Biologically active substances and their uses

Abstract

The present invention relates broadly to sarmentosin and its esters and their novel uses as MAO inhibitors and for the treatment or prevention of a variety of related medical conditions associated with MAO activity.

Description

[Technical field]

[0001] The present invention relates broadly to sarmentosin and its esters and their novel uses as MAO inhibitors and for the treatment or prevention of a variety of related medical conditions associated with MAO activity. [Background technology]

[0002] Monoamine oxidases (MAOs) are enzymes that catalyze the oxidative deamination of amines such as dopamine and serotonin. The enzymes appear as two isoenzymes, MAO-A and MAO-B. Monoamine inhibition can be reversible or irreversible and can either act nonselectively, affecting both isoforms, or selectively, affecting only one isoform. They play a major role in the metabolism of both dietary and endogenous monoamines (Yamada & Yasahura, 2004).

[0003] MAO enzymes are of interest as targets for dietary supplements, functional foods, and drug discovery because, for example, MAO-A has been implicated in psychiatric conditions and depression, and MAO-B has been linked to neurological disorders such as Parkinson's and Alzheimer's diseases.

[0004] In 1994, Delumeau et al. summarized that from various human and animal studies, MAO inhibitors appear to improve cognitive function and may also help prevent cognitive disorders such as Alzheimer's disease, and therefore such inhibitors, once identified, may exhibit real therapeutic value for their cognition-enhancing properties.

[0005] A further review by Zhiyou Cai in 2014 highlighted various MAO inhibitors that may represent therapeutic agents for Alzheimer's disease.

[0006] A further review by Carradoni et al. (2018) highlighted a range of MAO inhibitors in the patent literature from 2015 to 2017, including a large number of newly synthesized and naturally occurring chemicals derived from plant materials.

[0007] For example, phenelzine is an irreversible, nonselective MAO inhibitor that inhibits both MAO-A and B for up to three weeks.

[0008] In contrast, toloxatone, a reversible inhibitor of MAO-A, inhibits MAO-A for only 6 hours before activity returns to baseline values.

[0009] Selective and reversible inhibitors are preferred because they avoid a toxic buildup of dietary monoamines in the gastrointestinal tract, which, if continued for a long period of time, can lead to dangerous accumulation of tyramine and increased risk of hypertension. It is therefore important to identify reversible and / or selective MAO inhibitors.

[0010] Isocarboxazid, phenelzine, and tranylcypromine appear to have very similar properties (Mallinger & Smith 1991). They are readily absorbed, reaching peak concentrations in 1-2 hours. Elimination is also rapid, with half-lives ranging from 1.5 to 4 hours (tranylcypromine being particularly short).

[0011] Moclobemide, a newer MAO inhibitor, is readily absorbed, reaching peak plasma concentrations in approximately 1 hour, and is rapidly and completely metabolized with an elimination half-life ranging from 1 to 3 hours.

[0012] Deprenyl (also known as selegiline) is an MAO-B inhibitor that, at doses of 1.25 mg to 10 mg per day, is effective in treating depression and Parkinson's disease, but can have significant side effects, including suicidal ideation and high blood pressure.

[0013] Indeed, many MAO inhibitors have fallen out of favor for the treatment of depression due to side effects from harmful drug-diet interactions; they remain primarily used in the treatment of Parkinson's disease.

[0014] As a further example, US8367121B2 describes a dietary supplement-based approach to attenuating processes leading to Parkinson's disease that relies, in part, on the selection of known MAO inhibitors (see paragraphs

[0045] to

[0047] ).

[0015] Due to the role MAO plays in regulating dopamine and serotonin, the Mayo Clinic website (link) also highlights that MAO inhibitors continue to be useful tools for improving mental health conditions, mood, or addressing conditions such as depression or anxiety. A variety of MAO inhibitors have been approved by the FDA, including isocarboxazid (Marplan), phenelzine (Nardil), selegiline (Emsam), and tranylcypromine (Parnate).

[0016] Through studies in a variety of sources, including tobacco smoke and coffee, a class of amines known as β-carboline alkaloids have been discovered to be inhibitors of MAO (Herraiz, 2007).

[0017] A Madrid-based research team has shown that many fruits and fruit juices / extracts contain β-carbolines (βCs) (Herraiz, 2006, 2018). When the team compared a range of βCs, they found some with strong MAO activity.

[0018] There is some evidence supporting the effects of anthocyanin-rich blackcurrants on mood and cognition. Two studies have been published showing the effects of acute blackcurrant (Ribes nigrum) supplementation in healthy humans.

[0019] Blackcurrant juice is rich in anthocyanins and other flavonoids. Although these phenolics have been shown to have some MAO inhibitory activity, other unidentified constituents may also be present that may confer additional, possibly even stronger, MAO inhibitory activity and therefore may be of commercial and therapeutic importance. Watson et al. 2015 highlighted that MAO-B was not significantly inhibited after ingestion of anthocyanin-rich blackcurrant extracts, suggesting that removal or degradation of unknown bioactive substances may be responsible for the majority of MAO activity. Indeed, some βC in blackcurrants has previously been considered to be a strong candidate for the observed MAO inhibitory activity sometimes observed in BC juices (Budzikiewicz, 1994, and Herraiz, 2006, 2018).

[0020] In particular, there is a real interest in identifying compounds with MAO inhibitory activity in fruits that are naturally occurring and can then be utilized, isolated, protected, extracted, synthetically replicated, or recombinantly produced / expressed by cell fermentation, or by breeding or other known recombinant genetic techniques to enhance the levels of the compound(s) in the fruit or fruit juice.

[0021] It is an object of the present invention to address the aforementioned problems, or at least provide the public with a useful choice.

[0022] All references, including any patents or patent applications cited herein, are incorporated herein by reference. No admission is made that any reference constitutes prior art. The discussion in the references states what the authors assert, and applicants reserve the right to challenge the accuracy and pertinence of the cited documents. Although a number of prior art publications are referenced herein, it will be clearly understood that this reference does not constitute an admission that any of these documents form part of the common general knowledge in the art in New Zealand or any other country.

[0023] Throughout this specification, "comprise" or variations thereof (e.g., "comprises" or "comprising") will be understood to mean the inclusion of a stated element, integer, or step, or group of elements, integers, or steps, but not the exclusion of any other element, integer, or step, or group of elements, integers, or steps.

[0024] Further aspects and advantages of the present invention will become apparent from the following description which is given by way of example only. Summary of the Invention

[0025] Disclosure of the Invention According to a first aspect of the present invention there is provided the use of sarmentosin or an ester(s) thereof for inhibiting monoamine oxidase enzyme A (MAO-A) and / or monoamine oxidase enzyme B (MAO-B).

[0026] According to a first aspect of the present invention, there is provided use of sarmentosin or an ester(s) thereof for preventing or treating a disease or condition, or for maintaining or ameliorating a non-clinical cognitive condition or condition associated with MAO-A or MAO-B enzyme activity.

[0027] According to a further aspect of the present invention there is provided a composition formulated as a food or beverage, a pharmaceutical composition, a dietary supplement or an extract from botanical material, the composition comprising sarmentosin or an ester(s) thereof, the composition being used to inhibit monoamine oxidase enzyme A (MAO-A) and / or monoamine oxidase enzyme B (MAO-B).

[0028] According to a further aspect of the invention there is provided the use of sarmentosin or an ester(s) thereof, or plant material, extract or composition containing same, in the manufacture of a medicament for inhibiting monoamine oxidase enzyme A (MAO-A) and / or monoamine oxidase enzyme B (MAO-B) in a human in need thereof.

[0029] According to a further aspect of the invention, there is provided a method of inhibiting monoamine oxidase enzyme A (MAO-A) and / or monoamine oxidase enzyme B (MAO-B) by administering to a human in need thereof an effective amount of sarmentosin or its ester(s), or a plant material, extract, or composition containing same.

[0030] According to a further aspect of the present invention there is provided a black currant fruit, black currant juice, extract or isolate comprising an effective amount of sarmentosin or its ester(s) for use in inhibiting MAO-A or MAO-B.

[0031] As further discussed and exemplified below, a key aspect of the present invention is the newly identified use of sarmentosin and its ester(s) as MAO-A and / or MAO-B inhibitors. To the best of the applicant's knowledge, this important and unexpected activity of sarmentosin or its ester(s) was not previously known. Surprisingly, it was shown that the MAO inhibitory activity in black currant is not associated with anthocyanins / polyphenols and β-carbolines, as expected. This opens up the great potential of sarmentosin and its ester(s) as bioactive substances that can be used in purified form or as part of a product using natural sources such as black currant that ensure the retention of said bioactive substances, as a novel method of inhibiting MAO-A and / or MAO-B and many clinical and non-clinical diseases and conditions associated with these enzymes. Another key advantage of these bioactive substances is that they occur naturally and do not exhibit side effects like other commercially developed MAO inhibitors. Sarmentosin and its esters also appear to be reversible MAO inhibitors. The inventors first seek to discover the significant MAO activity in black currant that arises specifically from these bioactive substances, and therefore seek to utilize the specific use in black currant or other sources for MAO inhibition, or to improve, concentrate, enhance or protect sarmentosin for improved use, capacity or effect.This also opens new possibilities to purify sarmentosin in extracts or isolate as fully purified bioactive substances for new uses, and the opportunity to synthetically produce bioactive substances for beneficial use.The present invention will be further discussed in the following preferred embodiments and the detailed description of the invention.

[0032] definition Throughout this specification, the term "sarmentosin" shall be understood to mean γ-hydroxynitrile glycoside (chemical name: 4-(β-D-glucopyranosyloxy)-2-(hydroxymethyl-2-butenenitrile)), or a derivative thereof, as shown in the general chemical structure below: TIFF2025507640000002.tif41128

[0033] Throughout the present invention, the term "sarmentosin ester" or "ester thereof" shall be understood to mean niglumine-p-coumarate, niglumine caffeate and / or niglumine ferulate, or derivatives thereof, as shown in the general structure below, which are phenolic acid derivatives of sarmentosin. TIFF2025507640000003.tif53128

[0034] Throughout this specification, the term "extract" should be understood to mean a preparation containing sarmentosin or its esters that has been harvested, isolated, removed, purified, or otherwise extracted from a selected source in a more concentrated or purified form compared to that found in nature. For example, the extract may be in a liquid form, such as juice, juice concentrate, or in a dry form, such as powder, tablet, or capsule. The extract may contain other bioactive substances or components from the selected source, or may be combined with other extracts, ingredients, or products, as desired. Alternatively, the extract containing 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 the present invention.

[0035] Throughout this specification, the term "composition" should be taken to mean a combination of sarmentosin or its esters in a mixture with other ingredients or components. The composition may be in the form of, for example, a food or beverage, a pharmaceutical formulation, a dietary supplement, a supplement, or a natural extract, etc., without departing from the scope of the present invention.

[0036] Throughout this specification, the term "plant material" shall be taken to mean any biological vegetation, including roots, leaves, seeds, seedlings, fruits, stems, and the like.

[0037] Throughout this specification, the term "effective amount" should be taken to mean an amount of a compound sufficient to affect such treatment when administered to a human or other mammal to ameliorate, treat, prevent, or delay a condition, disorder, or disease state. Of course, the "effective amount" may vary depending on the compound, the particular disease state, and its severity, as well as the age, physical condition, or weight of the mammal being treated. Preferred effective amounts, including dosages, are discussed further below.

[0038] Throughout this specification, the term "monoamine oxidase," or "MAO" enzyme, should be taken to mean an enzyme that catalyzes the oxidative deamination of amines, such as dopamine and serotonin. The enzyme appears as two isoenzymes, MAO-A and MAO-B.

[0039] Throughout this specification, the term "MAO inhibitor" should be taken to mean a class of chemicals, drugs, substances, extracts, or bioactive substances that have partial or complete inhibitory activity against one or both monoamine oxidase enzymes, i.e., monoamine oxidase A (MAO-A) and monoamine oxidase B (MAO-B). This inhibition can be reversible or irreversible.

