[go: up one dir, main page]

WO2025048651A1 - Agents bioactifs et leurs utilisations - Google Patents

Agents bioactifs et leurs utilisations Download PDF

Info

Publication number
WO2025048651A1
WO2025048651A1 PCT/NZ2024/050012 NZ2024050012W WO2025048651A1 WO 2025048651 A1 WO2025048651 A1 WO 2025048651A1 NZ 2024050012 W NZ2024050012 W NZ 2024050012W WO 2025048651 A1 WO2025048651 A1 WO 2025048651A1
Authority
WO
WIPO (PCT)
Prior art keywords
mao
sarmentosin
blackcurrant
ester
juice
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.)
Pending
Application number
PCT/NZ2024/050012
Other languages
English (en)
Inventor
Andrew Belton Scholey
Stephen John BLOOR
Angus Ross BROWN
Samuel Christopher DODD
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.)
Arepa Ip Ltd
Original Assignee
Arepa Ip Ltd
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 Arepa Ip Ltd filed Critical Arepa Ip Ltd
Priority to AU2024287117A priority Critical patent/AU2024287117A1/en
Priority to US18/821,619 priority patent/US20250090564A1/en
Publication of WO2025048651A1 publication Critical patent/WO2025048651A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7032—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a polyol, i.e. compounds having two or more free or esterified hydroxy groups, including the hydroxy group involved in the glycosidic linkage, e.g. monoglucosyldiacylglycerides, lactobionic acid, gangliosides
    • 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)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Preparation or treatment thereof
    • A23L2/02—Non-alcoholic beverages; Dry compositions or concentrates therefor; Preparation or treatment thereof containing fruit or vegetable juices
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Preparation or treatment thereof
    • A23L2/52—Adding ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0087—Galenical forms not covered by A61K9/02 - A61K9/7023
    • A61K9/0095—Drinks; Beverages; Syrups; Compositions for reconstitution thereof, e.g. powders or tablets to be dispersed in a glass of water; Veterinary drenches

