group consisting of tablet, capsule, gel, ointment, lotion and suspension for oral, mucosal administration and also surface application.
Yet another objective of the present invention is to provide a formulation which shows no toxicity (acute/sub-chronic/chronic) on normal rodents (cells/tissues) both at effective dose (ED50) and at higher doses than ED50 values.
Yet another objective of the present invention is to provide a formulation which can lead to in vivo efficacy at very low effective dose (ED50) in rodents through oral administration.
Yet another objective of the present invention is to provide a process for preparation of the formulation of extracts of specific plants.
Yet another objective of the present invention is to provide a kit for the management of cancer.
BRIEF DESCRIPTION OF FIGURES AND ABBREVIATION:
Figure 1: Formulation did not show any toxicity up to -200 pg/ml in representative lung fibroblast cell line (WI-38).
Figure 2: Formulation reduced the growth of tumor in syngenic mice model of breast cancer given a representative image. Similar result was obtained with other types cancer models.
Figure 3: Formulation reduced the growth of tumor in mice xenograft model of ovarian cancer (using PA1 cell line) given a representative image.
Figure 4: Formulation reduced the growth of tumor in syngenic mice model ovarian cancer (using ID 8 cell line) given a representative image.
Figure 5: Reduced growth of melanoma tumor in C57BF6 mice model of melanoma (B16F10 cell line used)
Figure 6: Formulation induced apoptosis indicating by Annexin/PI positivity in representative ovarian cancer cell lines. Similar result was obtained with other types cancer cell lines
Figure 7: Formulation induces the nuclear fragmentation and condensation depicted by DAPI staining
Figure 8: Inhibition of autophagosome formation by formulation analysed by FACS in representative ovarian cancer cell line. Similar result was obtained with other types cancer models Figure 9: Formulation inhibits migration in representative ovarian cancer cells. Similar result was obtained with other types cancer cell lines.
Figure 10: Chronic toxicity study (90 days), body weights of mice: There is no weight loss observed during treatment period (90 days) indicating NO TOXICITY.
Figure 11: Chronic toxicity study (90 days), histopathology study of heart, liver, spleen and kidney by E and H staining.
ED50: median effective dose
MTD: maximum tolerated dose
EMT : epithelial to mesenchymal transition
IC50: half maximal inhibitory concentration
PI: Propidium Iodide
FACS: Fluorescence-activated cell sorting
CSC: cancer stem-like cell
SUMMARY OF THE INVENTION:
The present invention provides a synergistic formulation comprising extract of Murraya koenigii and extract of one or more than one plant selected from the group consisting of Alpinia galangal, Rubia cordifolia, Camellia sinensis, Aegle marmelos, Cinnamomum tamala, Spinacia oleracea, Ocimum tenuiflorum and Ipomoea aquatic, optionally along with one or more pharmaceutically acceptable preservative, lubricant, diluent and any other excipients for management of cancers.
In another embodiment of the invention, the formulation as described above, wherein the ratio of extract of Murraya koenigii: extract of one or more than one plant selected from the group consisting of Alpinia galangal, Rubia cordifolia, Camellia sinensis, Aegle marmelos, Cinnamomum tamala, Spinacia oleracea, Ocimum tenuiflorum and Ipomoea aquatic is in the range of 8-65: 35-92.
In another embodiment of the present invention, there is provided a synergistic formulation comprising Murraya koenigii, Alpinia galangal, Rubia cordifolia and Camellia sinensis, wherein the ratio of extract of Murraya koenigii, Alpinia galangal, Rubia cordifolia and Camellia sinensis is in the range of 10-40:10-60:18-25:12-25.
In another embodiment of the present invention, there is provided a synergistic formulation comprising Murraya koenigii, Alpinia galangal, Rubia cordifolia, Camellia sinensis and Aegle marmelos, wherein the ratio of extract of Murraya koenigii, Alpinia galangal, Rubia cordifolia, Camellia sinensis and Aegle marmelos is in the range of 8-30: 10-54: 16-30: 11-15:11- 15. Another embodiment of the present invention involves a synergistic formulation comprising extracts of all above mentioned 9 herbs Murraya koenigii, Alpinia galangal, Rubia cordifolia, Camellia sinensis, Aegle marmelos, Cinnamomum tamala, Spinacia oleracea, Ocimum tenuiflorum and Ipomoea aquatic mixed in the ratio of 10:30:20:10:10:7.5:7.5:2.5:2.5.
In another embodiment of the present invention, the formulation may be administered in different dosage forms selected from the group consisting of tablet, capsule, gel, ointment, lotion and suspension.
In another embodiment of the present invention, the formulation as described above is having anti-cancer and immunomodulatory activity.
In another embodiment of the present invention, the formulation is having growth inhibitory (Apoptosis/ Autophagy) effect in various cancers cells selected from the group consisting of ovarian, glioma, cervical, lung, pancreas, colon, breast, skin, oral, prostate, head and neck cancers, as well as different types of blood cancer including myeloid leukemia, T-cell/B- cell/Mixed T-cell and B-cell leukemia and cancer stem cells.
The present invention also provides a combination for the treatment of cancer including drug resistant cancer cells and cancer stem like cells comprising the formulation of plant extracts as aforementioned and any clinically approved anti -cancer drugs.
In another embodiment of the present invention, the anti-cancer drug is selected from the group consisting of cisplatin, paclitaxel and 5-fluorouracil.
In another embodiment of the present invention, the combination of formulation with clinically approved anti-cancer drugs showed good synergistic effect and thereby expected to reduces the side -effect and toxicity arising due to the high dosage of known anti-cancer drugs.
The present invention also provides a process for the preparation of formulation comprising the steps of
a) washing fresh plant parts of Murraya koenigii, drying, powdering and extracting with water for 2-3 hours at 40-60 °C,
b) macerating with a non-toxic solvent such as ethanol for 24-96 hours, filtering and drying at 40-60 °C,
c) mixing this extract of step (b) with the hydro-alcoholic (1:1) extract of one or more than one plant selected from the group consisting of Alpinia galangal, Rubia cordifolia, Camellia sinensis, Aegle marmelos, Cinnamomum tamala, Spinacia oleracea, Ocimum tenuiflorum and Ipomoea aquatic
to obtain the desired formulation. The present invention also provides a method of treating cancer by administering effective amount of the formulation or combination of formulation and clinically approved anti-cancer drug as aforementioned to human being or any other animal in need thereof.