[0040] Throughout this specification, the term "nootropic" shall be taken to mean a substance that improves or assists cognition, memory and / or facilitates learning.

[0041] Preferred Embodiments MAO inhibitory activity Preferably, sarmentosin, its esters, or extracts containing same provide reversible MAO inhibition.

[0042] The MAO-B inhibition profile of blackcurrant juice bears a striking similarity to pharmaceutical reversible MAO-B specific inhibitors such as lazabemide, showing rapid inhibition of MAO-B in platelets by over 90% 30 minutes after administration, with maximum inhibition subsiding 16 hours after administration and full recovery of enzyme activity returning 48 hours after administration of 100 mg lazabemide (Dingemanse et al., 1997).

[0043] Advantageously, blackcurrant supplementation, while exhibiting similar MAO inhibitory effects as pharmaceuticals, has not shown adverse side effects due to its MAO inhibition (Braakhuis et al., 2020), and appears to advantageously provide reversible inhibition of MAO enzymes. Furthermore, blackcurrant fruit, blackcurrant extract, or sarmentosin extracted or isolated from blackcurrant also appears to be particularly advantageous due to the strong safety history of blackcurrant as a food, suggesting that sarmentosin itself has a safe profile as an MAO inhibitor. In contrast, few MAO inhibitors are still in use due to their safety profile. To the inventors' knowledge, laxabemide has never been marketed.

[0044] Sarmentosin or its ester(s) preferably have both MAO-A and MAO-B inhibitory activity. This is supported and discussed in Example 2.

[0045] Having activity against both MAO enzymes is highly beneficial because they have strong affinities for various substrates. MAO-A shows greater affinity for hydroxylated amines such as noradrenaline and serotonin, while MAO-B shows greater affinity for non-hydroxylated amines such as benzylamine and β-phenylethylamine (PEA). Furthermore, dopamine and tyramine show similar affinities for each enzyme form, which means that inhibiting both may be beneficially required to control their availability.

[0046] Composition and format The composition may take a variety of forms without departing from the scope of the present invention.

[0047] Preferably, the sarmentosin or its ester(s) is present in a plant material, extract, or composition.

[0048] Particularly preferred formats are as a beverage or drink, or as a powdered supplement, however numerous other options are readily achievable by one of ordinary skill in the art.

[0049] Preferably, the sarmentosin or ester thereof is provided in a food or drink.

[0050] More preferably, the food or beverage is based on blackcurrant fruit, blackcurrant juice, blackcurrant extract (liquid or powder form), etc. It will be understood that any other fruit source containing sarmentosin or its ester(s) is applicable as well. Alternatively, one may choose to extract, isolate or synthetically produce sarmentosin without departing from the scope of the present invention, which can then be added to any such juice or composition type.

[0051] Since the present inventors have identified the presence and MAO inhibitory activity of sarmentosin and its esters in blackcurrant fruit (and its extracts), it is highly commercially desirable to utilize the existing biological activity for this new use in blackcurrant-based or blackcurrant-containing products.It should be understood that the existing levels of sarmentosin or its ester(s) can be used or can be enhanced, concentrated, or potentiated by additional amounts of sarmentosin.

[0052] Alternatively, sarmentosin or its ester(s) is provided in a dietary supplement or therapeutic format such as a powder, tablet, or capsule. Again, these formats may be derived from or contain blackcurrant material.

[0053] As previously highlighted, in another embodiment, the novel uses or methods described herein are achieved using whole fruits or parts thereof, or whole plant material or parts thereof (e.g., black currant) containing the sarmentosin or ester(s) thereof.

[0054] Alternatively, sarmentosin or its ester(s) is provided as an extract from a plant source.

[0055] The extract is preferably a polar fraction or component extracted from a selected source of sarmentosin.

[0056] Preferably the extract is non-polyphenolic.

[0057] Preferably, the extract containing MAO inhibitory activity from sarmentosin or its ester(s) also contains βC.

[0058] The extract is preferably a juice or a concentrated version thereof.

[0059] More preferably, the juice extract is derived from blackcurrant fruit.

[0060] In a further embodiment, one skilled in the art can appreciate that the present invention can be utilized by developing new plant varieties that contain sarmentosin or one of its esters at a higher level than normally or naturally occurs. For example, a plant variety can be of a blackcurrant plant (blackcurrant) in which sarmentosin and its esters are already naturally present, albeit in relatively small amounts, which is then selectively bred for increased levels of the bioactive substance(s) and then utilized for the uses described herein.

[0061] Preferred Concentrations 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 (or mg / g) of sarmentosin per gram of extract or composition.

[0062] This indicates the minimum levels of sarmentosin in the various blackcurrant juices tested by the inventors (see Example 4), however, it should be noted that there are differences in the levels of bioactive substances from the different juices and other samples tested. From experience, the inventors have found that various factors such as high temperature treatment (e.g. during pasteurization), time of the product in storage and storage conditions, seasonality of the fruit, and plant variety can all affect the levels of sarmentosin and its ester(s), which in some cases may be negligible or non-existent.

[0063] Therefore, there may be a need or advantage to concentrate, strengthen, add, or supplement sarmentosin in a composition or extract, or to select a plant or fruit that advantageously has a high level of sarmentosin.Similarly, it may be advantageous to stabilize, protect, restore, or even increase the level of sarmentosin beyond the level that is or was naturally present in black currant, or other sources thereof.Similarly, sarmentosin can ultimately be added to anything for the purposes described herein without departing from the scope of the present invention.

[0064] In a preferred embodiment, 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, 17.0, 18.0, 19.0, 20.0, 21.0, 22.0, 23.0, 24.0, 25.0, 26.0, 27.0, 28.0, 29.0, 30.0, 31.0, 32.0, 33.0, 34.0, 35.0, 36.0, 37.0, 38.0, 39.0, 40.0, 41.0, 42.0, 43.0, 44.0, 45.0, 46.0, 47.0, 48.0, 49.0, 50.0, 51.0, 52.0, 53.0, 54.0, 55.0, 56.0, 57.0, 58.0, 59.0, 60.0, 61.0, 62.0, 63.0, 64.0, 65.0, 66.0, 67.0, 68.0, 69.0, 70.0, 71.0 0.0, 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 of said sarmentosin.

[0065] More preferably, the extract or composition has about 0.005-70 mg of sarmentosin per gram of extract or composition (or mg / g).

[0066] As further discussed in the "Preferred Dosage" section, a preferred minimum dosage may be about 20 mg sarmentosin per day. If blackcurrant juice has 0.3 mg / ml sarmentosin, a serving size of 86 ml of the juice would achieve this minimum dosage. Those skilled in the art will appreciate that the concentration of sarmentosin can be suitably adjusted in the desired extract or composition using conventional methods and practices to achieve the effective amount / dosage and other criteria.

[0067] Similar embodiments can also be envisaged for the sarmentosin esters, e.g., as shown in Example 4. Although the amount of ester is low compared to sarmentosin, one skilled in the art will understand that there are means to increase the amount of ester in an extract or composition, if desired.

[0068] Source of Sarmentosine or its Esters Sarmentosin can be preferably extracted or isolated from a number of sources, including but not limited to: in the past, sarmentosin esters have been identified in black currant seeds (Lu et al., 2002), and sarmentosin has been identified in Kalanchoe species (Fernandes et al., 2021).

[0069] Now, the inventors have surprisingly found sarmentosin and its esters in blackcurrant fruit and fruit extracts (such as dried extracts or juices).Therefore, sarmentosin is preferably extracted or isolated from blackcurrant fruit.The extract can also contain other components from the source (such as blackcurrant) to provide additional benefits from bioactive substances or micronutrients.The inventors foresee that in an alternative embodiment of use, the whole fruit or plant material can be used to provide the beneficial MAO inhibitory effect from sarmentosin or its esters, as long as the material actually has this beneficial bioactive substance, which is the subject of this patent application.

[0070] Both CN102659860 and CN101974044B describe other methods of extracting sarmentosin from other plant sources. These provide alternative approaches to extract or isolate sarmentosin for the novel use of the present invention. While these may be suitable sources, it should be understood that other plants may also contain similar or greater amounts of sarmentosin or its esters, which are shown herein to have MAO inhibitory activity.

[0071] Alternatively, sources of sarmentosin or its esters can also be produced synthetically by known techniques, or by microbial fermentation production from microorganisms that either naturally express sarmentosin, or by genetic recombination using constructs encoded to recombinantly express sarmentosin in standard cell culture techniques.

[0072] Those skilled in the art will understand that there are other means to achieve commercial production of sarmentosin beyond that currently found in nature. This can be achieved by development of suitable plant varieties, either by genetic recombination, cross-breeding, or multiple selection procedures, to produce new varieties containing high amounts of sarmentosin or its esters. In other words, the present invention can be achieved by artificially producing sarmentosin or its esters, which are highly pure and can be provided, stored, or sold in purified form, or suitably provided in pharmaceutical compositions. This can be particularly useful for the pharmaceutical industry, where stringent production requirements are required.

[0073] In pharmaceutical embodiments, any suitable form including, but not limited to, transdermal creams or patches, parenteral or subcutaneous injections, pills, capsules, etc. are possible routes of administration.

[0074] Potential manufacturing options Blackcurrant juice can be produced according to standard practices, for example a juice concentrate can be obtained by evaporating the juice under vacuum to obtain a concentrate containing approximately 10 times the concentration of sarmentosin.

[0075] The juice in powder form can be produced by freeze drying or spray drying the juice. Thus, one skilled in the art will appreciate that the present invention can be provided in a variety of different dried formats, such as capsules, tablets, powders, etc.

[0076] Pure sarmentocin can be obtained by chromatographic separation of the juice or juice concentrate using reverse phase chromatography involving multiple steps until pure sarmentocin is obtained.

[0077] Those skilled in the art will appreciate that there are also standard techniques available for chemically synthesizing compounds such as sarmentosin or its esters, and such processes are encompassed by the present invention.

[0078] Similarly, cell expression systems are now routinely used to produce desired compounds on a large scale. Natural or engineered bacterial systems can, for example, be selected or produced that overexpress large amounts of sarmentosin, which can then be purified from the cell culture.

[0079] Further examples of methods for preparing compositions containing sarmentosine are described in the Detailed Description section of the specification.

[0080] Methods of Use and Treatment MAO inhibitors have already shown promise and are associated with a range of diseases and conditions, which are highlighted throughout this specification.Therefore, it is highly anticipated that sarmentosin or one of its identified esters (may be multiple) that is now found to have MAO inhibitory activity may very well have novel uses, both clinical and non-clinical, to treat or prevent these same or similar conditions.It should be understood that all MAO inhibitors generally share the same mechanism of action and are equivalent in their effectiveness.

[0081] Preferably, the uses or methods of treatment described herein regulate, maintain or increase a neurotransmitter selected from the group consisting of dopamine, serotonin, adrenaline (noradrenaline or norepinephrine), and tyramine.

[0082] Potential clinical applications The uses or methods of treatment described herein are preferably for treating or preventing a disease or condition, which is any neurological or psychiatric condition or disease associated with MAO-A or MAO-B enzyme activity, in a human in need thereof.

[0083] Sarmentosin or its ester(s) is preferably used to treat or prevent depression, atypical depression, panic disorder, social anxiety disorder, 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.

[0084] Non-clinical use Additionally, as discussed throughout this specification, MAO inhibitors have demonstrated potential for a range of non-clinical applications For example, Rhodiala, also known as golden root, contains MAO inhibitors and has been used to reduce mood, social anxiety, relieve fatigue, and improve athletic performance.