Definitions

  • Bioactives and their uses TECHNICAL FIELD This invention relates broadly towards sarmentosin and its esters and their new use as monoamine (MAO) inhibitors and use as treatment or prevention of various associated conditions related to MAO activity.
  • MAO monoamine oxidase
  • MAO-A is an enzyme which catalyses the oxidative deamination of amines such as dopamine and serotonin. The enzyme appears as two isozymes, MAO-A and MAO-B.
  • Monoamine oxidase inhibition can be reversible or irreversible and can either act non- selectively, affecting both isoforms, or selectively, affecting only one isoform.
  • MAO enzymes are of interest as targets for nutraceuticals, functional foods and drug discovery. This is because, for example, MAO-A is involved in psychiatric conditions and depression and MAO-B is linked to neurological disorders such as Parkinson’s and Alzheimer’s diseases.
  • MAO inhibitors appear to improve cognitive function as well as helping prevent cognitive disorders like Alzheimer’s disease, and therefore such inhibitors once identified, present real therapeutic benefit for their cognitive enhancing properties.
  • Isocarboxazid, phenelzine and tranylcypromine appear to have fairly comparable properties (Mallinger & Smith 1991). They are readily absorbed and reach peak concentrations in 1–2 hours. Elimination is also swift, with half-lives in the range of 1.5 to 4 hours (the half-life is especially short for tranylcypromine).
  • Moclobemide is a more recently developed MAO inhibitor drug and is readily absorbed and reaches peak plasma concentrations in approximately 1 hour. Metabolism is rapid and complete with an elimination half-life in the range of 1–3 hours.
  • Deprenyl also known as selegiline
  • MAO-B inhibitor drug with efficacy for treating depression and Parkinson’s disease at dosages between 1.25 mg to 10 mg per day.
  • many MAO inhibitor drugs have fallen out of favour for treatment of depression due to side effects from adverse drug and food interactions. Their primary clinical use largely remains in the treatment of Parkinson’s Disease.
  • US8367121B2 describes a nutraceutical-based approach to attenuating the underlying pathophysiological processes leading to Parkinson’s Disease, relying in part through selection of known MAO inhibitors (see para 0045-0047). Because of the role of MAO in modulating dopamine and serotonin, the Mayo Clinic website (https://www.mayoclinic.org/diseases-conditions/depression/in-depth/maois/art-20043992) also highlights the fact that MAO inhibitors have been useful tools to improve mental health conditions, mood or to address conditions like depression or anxiety.
  • MAO inhibitors have been approved by the FDA including isocarboxazid (Marplan), Phenelzine (Nardil), Selegiline (Emsam), and Tranylcypromine (Parnate).
  • isocarboxazid Marplan
  • Phenelzine Nardil
  • Selegiline Emsam
  • Tranylcypromine Tranylcypromine
  • MAO inhibition provides cardioprotection in numerous models of CVD, including ischemia/reperfusion, heart failure and diabetes (see also P I Adnitt, 1968 on the early identification of MAO inhibition and hypoglycemic action).
  • the review also highlights studies supporting the use of MAO inhibitors in treating patients with CVD.
  • This same paper also emphasises that reactive oxygen species (ROS) and oxidative stress play a role in cardiac injury, myocardial remodeling and heart failure. It further states that a major source of ROS in the mitochondria is from MAO through production of hydrogen peroxide (H2O2).
  • ROS reactive oxygen species
  • H2O2O2 hydrogen peroxide
  • Duarte et al, 2020 also reviews promising opportunities for MAO inhibitors in treating or preventing CVD.
  • MAO inhibitors represent promising targets for joint inflammation diseases such as rheumatoid arthritis (and associated joint pain or stiffness).
  • Ostadkarampour & Putnins (2021) also highlight further studies over the past 1-2 decades that support the use of MAO inhibitors for wide ranging conditions like hair growth, pain management (e.g. joint pain and stiffness), ocular disease, muscular dystrophy, sexual dysfunction, behavioral conditions, smoking cessation, and cancer (also see Wang et al.,2021, and Aljanabi et al., 2021).
  • MAO inhibitors like phenelzine also have shown application towards multiple sclerosis (Benson et al., 2013) MAO inhibitors have also been investigated for veterinary applications, although commercially, selegiline currently is the only commercially used MAO inhibitor for animals despite its considerable contradictions and potential side effects.
  • the Veterian Key website www.veteriankey.com/monoamine-oxidase-inhibitors
  • the website also highlights selegiline is used to treat and prevent cognitive dysfunction syndrome, anxiety and other behavioral issues in pets.
  • any compounds identified or developed with the ability to inhibit (preferably via selectively reversable inhibition) MAO enzyme activity are now being seen as viable routes beyond neurocognitive outcomes, for example for treating various cardiovascular diseases, diabetes, hyperglycemia, pre-diabetes, and/or preventing or reducing the risk of developing these conditions.
  • new MAO inhibitors and preferably those with lower, minimal, or no side effects comparable to commercially available MAO inhibitors, also represent very important opportunities to improve, reduce, maintain or inhibit the production of ROS or oxidative stress, which have implications towards CVD as well as a range of other commonly known disease states as discussed above.
  • any newly identified MAO inhibitor (and preferably one that is selectively reversable) that is shown to have a similar or efficacious potency (via in vitro or in vivo studies) to commercially known MOA inhibitors like selegiline, will be understood by those skilled in the art to have therapeutic and preventative application to these disease states.
  • studies on diverse sources such as cigarette smoke and coffee have found a class of amines known as beta-carboline alkaloids to be inhibitors of MAO (Herraiz, 2007).
  • beta-carboline alkaloids to be inhibitors of MAO (Herraiz, 2007).
  • a Madrid-based research team has shown that a number of fruits and fruit juices / extracts contain beta-carbolines (betaCs) (Herraiz, 2006, 2018).
  • Such components may potentiate the weak MAO inhibiting phenolics seen in berryfruits like blackcurrant. These newly identified compounds would therefore be of significant commercial interest.
  • 'Watson et al 2015 compared human MAO inhibition between two blackcurrant cultivars with equivalent levels of anthocyanins. The authors highlighted that there was significant MAO-B inhibition following consumption of one anthocyanin-rich blackcurrant extract but not the other. The MAO-B inhibition in the one cultivar was confirmed in further research (Watson, 2020).
  • the differential MAO inhibition suggests removal or degradation, in one preparation, of unknown bioactive(s) which may be important for achieving more potent MAO activity.
  • non-anthocyanin fraction is responsible for, or differentially enriches MAO inhibition in certain cultivars. Further, it is possible that non-anthocyanin and anthocyanin fractions interact to provide significant MAO inhibition. Indeed, it has been thought for some time that some betaCs in blackcurrant could be a strong candidate for the MAO inhibitory activity sometimes observed in BC juice (Budzikiewicz, 1994, and Herraiz, 2006, 2018).
  • compositions formulated as a food or beverage, pharmaceutical composition, pet food, pet supplement or veterinary therapeutic or composition, nutraceutical or an extract from a plant material, wherein the composition includes sarmentosin or an ester(s) thereof, and when the composition is used for inhibiting monoamine oxidase enzyme A (MAO-A) and/or monoamine oxidase enzyme B (MAO- B).
  • MAO-A monoamine oxidase enzyme A
  • MAO- B monoamine oxidase enzyme B
  • sarmentosin or an ester(s) thereof, or a plant material, an extract or a composition containing same in the manufacture of a medicament for the inhibition of monoamine oxidase enzyme A (MAO-A) and/or monoamine oxidase enzyme B (MAO-B) in a person or animal in need thereof.
  • MAO-A monoamine oxidase enzyme A
  • MAO-B monoamine oxidase enzyme B
  • MAO-A monoamine oxidase enzyme A
  • MAO-B monoamine oxidase enzyme B
  • sarmentosin or its ester(s) for improving the effects of another known MAO inhibitor (including but not limited to anthocyanin(s)).
  • a combination of sarmentosin or its ester(s) and at least one anthocyanin(s) when used to provide a MAO inhibition effect there is provided a blackcurrant fruit, blackcurrant juice, extract, or isolate including an effective amount of sarmentosin or ester(s) thereof, when used for inhibiting MAO-A or MAO-B.
  • an isolated compound comprising sarmentosin or an ester(s) thereof, or a combination thereof.
  • sarmentosin and its ester(s) as a MAO-A and/or MAO-B inhibitor.
  • this important and unexpected activity of sarmentosin or its ester(s) was not previously known.
  • the primary MAO inhibitory activity in blackcurrant was shown not to be associated with the anthocyanins/polyphenols nor ⁇ - carbolines.
  • the term “sarmentosin” should be taken as meaning a gamma hydroxy nitrile glycoside (chemical name: 4-(beta-D-glucopyranosyloxy)-2-(hydroxymethyl-2- butenenitrile)), or derivatives thereof as shown in the general chemical structure below:
  • the term “sarmentosin ester” or “ester thereof” should be taken as meaning nigrumin-p- coumarate, nigrumin caffeate and / or nigrumin ferulate or derivatives thereof as shown in the general structures below. These are phenolic acid derivatives of sarmentosin.
  • extract should be taken as meaning a preparation containing the sarmentosin or an ester thereof taken, isolated, removed, purified or otherwise extracted from a selected source in a more concentrated form or more purified form compared to how it is naturally found.
  • the extract may be in liquid form such as a juice, a juice concentrate, or a dried format such as a powder, tablet or capsule.
  • the extract may contain other bioactives or components from the selected source, or may be combined with other extracts, ingredients or products as desirable.
  • the extract containing the sarmentosin may be in a fully purified or semi purified form to the extent that it becomes a purified isolate, and still be considered an extract for the purposes of this invention.
  • composition should be taken as meaning a combination of the sarmentosin or an ester thereof, in an admixture together with other ingredients or constituents.
  • the composition for example, may be in the form of a food or beverage, pharmaceutical formulation, nutraceutical, supplement, or natural extract and so forth without departing from the scope of the invention.
  • plant material should be taken as meaning any biological vegetation including roots, leaves, seeds, seedlings, fruit, stalks and so forth.
  • effective amount should be taken as meaning the amount of a compound that, when administered to a human or other mammal for improving, treating, preventing or delaying a state, disorder or condition, is sufficient to affect such treatment.
  • the “effective amount” can vary based on the compound, the particular condition and its severity as well as the age, physical state, or weight of the mammal to be treated. Preferred effective amounts, including dosages, are discussed further below.
  • MAO monoamine oxidase
  • MAO-A and MAO-B two isozymes
  • MAO inhibitor should be taken as meaning a type of chemical, drug, substance, extract or bioactive that has partial or full inhibitory activity to one or both monoamine oxidase enzymes, namely monoamine oxidase A (MAO-A) and monoamine oxidase B (MAO-B). This inhibition may be reversible or irreversible.
  • nootropic should be taken as meaning a substance that improves or supports cognition, memory and/or facilitates learning.
  • the term “synergy” or “synergistic” or “synergism” should be taken as meaning a beneficial effect of combining two or more compounds, molecules, or bioactives together wherein the combination achieves a greater result than either would achieve individually.
  • the synergy may arise through various modes of action. Thes could include (but are not restricted to) through complexation for example to improve stability or bioavailability, through inhibition of, or competition for, metabolizing enzymes, via increased delivery of metabolic substrates (e.g., through increased blood flow) or through some other structural or functional mechanism that improves the potency or longevity of the MAO inhibition.