In another embodiment of the present invention, the formulation or combination of formulation of extracts and clinically approved anti-cancer drug can be administered by oral, mucosal or intra peritoneal administration.
In another embodiment of the present invention, the formulation showed no toxicity upto 2000 mg/kg body weight in normal rodents (maximum tolerated dose, MTD).
Another purpose of the present invention is to provide a kit for the treatment and management of cancer, wherein the kit comprising
(i) formulation of extracts optionally with one or more pharmaceutically acceptable preservative, lubricant, diluent and any other excipients
(ii) clinically approved anti-cancer drug
(iii) a pamphlet containing instruction of use
wherein the pamphlet contains instructions that administration of said combination provides synergistic effect in comparison with the administration of either formulation of extracts or clinically approved anti -cancer drug alone.
DETAILED DESCRIPTION OF THE INVENTION:
Natural products known to be the best source to treat many diseases like cancer. We have screened many plants sources for their anti-cancer activity and from that finally selected nine different herbs which having a potent anti-cancer activity. The selected plants include M. Koenigii, Alpinia galangal, Rubia cordifolia, Camellia sinensis, Aegle marmelos, Cinnamomum tamala, Spinacia oleracea, Ocimum tenuiflorum and Ipomoea aquatic.
The best source of M. Koenigii plant material used in the present invention was collected from Krishna District, Vijayawada-520002, Andhra Pradesh, India. Remaining eight plants were purchased from Jadavpur local market, near 8B bus stand, Kolkata-700032, West Bengal. These plant were submitted as specimen in Calcutta university Herbarium, Department of Botany, Calcutta University, West Bengal. Even though the starting materials are from a natural source, these are from renewable sources.
The present invention provides a synergistic formulation comprising extract of Murraya koenigii and extract of one or more than one plant selected from the group consisting of Alpinia galangal, Rubia cordifolia, Camellia sinensis, Aegle marmelos, Cinnamomum tamala, Spinacia oleracea, Ocimum tenuiflorum and Ipomoea aquatic, optionally along with one or more pharmaceutically acceptable preservative, lubricant and diluent for the management of cancers through growth inhibitory activity and immunomodulation.
The ratio of extract of Murraya koenigii: extract of one or more than one plant selected from the group consisting of Alpinia galangal, Rubia cordifolia, Camellia sinensis, Aegle marmelos, Cinnamomum tamala, Spinacia oleracea, Ocimum tenuiflorum, and Ipomoea aquatic in the formulation is in the range of 8-65:35-92.
More particularly, the formulation comprising Murraya koenigii and Aegle Marmelos, wherein the ratio of extract of Murraya koenigii: Aegle Marmelos is in the range of 23-33:67-77 (Table-3A, Example-6).
With respect to other formulation comprising Murraya koenigii and Camellia sinensis, wherein the ratio of the extract of Murraya koenigii: Camellia sinensis is in the range of 43- 65:35-57 (Table-3B, Example-6).
The ratio of extract of Murraya koenigii: Alpinia galangal in the present formulation comprising Murraya koenigii and Alpinia galangal is in the range of 8-27:73-92 (Table-3C, Example-6).
The ratio of extract of Murraya koenigii: Rubia cordifolia in the present formulation comprising Murraya koenigii and Rubia cordifolia is in the range of 27-60:40-73 (Table-3D, Example-6).
The present invention also describes about the synergistic formulation comprising 4 herbs i.e Murraya koenigii, Alpinia galangal, Rubia cordifolia and Camellia sinensis, wherein ratio of extracts of Murraya koenigii, Alpinia galangal, Rubia cordifolia and Camellia sinensis is in the range of 10-40:10-60:18-25:12-25 (Formulation 1, Example 7A , Table 3E).
The present invention further provides a synergistic formulation comprising 5 herbs i.e. Murraya koenigii, Alpinia galangal, Rubia cordifolia, Camellia sinensis and Aegle marmelos, wherein ratio of extracts of Murraya koenigii, Alpinia galangal, Rubia cordifolia, Camellia sinensis and Aegle marmelos is in the range of 8-30:10-54:16-30:11-15:11-15 (Formulation 2, Example 7B, Table 3F).
Apart from synergistic formulation 1 and formulation 2, another synergistic formulation comprising all 9 herbs is also prepared by mixing extracts of Murraya koenigii, Alpinia galangal, Rubia cordifolia, Camellia sinensis, Aegle marmelos, Cinnamomum tamala, Spinacia oleracea, Ocimum tenuiflorum and Ipomoea aquatic in the ratio of 10:30:20:10:10:7.5:7.5:2.5:2.5 (Example-7B, Table 3G). The formulation may be in different dosage forms selected from the group consisting of tablet, capsule, gel, ointment, lotion and suspension suitable for oral, mucosal or intra peritoneal administration.
The preservative used in the formulation is selected from the group consisting of methyl paraben or propyl paraben and it is preferably in 0.l-0.5%.
The lubricant used in the formulation is selected from the group consisting of magnesium stearate or stearic acid and it is preferably in 0.5 to 10%.
The binder used in the formulation is selected from the group consisting of sucrose or lactose and it is preferably in 0.5 to 10%.
The growth inhibitory effect of the present formulation was studied in various cancer cells including ovarian, glioma, cervical, lung, pancreas, colon, breast, skin, oral, head and neck cancers, myeloid leukemia, T-cell/B -cell/Mixed T-cell and B-cell leukemia through killing of cancer cells as determined by MTT and tryphan blue dye exclusion method by incubating cells with formulation by varying doses (0-30 pg/ml) and time (24-72 hours). The IC50 for values for all these cancer cells varied within the range of 10-20 pg/ml (Table 2) after exposure of formulation. However, no viable cells were observed at higher concentrations (~>30 pg/ml).
Under the similar conditions, formulation showed no toxicity towards normal cells (WI-38, Figure 1), even at >200 pg/ml dose. No inhibition in the cell proliferation was observed in solvent control. So, formulation was found to be a non-toxic agent under in vitro condition. Under indentical conditions, the IC50 for values for all these individuals plant extracts from nine different plants was also initially tested against cancer cells (Table 2).
After analyzing the in-vitro growth inhibitory effect, in vivo growth inhibitory effect of the formulation was studied in three representative cancer models namely, breast cancer (Figure 2), ovarian cancer (Figure 3-4) and melanoma (Figure 5) through oral administration of formulation. Reduced rate of tumor growth was observed after treatment with formulation (200- 400 mg/kg body weight for 10-20 days). Treated animal shows enhanced survivability through immunomodulation. Body weight (Figure 10), histopathology data of liver, heart, spleen and kidney (Figure 11) suggesting no adverse effect on these animals.