[0085] Sarmentosin or its ester(s) is preferably used to improve, restore or support non-clinical effects such as mood, anxiety, social anxiety, fatigue, cognitive performance, motor performance, attention / vigilance, calmness, mental clarity, executive function, working memory, secondary memory, mood, stress and / or stress reactivity, or to provide a nootropic effect.

[0086] Those skilled in the art will appreciate that other non-clinical applications related to cognitive and psychological support are also within the scope of the invention and its uses.

[0087] Sarmentosine may also be mixed with other nootropics or bioactive substances shown to be beneficial in treating neurological conditions or in preventing medical conditions or diseases.

[0088] Preferred Dosage The inventors have conducted preliminary studies (see Example 5) to investigate the likely effective dose of sarmentosin or its ester(s) for clinical use and also for non-clinical use. This was achieved by comparative enzyme inhibition analysis of a known commercial MAO-B inhibitor (deprenyl) used for clinical purposes. The data for deprenyl in Example 5 and extrapolation from the dosage guide (http: / / www.drugs.com / dosage / selegiline) suggest that for clinical use, an initial daily dose equivalent to 27.5 mg of sarmentosin may be used, and after 6 weeks, the daily dose may be increased to 65 mg if necessary.

[0089] Thus, for clinical uses similar to deprenyl, a daily dose of at least 20 mg of sarmentosin or its ester(s) may be preferred.

[0090] More preferably, the daily dose is 20 to 200 mg of sarmentosin or its ester(s).

[0091] More preferably, the daily dose 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 of sarmentosin or ester(s) thereof.

[0092] More preferably, the daily dose is 20 to 200 mg of sarmentosin or its ester(s).

[0093] Another preferred daily dose may be at least 110 mg / 60 kg body weight.

[0094] In a more preferred embodiment, the daily dose of sarmentosin or its ester is 110-130 mg / 60 kg of body weight.

[0095] In non-clinical settings, the preferred dose may be substantially lower than in clinical settings.

[0096] For non-clinical uses (e.g., for preventative, supportive, or maintenance effects on mood or cognition, as discussed above), the serving size / daily intake can be a minimum of 1 mg to 20 mg, although larger doses, similar to the preferred clinical dose, may also be useful and within the scope of the present invention.

[0097] Main advantages of this invention The present invention may include, but is not limited to, one or more of the following advantages: -Identification of novel applications of sarmentosin and its esters as MAO inhibitors, with potentially significant opportunities for therapeutic and preclinical applications. - Various embodiments of the present invention can advantageously take advantage of the fact that naturally occurring sources of sarmentosin or its esters are present in several plant materials, including black currant. -Preliminary evidence suggests that sarmentosin has minimal or no adverse side effects. - Preliminary evidence suggests that sarmentosin advantageously possesses both inhibitory activity against both MAO-A and MAO-B, opening up further applications. -Preliminary evidence suggests that sarmentosin beneficially and reversibly inhibits MAO-A and MAO-B. [Brief description of the drawings]

[0098] Further aspects of the invention will become apparent from the following description, given by way of example only, and with reference to the accompanying drawings, in which:

[0099] [Figure 1] βC reported from blackcurrant juice, see Example 1. Compound 1 (LHS) and Compound 2 (RHS). [Diagram 2]See Example 1, a third βC discovered in black currant (compound 3). [Diagram 3] LCMS analysis of test blackcurrant juice with reference to Example 1. [Figure 4A] 1 is a chromatogram of Neuroberry™ juice sample 1, see Example 1. [Figure 4B] 1 is a chromatogram of Neuroberry™ juice sample 2, see Example 1. [Figure 4C] 1 is a chromatogram of Neuroberry™ juice sample 3, see Example 1. [Diagram 5] Amount of 4HQ found in the fractions, see Example 3 (lower values ​​indicate greater inhibition). [Figure 6] 4 is a preparative HPLC trace (UV) with reference to Example 3. Fractions 18-23 are collected from 7-11 minutes. [Figure 7] Amount of 4HQ found in fractions of large scale preparative HPLC, see Example 3 (lower values ​​indicate greater inhibition). [Figure 8] 1 shows the chemical structure of sarmentosin with reference to Example 3. [Figure 9] 1 shows the chemical structure of the niglumine compound with reference to Example 3. [Figure 10] Activity versus sarmentosin content for preparative HPLC fractions 17-22, see Example 3. [Figure 11] FIG. 11 is a H NMR spectrum of a preparative HPLC fraction, see Example 3. [Figure 12] FIG. 1 is a H NMR spectrum detailing the 5-10 ppm region, referring to Example 3. [Figure 13] This shows the amount of 4HQ in the bioassay of reverse phase fractions rp-1 to rp-11, with reference to Example 3. [Figure 14] See Example 4, RP10 fraction 4HQ (S9 bioactivity relative to preparative HPLC fractions of RP10). [Figure 15] Coumaroyl and feruloyl sarmentosin content in RP10 HPLC fractions with reference to Example 4 (fractions 12-15 have higher peak areas than shown). [Figure 16] Caffeoyl Sarmentosine peak area content in RP10 HPLC fractions, see Example 4. [Figure 17A] Dilution series of Sarmentosin, see Example 5. 50% inhibition occurs at approximately 400 ng / ml. [Figure 17B] Dilution series of deprenyl, see Example 5. 50% inhibition occurs at approximately 10-15 ng / ml. [Figure 18] 1 is a plot of the concentration of sarmentosin (S) in samples versus the concentration of each sample required for 70% inhibition of MAO-b, with reference to Example 6. [Figure 19] FIG. 1 is a flow chart of progression through the phases of a randomized crossover study including two format groups, with reference to Example 7. [Figure 20] 1 shows the procedures carried out on trial days 1 and 2 of the intervention study with reference to Example 7. [Figure 21] Concentrations (mean ± SEM) of total anthocyanins (A), cyanidin derivatives (B, C, D), and delphinidin derivatives (E, F) in plasma before (0 min) and after blackcurrant beverage intervention with reference to Example 7. Data are presented as cyanidin 3-O-glucoside equivalents. [Figure 22] FIG. 11 shows plasma sarmentosin concentrations (mean±SEM) before (0 min) and after ingestion of a single dose of black currant juice or powder, see Example 7. [Figure 23] Platelet monoamine oxidase (MAO) B activity and blood glucose concentration before (0 min) and after ingestion of blackcurrant juice (A, C) or powder (B, D) and their corresponding placebos, with reference to Example 7. Values ​​are means ± SEM. * indicates significant difference from 0 min (p<0.05). ^ indicates significant difference from placebo intervention at the same time point. [Figure 24]Visual analogue scores for the Bond-Radar questionnaire items interesting / bored (A, B), kind / unkind (C, D), happy / sad (E, F), and calm / excited (G, H) from participants before (0 min) and after ingestion of blackcurrant juice or powder and their corresponding placebos, see Example 7. Values ​​are means ± SE. * indicates significant difference from placebo intervention at the same time point. [Diagram 25] FIG. 11 is a Spearman's rank sum correlation coefficient for neurotransmitters showing significant correlations (*p<0.05, **P<0.01) with MAO-B inhibition after taking either black currant juice intervention vs. placebo or black currant powder intervention vs. placebo, with reference to Example 7. [Figure 26-1] Differences in circulating plasma neurotransmitter concentrations (Δng / mL) (mean±SEM) between time (t)=0 and t=10, 20, 120, 240, and 480 minutes after ingestion of either blackcurrant juice intervention vs. placebo or blackcurrant powder intervention vs. placebo, with reference to Example 7. (A) 3,4-dihydroxyphenylacetic acid (DOPAC) juice intervention, (B) DOPAC powder intervention, (C) homovanillic acid (HVA) juice intervention, and (D) HVA powder intervention. [Figure 26-2] Differences in circulating plasma neurotransmitter concentrations (Δng / mL) (mean±SEM) between time (t)=0 and t=10, 20, 120, 240, and 480 minutes after ingestion of either blackcurrant juice intervention vs. placebo or blackcurrant powder intervention vs. placebo, see Example 7. (E) Vanillylmandelic acid (VMA) juice intervention, (F) VMA powder intervention, (G) dopamine (DA) juice intervention, and (H) DA powder intervention. [Figure 27]Circulating plasma neurotransmitter concentration differences (Δng / mL) (mean±SEM) between time (t)=0 and t=10, 20, 120, 240, and 480 minutes after ingesting either blackcurrant juice intervention vs. placebo or blackcurrant powder intervention vs. placebo, with reference to Example 7. (A) 5-hydroxyindoleacetic acid (5-HIAA) juice intervention, (B) and 5-HIAA powder intervention. * indicates significant difference between intervention and placebo at the same time point (p<0.05). [Figure 28] Differences in circulating plasma neurotransmitter concentrations (Δng / mL) (mean±SEM) between time (t)=0 and t=10, 20, 120, 240, and 480 minutes after ingestion of black currant powder intervention versus placebo, see Example 7. (A) xanthurenic acid (XA), (B) metanephrine (MN), (C) normetanephrine (NM), and (D) kynurenic acid (KA). [Figure 29] Circulating plasma neurotransmitter concentration differences (Δng / mL) (mean±SEM) between time (t)=0 and t=10, 20, 120, 240, and 480 minutes after ingestion of either blackcurrant juice intervention vs. placebo or blackcurrant powder intervention vs. placebo, with reference to Example 7. (A) 3-Methoxy-4-Hydroxyphenylglycol (MHPG) juice intervention, (B) MHPG powder intervention, (C) 3,4-Dihydroxyphenylglycol (DHPG) juice intervention, (D) DHPG powder intervention, (E) Phenylalanine (PHE) juice intervention, and (F) PHE powder intervention. * indicates significant difference between intervention and placebo at the same time point (p<0.05). [Diagram 30]FIG. 11 is a correlation circle plot of standardized neurotransmitter concentrations as dependent variables for blackcurrant (A) and placebo (B) beverages (combination of juice and powder) with reference to Example 7. Only the 12 variables that contribute most strongly to the definition of the principal components are displayed. Warm and cold colored variables are weighted heavily and lightly, respectively. The coordinates of the variables correspond to the value of their correlation coefficient, and the angle between two variables defines their relationship; if the angle between two variables is an acute angle, the correlation is positive, if the angle is an obtuse angle, the correlation is negative, and if the angle is a right angle, there is no correlation. EXAMPLES

[0100] Detailed Description Example 1 - Review of previous R&D and pilot studies of β-carbolines overview Previous studies have shown that blackcurrant (BC) juice intake affects the monoamine axis in human subjects. In 1994, Budzikiewicz showed that BC contains two types of β-carbolines (βC) - see Figure 1. This class of compounds is known to interact with MAO. As mentioned before, in 2006 and 2018, Herraiz et al. showed that various juices and fruits, including BC, contain β-carbolines (βC). When the team compared a range of βCs, they found strong MAO activity in some of the βCs. MAO-B activity was shown to be inhibitable by BC juice intake. The activity was reversible and nonselective, but was suggested to be possibly accompanied by greater MAO-B affinity. Although these βC compounds are present in very small amounts in juice, the inventors considered βCs as clear candidates for MAO inhibitory activity in BC juice.

[0101] Example 1 includes the following: Develop an analytical method to detect low amounts of β-carbolines in BC samples. This involves processing bulk BC juice or frozen berries (10 kg) and specifically extracting βC. -Preparing samples by enhanced βC using common precursor buffer reactions Using LC-MS and LC-HRMS to detect βC in juice and fortified samples.

[0102] Analytical methods and results of βC in BC. Analysis of these βCs is most conveniently performed using LCMS. Alkaloids tend to ionize well in mass spectrometry, and specific MRM analysis allows for more accurate analysis in complex mixtures. First, juice samples were extracted with diethyl ether, which gave ether extracts enriched in βCs. These were then run through LCMS, and peaks corresponding to the two known βC masses were identified (279 and 323, MH+ for 1 and 2).