  • MAO Inhibition Activity Preferably the sarmentosin, its esters thereof or extract containing same provides reversible MAO inhibition.
  • the profile of MAO-B inhibition of blackcurrant juice bears a notable similarity, to pharmaceutical reversible MAO-B specific inhibitors such as lazabemide, which have shown a rapid inhibition of MAO-B in platelets of >90% at 30 min post dose, with maximal inhibition subsiding 16 h post dose and full restoration of enzyme activity returning 48 h post dose following a 100 mg dose of lazabemide (Dingemanse et al., 1997).
  • blackcurrant supplementation whilst expressing similar MAO inhibitory effects as pharmaceutical drugs, blackcurrant supplementation has shown no shown detrimental side effects from its MAO inhibition (Braakhuis et al., 2020), and appears to advantageously offer reversible inhibition of MAO enzymes.
  • blackcurrant fruit, extracts from blackcurrant, or the sarmentosin extracted or isolated from blackcurrant also appears to be particularly advantageous because the strong safety history of blackcurrant as a food, further suggesting sarmentosin itself will have a safe profile as a MAO inhibitor.
  • very few MAO inhibitor drugs are still used due to safety profiles. To the best knowledge of the inventors, laxabemide was never marketed.
  • the sarmentosin or ester(s) thereof have, and/or are used, for both MAO-A and MAO- B inhibition activity.
  • Having activity to both MAO enzymes may be very beneficial as they have strong affinities for different substrates.
  • MAO-A shows greater affinity for hydroxylated amines such as noradrenaline and serotonin
  • MAO-B shows greater affinity for non-hydroxylated amines such as benzylamine and beta-phenylethylamine (PEA).
  • PDA beta-phenylethylamine
  • dopamine and tyramine show similar affinity for each enzyme form, meaning inhibition of both may be beneficially required to modulate their availability.
  • the sarmentosin or ester(s) thereof have, and/or are used, for either MAO-A or MAO-B inhibition activity alone. This is supported and discussed in the Examples.
  • Combinations with other MAO inhibitors such as anthocyanin(s)
  • sarmentosin or its ester(s) is combined with another MAO inhibitor.
  • the other MAO inhibitor may be any other commercially used MAO inhibitor. It may also be a MAO inhibitor not yet commercially used, or one undergoing research either now or in the future.
  • sarmentosin or its ester(s) is combined or used in combination with at least one anthocyanin(s).
  • the combination of sarmentosin or its esters with anthocyanin(s) appears to substantially improve its apparent MAO activity, even at low doses of anthocyanin(s).
  • the anthocyanin(s) is selected from cyanidin and/or delphinidin.
  • the anthocyanin(s) selected from the group consisting of Delphinidin 3-O-glucoside, Delphinidin 3-O-rutinoside, Cyanidin 3-O-glucoside, Cyanidin 3-O-rutinoside, Petunidin 3-O- rutinoside, Pelargonidin 3-O-rutinoside, Peonidin 3-O-rutinoside and combinations thereof.
  • anthocyanins were shown to be the most prominent anthocyanin(s) present in blackcurrant concentrate and powder extracts (See Table 6). However, one skilled in the art would appreciate there are many other similar or alternative anthocyanin(s) in blackcurrant or its extracts or alternative fruit or vegetable sources of anthocyanins (such as blueberry or boysenberry), and such anthocyanin(s) – currently known or unknown - should also be considered as viable options without limitation. One skilled in the art would appreciate the many different fruit or vegetables that contain anthocyanin(s) and would be expected to deliver the same or similar effect when combined with sarmentosin or it(s) esters.
  • anthocyanin(s) should also be considered as optionally / preferably including anthocyanin(s) to leverage the apparent synergistic effect observed.
  • blackcurrants have been shown to have some MAO inhibition (Watson et al 2020), but the bioactives responsible are still unknown.
  • Anthocyanins were thought to be responsible, but studies comparing two anthocyanin-rich blackcurrant extracts (Watson et al, 2015) have suggested that anthocyanins have low MAO inhibition activity, or may have lost some activity somehow during the processing of the extract.
  • Example 7 of the present specification exemplifies a beneficial synergy when sarmentosin (or its esters) is present together with anthocyanin(s) also from blackcurrant.
  • Both sarmentosin and anthocyanins were quantified in juice and powder used in the trial, and demonstrated a similar bioavailability time course in blood plasma tests following consumption.
  • the juice had a lower dosage of about 1 mg anthocyanins / kg weight compared to 7.8 mg anthocyanin(s) / kg weight in the powder dosage.
  • the powder format likely had 3 fold the amount of sarmentosin than the juice format.
  • the two extracts showed an unexpected similar MAO inhibition profile (or potentially even an improved in the juice compared to the powder, see Figure 23).
  • Figure 22 shows very similar sarmentosin bioavailability profiles in plasma tests of the subjects after consumption. This suggests that the sarmentosin may be advantageously increasing the MAO inhibition effect of anthocyanin(s) at a very low dosage of 1 mg / kg weight.
  • the anthocyanins may be synergistically improving the sarmentosin MAO inhibition effects.
  • composition and formats The composition may be in a variety of formats without departing from the scope of the invention.
  • the sarmentosin or its ester(s) thereof is within a plant material, an extract or a composition.
  • a particularly preferred format is a beverage or drink, or powder supplement; however, a number of other options may be achieved easily by a skilled person.
  • the sarmentosin or its esters are provided in a food or beverage. More preferably, the food or beverage is based on blackcurrant fruit, blackcurrant juice, blackcurrant extracts (liquid or powder format), and so forth. Similarly, one would appreciate that any other fruit source containing sarmentosin or its ester(s) would be applicable.
  • sarmentosin Alternatively, one may choose to extract, isolate or synthetically make sarmentosin, and then add it to any such juice or composition type and not depart from the scope of the invention.
  • the inventors have identified the presence and MAO inhibitory activity of sarmentosin and its esters in blackcurrant fruit (and its extracts), a strong commercial preference is to utilise the existing bioactivity for this new use in blackcurrant based or blackcurrant containing products. It should be appreciated that either the existing levels of sarmentosin or its ester(s) may be used, or further fortified, concentrated, boosted with additional amounts of sarmentosin.
  • the sarmentosin or its ester(s) are provided in a nutraceutical or therapeutic format such as a powder, tablet or capsule. Again, these formats may be derived from or contain blackcurrant material. As previously highlighted, in another embodiment the whole fruit or part thereof or whole plant material or part thereof (e.g. blackcurrant) containing said sarmentosin or its ester(s) is used to achieve the new uses or methods described herein.
  • the sarmentosin or its ester(s) is provided as an extract from a plant source.
  • the extract is a polar fraction or component extracted from the selected source of the sarmentosin.
  • the extract is non-polyphenolic.
  • the extract containing the MAO inhibition activity from sarmentosin or its ester(s) also includes BetaCs.
  • the extract is a juice or a concentrated version thereof. More preferably, the juice extract is from blackcurrant fruit.
  • the invention may be leveraged through the development of a new plant variety with sarmentosin or one of its esters with levels greater than normally or naturally present.
  • the plant variety may that of a blackcurrant plant (Ribes nigrum) which already has naturally occurring, albeit at relatively minimal levels, of sarmentosin and its esters which is then selectively bred to have increased levels of the bioactive(s) and then utilised for the applications described herein.
  • Preferred concentration of sarmentosin or ester(s) in the extract or composition Preferably, the extract or composition has at least about 0.005 mg, or more preferably 0.05 mg/g sarmentosin per gram (or mg/g) of the extract or composition.
  • the extract or composition has at least about 0.005, 0.01, 0.02, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0,15.0, 16.0, or 17.0, 18.0, 19.0, 20.0, 21.0, 22.0, 23.0, 24.0, 25.0, 30.0, 40.0, 50.0, 60.0, 70.0, 80.0, 90.0, 100.0 or 200.0 mg sarmentosin thereof per gram of extract or composition.
  • the extract or composition has at between about 0.005 – 70.0 mg sarmentosin per gram (or mg/g) of the extract or composition.
  • a preferred minimum dosage may be about 20 mg per day of sarmentosin. If a blackcurrant juice had about 0.3 mg / ml sarmentosin, a serving size of 86 ml of that juice would achieve this minimum dosage.
  • concentration of sarmentosin may be suitably adjusted in a desired extract or composition using conventional methods and practices to achieve effective amounts / dosages and other criteria. Similar embodiments may also be envisioned for the sarmentosin esters shown for example in Example 4.
  • sarmentosin Whilst the levels of the esters are lower comparatively to sarmentosin, someone skilled in the art would appreciate that there are means to increase the levels of these in an extract or composition if they so desired.
  • Sources of sarmentosin or its esters Preferably, sarmentosin may be extracted or isolated from a number of sources without limitation.
  • sarmentosin esters have been identified in blackcurrant seeds (Lu et al., 2002), and sarmentosin has been identified in Kalanchoe species (Fernandes et al, 2021).
  • sarmentosin and its esters in blackcurrant fruit, and fruit extracts (such as dried extracts or juices). Therefore, preferably sarmentosin is extracted or isolated from blackcurrant fruit.
  • the extract may also include other components from the source (such as blackcurrant) as this may provide additional benefits from bioactives or micronutrients.
  • a whole fruit or plant material may be used to provide the beneficial MAO inhibitory effect from sarmentosin or its esters, so long as the material indeed has this beneficial bioactive which is the subject of this patent application.
  • CN102659860 and CN101974044B both describe other methodologies for extracting sarmentosin from other plant sources. These present alternative approaches to extract or isolate sarmentosin for the new uses of the present invention. Whilst these may be suitable sources, it should be appreciated that other plants may also contain similar or higher levels of sarmentosin or its esters which here have been shown to have MAO inhibition activity. Alternatively, the source of sarmentosin or its esters may also be synthetically produced through known methodologies, or via microbial fermentation production either from microbes that naturally express sarmentosin, or via genetic modification using constructs that are coded to recombinantly express sarmentosin in standard cellular culture methodologies.
  • sarmentosin beyond what is naturally found currently. This may be through development of a suitable plant variety, either through genetic engineering, via cross-breeding or multi-generational selection procedures to produce new varieties with high levels of sarmentosin or its esters.
  • the present invention may be achieved through artificially producing sarmentosin or its esters at high purity, and provided, stored, or sold as a purified form or suitably provided within a pharmaceutical composition. This may be particularly useful for the pharmaceutical industry where stringent manufacturing requirements are required.
  • suitable formats including but not limited to transdermal creams or patches, parenteral or subcutaneous injection, pills, capsules and so forth are all possible routes of administration.
  • MAO inhibitors have been therapeutically used for a long time, and different routes of administration are routinely utilised.
  • Sabri, M et al., 2023 www.ncbi.nlm.nih.gov/books/NBK557395
  • highlights the most common route of administration of Selegiline is via transdermal patch, however it can also taken by mouth similar to other MAO inhibitors (isocarboxazid, phenelzine, and tranylcypromine).
  • MAO inhibitors such as Selegeline is typically given to the animal in tablet form.
  • the juice from the Ribes nigrum may be manufactured according to standard practice.
  • a juice concentrate may be obtained by evaporation under vacuum of the juice to yield a concentrate with approximately 10x the concentration of sarmentosin.