Understanding the mode of action is very important step to discover the targeted drug and it was found that formulation kills the cancer cells through programmed cell death (apoptosis; Figure 6). The cell death was confirmed by DAPI staining after treatment by flourescence microscopy. Nuclear fragmentation further proved that formulation induced cell death is an apoptosis (Figure 7). Similar result was observed using other cells lines from different types of cancer as mentioned in this document. The status of apoptotic related molecules were also determined. A significant drop was observed in Bcl2, caspase-3, caspase-9 and cleavage of PARP in formulation treated cancer cells.
The present formulation controls the cancer cells in a dual mode like both by inhibiting autophagy (Figure 8) as well as by inducing detachment induced cell death (anoikis) which are crucial for metastasis (secondary tumor formation which is incurable). Therefore, the present formulation not only kills the cancer cells (apoptosis), it could also manage the cancer metastasis and controls the metastasis through regulating anoikis (detachment induced cell death) and autophagy (self eating process; Figure 8). It has been observed that inhibition of migration and regulation of the epithelial to mesenchymal transition (EMT) is helpful to control tumour metastasis (Figure 9).
Hence taken together, description of the detailed mechanism exploring how the cancer cells goes through a programmed cell death (apoptosis), anoikis (detachment-induced cell death responsible for metastasis) and inhibition of autophagy (self eating process) after treatment with formulation exclusively establishes the death mechanism of cancer cells and also to control the metastasis.
Inhibition of autophagy, migration, EMT and inducing anoikis cell death in cancer cells after treatment with formulation indicates that the formulation could be used to manage tumor metastasis in patients and can prevent the secondary tumor formation.
The effect of formulation at maximum tolerated dose (MTD) in normal B alb/c mice was studied and it was found that formulation did not show any toxicity up to 2000 mg/kg body weight in normal rodents which is the maximum tolerated dose (Table 6).
The in vivo toxicity effect of the formulation was also examined in normal rodents at higher doses than ED50 values (200-400 mg/kg body weight) through oral administration. It was observed that formulation did not exhibit any acute (14 days), sub-acute (28 days) and chronic toxicity (90 days and 180 days) as corroborated by blood chemistry profile, histopathology studies, body weight and organ weights (Figure 10-11). The adverse effect of formulation towards body mass was also minimal.
The present invention further elaborates the immunomodulatory activity of formulation (Table-8A). The levels intra cellular cytokines IL-2 and IL-4 decreased and IL-10 and IL-12 increased in treated mice indicating enhanced immunomodulation and suggesting better therapeutic efficacy of this formulation.
Additionally, enhanced ratio of CD8:CD4 T-cells indicating the increased immunity of this treated animal to fight against cancer as reflected in the reduction of tumor size after 10-20 days. Therefore, treated mice maintains the homeostasis of immune system (Table-8A), suggesting that this formulation could be helpful for the management of cancer patients who are highly immunosuppressed. Formulation also showed a very good immunomodulatory activity in normal rodents suggesting that this can be in general used to boost immunity probably helpful for cancer prevention (Table-8B).
The present formulation showed synergistic effect with clinically approved drugs like cis- platin, paclitaxel and 5-fluorouracil in cancer cell lines (Table 4). Because of the synergistic effect, combination of formulation and clinically approved drug which reduced the dose of anti cancer drug. Dose reduction index (DRI) in Table-4 represents the fold reduction in dose of these clinically used anti-cancer drugs and thereby to reduce side effects/toxicity. Paclitaxol dose reduced to 1.5-2.0 fold, cisplatin dose reduced by 2.4-4.0 fold and 5-fluoruracil dose reduced by 1.5-2.5 fold.
This advancement (formulation in combination with clinically approved drugs) will be directly beneficial for treating various cancers including drug resistant cancer cells. The perfect combination of the different plant extracts in proper ratio present in this formulation may be the reason for improved synergistic efficacy.
The present invention further provides a process for the preparation of formulation, wherein the fresh plant parts of M.koenigii were washed with water, dried, ground into powder. The powder was then extracted with water for 2-3 hour at 40-60 °C. The dust so obtained was subjected to maceration with ethanol for 24-96 hour at 25 to 35 °C, filtered and dried at 40-60 °C. The extract was finally mixed with the hydro-alcoholic (water: ethanol 1:1) extract of one or more than one plant selected from the group consisting of Alpinia galangal, Rubia cordifolia, Camellia sinensis, Aegle marmelos, Cinnamomum tamala, Spinacia oleracea, Ocimum tenuiflorum and Ipomoea aquatic. The fresh plant parts used are selected from the group consisting of leaves, stem, root and flowers.
The stability of raw material was determined at different temperatures (4-25 °C) and at different time periods (2-4 years). A representative results of stability of M.koenigii plant material and extract was shown in (Table 5) which indicated that this raw material is highly stable. Similar experiments were performed with other eight plants and found to be stable for ~l-2 years.
Similar stability studies were performed for formulations and a representative data presented in Table-5 for formulation 1 which was found to be stable for >2 months at 40 °C which is equalent to 2 years at room temperature i.e 25 °C. So these formulations are stable for more than two years at room temperature (25 °C). Similar experiments was performed with other formulation and found to be stable for ~2 years at room temperature i.e 25 °C.
The present invention provides a kit for the treatment and management of cancer, wherein the kit comprising (i) formulation of extracts optionally with one or more pharmaceutically acceptable preservative, lubricant, diluent and any other excipients
(ii) clinically approved anti-cancer drug
(iii) a pamphlet containing instruction of use
wherein the pamphlet contains instructions that administration of said combination provides synergistic effect in comparison with the administration of either formulation of extracts or clinically approved anti -cancer drug alone.
The following examples given by way of illustration of the working of the invention in actual practice and should not be construed to limit the scope of the present invention in any way.