[0103] During this work, we also noted the presence of another βC with an MH+ of 231, which we identified as methyltetrahydro-β-carboline (see FIG. 2).

[0104] Compounds 1-3 (Figures 1-2) can be derived from tryptophan. Reacting tryptophan with aldehydes in the laboratory to give these types of compounds is known as the Pictet-Spengler reaction. Even under extremely mild conditions, such as in juice, tryptophan will react with aldehydes to give these products.

[0105] To aid in compound identification and samples required for activation testing, two of the compounds could be prepared. Compound 1 is prepared from ascorbic acid and tryptophan, while compound 3 is prepared from tryptophan and acetaldehyde. The ascorbic acid reaction likely involves conversion of ascorbic acid to an aldehyde first, resulting in low yields. The reaction with acetaldehyde proceeds more quickly. Compound 2 can be formed from hydroxymethylfurfural (HMF), but then requires an additional methylation step.

[0106] The preparation of compounds 1 and 3 was carried out on a small scale to provide sufficient documentation to confirm that the synthetic compounds were identical to the natural compounds and was also used to optimize MS analysis on LCMS. The compounds prepared were also prepared and used in Example 2.

[0107] Figure 3 shows the chromatogram of a sample of the test beverage containing BC. Only the channels for the three βCs are shown, the blue trace is compound 3, while the peak around 17 min is for compound 1 and the weaker peak around 13 min is compound 2. Although it was not possible to quantitate these compounds, compound 3 clearly shows a strong response in this analysis.

[0108] Using this method, other related samples could then be considered. Three samples of frozen Neuroberry™ blackcurrants were provided for analysis. Juice was also squeezed from a few berries of each, centrifuged, and diluted 1:1 with water before analysis. The chromatogram of the Neuroberry™ sample is shown in Figure 4. This shows a fairly high level of variation in the amount of βC in the three berry juice samples. For example, the peak areas of compound 3 were 3.8, 9.0, and 4.9 (relative peak areas in millions).

[0109] Samples of frozen BC juice concentrate and dried BC extract powder were also analyzed. The BC powder was a dried ethanol extract of BC. Both the dried ethanol concentrate and the frozen juice concentrate contained the three βCs identified above.

[0110] Fresh juice vs. dried extract As mentioned above, both the fresh juice and the dried ethanol extract contained βC. However, there are distinct differences between the juice and the ethanol concentrate. Most obvious is the absence of polar components in the ethanol extract. This is to be expected, since polar compounds such as sugars and food acids are not very soluble in ethanol. Two compounds of particular interest in this study are ascorbic acid and tryptophan. Tryptophan is the precursor of the aforementioned βC, and βC is known to take on a juice form, most likely during juice processing. Ascorbic acid is also important in this context, since some of the βC is formed from the reaction of ascorbic acid with tryptophan.

[0111] Analysis of the dried ethanol extract showed the absence of ascorbic acid and the presence of moderate amounts of tryptophan. High amounts of ascorbic acid and tryptophan were found in both the juice and the juice concentrate. The actual amounts of these components were not measured.

[0112] overview The present inventors have developed a method to screen blackcurrant juice products for low levels of specific β-carbolines. Three compounds have been identified in fresh juice, juice concentrate, and dried ethanol extract.

[0113] Two of the identified β-carbolines were prepared from tryptophan in combination with ascorbic acid or acetaldehyde. These types of compounds have been shown to be formed in juices and some fruits during processing.

[0114] Ethanol extracts of blackcurrant show an absence of ascorbic acid, which, and of other polar compounds, may reduce the formation of β-carbolines in vivo.

[0115] We conclude that more detailed analysis may be necessary to measure in vitro MAO activity in βC enriched juices and samples.

[0116] Example 2 - Bioassay In the previous Example 1, we identified several βC in BC samples and also prepared several βC enriched samples in preparations for our bioassays.

[0117] Most commercial MAO assay kits work by measuring the formation of hydrogen peroxide (produced when MAO deaminates monoamine substrates). This method can be subject to interference when antioxidants are present in the sample (antioxidants can inhibit the formation of peroxides). A study by Herraiz et al. (2018) showed that directly analyzing the deaminated substrate instead using HPLC is a viable option. Using this method, they showed that common dietary phenolic compounds such as quercetin and cyanidin are not active (rather, they inhibit peroxidase when used in commercial assays).

[0118] In Example 2, the inventors Testing for MAO-B and, optionally, MAO-A inhibition using in vitro assays using commercial kits; · HPLC assay was used to confirm activity in various samples including BC juice, dried ethanol extract (35% anthocyanins), and test βC prepared in Example 1.

[0119] Methods and Results In this example, we focused on the in vitro evaluation of the effects of BC juice using a commercial kit-based MAO assay.

[0120] The kits used were from Sigma-Aldrich [MAK295 / 296] for MAO-A and MAO-B enzymes. The kit method detects the formation of peroxides (resulting from MAO activity) by the production of a fluorescent product. Because the BC samples are complex compounds containing several fluorescent components and a highly colored background, only a limited number of samples could be assayed with each kit (due to the need for multiple control and background samples).

[0121] In conducting the initial kit assay, the inventors investigated samples of freshly extracted blackcurrant juice, a dried extract, a test beverage containing blackcurrant juice (aged), and a mixture of two of the β-carboline samples prepared in Example 1 (compounds 1 and 3).

[0122] The results are shown in Table 1 below. The values ​​in the table (mg / ml) are estimates of juice content based on anthocyanin comparison. This was determined by measuring the anthocyanin concentration of the dried extract and juice concentrate (UV-Vis) versus freshly squeezed juice. Thus, the juice concentrate is 4.5 times the concentration of the juice and the dried extract is 73 times the concentration of the juice. The test beverages are known to be 1:1 dilutions of the juice.

[0123] Table 1: Results of the first round of assays TIFF2025507640000004.tif110170

[0124] These results are very encouraging. Good MAO-A and MAO-B inhibition is observed by the juice samples and juice concentrates. Inhibition is also observed by both the dry extracts and the beverage products tested.

[0125] However, almost no inhibition was observed with the β-carboline mix. Although this latter result is somewhat disappointing, this represents only two out of a range of possible βCs, and published studies show a large variability in βC activity according to structure.

[0126] In general, both MAO-A and MAO-B inhibition was observed for active samples, with the optimal test rate being approximately 10 mg / ml juice equivalent.

[0127] A second round of kit testing was conducted (Table 2 below). In this round, the juice was retested at three dilutions: 20, 10, and 5 mg / ml. These samples showed MAO-A results consistent with the previous set, with good order of activity at the 5 and 10 mg / ml juice equivalents.

[0128] Due to ongoing concerns that activity is consistent with flavonoid / anthocyanin polyphenols in the juice, a range of functions were also tested in an attempt to separate activity from polyphenols, and the results are shown in Table 2.

[0129] Extracts E1, E2 and E3 are samples prepared by diethyl ether extraction of the juice concentrates (diluted in water), E2 is an ether extract of E1 after it has been made alkaline and E3 is an ethyl acetate extract of E2.

[0130] None of these extracts were active, indicating that the activity was not associated with the non-polar portion of the extract and was unlikely to be related to known β-carbolines.

[0131] Another way to separate the polyphenols from the "other" components is to pass the extract through a polyamide column. The results are also shown in Table 2 below. In this case, a small sample of the juice (from the concentrate) was prepared with 0.25% acetic acid and loaded onto the polyamide column, PA1 being the load fraction, PA2 being the polar non-phenolic fraction eluted with 0.25% acetic acid, PA3 being the main polyphenol fraction (colored) eluted with 1:1 methanol:water, while PA4 is eluted with neat methanol. Most of the activity is found in fraction PA2, which is the polar material separated from the main anthocyanin / polyphenol fraction (PA3). This tells us that the main activity is surprisingly not associated with anthocyanins / polyphenols, and not associated with β-carbolines, as expected.

[0132] (Table 2) Results of the second round of assays. TIFF2025507640000005.tif60170

[0133] In this second round of assays, the MAO-A results were generally on scale, while MAO-B appeared to be more sensitive, with most results exceeding 100%, but still confirmed that MAO-B activity was consistent with MAO-A, suggesting that only one of the assays need be run to track activity in later examples.

[0134] overview Activity testing using commercial kits from Sigma-Aldrich shows consistent MAO-A and MAO-B activity for juice, juice concentrate, and dried juice extract. Activity is concentration dependent. Because MAO-A and MAO-B activity appear to track together, a single (MAO-A) assay is suggested in future examples for efficiency.

[0135] Testing several fractions that separate polar and non-polar components from the major juice polyphenols showed that the activity was associated with the polar non-polyphenol fraction from the juice, suggesting that the activity was not due to known β-carbolines, as it was enriched in the non-polar fractions. This work led to Example 3, which aims to identify the specific fraction and component(s) responsible for the MAO inhibitor activity.

[0136] Example 3: Bioassay-Guided Compartmentation Overview In Example 3, we investigated the fractionation process and sought to narrow down the identity of the active ingredient(s).

[0137] Methods and results. To allow for the screening of a large number of fractions / samples, an alternative assay to the commercial assay kit from Sigma-Aldrich used in Example 2 was used in Example 3. Porcine S9 liver microsomal fraction was used as the source of MAO-A and MAO-B enzymes. This method allowed for the screening of a much larger number of samples than was possible with the kit-based assay. S9 porcine liver microsomes were isolated from macerated porcine liver.

[0138] The S9 porcine fraction was mixed with a specific MAO substrate (kynuramine) and mixed with the test sample and buffer. After 1 hour at 37°C, the reaction was stopped by cooling and adding 2N NaOH and acetonitrile. The centrifuged samples were then analyzed using LCMS to determine the amount of 4-quinolol (4HQ) product.

[0139] The method was performed according to the procedure described in Ghosal.,(2020)(title: "Evaluation of the clearance mechanism of non-CYP-mediated drug metabolism and DDI as a victim drug"). This method utilizes the natural formation of 4-quinolol from oxidized kynuramine. The amounts of kynuramine and 4-quinolol were investigated by LCMS, and the absence of small amounts of 4-hydroxyquinolol / 4-hydroxyquinolol indicates inhibition of MAO.

[0140] This method has proven to be very useful for monitoring MAO inhibitory activity through multiple manipulative steps.

[0141] Preparative separation using column chromatography (CC). Three modes of CC were used.

[0142] 1. Reversed Phase Chromatography (RPCC) First, a small-scale column was run with a mini-RP column and eluted with water, followed by increasing proportions of methanol.

[0143] MAO inhibitory activity was only seen in the most polar fractions, ie those eluting at the beginning.

[0144] After this, the juice concentrate (5 g) of the larger sample was dissolved in water and the pH adjusted to 5.5 with 2N NaOH. This extract was applied to a larger C18 silica RP column and eluted with water (2 fractions), followed by acidified water (2 fractions), followed by a solution of 15% ethanol in water, and finally with ethanol.

[0145] Assay of these larger fractions indicated that the major activity was restricted to the first two aqueous fractions.

[0146] 2. Cation Exchange Chromatography Small scale chromatography using a commercial preparative column (SPE type). A sample of juice concentrate was dissolved in pH 8 buffer and loaded onto the column. The column was then washed with 0.1N HCl, followed by water and then NaOH.

[0147] The MAO inhibitory activity was concentrated in the load fraction, ie, it did not remain on the column.

[0148] 3. Anion Exchange Chromatography Some tests were performed using anion exchange material. Initially, activity appeared to be retained on the column and eluted with the acid. However, the assay proved to be somewhat sensitive to traces of acid, and once the sample was dried down and made up with water, activity was restricted to the load fraction.

[0149] These results indicated that the actives in the BC juice concentrate were polar (eluted early on the PR CC) and were neither acidic nor basic in nature (not retained on anion or cation exchange media).