  • a powdered form of the juice may be produced by freeze-drying or spray drying the juice. Therefore, one skilled in the art would appreciate that the invention may be provided in various different dried formats, such as capsules, tablets, powders, and so forth.
  • Pure sarmentosin may be obtained by chromatographic separation of the juice or juice concentrate using reversed phase chromatography with multiple steps until pure sarmentosin is obtained.
  • compositions containing sarmentosin may be prepared are in the Best Modes section of the specification. Methods of use and treatments MAO inhibitors have already shown promise and are associated with a range of diseases and conditions as highlighted throughout this specification.
  • the use or methods of treatment as described herein modulates, maintains or increases a neurotransmitter selected from the group consisting of dopamine, serotonin, adrenalin (epinephrine) or noradrenalin (norepinephrine) and tyramine.
  • MAO inhibitors have application not only towards human clinical and non-clinical uses, but also towards veterinary therapeutic applications in animals. Therefore in one embodiment, sarmentosin or its ester(s) are used as a MAO inhibitor (or towards clinical or non-clinical applications linked to MAO inhibition) either in a human or animal in need thereof.
  • the animal may be a companion animal or such as a dog, cat, horse, rabbit or bird.
  • the animal may be a farmed animal such as a cow, sheep, pig or the like.
  • the use or methods of treatment as described herein are for the treatment or prevention of a disease or condition is any neurological or psychiatric condition or ailment associated with MAO-A or MAO-B enzyme activity in a person or animal in need thereof.
  • the sarmentosin or its ester(s) are used as an adjunct to a compound, pharmaceutical or nutraceutical already known or used for treating or preventing any of the conditions or diseases discussed below or within this specification .
  • the sarmentosin or its ester(s) are used for treating or preventing depression, atypical depression, panic, social anxiety, Generalised Anxiety Disorder (GAD), bipolar disorder (especially the depressive phase), post-traumatic stress disorder (PTSD), Obsessive Compulsive Disorder (OCD), adult ADHD, Alzheimer’s disease, Dementia, Parkinson's disease and/or Huntington’s disease in a person or animal in need thereof.
  • GAD Generalised Anxiety Disorder
  • PTSD post-traumatic stress disorder
  • OCD Obsessive Compulsive Disorder
  • adult ADHD Alzheimer’s disease, Dementia, Parkinson's disease and/or Huntington’s disease in a person or animal in need thereof.
  • the sarmentosin or its ester(s) are used for treating or preventing any one or more of any other clinical / therapeutic condition or disease in humans or other animals associated with MAO activity / inhibition as discussed below.
  • the sarmentosin or it(s) esters thereof are used to treat or prevent ocular disease.
  • the sarmentosin or it(s) esters thereof are used to treat or prevent muscular dystrophy.
  • the sarmentosin or it(s) esters thereof are used to treat or prevent multiple sclerosis.
  • the sarmentosin or it(s) esters thereof are used to treat or prevent sexual dysfunction.
  • the sarmentosin or it(s) esters thereof are used to treat or prevent hair loss, and/or used to induce hair growth.
  • the sarmentosin or it(s) esters thereof are used to increase life span and provide anti-aging properties.
  • MAO inhibitors have shown potential application to a range of non-clinical uses both in humans and other animals, as discussed throughout this specification.
  • Rhodiola also known as golden root Rhodiala, which contains MAO inhibitors
  • the sarmentosin or its ester(s) are used for treating or preventing any one or more of any other clinical / therapeutic condition or disease in humans or other animals associated with MAO activity / inhibition as discussed below.
  • the sarmentosin or its ester(s) are used for improving, restoring or supporting mood, anxiety, social anxiety, behavioral conditions, fatigue, cognitive performance, exercise performance, attention / alertness, calmness, mental clarity, executive function, working memory, secondary memory, mood, stress and/or stress reactivity or to provide a nootropic effect.
  • the sarmentosin or its ester(s) are used for helping or achieving smoking cessation.
  • the sarmentosin or its ester(s) are used for treating or preventing cognitive dysfunction syndrome, anxiety and other behavioral issues in animals such as companion pets.
  • a skilled person in the art would appreciate other non-clinical uses related to cognitive and mental support would also fall within the scope of the invention and its uses.
  • the sarmentosin may also be mixed with other nootropics or bioactives that are known to be beneficial for neurological state or prevention of conditions or disease.
  • Preferred Dosage The inventors have conducted preliminary tests (see Example 5) to assess the likely efficacious dosage of sarmentosin or its ester(s) for clinical uses, and potentially also a guide for non-clinical uses too. This was achieved through comparative enzyme inhibition analysis to a known commercial MAO-B inhibitor (Deprenyl, otherwise known as Selegiline) used for clinical purposes.
  • the daily dosage may preferably be at least 20 mg sarmentosin or its ester(s).
  • the daily dosage is about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105,110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 mg sarmentosin or its ester(s) per day. More preferably, the daily dosage is between 20 to 200 mg sarmentosin or its ester(s) per day. Another preferred daily dosage may be in the order of at least 110 mg per day. In a more preferred embodiment, the daily dosage of sarmentosin or its esters thereof is between 110-130mg per day.
  • the efficacious dosage may be substantially less, either due to potential synergy, or because of sufficient background MAO inhibition activity of the other MAO inhibitor.
  • the dosage of sarmentosin may be as low as 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 mg per day. In a non-clinical setting, preferred dosages of sarmentosin or its ester(s) may be substantially less than in clinical settings.
  • a serving size / daily serving may be as low as 1 mg – 20 mg per day, although higher dosages similar to the preferred clinical dosages may also be useful and within the scope of the invention.
  • the dosage of anthocyanin(s) is less than 7.8 mg per day.
  • the sarmentosin dose may be less than 1 mg / kg weight per day due to the likely synergistic effect observed.
  • the daily dosage of anthocyanin(s) may be about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 15.0, 20.0, 30.0, 40.0, 50.0 mg per day, when used in combination with sarmentosin or its ester(s).
  • the present invention may include one or more of the following advantages, without limitation: - Identification of a new use of sarmentosin and its esters as a MAO inhibitor with potentially significant opportunity for therapeutic and non-clinical uses, both in humans and other animals.
  • Various embodiments of the present invention may beneficially leverage a naturally occurring source of sarmentosin or its esters, owing to the fact it is present in several plant materials including blackcurrants.
  • - Preliminary evidence suggests the sarmentosin has minimal or no disadvantageous side effects.
  • - Preliminary evidence supports that sarmentosin or its esters may synergically improve MAO inhibition when used in combination with another MAO inhibitor such as anthocyanin(s), which are considered to have weak MAO inhibition.
  • sarmentosin advantageously has both inhibitory activity towards both MAO-A and MAO-B, opening up additional uses towards conditions associated with either one or both of these enzymes.
  • FIG. 1 Referring to Example 1, BetaCs reported from blackcurrant juice. Compound 1 (LHS) and compound 2 (RHS).
  • Figure 2 Referring to Example 1, a third betaC found in blackcurrant (Compound 3).
  • Figure 3 Referring to Example 1, LCMS analysis of test blackcurrant juice.
  • Figure 4 Referring to Example 1, Chromatograms of Neuroberry TM juice samples, 1, 2 and 3 (top to bottom).
  • Figure 5 Referring to Example 3, levels of 4HQ seen in fractions (lower value indicates more inhibition)
  • Figure 6 Referring to Example 3, prep HPLC trace (UV). Fractions 18-23 are collected from 7-11 minutes.
  • Figure 7 Referring to Example 3, levels of 4HQ seen in fractions from large scale prep hplc (lower value indicates more inhibition)
  • Figure 8 Referring to Example 3, showing the chemical structure of sarmentosin.
  • Figure 9 Referring to Example 3, showing the chemical structures of nigrumin compounds.
  • Figure 10 Referring to Example 3, activity vs sarmentosin content for fractions 17-22 from prep HPLC.
  • Figure 11 Referring to Example 3, H NMR spectrum of prep HPLC fractions.
  • Figure 12 Referring to Example 3, H NMR spectra, detail of 5-10 ppm region.
  • Figure 13 Referring to Example 3, levels of 4HQ in bioassay of reversed phase fractions rp-1 through 11.
  • Figure 14 Referring to Example 4, RP10 fractions 4HQ (S9 Bioactivity for preparative HPLC fraction from RP10)
  • Figure 15 Referring to Example 4, Caumaroyl and feruloyl sarmentosin content in RP10 HPLC fractions (fractions 12-15 have higher peak areas than shown)
  • Figure 16 Referring to Example 4
  • Figure 17 Referring to Example 5 A) Dilution series for sarmentosin.50% inhibition occurs at about 400 ng/ml.
  • FIG. 22 Referring to Example 7, Sarmentosin concentrations (mean ⁇ SEM) in plasma before (0min) and after consuming a single dose of blackcurrant juice or powder.
  • Figure 23 Referring to Example 7, Platelet monoamine oxidase (MAO)-B activity and blood glucose concentrations before (0 min) and after consuming blackcurrant juice (A, C) or powder (B, D) and their corresponding placebos. Values are mean ⁇ SEM. * Indicates significant difference from 0 min (p ⁇ 0.05). ⁇ Indicates significant difference from placebo intervention at the same time point.
  • MAO Platelet monoamine oxidase
  • Figure 24 Referring to Example 7, Visual Analogue scores for Interested/Bored (A, B), Friendly/Unfriendly (C, D), Happy/Sad (E, F) and Calm/Excited (G, H) items from the Bond-Lader Questionnaire given by participants before (0 min) and after consuming blackcurrant juice or powder and their corresponding placebos. Values are mean ⁇ SE. * Indicates significant difference placebo intervention at the same time point.
  • Figure 25 Referring to Example 7, Spearman’s rank-sum correlation coefficients for neurotransmitters showing a significant correlation (*p ⁇ 0.05, **P ⁇ 0.01) with MAO-B inhibition after consumption of either a blackcurrant juice intervention vs a placebo, or a blackcurrant powder intervention vs a placebo.
  • A 5-hydroxyindoleacetic acid (5-HIAA) juice intervention
  • B 5-HIAA powder intervention. * indicates significant difference between the intervention and the placebo at the same time point (p ⁇ 0.05).
  • XA xanthurenic acid
  • MN metanephrine
  • NM normetanephrine
  • KA kynurenic acid
  • MHPG 3-methoxy-4-hydroxyphenylglycol
  • DHPG 3,4- dihydroxyphenylglycol
  • D DHPG powder intervention
  • E phenylalanine (PHE) juice intervention
  • F PHE powder intervention.
  • FIG. 30 Referring to Example 7, Correlation circle plots of standardised neurotransmitter concentrations as dependent variables for blackcurrant (A) and placebo (B) beverages (juice and powder combined). Only the 12 variables with the strongest contributions to the definition of the principal components are displayed. Variables with warm and cool colours higher and lower weights, respectively. The coordinates of the variables correspond to their correlation coefficient values and the angle between two variables defines their relationship: If the angle between two variables is sharp the correlation is positive, if the angle is obtuse the correlation is negative and if the angle is right the correlation is null.
  • FIG 31 Schematic representation of the tyrosine and tryptophan metabolic pathways
  • Figure 32 Sarmentosin in in vitro digestion samples, prepared via addition of methanol to precipitate any protein.
  • Sarm Sarmentosin
  • SCE coumaroyl ester
  • SFE2 feruloyl ester
  • Figure 33 Sarmentosin in in vitro digestion samples, prepared via direct sampling of neat solution after centrifugation.
  • BC contains two beta- carbolines (betaCs) - see Figure 1.
  • This class of compounds are known to interact with MAOs.
  • Herraiz et al. showed that a variety of juices and fruits, including BC contain beta carbolines (betaCs). When they compared a range of betaCs they found potent MAO activity in some of the betaCs.