Example 1: Source of all nine plants
All these nine plants are available all over the India.The best source of Murraya koenigii plant material used in the present invention was collected from Krishna District, Vijayawada- 520002, Andhra Pradesh, India. These plants were submitted as specimen in Calcutta university Herbarium, Department of Botany, Calcutta University, West Bengal.
Remaining eight plants were purchased from Jadavpur local market, near 8B bus stand, Kolkata-700032, West Bengal, India. These plants specimen were also submitted in Calcutta university Herbarium (CUH), Department of Botany, Calcutta University, West Bengal. Even though the starting materials are from a natural source, these are from renewable sources.
Example 2: Standardization of seasonal variation for collection of raw material
To optimize the season of raw materials collection, the fresh plant parts was collected during various seasons (for every 2 months) and prepared the formulation according to protocol described in Example-3 and analyzed for its biological activity by MTT assay. We found that July-December is best time to collect all these plant materials.
Example 3: Preparation of formulation
The fresh plant parts of all nine plants were washed with water and dried. Initially, dried M. koenigii material was extracted with water followed by extraction with ethanol for 72 hour. The remaining eight plants powders were also individually soaked in ethanol: water (1:1) for 72 hour and filtered. All these solvent extractions were dried and mixed in different ratios and checked for their biological activity.
Example 4 Culturing of different human cancer cell lines
PA1, ovarian cancer, MIAPaCa-2 pancreatic cancer, A549 (lung carcinoma), B16F10 (mouse melanoma), HeLa (cervical), HCT116 (colon), WI-38 (human lung fibroblasts), U87MG (glioma), K562 (leukemia) and many other cell lines collected from American Type Culture Collection, Manassas, VA, USA, were cultured in MEM or RPMI-1640 or IMDM with 10% FCS with antibiotic and antimycotic. These cells were cultured till 80% confluence under humidified atmosphere of 95% air, 5% Carbon dioxide at 37°C. Cells were washed once with IX PBS and harvested with Trypsin-EDTA (0.05%) for future purpose. Additionally, U87MG stems like cells was also used.
Example 5A: Biological activity of individual extracts by MTT assay
A representative ovarian cancer cell line was incubated with individual extracts (10-200 pg/ml) from nine different plants for 48 hour separately and cell viability was analysed by MTT assay (Table 1). Briefly, 10,000 cells were plated in triplicate in 96 well plates and incubated in complete media in presence of varying dose of extract. 100 pg MTT was added in each well. Incubated the plate for 2-4 hours at 37 °C and humidified under 5% C02 atmosphere. Absorbance was recorded at 550 nm with 96 well plate readers. Viability and IC50 values were calculated and tabulated in Table 1.
Table-1 cytotoxicity effect of nine different plant extracts in representative cell line of ovarian cancer
Figure imgf000015_0001
Example 5B: Biological activity of formulation of extracts by MTT assay A representative cell lines from different types of cancers (as mentioned in Example 4) were incubated with formulation 1 (10-20 pg/ml) for 48 hours separately and cell viability was analysed by MTT assay (Table 2). Briefly, 10,000 cells were plated in triplicate in 96 well plates and incubated in complete media in presence of varying dose of extract. 100 pg MTT was added in each well. Incubated the plate for 2-4 hours at 37°C and humidified under 5% C02 atmosphere. Absorbance was recorded at 550 nm with 96 well plate readers. Viability and IC50 values against different types of cancer cell lines were similarly analysed by MTT assay (10- 20pg/ml) as tabulated in Table 2. Similarly experiment was performed with other formulations and found to have equal or slightly higher activity.
Table 2: Growth inhibitory effect (IC50) of Formulation 1 in a representative cell line from different types of cancers
Figure imgf000016_0001
Figure imgf000017_0003
Example 6: Beneficial effects between combinations of different plant extracts
During cancer progression, many different signaling pathways simultaneously involve. To combat the cancer a combination of plant extracts will be helpful as these extacts can target different signaling pathways simultaneously and may stop the progression of cancer. Here we have combined different plant extracts which individually showed a potent anti-cancer effect (Example 5A, Table-1).
Combined M. koenigii and A. Marmelos extracts each other in different proportion (23- 33:61-11 , Table-3A) and found to have additional or synergistic activity. Likewise, we have combined M. koenigii with Camellia sinensis extracts (43-65:35-57, Table-3B), M. koenigii with Alpinia galangal extracts (8-27: 73-92, Table-3C) and M. koenigii with Rubia cordifolia extracts (27-60:40-73, Table-3D). All these combinations showed enhanced cell death indicating the beneficial effect of combing the different extracts for treatment of cancer. Similarly, we have combined remaining plants in various ratios and found to have increased cell death at lower concentration.
Table-3A Combining M. koenigii and A. Marmelos extracts decreased cell viability in ovarian cancer
Figure imgf000017_0001
Table-3B Camellia sinensis and M. Koenigii extracts combination shows a synergistic decreased cell viability in ovarian cancer
Figure imgf000017_0002
Figure imgf000018_0001
Table-3C Synergistic combination of Alpinia galangal and M. koenigii extracts increased their cytotoxicity effect in ovarian cancer by exhibiting decreased cell viability
Figure imgf000018_0002
Table-3D Beneficial effects of combining Rubia cordifolia extract with M. koenigii extracts in ovarian cancer
Figure imgf000018_0003
Example 7A: Synergistic enhancement of cytotoxicity different extracts after combination
Each of the nine plant extracts showed anti-cancer effect (Table-2). All extracts are mixed in various combinations and many of these combinations displayed additive or synergistic effects on the cancer cell lines as explained in Example-6. To see at which extend it is possible to increase the anti-cancer effect, we mixed Murayya koenigii extract with three other extracts ( Alpinia galangal, Rubia cordifolia and Camellia sinensis extracts ) in the ratio of 10:60:18:12 and this combination showed good synergistic activity. Similarly different combinations of other extracts also checked and found to have beneficial effects in increasing cell death which leads to decreased concentration indicating synergistic activity. Table-3E Synergistic composition of different extracts in ovarian
Figure imgf000019_0001
Example 7B: More snergistic enhancement of cytotoxicity of five different extracts after combination