[0150] Preparative HPLC Preparative HPLC was the next step to further refine the activity. Initially, a chromatographic run was performed using a 250 x 10 mm Luna C18 column. Fractions were collected every minute and an acetonitrile gradient in acidified water was used. Subsamples of each fraction were taken and dried prior to assay. The assay profile is shown in Figure 5. As shown, activity was restricted to early fractions during the HPLC run, primarily fractions 3 and 4 (but also 8).

[0151] To further narrow down the activity, a second preparative HPLC run was performed (see FIG. 6), which used a larger column (Supelco Ascentis C18, 250×22 mm) and more fractions were collected around the region of interest (fractions 18-23 were collected between 7-11 min).

[0152] As shown in Figure 7, the most active fractions in the assay were 19 and 20. LCMS analysis indicated that these fractions likely represent a single compound with a molecular weight of 276. High resolution MS revealed that C 11 H 17 O 7 The formula for this compound was given as N. The major ion seen in negative mode is the formate adduct (m / z 320). Fractions 19 and 20 were combined and an NMR spectrum obtained. The NMR data indicated the presence of a sugar, most likely glucose. A search of the literature using the identified formula and the presence of a sugar allowed the most likely candidate to be identified as sarmentosin (shown in FIG. 8), a nitrile glycoside previously isolated from plants such as Kalanchoe species. Comparing the NMR data to that of sarmentosin confirmed this indication.

[0153] To the best of the inventors' knowledge, MAO activity has never been identified or discovered from sarmentosin. Thus, the present invention relates to the novel use of sarmentosin from natural sources, which may be derived from, for example, black currant or Kalanchoe species, or which may be derived in other ways, such as by cell-based fermentation and purification techniques, or synthetic production of the compound, as an MAO inhibitor (and related uses, as described elsewhere herein).

[0154] Sarmentosin can be classified as a gamma-hydroxynitrile glycoside, which is an unusual class of hydroxynitrile glycoside since most known hydroxynitrile glycosides are alpha-hydroxynitrile glycosides (e.g., prunasin from apple seeds).

[0155] Further HPLC analysis of the majority of the active fractions appears to show the greatest amount of sarmentosin in fractions 18-21, as shown in Figure 10. This would appear to be consistent with sarmentosin being the active component.

[0156] We also investigated the NMR spectra of the immediately preceding fraction and the fraction containing sarmentosin (see FIG. 11). Fractions 19 and 20 combined show the major NMR signal for sarmentosin and only trace amounts for other compounds. Fraction 18 also has several small peaks in the 5.5-9 ppm region. Fraction 17 has a small amount of sarmentosin, but more other peaks in the 5.5-9 ppm region (detailed in FIG. 12). Fraction 16 (inactive) has no sarmentosin and also misses other signals in the 5.5-9 ppm region. This is consistent with sarmentosin being the source of activity in these fractions.

[0157] Column chromatographic fractionation revisited. Due to the high likelihood of activity in the identified polar fractions, it was decided to check for any other activity in the fractions from the reversed phase C18 column. A new column was run with 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 later eluting non-polar fraction (see Figure 13). Fraction 10 is active. The major sarmentosin fraction is fraction 2.

[0158] Analysis of fraction 10 shows the presence of a set of flavonol glycosides and several other phenols. Among these phenols, LCMS extraction of the ions identified the presence of two nilumine ester derivatives of sarmentosin, as shown in Figure 9. These two related compounds were previously isolated from blackcurrant seeds (Lu et al., 2002), although again they were not identified as having MAO inhibitory activity. These are nilumine-p-coumarate and nilumine ferulate (Figure 9). These are phenolic acid derivatives of sarmentosin.

[0159] A sample of fraction 10 was run on a RP preparative HPLC and the fractions were assayed using the previously used S9 assay. The results (shown in Figure x) show three active bands, fractions 9 / 10, fractions 12-16 and a later band (fraction 21).

[0160] These active fractions were analyzed using LCMS (as described above). Coumaroyl and feruloyl appear to be enriched in fractions 12-16 (Figure x, peak area vs. fraction number). This is consistent with the activity shown in the fraction bands. Fractions 9 and 10 shown also show some characteristic mass spectral peaks, indicating the presence of an additional sarmentosin ester. This was found to be the caffeoyl ester of sarmentosin by the characteristic MRM transition (m / z 436->179). This is likely the compound responsible for the activity observed in fractions 9 and 10 (Figure x).

[0161] It should be noted that the HPLC fractions are primarily mixtures of compounds. In many of these fractions, especially fractions 8-12, there are significant amounts of other components, primarily flavonoids.

[0162] Fractions 13 and 14 are dominated by ester compounds.

[0163] With access to purified samples of the two niglumine esters from historical studies and the S9-LCMS assay, the niglumine mixture was active at approximately the same level as sarmentosin. This strongly supports that the active compound in the S9 assay is sarmentosin. The sarmentosin esters (ferulic / coumaric acid niglumine) may be hydrolyzed to sarmentosin by the S9 enzyme.

[0164] Comparison of extracts In human studies, ethanol extract powder (DelCyan) was not very effective in altering MAO activity. In this study, we analyzed the provided blackcurrant powder and found only very small amounts of sarmentosin (compared to juice or juice concentrate). This is also evident in the S9 LCMS bioassay, where the juice concentrate is more active than the dry extract when compared to the same amount of total anthocyanins (73 times the juice of the dry extract, 4.5 times the juice of the concentrate). The dry extract is expected to still contain niglumine esters.

[0165] overview Conclusion: · MAO inhibitory activity is predominantly traced to sarmentosin and to a lesser extent to sarmentosin esters. Activity is most easily observed using crude liver S9 fraction containing the enzyme mix.

[0166] Example 4: Further analysis of sarmentosin activity in samples Overview In Example 4, samples of active compounds were purified from juice concentrates (a scaled-up study from Example 3) to obtain approximately 10 mg of each active compound. Furthermore, the content of these actives in juices or other berry samples (e.g., blueberry, and various black currant samples) was analyzed. Finally, the ester-containing fractions were subjected to fractionation and bioassays (S9 assay) to confirm activity against the esters.

[0167] result 1. Purification of active compounds. Approximately 2 kg of frozen berries were blended with an equal volume of 1:1 ethanol:water. The liquid was separated from the remaining solids using a fine nylon cloth as a filter. Ethanol was removed from the filtrate by rotary evaporation. Half of the liquid extract was then applied to a reverse phase chromatography column plug (dimensions: 9 cm diameter and 4 cm depth). After loading, the column was eluted with water containing 0.1% formic acid and increasing percentages of ethanol in water.

[0168] Fractions were analyzed using LCMS. Sarmentocin was concentrated in fractions eluted with water and up to 5% ethanol, while sarmentocin esters were found in fractions eluted with 40-60% ethanol. The remainder of the extract was chromatographed in a second run.

[0169] Fractions containing sarmentosin were combined, lyophilized and rechromatographed using the same column. The sarmentosin-rich fractions from this separation were subsequently used for preparative HPLC. Preparative HPLC was performed using a Gilson preparative system, eluting with acetonitrile and 0.1% formic acid in water, using a gradient starting with 1% acetonitrile and ending with 15% acetonitrile. The purity of the sarmentosin fractions was investigated using LCMS and 1H NMR.

[0170] Similarly, the sarmentosine ester-rich fractions were combined, rechromatographed using a RP plug, and finally purified using preparative HPLC (same system as above, but with a modified gradient of 5-40% acetonitrile). The purity of the ester was investigated using LCMS.

[0171] 2. Analysis of samples. Four comparative blackcurrant juice beverages, labeled Samples 1-4, were used and compared with Applicant's test blackcurrant juice samples provided as a pure Neuroberry™ blackcurrant juice extract (Sample 5), as well as in concentrated juice (Sample 6), and in commercial form (Sample 7).

[0172] In addition, two extracts from freeze-dried black currants (samples 8 and 9) were tested which served as a useful comparison to Applicant's experimental dried extract (sample 10) which contained black currant material.

[0173] The samples were analyzed using LCMS. Standards were prepared for sarmentocin, feruloyl sarmentocin, and coumaroyl sarmentocin. The analysis used the specific mass data (MRM's) for each of the analytes, and compared the peak areas for the samples to the peak areas for the standards.

[0174] Juices were analyzed either neat or as a 1:1 dilution. Freeze-dried berries were extracted with water or ethanol:water (1:1).

[0175] The values ​​were then compared with those obtained for samples 5-7.

[0176] These results are shown in Table 3. Note that the sarmentosin content is given in mg / g of sample, while the esters are given in ug / g. From this data, it can be seen that sample 5 has about 15 times more sarmentosin compared to each of the esters. The amounts of the two esters are similar.

[0177] Based on these initial results, the inventors believe that the typical amount of sarmentosin in different types of blackcurrant juice / extracts may vary substantially, ranging from about 5 to 1300 μg / g. Thus, in one aspect of the invention, the inventors envisage using juice extracts or other formats with sarmentosin or ester(s) either within this range for novel uses in MAO inhibition, or advantageously increased beyond this amount to increase the potency or effectiveness of the composition, dietary supplement, or extract.

[0178] It can also be concluded that the freeze-dried berry extract samples 8 and 9 have approximately the same content as sample 5. Juice sample 2 is approximately the same as sample 5, although the amount of sarmentosin is slightly less. In juice sample 4, the sarmentosin content is consistent with the juice content of 5.7%. Test beverage product 7 was from an aged sample stored at room temperature, so some sarmentosin may have been lost over time. As this test beverage is a 1:1 dilution of juice, a fresher sample would typically be expected to contain approximately 0.7-0.8 mg / g sarmentosin. Both the concentrated beverage sample 6 and the dried powder (sample 5) are very high in sarmentosin compounds, but the dried extract has proportionally less sarmentosin (the esters are approximately 3 times as high in sample 6, but less sarmentosin). This is likely an effect of using ethanol, which favors the extraction of less polar esters.

[0179] Table 3: Analysis of sarmentosin and ester content in various blackcurrant juice products. TIFF2025507640000006.tif103169

[0180] Fractionation and bioassay of the ester-rich fraction In Example 3, it was determined that a less polar fraction from reverse phase chromatography was also active in the S9 bioassay. This fraction (RP10) showed good activity and appeared to contain the sarmentosine esters described above.

[0181] A sample of RP10 was run on a RP prep HPLC and fractions were assayed using the previously used S9 assay. The results (shown in Figure 14) show three active bands, fractions 9 / 10, fractions 12-16, and a late band (fraction 21).

[0182] These active fractions were analyzed using LCMS (as described above). Coumaroyl and feruloyl appear to be enriched in fractions 12-16 (Figure 15, peak area vs. fraction number). This is consistent with the activity exhibited by the fraction bands. Fractions 9 and 10 shown also show some characteristic mass spectral peaks, indicating the presence of an additional sarmentosin ester. This was found to be the caffeoyl ester of sarmentosin by the characteristic MRM transition (m / z 436->179). This is likely the compound responsible for the activity observed in fractions 9 and 10 (Figure 16).

[0183] It should be noted that the HPLC fractions are primarily mixtures of compounds. In many of these fractions, especially fractions 8-12, there are significant amounts of other components, primarily flavonoids. Fractions 13 and 14 are dominated by ester compounds. The later active fraction (22) did not show any peaks by LCMS to distinguish it from fractions 20 or 22.

[0184] conclusion This study on blackcurrant juice showed that sarmentosin was present in relatively high amounts in the juice samples. Small amounts of phenolic esters of sarmentosin were also present and these were also active. Although coumaroyl and feruloyl esters were the predominant esters, caffeoyl esters were also identified in the active fractions.

[0185] Example 5: Comparison of MAO-B activity of Sarmentosin with Deprenyl Overview In this example, the MAO-B activity of sarmentosin is compared to selegiline (also known as deprenyl or L-deprenyl, sold under trade names such as Eldepryl and Emsam, a known MAO-B inhibitor used to treat Parkinson's disease and major depressive disorder), which helps to identify effective doses of sarmentosin alone or, for example, in an extract (such as juice).