  • MAO-B activity was shown to be inhibited by consumption of the BC juice. The activity was suggested to be reversible and non-selective but with possible greater MAO-B affinity.
  • Example 1 includes: - developing analytical methods to detect low levels of beta-carbolines in BC samples. This would involve processing of a bulk BC juice or frozen berries (10 kg) to specifically extract the betaCs. - preparing samples with enhanced betaCs using buffered reactions of common precursors - use of LC-MS and LC-HRMS to detect betaCs in juice and enhanced samples Method and Results of analysis of betaCs in BC. Analysis of these betaCs is most conveniently performed using LCMS. Alkaloids tend to ionise well in mass spectrometry and specific MRM analysis allows more precise analysis in complex mixtures.
  • Example 2 Bioassays
  • Most commercial MAO assay kits work by measuring hydrogen peroxide formation (produced when MAO deaminates a monoamine substrate). This method can suffer interference when antioxidants are present in the sample (antioxidants can inhibit the peroxide formation).
  • Work by Herraiz et al (2018) has shown that an alternative direct analysis of the deaminated substrate using HPLC is a viable option. Using this method, they showed common food phenolic compounds such as quercetin and cyanidin are not active (rather they inhibit the peroxidase used in the commercial assay).
  • Example 2 the inventors: - used an in vitro assay using a commercial kit to test for MAO-B and possibly MAO-A inhibition; - used the HPLC assay method to confirm activity in various samples including BC juice, dried ethanol extract (35% anthocyanins), and test betaCs produced in Example 1.
  • Methods and Results In this Example we have focused on in vitro evaluation of the effect of BC juice using commercial- kit-based MAO assays.
  • the kit used was from Sigma-Aldrich [MAK295/296] for the MAO-A and MAO-B enzymes.
  • the kit method detects peroxide formation (resulting from MAO activity) via production of a fluorescent product.
  • the juice concentrate is 4.5x the concentration of the juice and the dried extract is 73x the concentration of the juice.
  • the test drink beverage is known to be a 1:1 dilution of the juice. Table 1. Results from the first round of assays These results were very encouraging. We saw a good MAO-A and MAO-B inhibition with the neat juice sample and juice concentrate. We also see inhibition with both the dried extract and the test drink product. However, we see little inhibition with the ⁇ -carboline mix. While this latter result is somewhat disappointing it is only two from a range of possible betaCs and published work shows huge variability in betaC activity according to structure. In general, both MAO-A and MAO-B inhibition is seen for the active samples with optimal testing rate around 10 mg/ml juice equivalent.
  • Extracts E1, E2 and E3 are samples prepared by diethyl ether extraction of juice concentrate (diluted in water).
  • E2 is an ether extraction of E1 after it was made alkaline
  • E3 is an ethyl acetate extraction of E2. None of these extracts were active, indicating that the activity is not associated with a non-polar part of the extract and so is unlikely to be associated with the known ⁇ -carbolines.
  • Another way to separate the polyphenols from “other” components is to pass the extract through a polyamide column. The results of this are also shown in Table 2 below.
  • Example 3 Bioassay guided fractionation Overview
  • the inventors looked at fractionation processes to try to narrow down the identity of the active component(s). Methods and Results. To allow for screening of larger numbers of fractions/samples, an alternative assay was used in Example 3 to the Sigma-Aldrich commercial assay kit used in Example 2.
  • a porcine S9 liver microsome fraction was used as the source of the MAO-A and MAO-B enzymes. This method allowed the screening of many more samples than possible with the kit-based assays.
  • a S9 pig liver microsome was isolated from macerated pig livers.
  • the S9 porcine fraction was mixed with a specific MAO substrate (kynuramine) and mixed with the test sample and buffer. After 1 hr at 37°C the reaction was halted by cooling and addition of 2N NaOH and acetonitrile. The centrifuged sample was then analysed using LCMS and the level of 4-quinolol (4HQ) product measured.
  • the method was performed according to the protocol described in Ghosal., (2020), entitled “ Evaluation of the clearance mechanism of non-CYP-mediated drug metabolism and DDI as a victim drug.”
  • This method takes advantage of the spontaneous formation of 4-quinolol from the oxidised kynuramine.
  • the levels of kynuramine and 4-quinolol are assessed by LCMS and the low level/absence of 4hydroxyquinolol indicates inhibition of MAO.
  • This method proved to be very useful for following the MAO inhibition activity through a number of separation steps. Preliminary separation using column chromatography (CC). Three modes of CC were used.
  • the present invention relates to the new use of sarmentosin as a MAO inhibitor (and associated uses thereof as described elsewhere in this specification) from a natural source for example could be from the likes of blackcurrant or Kalanchoe species, or derived in other ways such as through cell-based fermentation and purification technologies, or synthetic production of the compound.
  • Sarmentosin can be classified as a gamma hydroxy nitrile glycoside. This is an unusual class of hydroxy nitrile glycosides as most known hydroxy nitrile glycosides are alpha hydroxy nitrile glycosides (e.g. prunasin from apple seeds).
  • Fraction 16 (inactive) has no sarmentosin and is also missing the other signals in the 5.5-9 ppm region. This is consistent with sarmentosin being the source of the activity in these fractions. Revisiting column chromatography fractions. With the likely activity in the polar fractions identified we decided to check for any other activity in fractions from a reversed phase C18 column. A fresh column was run using 5 g of concentrate and 11 fractions were collected. When these fractions were assayed using the S9- LCMS method a second band of activity was observed in the late eluting non-polar fractions (See Figure 13). Fraction 10 is active. The main sarmentosin fraction is Fraction 2.
  • the sarmentosin esters are possibly hydrolysed to yield sarmentosin by the S9 enzymes.
  • Comparison of extracts In the human studies the ethanolic extract powder (DelCyan) was less effective at altering MAO activity. In this work the inventors have analysed the provided blackcurrant powder and found very low levels only of sarmentosin (compared to juice or juice concentrate). This also is evident in the S9 LCMS bioassay where the juice concentrate is more active than the dried extract when compared at the same level of total anthocyanins (dried extract 73 X juice, concentrate 4.5 X juice). The dried extract would be expected to still contain the nigrumin esters.
  • Example 4 Further analysis of sarmentosin activity in samples Overview
  • samples of the active compounds were purified from the juice concentrate (scale up of work from Example 3) to give ca.10 mg of each active compound.
  • juice or other berry samples e.g. blueberry, and various blackcurrant samples
  • S9 assay bioassay
  • sarmentosin content is shown in mg/g of sample while the esters are ug/g. From this data we can see that Sample 5 has about 15x as much sarmentosin as each of the esters. The amount of the two esters is similar. Based on these initial results, the inventors consider a typical level of sarmentosin in a different types of blackcurrant juices / extracts may range substantially from about 5 to 1300 ⁇ g/g.
  • the inventors foresee using a juice extract or other format with sarmentosin or ester(s) either within this range for the new use towards MAO inhibition, or advantageously increased above this level to increase the potency or effectiveness of a composition, nutraceutical or extract.
  • the freeze-dried berry extract samples 8 and 9 have approximately the same content as Sample 5.
  • Juice sample 2 is roughly similar to Sample 5 although the sarmentosin level is somewhat lower.
  • sarmentosin content is consistent with a 5.7% juice content.
  • the test drink product 7 was from an aged sample stored at room temperature so may have lost some of the sarmentosin over time.
  • This test drink is juice at 1:1 dilution so it would be expected to normally contain about 0.7-0.8 mg/g sarmentosin in a fresher sample.
  • the concentrated beverage sample 6 and dried powder (Sample 5) are both very high in sarmentosin compounds however the dry extract has proportionately lower levels of sarmentosin (esters are about three times the level in Sample 6 but sarmentosin is lower). This is likely the effect of the use of ethanol which would favour the extraction of the less polar esters.
  • Table 3 Analysis of sarmentosin and esters content in various blackcurrant juice products.
  • Example 5 Comparison of sarmentosin MAO-B activity vs Deprenyl Overview This example compares MAO-B activity of sarmentosin to Selegeline (also known as Deprenyl or L-deprenyl, and sold under brand names such as Eldepryl and Emsam is a known MAO-B inhibitor and is used to treat Parkinson’s disease, and major depressive disorder). This helps to validate effective dosages of sarmentosin alone, or for example within an extract (such as a juice). Results Deprenyl was purchased from Sigma-Aldrich. (R-(-)-deprenyl, this compound is also known as selegiline).
  • the sample of sarmentosin was from material purified in the earlier work. The concentration of the sarmentosin was confirmed using NMR with a quantitative NMR standard.
  • the S9 liver microsome fraction containing the crude enzyme mix was from a frozen bulk sample derived from porcine liver. Samples were prepared in a 6-well plate (deep well format with 1.1 ml strip tubes). The assay was performed by diluting the crude S9 fraction with buffer (PBS, pH 7.4, diluted 1 in 5, 150 ⁇ l) and adding the test sample dissolved in buffer (75ul). An aliquot of kynuramine was added (10 ⁇ l of 1 mg/ml) and the plate was placed in a 37 C water bath for 1 hr.
  • the plate was cooled in ice water before addition of 20 ⁇ l 2N NaOH followed by 250 ⁇ l acetonitrile.
  • the individual strip tubes were centrifuged, and samples taken from each for LCMS analysis.
  • the LCMS analysis was performed using a Cyano HPLC column eluted with 0.1% formic in water and acetonitrile.
  • the LC run was isocratic mode (40% Acetonitrile) and the relative levels of 4 hydroxy quinoline (4HQ) and kynuramine determined from peak areas. The % inhibition was determined by comparing the amount of 4HQ with that of a buffer-only sample well. Kynuramine is converted to 4HQ by the MAO-B enzyme. The results of this analysis are described below, with reference to Figure 17.
  • the sarmentosin 50% inhibition is seen at around 400 ng/ml, which is equivalent to a concentration of 1.45 ⁇ M.
  • the Deprenyl 50% inhibition is seen at around 15 ng/ml, or 67nM.
  • a recommended minimum starting dose of Deprenyl (selegiline) is 1.25 mg/day and increased to 2.5 mg/day after 6 weeks (see https://www.drugs.com/dosage/selegiline.html).
  • a preferred dosage of sarmentosin for MAO-B inhibition for cognitive diseases or preventative / improvement in mental health is at least about 27500 ⁇ g sarmentosin / day. Therefore, for a blackcurrant juice that includes for example 320 ⁇ g / g sarmentosin, a daily serving of 86 ml of this blackcurrant juice is likely to provide about 27500 ⁇ g sarmentosin which may provide particularly preferred efficacious dose when used to achieve MAO-B inhibition (i.e. when applying the 22x conversion, would be equivalent to an efficacious dose of 1.25 mg Deprenyl).
  • Example 6 Analysis of sarmentosin content / MAO activity in a range of blackcurrant berries, juice and juice concentrates Overview This example assesses a range of blackcurrant cultivars for both MAO activity (S9 assay as above) and sarmentosin content (LCMS analysis). Results As part of ongoing improvements to the analysis method the LCMS method employed for the previous work was adapted. The previous method suffered from an overlap of metabolites eluting around the same time as sarmentosin. This led to interactions between the metabolites giving suppression of the sarmentosin signal and hence incorrect estimations of concentrations.
  • the HPLC method was improved by the use of 10 mM ammonium formate in the aqueous solvent (instead of 0.1 % formic acid). This change moved the sarmentosin peak to elute later than the food acids and sugars and gave more consistent results for the samples and standards.