Mixed Murraya koenigii extract with four other extracts ( Alpinia galangal, Rubia cordifolia, Camellia sinensis and Aegle marmelos ) in the ratio of 8:54:16:11:11 and this combination showed good synergistic activity slightly more than the formulation 1 (Table-3F).
Similarly different combinations of other extracts also checked and found to have beneficial effects in increasing cell death which leads to decreased concentration indicating synergistic activity.
Table-3F Synergistic composition of different extracts in ovarian
Figure imgf000019_0002
Mixed extract of Murraya koenigii with extracts of remaining 8 herbs ( Alpinia galangal, Rubia cordifolia, Camellia sinensis, Aegle marmelos, Cinnamomum tamala, Spinacia oleracea, Ocimum tenuiflorum and Ipomoea aquatic) in the ratio of 10:30:20:10:10:7.5:7.5:2.5:2.5 and this combination also showed synergistic activity slightly higher than formulation 1 and formulation 2 (Table 3G).
Table-3G Synergistic composition of different extracts in ovarian cancer cells
Figure imgf000020_0001
Example 8: Measurement of growth inhibition in normal cell lines by MTT assay
Normal cells [Lung fibroblast cells, WI-38] were processed similarly and found to be less sensitive towards formulation indicated by in vitro MTT assay under identical conditions (Figure 1). Example 9: Beneficial effects of formulation with clinically approved drugs
Drugs like cisplatin, paclitaxel and 5-flourouracil are known for their good anti-cancer activity. But over the period of time, patient becomes resistance to these drugs, which can be overcome by giving with combination of other drugs. Combination of Formulation 1 (Example 7 A) with clinically approved drugs showed enhanced activity depicted by combination index (Cl)
(Table- 4). The synergistic effect of formulation 1 when used in combination with cisplatin or paclitaxel or 5-flourouracil suggests that formulation 1 decreased the dose of these commercially used anti-cancer drugs. DRI in Table -4 represents the fold reduction in dose of these clinically used anti-cancer drugs and thereby to reduce side effects/toxicity. Paclitaxel dose reduced to 1.5 fold in ovarian cvancer and 2.0 fold in melanoma, cisplatin dose reduced by 2.4 fold in ovarian cancer and 5-fluoruracil dose reduced by 1.5 fold in colon cancer. Similar results were found with other formulations.
When Cl is less than 1 , it indicates synergistic effect which means extract/formulation is expected to decrease the dose of these commercially available drugs. Whereas Cl = 1, it indicates an additional effect. Similar results were found with other formulations.
Table-4: Beneficial effects of formulation 1 with clinically approved drugs in a representative cancer cells
Figure imgf000021_0001
Example 10.· Stability of Formulation at different temperatures
To know the stability of formulation 1, we performed stability studies. The product was kept at different temperatures from 4 °C to 40 °C for 2 months. The growth inhibitory activity i.e. IC50 values was analyzed by in vitro by MTT assay using different cancer cell lines in comparison with a freshly prepared formulation. No significant differences in IC50 values between these extracts was observed suggesting that formulation is stable for 2 months at 40 °C. In such accelerated stability studies, this is considered to be equal to 2 years at 25°C (Table-5). Similar experiment performed for other formulations and are also found to be stable.
Table 5: Stability studies at different temperatures using ovarian cancer cells
Figure imgf000022_0001
Example 11: Stability of raw material
To know the stability of raw material for preparation of formulation, we dried plant materials at different temperatures from 4 °C to 25 °C for 2-4 years. The formulation was prepared using these raw materials which were kept at different conditions. Next bioactivity of each formulation was analyzed by in vitro by MTT assay using cancer cells in comparison with a formulation which was prepared from freshly collected plant materials. A representative data of M.koenigii raw material was presented in Table-5.
No significant differences in the growth inhibitory activity (IC50 values) between these formulations were observed suggesting that raw materialis stable at 4-25 °C for ~2-4 years. Stability of other plant materials were found to be ~ 1-2 years at 4-25 °C.
Example 12A: Acute toxicity studies in normal rodents
Animals were examined for 14 days after the administration of single acute toxic dose (400- 2000 mg/kg/day). Toxicity was analyzed by taking the weight, serum chemistry and also by examination of tissue by H and E staining. And in this in vivo experiment, there was no indication of any toxicity even in highest dose (2000 mg/kg) treated animals (Table-6). Similar results were obtained with other formulations.
Table-6: Maximum tolerated dose (MTD) of formulation in single dose orally fed mice
Figure imgf000022_0002
Figure imgf000023_0001
Example 12B: Sub-acute toxicity studies
Animals were examined for 28 days after the administration of formulation 1 (250 mg/kg/day) for every day. Toxicity was analyzed by taking changes in behavior, feeding habit, total body mass, serum chemistry and in number of biochemical and hematological parameters and also by examination of tissue by H and E staining. And in this in vivo experiment, there was no indication of any toxicity in treated animals (Figure 10-11, Table-7A). Similar results were obtained with other formulations.
Chronic toxicity studies for 90 days
Similar studies were carried out to understand the chronic toxicity of the formulation 1 (Figure 10-11). Animals were examined for 90-180 days after the administration of dose (250 mg/kg/day) for every alternative day. No chronic toxicity was observed. It was observed that there was no indication of any toxicity in formulation treated rodents. Similarly other formulations were also found to be non toxic for 90 days.
Chronic toxicity studies for 180 days
Animals were examined for 180 days after the administration of dose (250 mg/kg/day) for every alternative day. No toxicity was observed with any formulations (Table 7B).
Table-7A: Serum biochemistry of 90 days fed mice
Figure imgf000024_0001
Table-7B: Serum Biochemistry of 180 days fed mice with formulation 1
Figure imgf000024_0002
Figure imgf000025_0001
Example 13: Effect of formulation 1 on breast cancer syngenic mice model
Female BALB/c mice were injected subcutaneously with mouse breast cancer cell lines 4T1. Tumors (450-200 mm3) were generated within 7 days. Mice were administered orally with formulation for 36 days. Mice weight/tumor volume/weight was calculated (Figure 2). Similar results were obtained with other formulations.
Example 14: Effect of formulation 1 on ovarian cancer xenograft model