[0186] result Deprenyl was purchased from Sigma-Aldrich. (R-(-)-deprenyl, this compound is also known as selegiline). Sarmentosin samples were from material purified in a previous study. Sarmentosin concentrations were confirmed using NMR with quantitative NMR standards. S9 liver microsomal fraction, containing the crude enzyme mix, was from a frozen bulk sample from porcine liver.

[0187] Samples were prepared in 6-well plates (deep-well format in 1.1 ml strip tubes). The assay was performed by diluting the crude S9 fraction with buffer (PBS, pH 7.4, diluted 1:5, 150 μl) and adding the test sample dissolved in buffer (75 ul). An aliquot of kynuramine (10 μl at 1 mg / ml) was added and the plate was placed in a 37° C. water bath for 1 hour. After cooling the plate in ice water, 20 μl of 2N NaOH was added, followed by 250 μl of acetonitrile. Individual strip tubes were centrifuged and samples were taken from each for LCMS analysis.

[0188] LCMS analysis was performed using a Cyano HPLC column eluted with 0.1% formic acid in water and acetonitrile. LC was run in isocratic mode (40% acetonitrile) and the relative amounts of 4-hydroxyquinoline (4HQ) and kynuramine were determined from the peak areas. Percent inhibition was determined by comparing the amount of 4HQ to that in a buffer only sample well. Kynuramine is converted to 4HQ by the MAO-B enzyme.

[0189] The results of this analysis are described below with reference to FIG. - 50% inhibition of sarmentosin is seen at approximately 400ng / ml, which is equivalent to a concentration of 1.45μM. -50% inhibition of deprenyl is seen at approximately 15 ng / ml, or 67 nM. This indicates that -deprenyl is a 22-fold more potent inhibitor of MAO-B than sarmentosin. -The minimum recommended starting dose of Deprenyl (selegiline) is 1.25 mg / day, increased to 2.5 mg / day after 6 weeks (see https: / / www.drugs.com / dosage / selegiline.html).

[0190] Consideration: Based on these results, a preferred dose of sarmentosin for MAO-B inhibition for cognitive disorders or prevention / improvement of mental health is at least about 27,500 μg of sarmentosin / day.

[0191] Thus, for example, for blackcurrant juice containing 320 μg / g sarmentosin, a daily intake of 86 ml of this blackcurrant juice would likely provide approximately 27,500 μg sarmentosin, which may provide a particularly favorable effective dose when used to achieve inhibition of MAO-B (i.e. equivalent to an effective dose of 1.25 mg deprenyl when applying a 22-fold conversion).

[0192] Example 6: Analysis of sarmentosin content / MAO activity in a series of blackcurrant berries, juices, and juice concentrates Overview In this example, a range of blackcurrant varieties are investigated for both MAO activity (S9 assay as described above) and sarmentosin content (LCMS analysis).

[0193] result As part of the ongoing improvements to the analytical method, the LCMS method used in the previous study was adapted. The previous method suffered from overlapping metabolites that eluted at approximately the same time as sarmentosin. This led to metabolite-metabolite interactions, resulting in suppression of the sarmentosin signal and therefore inaccurate estimation of concentration. The HPLC method was improved by using 10 mM ammonium formate in aqueous solvent (instead of 0.1% formic acid). This change caused the sarmentosin peak to elute after the food acids and sugars, resulting in more consistent results for samples and standards.

[0194] As part of this study, the sarmentocin standard was also re-examined. Previously, the standard was a sample of sarmentocin isolated from preparative HPLC and shown to be "pure" using NMR.

[0195] However, when the purity was examined using quantitative NMR, the sample was found to be less pure, and all subsequent analyses were based on this new NMR standard.

[0196] A variety of juice samples, concentrates, powders, and whole fruit were tested. Each sample was diluted with water to obtain a sarmentosin peak at approximately the appropriate concentration for LCMS analysis. From the results of Example 5, it was possible to estimate the appropriate concentration of sarmentosin required to obtain 20-80% inhibition in the S9 assay. The assay samples were diluted to this appropriate concentration by dilution with water, and assays were performed in a two-fold dilution series, using four wells for each sample. Due to limited reagent supplies, assays were performed only once for each sample.

[0197] The results are shown below in Table 4, along with the amount of sarmentosin in each sample, followed by the sample concentration (mg / g) that produced either 70 or 80% inhibition in the assay.

[0198] Ethanol extract is a powder that is an ethanol extract of berries. Whole berry sample is a water extract of whole berries. Viberi® sample is a prepackaged freeze-dried commercial sample of whole berries.

[0199] Consideration These results are consistent with sarmentosin being the effective active agent in the samples. Figure 17 is a plot of the concentration required for 70% inhibition versus sarmentosin concentration, showing a direct correlation between sarmentosin content and inhibitory activity.

[0200] Table 4: Sarmentosin and relative MAO-B inhibitory activity for a range of black currant samples. TIFF2025507640000007.tif84169

[0201] Example 7: Effects of Ingesting a Single Dose of Blackcurrant Juice or Lyophilized 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 randomized, double-blind, two-arm, placebo-controlled, crossover human intervention study to investigate 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. Findings from this study showed that: Anthocyanin bioavailability in plasma was detectable 10 min after ingestion of the blackcurrant intervention, peaked at 120 min, and decreased at subsequent time points. Greater anthocyanin bioavailability was measured in the blackcurrant powder intervention than in the juice concentrate intervention, possibly due to the relatively higher anthocyanin dose ingested by participants assigned to the powder intervention. The bioavailability profile of sarmentosin after ingestion of blackcurrant powder and juice was similar to the anthocyanin profile. Despite differences in the anthocyanin doses ingested by participants assigned to the blackcurrant powder and juice groups, there were no differences in plasma sarmentosin concentrations between the formats at any time point. Both black currant formats were similarly effective in reducing peripheral platelet MAO-B enzyme activity by -89.6% ± 2.2 and -90.5% ± 2.8, respectively. Significant enzyme inhibition was observed as early as 10 minutes after treatment ingestion in both the juice concentrate and powder formats and persisted until the final time point of the study day (480 minutes after ingestion). Inhibition of platelet MAO-B by both black currant forms corresponded with a significant reduction in circulating concentrations of the monoamine metabolites DOPAC (a metabolite of dopamine), 5-HIAA (a metabolite of serotonin), and MHPG (a metabolite of norepinephrine).A significant reduction in circulating vanillylmandelic acid (VMA) and 3,4-dihydroxyphenylglycol (DHPG) was observed after ingestion of black currant juice but not after ingestion of the powder, indicating a difference in the regulatory effect between the two black currant forms. Subjective mood data collected during the study suggest the effectiveness of both black currant forms in improving vigilance, as well as stress and mental fatigue. A better improvement was observed in the black currant powder intervention, which may be due to the higher dose taken by volunteers assigned to the powder intervention.

[0202] background Previous studies comparing the bioactivity of blackcurrant juice and blackcurrant powder suggested that the delivery format (i.e., extract, freeze-dried, juice) may affect the bioactivity of blackcurrant in supporting cognition. Despite participants receiving relatively the same polyphenol dose for both interventions, no significant MAO-B inhibition was observed following blackcurrant juice ingestion when an anthocyanin-rich extract was ingested (Watson et al. 2015). The difference in efficacy between the two food formats may be contributed to the removal or degradation of key bioactive compounds during the processing steps that produce the extract format. Therefore, food format and processing conditions are important to consider when preparing blackcurrant-based foods for nootropic effects.

[0203] Goals and Objectives Primary objective: To characterize temporal changes in platelet MAO-B enzyme activity after participants ingested a single dose of two different black currant formulations.

[0204] Secondary objectives: To characterize the temporal bioavailability of blackcurrant polyphenols, bioavailability of sarmentosin, circulating neurotransmitter concentrations, and mood parameters after participants ingest a single dose of two different blackcurrant formulations.

[0205] methodology Test beverage Two Neuroberry® Blackcurrant (BC) product formats were tested in this study: juice concentrate and freeze-dried powder. Each BC format was compared to a placebo (PL) beverage that matched as closely as possible the appearance, flavor, and texture of the corresponding BC beverage. The BC powder and matching PL powder were supplied ready-to-use by AlphaGen Limited. The BC and PL juice concentrates were blended by The New Zealand Institute for Plant and Food Research Limited (PFR) in a food-safe laboratory with ingredients supplied by AlphaGen and Sensient Technologies. The BC and placebo blends were weighed and dispensed into single servings in amber bottles, stored at -20°C, and then served to participants as 300 mL beverages.

[0206] The total amount of BC anthocyanins in the two BC beverages differed between the two formats, as previously agreed upon by AlphaGen and PFR. The anthocyanin dose in the BC powder beverage (BC-P) was standardized to each participant's body weight, so participants consumed a weight of BC powder equal to a total of 7.8 mg anthocyanins / own body weight, reconstituted with 300 mL of water. The placebo powder beverage (PL-P) was a placebo powder equivalent to the BC powder in 300 mL of water. Apple juice concentrate and clove extract were added to both the BC and PL concentrate formulations to increase flavor complexity. Citric acid was added to both concentrate formulations, but in greater amounts in the placebo concentrate beverage (PL-JC), to match the sourness of BC-JC. The flavor and color of PL-JC were matched to BC-JC using BC flavorings, Allura Red, Raspberry, and Blue Solution (Sensient Technologies) (Table 5). For PL-JC, sucrose solution (65°Brix) was used instead of BC juice concentrate.

[0207] Table 5. Composition of blackcurrant and placebo juice concentrate blends presented as single servings. TIFF2025507640000008.tif61150N / A=Not applicable

[0208] Microbial Pathogen Testing The received powder and blended juice concentrate were sent to AsureQuality Limited for microbial pathogen testing.

[0209] Beverage preparation An independent researcher prepared the test beverages. Amber bottles required for stored formulations were transferred to 4° C. the night before the study day. Powdered beverages were prepared by blending the powder with 300 mL of water in a NutriBullet®. Water was added to the juice concentrate to make 300 mL and stirred to mix. Test beverages were consumed by participants within 1 hour of preparation.

[0210] Anthocyanin quantification in beverage formulations Anthocyanin concentrations of the blended test juice concentrates and powder feeds 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. Blended juice concentrates were diluted 1 / 1 with 5 / 95 formic acid / water (v / v). Weighed powder samples were dissolved in 5 / 95 formic acid / water (v / v) to obtain aqueous solutions with concentrations of 20-21 mg / mL. Detected anthocyanins were quantified using pure standards of cyanidin 3-O-glucoside, and all results for individual and total anthocyanins are expressed as cyanidin 3-O-glucoside equivalents.

[0211] Clinical Trials Recruitment and Clinical Trial Enrollment Thirteen healthy individuals aged 26-39 years, recruited from the greater Palmerston North community, provided informed consent to participate in the study. During recruitment, potential participants completed a health screening questionnaire and were excluded if they had a chronic illness (e.g., heart disease, cancer), known blood-borne disease (e.g., hepatitis), recent viral or bacterial illness, were pregnant, were taking thrombotic or mood-affecting medications, had an intolerance to blackcurrant, or had a known strong reaction to acupuncture. All methods and procedures were reviewed and approved by New Zealand's Northern B Health and Disability Ethics Committee (2021 EXP 11576).

[0212] Sample size A power analysis from a previous blackcurrant intervention trial measuring peripheral MAO-B activity was used to calculate the number of volunteers in this study. The analysis revealed that three participants per intervention group were needed to detect a difference in MAO-B inhibition of 20% with 80% power. To account for the possibility of participants dropping out, at least six participants were recruited per intervention group.