  • the sarmentosin standard was also reassessed as part of this work. Previously the standard was the sample of sarmentosin isolated from preparative HPLC and shown to be “pure” using NMR. However, when the purity was assessed using a quantitative NMR standard the sample was found to be of lower purity. All subsequent analysis is based on this new NMR standard. Various juice samples, concentrates, powders and whole fruit were tested.
  • each of the samples was diluted with water to give a sarmentosin peak that was around the appropriate concentration for the LCMS analysis. From the results from Example 5 we could estimate the approximate concentration of sarmentosin required to give a 20-80% inhibition in the S9 assay.
  • the samples for the assay were diluted to this approximate concentration by dilution with water and the assay performed using 4 wells for each sample in a 2X dilution series. Due to constrictions of supply of reagents the assays were only performed once for each sample. The results are shown below in Table 4, with the amount of sarmentosin in each sample and then the concentration of sample in mg/g which gives either 70 or 80% inhibition in the assay.
  • the ethanolic extract is a powder which is an ethanolic extract of the berries.
  • the whole berry sample is a water extract from whole berries.
  • the Viberi ® sample is a commercial sample of packaged freeze-dried whole berries. Discussion These results are consistent with the sarmentosin being the effective active in the samples.
  • Figure 17 is a plot of the concentration needed for 70% inhibition vs the sarmentosin concentration. This indicates a direct correlation between the sarmentosin content and the inhibitory activity. Table 4. Sarmentosin and relative MAO-B inhibition activity for a range of blackcurrant samples.
  • Example 7 The effects of consuming a single dose of blackcurrant juice or freeze-dried powder on phytochemical bioavailability, platelet MAO-B activity, plasma neurotransmitter concentrations and mood in healthy adults Commissioned by Alphagen NZ Limited, Plant and Food Research Limited conducted a randomised, double-blind, two-arm, placebo-controlled, cross-over human intervention study to evaluate temporal platelet monoamine oxidase B (MAO-B) enzyme activity, phytochemical bioavailability, circulating neurotransmitters, and subjective markers of mood after participants consumed beverages prepared from two blackcurrant product formats: juice concentrate and freeze-dried powder.
  • MAO-B platelet monoamine oxidase B
  • the BC and PL juice concentrates were formulated by The New Zealand Institute for Plant and Food Research Limited (PFR), within its food- safe laboratory, with ingredients supplied by AlphaGen and Sensient Technologies.
  • the BC and placebo formulations were weighed and portioned into single-serve quantities in amber bottles, stored at -20°C, then served to participants as a 300-mL beverage.
  • the total amount of BC anthocyanins contained within the two BC beverages differed between the two formats, as previously agreed on by AlphaGen and PFR.
  • the anthocyanin dose of the BC powder beverage (BC-P) was standardised to each participant’s weight, so that they consumed a weight of BC powder equivalent to 7.8 mg total anthocyanin/kg of their bodyweight, reconstituted in 300 mL water.
  • the placebo powder beverage (PL-P) was an equivalent weight of placebo powder as the BC powder, in 300 mL water. Apple juice concentrate and clove extract were added to both BC and PL concentrate formulations to increase flavour complexity. Citric acid was added to both concentrate formulations, but in greater quantity for the placebo concentrate beverage (PL-JC), to match the sourness of BC-JC.
  • the flavour and colour of the PL-JC was matched to BC-JC using BC flavouring, Allura Red, Raspberry, and Blue colour solutions (Sensient Technologies) (Table 5).
  • a sucrose solution 65 °Brix
  • was used in place of the BC juice concentrate for PL-JC. Table 5. Constituents of the blackcurrant and placebo juice concentrate formulations presented as single-serve quantities. N/A not applicable.
  • Microbial pathogen testing Received powders and formulated juice concentrates were sent to AsureQuality Limited for microbial pathogen testing. Beverage preparation An independent researcher prepared the trial beverages. The required amber bottles of stored formulations were moved to 4°C the night before a trial day. The powder drinks were prepared by blending the powders and 300 mL of water together in a NutriBullet®. Water was added to the juice concentrates to equal 300 mL and stirred to combine. Trial beverages were consumed by the participants within 1 h of being prepared.
  • Anthocyanin quantitation of beverage formulations Anthocyanin concentrations of formulated trial juice concentrates and supplied powders were measured using a Dionex Ultimate 3000 Series UHPLC (ThermoFisher Scientific, San Jose, CA, USA) with PDA (photodiode array) detection at 520 and 530 nm.
  • the formulated juice concentrates were diluted 1/1 with 5/95 formic acid/water v/v. Weighed quantities of the powder samples were dissolved in 5/95 formic acid/water v/v to give aqueous solutions of concentration between 20 and 21 mg/mL.
  • Clinical trial Recruitment and trial registration Thirteen healthy individuals between 26 and 39 years old, who were recruited from the wider Palmerston North community, provided informed consent to participate in this study. During recruitment, prospective participants completed a health screening questionnaire to exclude those who had a chronic disease (e.g. heart disease, cancer), known blood-borne diseases (e.g. hepatitis), a recent viral or bacterial illness, were pregnant, were taking medication that affected blood-clotting properties or mood, had a known intolerance to blackcurrants, or a known strong reaction to needles.
  • chronic disease e.g. heart disease, cancer
  • known blood-borne diseases e.g. hepatitis
  • a recent viral or bacterial illness were pregnant, were taking medication that affected blood-clotting properties or mood, had a known intolerance to blackcurrants, or a known strong reaction to needles.
  • the trial participants were to complete two trial days, where they would consume one of the two intervention drinks (blackcurrant or placebo), with at least one-week washout between them.
  • Subjective feeling questionnaires and venous blood samples were collected at various timepoints over 8 h.
  • the participants were randomly allocated (ratio 1:1) into one of the two format groups: juice concentrate, or powder, and then randomly allocated (ratio 1:1) for the order in which they received the intervention beverages.
  • Participants and trial coordinators were masked as to which intervention drink participants consumed. Before each trial day, participants excluded foods and supplements high in polyphenol compounds from their diet, for 24 h. They fasted for at least 10 h before consuming an Almond with Vanilla One Square Meal bar® (Cookie Time Ltd) for breakfast, 2 h before the start of the trial.
  • VAS visual analogue scale
  • Bond-Lader Visual Analogue Mood Scales Questionnaire allows self-evaluation of mood. In total, 16 dimensions of mood are given: Alert-Drowsy, Calm-Excited, Strong-Feeble, Muzzy-Clear headed, Well Coordinated-Clumsy, Lethargic-Energetic, Contented-Discontented, Troubled- Tranquil, Mentally Slow-Quick Witted, Tense-Relaxed, Attentive-Dreamy, Incompetent-Proficient, Happy-Sad, Antagonistic-Friendly, Interested-Bored, Withdrawn-Social. The participants were required to mark on a 100-mm line to what extent each described state was appropriate to them at that moment in time.
  • Platelet MAO-B activity Platelets were isolated and prepared using previously described methods (Watson et al.2015). In brief, platelets were isolated from whole blood (10 mL in 10% disodium EDTA solution), by centrifugation at 600 g at 22°C for 3 min without a break. Platelets were stored at -80°C until required. The protein concentration of the platelet samples was determined using the BCA Protein Assay Kit (23225, PierceTM). MAO-B activity was measured using the Amplex® Red Monoamine Oxidase Assay Kit (A12214, Invitrogen), as per the manufacturer’s instructions.
  • Plasma was separated from whole blood collected in lithium heparin tubes from participants by centrifugation at 4000 x g for 10 min at 4°C.
  • Plasma (1 mL) was spiked with 30 ⁇ L of 50% formic acid and 100 ⁇ L of 10 mmoL ascorbic acid then stored at -80°C prior to shipping on dry ice to the Physiological Chemistry laboratory.
  • the plasma samples (350 ⁇ L) were spiked with malvidin 3-O-galactoside and further acidified with phosphoric acid prior to clean up on a SOLA ⁇ TM Solid Phase Extraction plate.
  • the methodology for neurotransmitter analysis utilises a MS-probe and stable isotope coding LCMS method developed in- house at Plant & Food Research, optimised for plasma samples (Parkar et al. 2020; Watson et al.2020).
  • the neurotransmitter methodology includes extensive coverage of inhibitory and excitatory neurotransmitters from the tyrosine, tryptophan and glutamate metabolic pathways involved in the gut- brain axis.
  • metabolites measured in the tyrosine metabolic pathway were phenylethylamine (PEA), tyrosine (TYR), 3,4- dihydroxyphenylalanine (L-DOPA), dopamine (DA), 3-methoxytyramine (3-MT), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), norepinephrine (NE), 3,4-dihydroxyphenylglycol (DHPG), 3-methoxy-4- hydroxyphenylglycol (MHPG), normetanephrine (NM), epinephrine (E), metanephrine (MN), and vanillylmandelic acid (VMA).
  • PEA phenylethylamine
  • TYR 3,4- dihydroxyphenylalanine
  • DA dopamine
  • 3-MT 3-methoxytyramine
  • DOPAC 3,4-dihydroxyphenylacetic acid
  • HVA homovanillic acid
  • NM no
  • tryptophan metabolic pathway were tryptophan (TRP), kynurenine (KYN), kynurenic acid (KA), xanthurenic acid (XA), quinolinic acid (QA), 5- hydroxytryptophan (5-HTP), serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA) and melatonin (MT).
  • TRP tryptophan
  • KYN kynurenine
  • KA kynurenic acid
  • XA xanthurenic acid
  • QA quinolinic acid
  • 5-HTP serotonin
  • 5-hydroxyindoleacetic acid 5-HIAA
  • MT melatonin
  • glutamate metabolism the metabolites measured were glutamic acid (GLU), alpha-aminobutyric acid (AABA) and gamma-aminobutyric acid (GABA).
  • IS-XdP a labelled IS for each analyte was prepared (IS-XdP) by derivatising a mixed neurotransmitter standard, as described for the samples, with the exception that deuterated acetic anhydride [d6] and deuterated trifluoroethanol [d3] were used in place of unlabelled acetic anhydride and unlabelled trifluoroethanol.
  • the IS-XdP standard was added to the samples after sample work-up. Briefly, acetic anhydride, sodium bicarbonate/carbonate buffer, acetonitrile and the mixed IS comprising of deuterated analytes was added to each plasma sample (100 ⁇ L).
  • Samples were evaporated to dryness overnight then derivatised with acetic anhydride and trifluoroethanol at 70°C for 4 h and the temperature reduced to 50°C before being left to react overnight. Samples were once again taken to dryness and further derivatised with acetic anhydride before the addition of water, acetonitrile and the IS-XdP. Samples were filtered through a 96-well glass fibre plate and collected in a 96-deepwell polypropylene plate prior to analysis by LCMS.
  • LCMS experiments were carried out on a 7500 QTrap triple quadrupole/linear ion trap (QqLIT) mass spectrometer equipped with a Turbo VTM ion source and electrospray ionisation (ESI) probe (AB Sciex, Concord, ON, Canada) coupled to a Shimadzu Nexera LC40 UHPLC (Shimadzu, Tokyo, Japan).
  • MS data was acquired in the positive mode using a multiple reaction monitoring (MRM) method. Blood glucose Blood glucose concentrations of whole blood was measured using a HemoCue® 201 DM System (HemoCue®, ⁇ ngelholm, Sweden) blood glucose analyser.
  • MRM multiple reaction monitoring
  • Monoamine oxidase-B activity, subjective measures and glucose data are presented as means ⁇ standard errors of the mean. Comparisons of means between timepoints, treatments and formats were made using Analysis of Variance (ANOVA) from a linear mixed effects model, with fixed effects for format, study day, treatment, time, and their interactions, and random effects for participant, participant x study day, participant x treatment and participant x time-point. The models were fitted with R package lmerTest, and least significant differences (LSDs) for comparing means were calculated post hoc using the R package predictmeans. Statistical significance for all indices was set at p ⁇ 0.05 with a confidence level of 95%. Neurotransmitter data analysis R version 4.2.1 was used for analysis and data visualisation of neurotransmitter results.