Female nude mice were injected subcutaneously with human ovarian cancer cell (PA1) for xenograft model. Tumors (400 mm3) were generated within 10 days. Mice were administered orally with formulation for 35 days. Mice weight/tumor volume/weight was calculated (Figure 3). Other formulations were also reduced tumor size similarly.
Example 15: Effect of formulation on ovarian cancer syngenic mice model
Female BALB/c mice were injected subcutaneously with mouse ovarian cancer cell (ID8) for syngenic model. Tumors (450-200 mm3) were generated within 7 days. Mice were administered orally with formulation for 14 days. Mice weight/tumor volume/weight was calculated (Figure 4). Similar results were obtained with other formulations.
Example 16: Effect of formulation on melanoma syngenic mice model
Female C57BL6 mice (h=10, 4-6 weeks old) were injected subcutaneously with mouse melanoma cell (B16F10). Tumors were generated within 5 days. Mice were administered orally with formulation for 14 days. Mice weight was monitored during the experiment. Tumor volumes/weights were reduced. The tumor cells were tested for apoptosis/autophagy and ROS production (Figure 5). Similar experiment was performed with other formulations and found to reduce the tumor size.
Example 17: Formulation 1 induced homeostasis of immune system in ovarian cancer mice model
Balb/c mice with ovarian tumors were orally fed for 14 days. Intracellular cytokines (IL- 2/IL-4/IIL-10/IL-12) and CD4/CD8 percent positive cells indicated the formulation 1 maintains the homeostasis of immune system in cancer mice after treatment (Table -8A). Similar results were obtained with other formulations.
Table- 8A: Immunomodulation by formulation 1 in ovarian cancer mouse model:
Figure imgf000026_0001
Normal Balb/c mice were orally fed for 14 days with the formulation. Intracellular cytokines (IL-2/IL-4/IIL-10/IL-12) and CD4/CD8 percent positive cells indicated the formulation maintains the homeostasis of immune system in normal mice after treatment. Therefore, formulation 1 is also showed a very good immunomodulatory activity in normal rodents suggesting that this can be in general used to boost immunity probably helpful for cancer prevention (Table-8B).
Table- 8B: Iimnunomod illation by formulation in normal rodent:
Figure imgf000026_0002
Example 18A: Flow cytometric analysis of apoptosis
Human ovarian cancer cell line is cultured in presence or absence of formulation 1 (10-30 pg/ml) 24 hours. Cells were incubated with PI and annexin V for 30 minutes at 4 °C and washed with annexin V staining buffer and resuspend in the same buffer. Data acquisition was done on a FACSCalibur flow cytometer at excitation wavelength at 488 nm and emission wavelength at 647 nm and analyzed with CellQuestPro software. The experiment was performed using different types of human cancer cells (Figure 6). Similar results were obtained with other formulations. Example- 18B: Flow cytometric analysis of apoptosis by DAPI staining
Human ovarian cancer cell line was plated on cover slip and then treated with formulation 1 (10-30 pg/ml) for 24 hours. These adhered cells were washed with PBS and stained with 1 pg/ml DAPI in methanol for 5 minutes at room temperature (-25° C) and washed thrice with PBS. These slides were mounted on glass slide and images were capture with microscope (Figure 7). Other formulations also induced apoptosis. The experiments were performed using different types of human cancer cells which exhibited similar result.
Example 19: Quantification of autophagosomes
PA1 (lxlO6) were treated with formulation 1 for 24 hours. Then green detection reagent was added and incubated at 37 °C for 30 minutes. Cells were washed and analyzed by FACS. The experiment was performed in a representative human cancer cell line PA1 (Figure 8). Other formulations were also inhibited autophagosomes.
Example 20: Scratch-wound Assay
PA1 (lxl06/ml/well) were cultured to >90% confluence and scratch-wounds were made and cultured with formulation 1 for 24 hours. Wound size was calculated and expressed in relative percent compared to untreated cells. The experiment was performed using different types of human cancer cells (Figure 9). Similar results were obtained with other formulations.
Example 21
A pharmaceutical formulation (capsule of 300 mg single dose) comprising of formulation 1 (94.3%), Lubricant: stearic acid (0.5%), diluent: Sucrose (5%) and preservative: methyl paraben (0.2%) showed the anti-cancer activity in vitro (10-16 pg/ml in various cancer cells). Same composition with other formulations also prepared and found to be active.
ADVANTAGES:
1. This formulation is a commercially viable product.
2. All these plants are from a renewable abundant source available all geographical locations of India.
3. Starting materials can be collected throughout the year.
4. Process involved only non-toxic solvents water and ethanol.
5. Procedure for preparation time is short(~4-8 days).
6. Formulation containing combination of biologically active extracts and well-known clinically approved widely used anti-cancer drugs gives synergistic activity which leads to decreased dose thereby toxicity. Therefore, such combination therapy with formulation reduced dose of highly toxic clinically approved anti -cancer drugs.
7. Stability of raw materialsare very high at 4-25°C for ~2-4 years.
8. Final product is stable at different temperatures (4-25°C) for various lengths of days/months/years (2-6 years).
9. Exhibiting its biological activity in many different cancer cell lines with different mutations through inducing apoptosis and detachment-induced cell death (Anoikis) as well as inhibiting autophagy. This formulation showed 50% growth inhibition at 10- 20 pg/ml concentration.
10. Normal cells are less sensitive.
11. Minimum/no toxic effects on normal animals after oral administration of formulation.
12. Maximum tolerated dose (MTD) is very high, -2000 mg/kg/day.
13. Formulation did not exhibit any acute/sub -chronic/chronic toxicity after oral administration in normal rodents.
14. Elemental analysis exhibited absence of toxic metals in the formulation.
15. Formulation inhibits migration and regulates epithelial to mesenchymal transition which is helpful for management of tumor metastasis.
16. Effective against tumor bearing mice using different cancer models which decrease the growth of cancer and increases the survivability of the mice at effective dose (200- 400 mg/kg/day), this dose is equivivalent to 1/12* dose in human (16-34 mg/kg/day).
17. It can be administered through oral administration (tablet/capsule).
18. It showed good immunomodulatory activity in normal rodents which can be used as prevention of disease.
19. It showed good immunomodulatory activity in animals with cancer which can be used to treat many diseases including cancers especially during clinical remission/relapse for the management of cancer.
20. This may be available as tablet/capsule/ointment or any other form.