[0213] research design The study followed a randomized, double-blind, two-arm, placebo-controlled, crossover human intervention design. Figure 19 shows a flow chart of the study procedures, and Figure 20 shows the procedure for each study day. Study participants were scheduled to complete two study days, with at least one week washout between the study days, during which participants consumed one of two intervention beverages (black currant or placebo).

[0214] At various time points after the 8-hour period, subjective sensory questionnaires and venous blood samples were collected. Participants were randomly assigned (1:1) to one of two formats: juice concentrate or powder, and then randomly assigned (1:1) to the order in which they received the intervention beverages. Participants and the study coordinator were masked as to which intervention beverage they consumed. Prior to each study day, participants removed foods and supplements high in polyphenolic compounds from their diet for 24 hours. Participants fasted for at least 10 hours, then consumed an almond-containing Vanilla One Square Meal bar® (Cookie Time Ltd) for breakfast, 2 hours after which they began the study. Participants arrived at the PFR clinical facility and made themselves comfortable here. After a 5-minute break, participants completed a visual analog scale (VAS) subjective sensory questionnaire and donated a venous blood sample. Participants were then provided with the intervention beverage and instructed to consume it as quickly as possible. A VAS mood questionnaire was completed and venous blood samples were taken 10, 20, 120, 240, and 480 min after finishing the beverage.

[0215] Besides the standardized breakfast and intervention beverage, participants had only water and a small low-polyphenol lunch (white bread with butter, mayonnaise, and poached chicken or mashed boiled egg) throughout the 480 min. After the 20 min time point, participants were allowed to leave the clinical site and return for subsequent time points if they wished.

[0216] Subjective mood questionnaire The Bond-Lader Visual Analogue Mood Scales Questionnaire allows the self-assessment of mood. In total, 16 mood traits are given: alert-sleepy, calm-excited, strong-weak, foggy-clear-minded, well-conditioned-awkward, lethargic-energetic, contented-unsatisfied, troubled-calm, poor mental functioning-attractive, tense-relaxed, alert-dreamy, incompetent-skilled, happy-sad, hostile-kind, interested-bored, withdrawn-social. Participants had to mark on a 100 mm line how appropriate each described state was for that moment. The individual responses from the 16 mood scales were combined to create three affective traits: alert, contented, and calm (Bond and Lader 1974). Participants' current feelings of stress, anxiety, and mental fatigue were measured using visual analogue mood scales for stress, anxiety, and mental fatigue, with a 100mm bar anchored at one end of the scale as "not at all true" and at the other end as "extremely true."

[0217] biochemical analysis Venous blood was collected from each participant before (0 min) and 10, 20, 120, 240, and 480 min after beverage ingestion and placed into 1 × 10 mL EDTA vacutainers for platelet MAO-B activity and blood glucose measurements and into 1 × 10 mL lithium-heparin tubes for plasma anthocyanin quantification.

[0218] Platelet MAO-B activity Platelets were isolated and prepared using previously described methods (Watson et al. 2015). Briefly, platelets were isolated from whole blood (10 mL in 10% disodium EDTA solution) by centrifugation at 600 g for 3 min at 22°C without disruption. Platelets were stored at -80°C until required. Protein concentration of platelet samples was measured using a BCA Protein Assay Kit (23225, Pierce™). MAO-B activity was measured using the Amplex® Red Monoamine Oxidase Assay Kit (A12214, Invitrogen) according to the manufacturer's instructions.

[0219] Analysis of anthocyanins and sarmentosines Plasma was collected from participants' whole blood into lithium heparin tubes by centrifugation at 4000xg for 10 min at 4° C. Plasma (1 mL) was spiked with 30 μL of 50% formic acid and 100 μL of 10 mmol ascorbic acid and then stored on dry ice at −80° C. before shipping to the Physiological Chemistry laboratory.

[0220] Plasma samples (350 μL) were spiked with malvidin 3-O-galactoside and further acidified with phosphoric acid before cleanup on SOLAμ™ solid phase extraction plates. After washing with water and acetic acid, the retained anthocyanins were eluted with methanol:formic acid (95:5) and evaporated to dryness, then reconstituted in acetonitrile:formic acid:water (5:3:92) and analyzed by liquid chromatography-mass spectrometry (LC-MS). Quantification of individual and total anthocyanins was performed using an internal standard ratio method using MultiQuant software, and all results are reported as cyanidin 3-O-glucoside equivalents. Quantification of sarmentosin was performed using an external reference standard provided by Stephen Bloor (Callaghan Institute).

[0221] Neurotransmitter analysis Overall, 111 plasma samples were analyzed, with the total number of samples available for each intervention type / intervention format / time point ranging from n=3 to n=7. Neurotransmitter analysis methodology utilizes MS probes and stable isotope-coded LCMS methods developed in-house by Plant & Food Research, optimized for plasma samples (RParkar et al. 2020; Watson et al. 2020). Neurotransmitter methodology comprehensively includes inhibitory and excitatory neurotransmitters from tyrosine, tryptophan, and glutamate metabolic pathways involved in the gut-brain axis. Briefly, 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). In the tryptophan metabolic pathway, 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) were present. Additionally, metabolites measured for glutamate metabolism were glutamic acid (GLU), α-aminobutyric acid (AABA), and γ-aminobutyric acid (GABA).

[0222] Other endogenous analytes associated with the gut-brain axis measured were glycine (GLN), serine (SER), histamine (HIS), adenosine (ADO), and cortisol (CORT). Prior to LC-MS analysis, samples were derivatized in three steps to acetylate alcohol, amine functional groups, and alkylate carboxylic acid groups. Multiple derivatization strategies were found to be necessary to acetylate the less abundant reactive alkyl hydroxyl and carboxylic acid groups. To correct for recovery, deuterated internal standards (IS) for the analytes were added at the beginning of the sample workup.

[0223] Labeled IS was prepared for each analyte by derivatizing mixed neurotransmitter standards as described for the samples, except that deuterated acetic anhydride [d6] and deuterated trifluoroethanol [d3] were used instead of unlabeled acetic anhydride and unlabeled trifluoroethanol to facilitate quantitation and correct for matrix effects during the analysis (IS-XdP). The IS-XdP standards were added to the samples after sample workup.

[0224] Briefly, acetic anhydride, sodium bicarbonate / carbonate buffer, acetonitrile, and mixed IS containing deuterated analytes were added to each plasma sample (100 μL). Samples were evaporated to dryness overnight and then derivatized with acetic anhydride and trifluoroethanol at 70° C. for 4 h and then cooled to 50° C. and allowed to stand overnight. Samples were evaporated to dryness once more and further derivatized with acetic anhydride, followed by addition of water, acetonitrile, and IS-XdP. Samples were filtered through a 96-well glass fiber plate and collected in a 96-deep well polypropylene plate before analysis by LCMS. LCMS experiments were performed on a 7500 QTrap triple quadrupole / linear ion trap (QqLIT) mass spectrometer equipped with a Turbo V™ ion source and electrospray ionization (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 multiple reaction monitoring (MRM) techniques.

[0225] blood sugar Whole blood glucose concentrations were measured using a HemoCue® 201 DM System (HemoCue®, Angelholm, Sweden) blood glucose analyzer.

[0226] statistics Chemical data presented in this report are means ± standard error of the mean. Monoamine oxidase B activity, subjective measures, and glucose data are expressed as means ± standard error of the mean. Comparisons of means between time points, treatments, and formats were performed using analysis of variance (ANOVA) from a linear mixed-effect 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. Models were fitted with the R package lmerTest, and least significant differences (LSDs) for mean comparisons were calculated subsequently using the R package predictor. Statistical significance for all indices was set at p<0.05 with a 95% confidence level.

[0227] Analysis of neurotransmitter data R version 4.2.1 was used for analysis of neurotransmitter results and data visualization. All observations were standardized as the difference relative to the first measurement at time-course (0 min). Spearman rank sum correlation matrices of standardized neurotransmitter concentrations and MAO-B inhibition were calculated for each condition using the package "PerformanceAnalytics". One-dimensional plots of variables showing significant correlation with MAO-B inhibition in the intervention format and their corresponding correlation coefficients in the placebo format were visualized using R-based functions. A multivariate approach was used using standardized median-centered and scaled neurotransmitter concentrations as variables. Principal component analysis was performed using the "factoMineR" package and variable loadings with the strongest contributors to components 1 and 2 were visualized as correlation circle plots.

[0228] For univariate statistical analysis of standardized neurotransmitters, linear mixed effects models were computed using the R package "lmerTest" to account for both fixed and random effects of multiple factors. The fitted model had fixed effect coefficients for formulation (powder vs. juice), treatment (black currant vs. placebo), and time. Random effects were participant, participant x treatment, and participant x time.

[0229] A random effect of participant x formulation was not required because each participant received only one formulation. If the ANOVA indicated there was a significant (p<0.01) treatment effect or interaction, predicted means were obtained and pairwise comparisons were performed using Fisher's least significant difference (LSD, α=0.05) and performed using the "predictmeans" package.

[0230] result Anthocyanin content of juice and powder The blackcurrant anthocyanin content of the blended test juice concentrate and powder before adding water (to make 300 mL beverage) is shown in Table 6. Based on the total anthocyanin results, the blackcurrant juice concentrate contained 2179.4 μg / g wet weight total anthocyanins and the blackcurrant powder contained 33720.4 μg / g dry weight total anthocyanins. The placebo juice concentrate and powder form contained no detectable blackcurrant anthocyanins.

[0231] Table 6: Content of blackcurrant anthocyanins in blackcurrant and placebo juice concentrates and blackcurrant and placebo powders. Juice concentrates are reported in μg / g wet weight and powders are reported in μg received / g dry weight. nd: not detected. TIFF2025507640000009.tif84170

[0232] Clinical trial participants Of the 13 recruited, 10 participants completed both study days (Figure 19).

[0233] Data from the three participants who did not complete both study days were not included in the analyses in this report. Anthropometric data for the 10 participants are shown in Table 7. Of these 10, five were assigned to the juice concentrate format and five were assigned to the powder format. Although some participants declined, the number who completed both study days was within the range needed to detect statistically significant MAO-B enzyme inhibition from intake of the blackcurrant format.

[0234] Participant withdrawal and reactions Table 7: Anthropometric characteristics of participants for the two format groups: juice concentrate and powder. Data are mean ± SEM and minimum and maximum values ​​from n=5 individuals per group 1. TIFF2025507640000010.tif45170

[0235] Of the three participants who did not complete both study days, one asked to withdraw after the first day, one withdrew after having a severe reaction to the needle, and the other withdrew after having a transient adverse gastrointestinal reaction to the intervention drink.

[0236] Total Anthocyanin Dose The absolute anthocyanin doses taken by participants in the two Neuroberry® Blackcurrant interventions were not equal (Table 8), which was due to the different dose standardization used for each format. All participants in the BC-JC group consumed 300mL of single-strength blackcurrant juice, while participants in the BC-P group consumed a standardized dose of anthocyanins (7.8mg total anthocyanins / kg body weight). As a result, participants in the PC-P group consumed approximately 7.8 times more anthocyanins per kg body weight than volunteers in the BC-JC group.

[0237] Table 8: Mean intake of blackcurrant blends and total anthocyanins per format group. Data are mean ± SEM from n=5 individuals per format group. TIFF2025507640000011.tif28170*No additional water.

[0238] Bioavailability of Anthocyanins and Sarmentosin Concentrations of total anthocyanins, cyanidin, methyl ester glucuronide, cyanidin 3-O-rutin oside, cyanidin 3-O-glucoside, delphinidin 3-O-rutin oside, and delphinidin 3-O-glucoside were measured in plasma after ingestion of blackcurrant intervention (Figure 21). Blackcurrant anthocyanins were not detected in plasma of placebo (PL) intervention (data not shown). At all time points measured after ingestion, the concentration of anthocyanins was higher in BC-P plasma than in BC-JC. The maximum total anthocyanin concentrations in plasma were 25.7±4.1 nM and 3.6±0.4 nM for BC-P and BC-JC, respectively. The peak concentration of total anthocyanins was measured at 120 min for both formats.