  • Anthocyanin content of juice and powders The blackcurrant anthocyanin contents of the formulated trial juice concentrates and powders before water was added (to make 300-mL beverages), are shown in Table 6. Based on the total anthocyanin results, the blackcurrant juice concentrate contained 2179.4 ⁇ g/g wet weight total anthocyanins, and the blackcurrant powder contained 33720.4 ⁇ g/g dry weight total anthocyanins. The placebo juice concentrate and powder formulation contained no detectable blackcurrant anthocyanins. Table 6. Blackcurrant anthocyanin content in the blackcurrant and placebo juice concentrates, and blackcurrant and placebo powders.
  • Juice concentrates are reported in ⁇ g/g wet weight, and powders reported in ⁇ g/g dry weight as received. nd: not detected.
  • w der Trial participants Ten participants completed both trial days out of the 13 who were recruited ( Figure 19). Data of the three participants who did not complete both trial days were not included in the analyses for this report. The anthropometric data of the ten participants are shown in Table 7. Of these ten, five were allocated to the juice concentrate format, and five were allocated to the powder format. Despite withdrawals by some participants, the number that completed both trial days were within what was required to detect statistically significant MAO-B enzyme inhibition from consuming the blackcurrant formats. Participant withdrawals and reactions Table 7. Anthropometric characteristics of participants of the two format groups: juice concentrate and powder.
  • Anthocyanin and sarmentosin bioavailability The concentrations of total anthocyanins, cyanidin methyl ester glucuronide, cyanidin 3-O- runtinoside, cyanidin 3-O-glucoside, delphinidin 3-O-runtinoside, and delphinidin 3-O-glucosides measured in plasma following the consumption of the blackcurrant interventions were measured ( Figure 21). No blackcurrant anthocyanins were detectable in the plasma of the placebo (PL) interventions (data not shown). The concentrations of the anthocyanins were higher in the BC-P plasma than in the BC-JC for all timepoints measured post-consumption.
  • Alertness, calmness and contentment scores (from Bond-Lader Questionnaire) after consuming blackcurrant juice or powder and their corresponding placebos. Values are means ⁇ SEM. * Denotes significant difference from 0 min (p ⁇ 0.05). ⁇ Denotes significant difference from placebo at corresponding time point (p ⁇ 0.05). Participants’ alertness scores significantly increased 20 min after consuming the PL-JC intervention, then declined thereafter, so that alertness scores at 480 min were significantly lower than baseline (0 min) scores (Table 11). Alertness scores did not significantly change from baseline (0 min) after BC-JC consumption at any timepoints. For the powder formats, alertness scores were significantly higher than baseline (0 min) scores 20 min after consuming the PL-P intervention.
  • BC-P alertness scores 120 min post-consumption were significantly higher than placebo (PL-P) scores at this timepoint.
  • Mean calmness was significantly higher 480 min after consuming the juice placebo beverage (PL-JC), while significantly lower calmness was measured at this timepoint after drinking the blackcurrant juice (BC-JC), so that the calmness scores between treatment groups at this timepoint were significantly different (p ⁇ 0.05) (Table 11).
  • a significant increase in calmness was measured 20 min after PL-P consumption, while no change in calmness was measured in the BC-P intervention group. Calmness scores in the PL-P were significantly higher 20 min after consumption than in the BC-P intervention at this timepoint.
  • ⁇ Denotes significant difference from placebo at corresponding time point (p ⁇ 0.05). Circulating neurotransmitter concentrations Concentrations of 32 endogenous neurotransmitters and other compounds associated with the gut brain axis were measured in plasma for 13 participants. Of the ten participants who completed both trial days, comprising the blackcurrant intervention and placebo interventions, five participants (four receiving the juice format, and one receiving the powder format) had samples taken for each time point. Three further participants, all receiving the powder format, had one missing time point for each trial day. The remaining five participants completed only the blackcurrant intervention trial day, two participants receiving the juice format and three the powder format.
  • GABA gut brain axis
  • these neurotransmitters are MAO- derived metabolites, and suggests the downstream effect of BC-induced MAO-B inhibition on circulating neurotransmitter concentrations. Aside from DOPAC, these same neurotransmitters also showed significant positive correlations with MAO-B inhibition following consumption of the blackcurrant powder, that were not observed for the placebo. For the powder intervention format only, significant correlations with MAO-B inhibition were also observed for XA, MN, NM and KA, metabolites from the tryptophan metabolic pathway. While NM is a substrate for MAO-A/B, XA, MN and KA are not substrates or metabolic products of MAO-A/B.
  • Plasma DOPAC was significantly lower 120 min post BC-JC and BC-P consumption compared with the placebo group at the same timepoint ( Figure 26A-B).
  • Plasma VMA concentrations were significantly lower 120 min and 240 min after BC-JC, but not BC-P, consumption compared with the placebo group at these timepoints ( Figure 26E-F).
  • plasma HVA and DA concentrations tended to be lower following BC-JC and BC-P consumption compared with their corresponding placebos, no significant treatment differences were measured at any of the timepoints for these neurotransmitters.
  • Figure 30 display the correlations between the top 12 variables with the strongest contributions to the principal components.
  • Figure 30A shows a primary contribution of 5-HIAA to drive variation in neurotransmitter concentrations during the time-course experiments associated with the blackcurrant intervention (juice and powder combined). It further suggests a strong interaction between 5-HIAA, HVA, VMA, and association with DOPAC and MHPG. These associations were not observed for the placebo intervention (Figure 30B).
  • MAO-B inhibition correlated with the increasing bioavailability of anthocyanins in plasma, while the decline in bioavailable anthocyanins following 120 min after BC consumption was concomitant with reduced MAO-B inhibition.
  • Consuming a single dose of a BC anthocyanin-enriched extract was previously shown to have no significant effect in reducing platelet MAO-B activity (Watson et al., 2015). While anthocyanins may have some minimal MAO inhibition activity, the present application supports that sarmentosin or its ester(s) is providing a significant MAO-B inhibition effect.
  • the sarmentosin is having a synergistic effect on the anthocyanin(s) by somehow further boosting the MAO inhibitory effects of anthocyanin(s).
  • Another interesting finding is the equivalent MAO-B inhibitory activity of both blackcurrant powder and juice formats, despite the large difference in anthocyanin dose consumed by participants in each format.
  • an effective anthocyanin dose for platelet MAO-B inhibition may have already peaked at approximately 1 mg total anthocyanin/kg bodyweight, and thus consuming a greater dose may have no additional effect in inhibiting platelet MAO-B.
  • Analysis of the subjective data indicate a potential effect of the BC beverages in modulating mood in parallel with increased sarmentosin concentration plasma levels, and also MAO enzyme inhibition. Consumption of blackcurrant juice and powder variably reduced stress and anxiety of participants, reduced participant calmness compared to placebo, and either maintained or significantly reduced subjective scores of mental fatigue. Greater reductions of stress, anxiety, and mental fatigue were measured in the BC-P group compared with the BC-JC group.
  • MAOs are present as two isoforms, MAO-A and MAO- B, which display regional differences in enzyme activity, substrate specificity and distribution in the brain and periphery (Yeung et al.2019).5-HT is reported to be preferably degraded by MAO-A, whereas MAO-B exhibits higher affinity towards benzylamine and PEA.
  • Catecholamines such as DA, E, NE, tryptamine and 3-MT are substrates for both isoforms (Goldstein et al.2021). Further, plasma concentrations of DHPG and MHPG have been described as sensitive indicators of MAO-A dependent metabolism of NE (Scheinin et al.1991).
  • Anthocyanins are also known to modulate blood glucose concentrations (Kim et al.2016), which may partly explain the reduction in blood glucose concentrations observed in the anthocyanin- rich BC-JC intervention compared with the PL-JC intervention.
  • Conclusions and future work The results showed very similar anthocyanin bioavailability profiles between the blackcurrant juice concentrate and powder interventions, although at different magnitudes.
  • the temporal bioavailability of sarmentosin, a specific metabolite of interest for this study also followed a very similar bioavailability profile between the two blackcurrant formats up to 480 min after consumption.
  • the MAO-B activity data in conjunction with the bioavailability data indicate that a lower dose of blackcurrant anthocyanins, when in combination with sarmentosin, causes an equivalent MAO-B inhibition to that of a higher anthocyanin dose from a blackcurrant extract.
  • the inhibition of platelet MAO-B activity following consuming a single dose of BC juice or powder was also concomitant with significant transient reduction of circulating monoamine neurotransmitters DOPAC, 5-HIAA and MHPG.
  • monoamine neurotransmitters were also concomitant with significant transient reduction of circulating monoamine neurotransmitters DOPAC, 5-HIAA and MHPG.
  • monoamine neurotransmitters were also concomitant with significant transient reduction of circulating monoamine neurotransmitters DOPAC, 5-HIAA and MHPG.
  • Example 8 Further in vitro bioactivity studies on sarmentosin In this Example, two studies were completed to further demonstrate MAO inhibition activity of sarmentosin.
  • the sarmentosin ester(s) appear less stable, potentially subject to greater degradation and may be less bioavailable compared to sarmentosin.
  • Study 2 Comparison with commercial standard of sarmentosin A commercial sample of natural sarmentosin was purchased from BOCSci (USA), as CAS No 71933-54-5 (Molecular Formula C11H17NO7, MW 275.25, > 97% purity). The sample was an oil. For this work the sample weight was assumed to be 5 mg as noted on the certificate of analysis. This 5 mg was dissolved in water and a subsample taken for further work. The remaining sample is stored frozen.
  • samples were combined with the kit’s luminogenic MAO substrate solution and human enzyme (MAO-A or MAO-B at a final concentration of 20 ⁇ g/mL) and incubated at room temperature for 1 h. Following incubation, the luciferin detection reagent were added to each sample and the change in luminescence was measured at room temperature over 40 min in a FLUOstar Omega plate reader (BMG FluoStar Optima, Alphatech Systems, Auckland, New Zealand). Results were corrected using a negative control with no enzyme added and expressed as a percentage inhibition of enzymatic activity compared with no inhibition (positive control). All samples were analysed in triplicate on each plate. Results: The results are shown in Figure 35.
  • the inhibitory effect clorgyline and deprenyl, known pharmaceutical inhibitors of MAO-A and MAO-B, respectively, were included to verify the accuracy of the assay and provide a comparison for the efficacy of sarmentosin in inhibiting both MAO isoforms.
  • the IC 50 values for clorgyline was 6 ⁇ 10 -4 ⁇ M and 1 ⁇ M for deprenyl based on the dose response curve of this compound in inhibiting MAO-A and MAO-B, respectively.
  • the IC 50 of sarmentosin in inhibiting MAO-A was 3.55 ⁇ M.
  • the MAO inhibitor phenelzine can improve functional outcomes in mice with established clinical signs of experimental autoimmune encephalomyelitis (EAE). Behav Brain Res.1; 252:302-11. Wang X, et al 2021. Targeting monoamine oxidase A for T cell-based cancer immunotherapy. Science Immunology. Vol 6, Issue 59. Aljanabi R, Alsous L, Sabbah D, Gul H, Gul M, Bardaweel S, 2021. Monoamine oxidase (MAO) as potential target for anticancer drug design and development. Molecules.26(19): 6019. Sidhu G, Marwaha R. Phenelzine. InStatPearls [Internet] 2022 Mar 12. StatPearls Publishing.