Claims

We claim:
1. A synergistic formulation comprising extract of Murray a koenigii and extract of one or more than one plant selected from the group consisting of Alpinia galangal, Rubia cordifolia, Camellia sinensis, Aegle marmelos, Cinnamomum tamala, Spinacia oleracea, Ocimum tenuiflorum and Ipomoea aquatic, optionally along with one or more pharmaceutically acceptable preservative, lubricant, diluent and any other excipients.
2. The formulation as claimed in claim 1, wherein the ratio of extract of Murray a koenigii: extract of one or more than one plant selected from the group selected from the group consisting of Alpinia galangal, Rubia cordifolia, Camellia sinensis, Aegle marmelos, Cinnamomum tamala, Spinacia oleracea, Ocimum tenuiflorum and Ipomoea aquatic is in the range of 8-65:35-92.
3. The formulation as claimed in claim 1 comprising Murray a koenigii and Aegle Marmelos, wherein the ratio of extract of Murraya koenigii: Aegle Marmelos is in the range of 23- 33:67-77.
4. The formulation as claimed in claim 1 comprising Murraya koenigii and Camellia sinensis, wherein the ratio of extract of Murraya koenigii: Camellia sinensis is in the range of 43-65:35-57.
5. The formulation as claimed in claim 1 comprising Murraya koenigii and Alpinia galangal, wherein the ratio of extract of Murraya koenigii: Alpinia galangal is in the range of 8- 27:73- 92.
6. The formulation as claimed in claim 1 comprising Murraya koenigii and Rubia cordifolia, wherein the ratio of extract of Murraya koenigii: Rubia cordifolia is in the range of 27-60: 40-73.
7. The formulation as claimed in claim 1 comprising Murraya koenigii, Alpinia galangal, Rubia cordifolia and Camellia sinensis, wherein the ratio of extract of Murraya koenigii, Alpinia galangal, Rubia cordifolia and Camellia sinensis is in the range of 10-40:10- 60:18-25:12-25.
8. The formulation as claimed in claim 1 comprising Murraya koenigii, Alpinia galangal, Rubia cordifolia, Camellia sinensis and Aegle marmelos, wherein the ratio of extract of Murraya koenigii, Alpinia galangal, Rubia cordifolia, Camellia sinensis and Aegle marmelos is in the range of 8-30:10-54:16-30:11-15:11-15.
9. A synergistic formulation as claimed in claim 1, wherein the ratio of extracts of Murraya koenigii, Alpinia galangal, Rubia cordifolia, Camellia sinensis, Aegle marmelos, Cinnamomum tamala, Spinacia oleracea, Ocimum tenuiflorum and Ipomoea aquatic is 10:30:20:10:10:7.5:7.5:2.5:2.5.
10. The formulation as claimed in claim 1, wherein the preservative used is selected from the group consisting of methyl paraben or propyl paraben.
11. The formulation as claimed in claim 1, wherein the lubricant used is selected from the group comprising of magnesium stearate or stearic acid.
12. The formulation as claimed in claim 1, wherein the binder used is selected from the group comprising of sucrose or lactose.
13. The formulation as claimed in claim 1, wherein the formulation may be in different dosage forms selected from the group consisting of tablet, capsule, gel, ointment, lotion and suspension.
14. The formulation as claimed in claim 1, wherein the formulation is having anti-cancer and immunomodulatory activity.
15. The formulation as claimed in claim 1, wherein the formulation is having growth inhibitory
(Apoptosis/ Autophagy) effect in various cancers cells selected from the group consisting of ovarian, glioma, cervical, lung, pancreas, colon, breast, skin, oral, prostate, head and neck cancers, as well as different types of blood cancer including myeloid leukemia, T-cell/B- cell/Mixed T-cell and B-cell leukemia and cancer stem cells.
16. A combination for the treatment of cancer including drug resistant cancer cells and cancer stem-like cells comprising the formulation as claimed in claim 1 and any clinically approved anti-cancer drug.
17. The combination as claimed in claim 16, wherein the combination shows good synergistic activity and reduces the dose of anti-cancer drug by upto about 4.0 fold.
18. The combination as claimed in claim 16, wherein the anti-cancer drug is selected from the group consisting of cisplatin, paclitaxel and 5-fluorouracil.
19. The combination as claimed in claim 16, wherein the combination is having anti-cancer and immunomodulatory activity.
20. The combination as claimed in claim 16, wherein the combination of extracts as claimed in claim 1 and clinically approved anti-cancer drug is having growth inhibitory (Apoptosis/ Autophagy) effect in various cancers cells selected from the group consisting of ovarian, glioma, cervical, lung, pancreas, colon, breast, skin, oral, prostate, head and neck cancers, as well as different types of blood cancer including myeloid leukemia, T-cell/B- cell/Mixed T-cell and B-cell leukemia and cancer stem-like cells.
21. A process for preparation of the formulation as claimed in claim 1, wherein the process comprises the steps of
a) washing fresh plant parts of Murraya koenigii, drying, powdering and extracting with water for 2-3 hour at 40-60 °C, b) macerating with a non-toxic solvent such as ethanol for 24-96 hours, filtering and drying at 40-60 °C,
c) mixing this extract of step (b) with the hydro-alcoholic (1:1) extract of one or more than one plant selected from the group consisting of Alpinia galangal, Rubia cordifolia, Camellia sinensis, Aegle marmelos, Cinnamomum tamala, Spinacia oleracea, Ocimum tenuiflorum and Ipomoea aquatic
to obtain the formulation.
22. The process as claimed in claim 21, wherein the fresh plant parts are selected from the group consisting of leaves, stem, root and flowers.
23. A method of treating cancer by administering effective amount of the formulation or combination of formulation and clinically approved anti-cancer drug as claimed in any of the preceding claims to human being or any other animal in need thereof.
24. The method of treating cancer as claimed in claim 23, wherein the formulation or combination of formulation of extracts and clinically approved anti -cancer drug can be administered by oral, mucosal or intra peritoneal administration.
25. The method of treating cancer as claimed in claim 23, wherein the formulation or combination of formulation of extracts with clinically approved anti-cancer drug exhibits inhibition of migration and regulation of epithelial to mesenchymal transition helpful for controlling tumor metastasis.
26. A kit for the treatment and management of cancer, wherein the kit comprising;
(i) formulation of extracts optionally with one or more pharmaceutically acceptable preservative, lubricant, diluent and any other excipients
(ii) clinically approved anti-cancer drug
(iii) a pamphlet containing instruction of use
wherein the pamphlet contains instructions that administration of said combination provides synergistic effect in comparison with the administration of either formulation of extracts or clinically approved anti -cancer drug alone.
PCT/IN2018/050835 2017-12-14 2018-12-13 Pharmaceutical formulations for management of cancers Ceased WO2019116391A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IN201711044953 2017-12-14
IN201711044953 2017-12-14