Landscapes

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

Abstract

La présente invention concerne de manière générale la sarmentosine et ses esters et leur nouvelle utilisation en tant qu'inhibiteurs de monoamines (MAO) et en tant que méthodes de traitement ou de prévention de diverses pathologies associées liées à l'activité de MAO.
PCT/NZ2024/050012 2022-02-28 2024-02-09 Agents bioactifs et leurs utilisations Pending WO2025048651A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU2024287117A AU2024287117A1 (en) 2023-08-25 2024-02-09 Bioactives and their uses
US18/821,619 US20250090564A1 (en) 2022-02-28 2024-08-30 Bioactives and their uses

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
NZ80311423 2023-08-25
NZ803114 2023-08-25

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US18/821,619 Continuation-In-Part US20250090564A1 (en) 2022-02-28 2024-08-30 Bioactives and their uses

Publications (1)

Publication Number Publication Date
WO2025048651A1 true WO2025048651A1 (fr) 2025-03-06

Family

ID=94820078

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/NZ2024/050012 Pending WO2025048651A1 (fr) 2022-02-28 2024-02-09 Agents bioactifs et leurs utilisations

Country Status (2)

Country Link
AU (1) AU2024287117A1 (fr)
WO (1) WO2025048651A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0531155A1 (fr) * 1991-09-06 1993-03-10 Merz & Co. GmbH & Co. Compositions d'activation cérébral
CN101974044A (zh) * 2010-10-19 2011-02-16 南京荣世医药科技有限公司 一种垂盆草苷的制备方法
CN102659860A (zh) * 2012-04-19 2012-09-12 南京泽朗医药科技有限公司 一种从垂盆草中提取纯化垂盆草苷的方法
CN104771451B (zh) * 2015-04-24 2018-01-30 中国人民解放军第二军医大学 一种具有抗抑郁作用的中药提取物及其应用
WO2023163606A1 (fr) * 2022-02-28 2023-08-31 Arepa Ip Limited Agents bioactifs et leurs utilisations

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0531155A1 (fr) * 1991-09-06 1993-03-10 Merz & Co. GmbH & Co. Compositions d'activation cérébral
CN101974044A (zh) * 2010-10-19 2011-02-16 南京荣世医药科技有限公司 一种垂盆草苷的制备方法
CN102659860A (zh) * 2012-04-19 2012-09-12 南京泽朗医药科技有限公司 一种从垂盆草中提取纯化垂盆草苷的方法
CN104771451B (zh) * 2015-04-24 2018-01-30 中国人民解放军第二军医大学 一种具有抗抑郁作用的中药提取物及其应用
WO2023163606A1 (fr) * 2022-02-28 2023-08-31 Arepa Ip Limited Agents bioactifs et leurs utilisations

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BJARNHOLT, N. ; ROOK, F. ; MOTAWIA, M.S. ; CORNETT, C. ; JORGENSEN, C. ; OLSEN, C.E. ; JAROSZEWSKI, J.W. ; BAK, S. ; MOLLER, B.L.: "Diversification of an ancient theme: Hydroxynitrile glucosides", PHYTOCHEMISTRY, vol. 69, no. 7, 1 May 2008 (2008-05-01), pages 1507 - 1516, XP025533565, DOI: 10.1016/j.phytochem.2008.01.022 *
LOMIWES DOMINIC ET AL.: "Timed consumption of a New Zealand blackcurrant juice support positive affective responses during a self-motivated moderate walking exercise in healthy sedentary adults", JOURNAL OF THE INTERNATIONAL SOCIETY OF SPORTS NUTRITION, vol. 16, no. 1, 2019, pages 33, XP093088428, DOI: 10.1186/s12970-019-0300-0 *
SOJKA M ET AL.: "Composition and properties of purified phenolics preparations obtained from an extract of industrial blackcurrant (Ribes nigrum L.) pomace", THE JOURNAL OF HORTICULTURAL SCIENCE AND BIOTECHNOLOGY, vol. 84, no. 6, 2009, pages 100 - 106, XP093088430, DOI: 10.1080/14620316.2009.11512604 *
WATSON ANTHONY W ET AL.: "Acute supplementation with blackcurrant extracts modulates cognitive functioning and inhibits monoamine oxidase-B in healthy young adults", JOURNAL OF FUNCTIONAL FOODS, vol. 17, 2015, pages 524 - 539, XP093088427, DOI: 10. 1016/j jff. 2015.06.00 5 *
WATSON ANTHONY W. ET AL.: "he pharmacodynamic profile of ''Blackadder", NUTRITIONAL NEUROSCIENCE, vol. 23, no. 7, 2020, pages 516 - 525, XP009548473, DOI: 10.1080/1028415X.2018.1525950 *

Also Published As

Publication number Publication date
AU2024287117A1 (en) 2025-03-13

Similar Documents

Publication Publication Date Title
Li et al. Young apple polyphenols postpone starch digestion in vitro and in vivo
Durak et al. Coffee with cinnamon–Impact of phytochemicals interactions on antioxidant and anti-inflammatory in vitro activity
Turski et al. An exceptionally high content of kynurenic acid in chestnut honey and flowers of chestnut tree
Figueiredo-González et al. Evaluation of the neuroprotective and antidiabetic potential of phenol-rich extracts from virgin olive oils by in vitro assays
de Freitas et al. Biological evaluation and quantitative analysis of antioxidant compounds in pulps of the Amazonian fruits bacuri (Platonia insignis Mart.), ingá (Inga edulis Mart.), and uchi (Sacoglottis uchi Huber) by UHPLC‐ESI‐MS/MS
BRPI0721648A2 (pt) composiÇço que tem atividade lipolÍtica, mÉtodo de produÇço da mesma e uso da composiÇço
Gonçalves et al. Bioactive properties of Chamaerops humilis L.: antioxidant and enzyme inhibiting activities of extracts from leaves, seeds, pulp and peel
AU2025252522A1 (en) Bioactives and their uses
Gonçalves et al. Serotonin and melatonin: plant sources, analytical methods, and human health benefits
Ripari Garrido et al. Nutritional and bioactive characterization of pistachio—a review with special focus on health
WO2018070545A1 (fr) Composition comprenant un ester de choline destinée à une ingestion orale
Gopi et al. Natural sports supplement formulation for physical endurance: a randomized, double-blind, placebo-controlled study
Lomiwes et al. Identification of Sarmentosin as a Key Bioactive from Blackcurrants (Ribes nigrum) for Inhibiting Platelet Monoamine Oxidase in Humans
Cano‐Lamadrid et al. Inhibition of enzymes associated with metabolic and neurological disorder by dried pomegranate sheets as a function of pomegranate cultivar and fruit puree
US20250090564A1 (en) Bioactives and their uses
WO2025048651A1 (fr) Agents bioactifs et leurs utilisations
Hithamani et al. Bioavailability of finger millet (Eleusine coracana) phenolic compounds in rat as influenced by co-administered piperine
Avila et al. Chemical and biological potential of passiflora vitifolia fruit byproducts collected in the Colombian central Andes
JP7070933B2 (ja) アセトアルデヒド毒性を低減するためのβ－エスシンを含有する経口組成物
JP2021517120A (ja) 肥満および体重過多の治療のための相乗的な草本組成物
Frías-Zepeda et al. Ethanolic extract of Lippia graveolens stem reduce biochemical markers in a Murine Model with Metabolic Syndrome
Giurleo A phytochemical exploration of Griffonia simplicifolia seeds and leaves
Alshammari et al. Maturation-stage effects of Ziziphus spina-christi fruits and leaves on digestive, inflammatory, antioxidant, and glucose metabolism enzymes in obese rats with type 2 diabetes
JP2023554187A (ja) 身体運動パフォーマンスを改善するため方法
KR100526631B1 (ko) 모노아민산화효소 저해활성을 갖는 상심자 추출물을 함유한조성물

Legal Events

Date Code Title Description
ENP Entry into the national phase

Ref document number: 2024287117

Country of ref document: AU

Date of ref document: 20240209

Kind code of ref document: A

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

Ref document number: 24860546

Country of ref document: EP

Kind code of ref document: A1