Publications (1)

Publication Number Publication Date
WO2019116391A1 true WO2019116391A1 (en) 2019-06-20

Family

ID=66819617

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IN2018/050835 Ceased WO2019116391A1 (en) 2017-12-14 2018-12-13 Pharmaceutical formulations for management of cancers

Country Status (1)

Country Link
WO (1) WO2019116391A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2597268A (en) * 2020-07-17 2022-01-26 Maxstim Ltd Composition and method of treating plant material
US11400043B2 (en) 2019-12-10 2022-08-02 Mary Kay Inc. Cosmetic composition

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130065932A1 (en) * 2011-03-11 2013-03-14 Council Of Scientific And Industrial Research Process for the isolation of organic compounds useful for the treatment of cancer
WO2016123475A1 (en) * 2015-01-31 2016-08-04 Constance Therapeutics, Inc. Methods for preparation of cannabis oil extracts and compositions

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130065932A1 (en) * 2011-03-11 2013-03-14 Council Of Scientific And Industrial Research Process for the isolation of organic compounds useful for the treatment of cancer
WO2016123475A1 (en) * 2015-01-31 2016-08-04 Constance Therapeutics, Inc. Methods for preparation of cannabis oil extracts and compositions

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
"Maha Mega Rasangam; Knowledge known since: 1000 years", [retrieved on 20190315] *
D. SUSANNA ET AL.: "Study of synergistic anti-inflammatory activity of Murraya koenigii and Aegle marmelos", ANNALS OF BIOLOGICAL RESEARCH, vol. 6, no. 6, 2015, pages 33 - 38, XP055618710 *
LIAT LOMNITSKI ET AL.: "Composition, Efficacy, and Safety of Spinach Extracts", NUTRITION AND CANCER, vol. 46, no. 2, 2003, pages 222 - 231, XP009041973 *
NIDHI AGARWAL ET AL.: "Natural Herbs as Anticancer Drugs", INTERNATIONAL JOURNAL OF PHARMTECH RESEARCH, vol. 4, no. 3, 2012, pages 1142 - 1153 *
PANDEY GOVIND: "Some Important Anticancer Herbs: A Review", INTERNATIONAL RESEARCH JOURNAL OF PHARMACY, vol. 2, no. 7, 2011, pages 45 - 52, XP055618713 *
TSUN-THAI CHAI ET AL.: "Edible freshwater macrophytes: a source of anticancer and antioxidative natural product-a mini-review", PHYTOCHEMISTRY REVIEWS, vol. 14, no. 3, 2015, pages 443 - 457, XP055618717 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11400043B2 (en) 2019-12-10 2022-08-02 Mary Kay Inc. Cosmetic composition
US11684566B2 (en) 2019-12-10 2023-06-27 Mary Kay Inc. Cosmetic composition
US12059491B2 (en) 2019-12-10 2024-08-13 Mary Kay Inc. Cosmetic composition
GB2597268A (en) * 2020-07-17 2022-01-26 Maxstim Ltd Composition and method of treating plant material
GB2597268B (en) * 2020-07-17 2023-07-19 Maxstim Ltd Composition and method of treating plant material

Similar Documents

Publication Publication Date Title
JP5300195B2 (en) Composition of botanical extract for cancer treatment
CN113262258B (en) Flos Potentillae Anserinae extract, its medicine, and its preparation method and application
JP6961100B2 (en) An immunopotentiator, an immunoanticancer agent, and a side effect relieving agent for an anticancer agent containing an anthocyanin-fucoidan complex as an active ingredient.
WO2009109109A1 (en) Use of tea polyphenols in preparing medicaments for prevention or treatment of tumors
WO2019116391A1 (en) Pharmaceutical formulations for management of cancers
ES2279157T3 (en) EXTRACT WITH ANTITUMORAL AND ANTIVENOUS ACTIVITY.
CN105327071A (en) Antineoplastic traditional Chinese medicinal composition and application thereof
CN101143148B (en) Application of papaya saponin Ⅰ and its derivatives
CN104623215B (en) A kind of antitumor medicine composition
CN102908340A (en) Isolicoflavonol-containing antitumor drug and application thereof
CN102335180B (en) Application of ursane compounds in preparing antitumor drugs
CN102309656B (en) Traditional Chinese medicine composition with effect of preventing and treating cancers and medicinal application of traditional Chinese medicine composition
KR102694803B1 (en) Combination of A-nor-5α-androstane compound drugs and anticancer agents
Ezejindu et al. Antibacterial activity, phytochemical properties and mineral content of “Aju Mbaise” decoction administered to nursing mothers
JP6627141B2 (en) Method for preparing safflower buckwheat extract, extract prepared thereby, and use of extract
Punitha et al. Potent in vitro cytotoxic effect of Gmelina arborea Roxb.(Verbenaceae) on three human cancer cell lines
CN109320409A (en) A kind of preparation method and application of anthraquinone dimer compound with antifungal and antitumor activity
CN113214154B (en) Tribenzyl isoquinoline alkaloid, preparation method, pharmaceutical composition and application thereof
CN102114065B (en) Composition of Desmodium and Tripterygium wilfordii traditional Chinese medicine with anti-cancer effect and application thereof
CN101732377A (en) Method for preparing anti-cancer total saponin extracts of oplopanax elatus nakai leaves
AU2009258014A1 (en) Anticancer methods using extracts of Anemarrhena asphodeloides Bunge
CN101289453A (en) Ellagic acid compound and its preparation and application in anticancer drugs
KR100726006B1 (en) Pharmaceutical composition for inhibiting cancer disease metastasis comprising polysaccharides derived from Angelica gigas
NL1013004C1 (en) Herbal composition suitable for the preparation of a pharmaceutical preparation for the treatment of cancer, an extract and a pharmaceutical preparation.
CN101690738A (en) New use of gooseberry and its extract in preparing antitumor drugs

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18887283

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18887283

Country of ref document: EP

Kind code of ref document: A1