[0239] The bioavailability of sarmentosin in plasma after ingestion of the BC beverage is shown in FIG. 22. Similarly, for anthocyanin bioavailability, the peak concentration of sarmentosin was measured at 120 minutes. The bioavailability profile of sarmentosin is similar in both formats. However, it should be noted that the analytical methodology was not optimized for sarmentosin, and the cleanup procedure did not adequately recover sarmentosin. Thus, although the total relative concentrations of sarmentosin measured for the two BC formats can be used to inform the bioavailability profile, the absolute concentrations of sarmentosin are likely underreported. Any further analysis will require the use of stepwise cleanup procedures and separate LC-MS analysis for anthocyanins and sarmentosin.

[0240] MAO-B Inhibition and Blood Glucose Blood glucose concentrations and platelet MAO-B enzyme activity after Neuroberry® black currant and placebo interventions are shown in Figure 23. ANOVA using a mixed effects model (Table 9) revealed that format (i.e., powder or juice) had no significant effect on MAO-B enzyme activity (p=0.561). However, significant treatment (BC vs. placebo) (p=0.011), time (p<0.001), and treatment x time (p<0.001) interactions were detected.

[0241] Table 9: Analysis of variance (ANOVA) results from a mixed effects model to measure the effects of format, treatment, time, and treatment x time interaction on MAO-B enzyme activity and blood glucose after ingestion of the test beverage. Data are F values ​​and p values, with p<0.05 indicating a significant effect. TIFF2025507640000012.tif35170

[0242] MAO-B activity decreased after ingestion of all test beverages, but there was a significant decrease in MAO-B activity from baseline (0 min) 10 min after ingestion of the blackcurrant beverage (-76.1% ± 7.9 and -75.9% ± 9.7, respectively; BC-JC, BC-P). Maximum MAO-B inhibition was measured in both formats 20 min after ingestion (-89.6% ± 2.2, -90.5% ± 2.8, respectively; BC-JC, BC-P) and this level was maintained for up to 120 min. Significant inhibition of MAO-B persisted for the duration of the study, so that enzyme activity decreased significantly in both blackcurrant formats 480 min after ingestion (-35.3% ± 11.9 and -38.3% ± 11.7, respectively; BC-JC, BC-P).

[0243] Small but significant decreases in MAO-B activity from baseline were measured after ingestion of both placebo formulations, with maximum inhibition for these drinks being -8.6% ± 6.2 and -21.0 ± 6.3 for PL-JC and PL-P at 480 and 240 minutes, respectively.

[0244] Statistical analysis (Table 9) revealed a significant effect of time on blood glucose concentration (p=0.043). On average, blood glucose concentrations generally increased after ingestion of the test beverage (10 and 20 min) and remained above baseline concentrations for the remaining time points. No significant changes in blood glucose were observed after ingestion of the powdered beverages (BC-P and PL-P). In comparison, significant increases in blood glucose from baseline (0 min) concentrations were measured after ingestion of PL-JC (10 min and 20 min) and BC-JC (20 min, 120 min, and 480 min).

[0245] VAS subjective mood score Bond Radar Survey Table 10: Analysis of variance (ANOVA) results from a mixed effects model to measure the effects of format, treatment, time, and their interactions on the outcome measures related to vigilance after ingesting the test beverage. Data are F-values ​​and p-values, with p<0.05 indicating a significant effect. TIFF2025507640000013.tif23351

[0246] Table 11: Analysis of variance (ANOVA) results from a mixed effects model to measure the effect of format, treatment, time, and their interactions on outcomes related to well-being and calmness (tension / excitement, and tension / relaxation) after ingesting the test beverage. Data are F-values ​​and p-values, with p<0.05 indicating a significant effect. TIFF2025507640000014.tif23343

[0247] For the vigilance-related descriptors, a significant format × treatment × time interaction was observed for the apathy / energetic score and a significant format × treatment × time interaction was observed for the interest / boredom score. No significant effects of format, time, or treatment were observed, and these interactions were observed for the rest of the vigilance-related items.

[0248] A significant treatment × time interaction was measured and observed for the unkind / kind score.Significant format × treatment × time interactions were also measured for the satisfaction-related items happy / sad and unkind / kind.A significant format × treatment × time interaction was also observed for the satisfaction-related descriptor calm / excited score.

[0249] Descriptive items from the Bond Radar Questionnaire that were found to have significant Format × Treatment × Time interactions are plotted in Figure 24. Significantly (p<0.05) higher interest / boredom and calm / excitement scores were observed with the BC-JC compared to the PL-JC intervention at 120 and 480 minutes, respectively. No significant differences between BC-P and PL-P for these items were observed at the same time points.

[0250] Table 12: Analysis of variance (ANOVA) results from a mixed effects model to measure the effects of format, treatment, time, and their interactions on measures related to alertness, calmness, and satisfaction after ingesting the test beverage. Data are F-values ​​and p-values, with p<0.05 indicating a significant effect. TIFF2025507640000015.tif44161

[0251] When items from the Bond Radar Questionnaire were categorized to yield composite scores for vigilance, calmness, and satisfaction, significant effects of time were observed for vigilance but not for calmness and satisfaction. No significant format, treatment, treatment × time, or format × treatment × interactions were observed for these categories.

[0252] Table 13: Alertness, calmness, and satisfaction scores (Bond-Leder questionnaire) after ingestion of blackcurrant juice or powder and their corresponding placebos. Values ​​are means ± SEM. TIFF2025507640000016.tif89167* indicates significant difference from 0 min (p<0.05). ^ indicates significant difference from placebo at the corresponding time point (p<0.05).

[0253] Participants' vigilance scores increased significantly 20 minutes after taking the PL-JC intervention and then decreased, such that the vigilance scores at 480 minutes were significantly lower than the baseline (0 minutes) scores (Table 11). Vigilance scores did not change significantly from baseline (0 minutes) after taking BC-JC at any time point. For the powder format, vigilance scores were significantly higher than the baseline (0 minutes) scores 20 minutes after taking the PL-P intervention. Significant increases in vigilance were also observed 10 minutes, 20 minutes, and 120 minutes after taking BC-P. Furthermore, the vigilance scores of BC-P 120 minutes after taking were significantly higher than placebo (PL-P) at this time point.

[0254] There was a significant difference in calmness scores between the treatment groups at this time point, as the mean calmness was significantly higher 480 minutes after ingestion of the juice placebo drink (PL-JC), whereas significantly lower calmness was measured at this time point after drinking blackcurrant juice (BC-JC) (p<0.05) (Table 11). A significant increase in calmness was measured 20 minutes after ingestion of PL-P, whereas no change in calmness was measured in the BC-P intervention group. Calmness scores in PL-P were significantly greater 20 minutes after ingestion than in the BC-P intervention at this time point.

[0255] A sharp increase in satisfaction from baseline (0 min) scores was measured in the PL-JC intervention group (Table 11). This trend was also observed in the BC-JC group, which had significantly higher satisfaction at 20 and 480 min after beverage ingestion. As for the powdered forms, none of the beverages (PL-P or BC-P) produced a significant change in satisfaction scores at any of the measured time points.

[0256] Stress, anxiety, and mental fatigue Table 14: Analysis of variance (ANOVA) results from a mixed effects model to measure the effects of format, treatment, time, and their interactions on scores related to stress, anxiety, and mental fatigue after consuming the test beverage. Data are F-values ​​and p-values, with p<0.05 indicating a significant effect. TIFF2025507640000017.tif42164

[0257] No significant format, treatment, or time effects were observed on subjective measures of stress, anxiety, or mental fatigue (Table 14). However, there were significant treatment x time effects for stress (p=0.021) and mental fatigue (p=0.013). Nearly significant format x treatment x time interactions were detected for stress (p=0.051) but not for anxiety and mental fatigue.

[0258] Both forms of placebo interventions tested in this study (PL-JC and PL-P) had no significant effect on subjective stress scores. Significant reductions in stress scores were observed after ingestion of BC-JC (20 and 240 min) and after ingestion of BC-P (20, 120, and 480 min). No significant treatment differences in stress scores were observed between PL-JC and BC-JC at any of the measurement time points. In comparison, stress scores measured at baseline (0 min), 10 min, 20 min, and 240 min were significantly higher in the BC-P group than in the PL-P group.

[0259] No significant changes in anxiety scores were observed after ingestion of either powdered intervention (PL-P or BC-P). However, anxiety scores were significantly lower than baseline (0 min) at 10 and 480 min after ingestion of PL-JC and BC-JC. In the BC-P group, anxiety scores were measured at baseline (0 min), 10 and 20 min than the corresponding placebo intervention (PL-P) at these time points.

[0260] No significant change in the scores for mental fatigue was observed after ingestion of BC-JC, whereas a significant increase was observed in this parameter 240 and 480 minutes after ingestion of the placebo intervention (PL-JC). Conversely, ingestion of the placebo powder (PL-P) had no significant effect on the mental fatigue scores, whereas a significant decrease from the baseline (0 min) scores was observed 20, 120, and 240 minutes after ingestion of the BC-P intervention. In addition, the mental fatigue scores at baseline (0 min) and 480 minutes after ingestion of BC-P were significantly higher than the scores after ingestion of the placebo (PL-P) at these time points.

[0261] Table 15: Stress, anxiety, and mental fatigue responses (on a 100 mm visual analog scale (VAS) anchored at either end by "not at all true" and "extremely true") following ingestion of blackcurrant juice or powder and their corresponding placebos. Values ​​are the mean ± SEM for each parameter. TIFF2025507640000018.tif90170* indicates significant difference from 0 min (p<0.05). ^ indicates significant difference from placebo at the corresponding time point (p<0.05).

[0262] Circulating neurotransmitter concentrations Concentrations of 32 endogenous neurotransmitters and other compounds associated with the gut-brain axis were measured in the plasma of 13 participants. Of the 10 participants who completed both study days, including the black currant and placebo interventions, 5 participants (4 receiving the juice format and 1 receiving the powder format) had samples taken at each time point. All received the powder format, with 3 additional participants skipping a time point on each study day. The remaining 5 participants only completed the black currant intervention day, 2 receiving the juice format and 3 receiving the powder format.

[0263] Analysis of variance revealed significant (p<0.01) treatment x time interactions for five neurotransmitter metabolites associated with the tyrosine metabolic pathway (PHE, DOPAC, DHPG, MHPG, and VMA) and for 5-HIAA, a neurotransmitter from the tryptophan metabolic pathway (Table 16). Form x treatment x time interactions were also detected for PHE, NE, KA, and GLU.

[0264] Table 16: Analysis of variance (ANOVA) results from a mixed effects model to measure the effects of treatment and time, as well as their interactions, on circulating neurotransmitters associated with the tyrosine, tryptophan, and lutamate metabolic pathways following ingestion of the test beverage. Data are F-values ​​and p-values. Treatment x time effects significant at p<0.01 are in bold and highlighted in red. TIFF2025507640000019.tif189168Phenylethylamine, PEA;Phenylalanine, PHE;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-Hydroxy Phenylglycol, MHPG; normetanephrine, NM; metanephrine, MN; vanillylmandelic acid, VMA; 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; melatonin, MT; glutamic acid, GLU; α-aminobutyric acid, AABA; and γ-aminobutyric acid, GABA. Other endogenous analytes associated with the gut-brain axis (GBA) that were measured were glycine, GLN; serine, SER; histamine, HIS; adenosine, ADO; and cortisol, CORT.

[0265] To identify whether any modulation of plasma neurotransmitter pollution may be correlated with MAO-B inhibition, paired rank-sum correlation tests were performed. Four neurotransmitters of the tyrosine metabolic pathway, namely DOPAC, VMA, HVA, and DA, and one neurotransmitter of the tryptophan metabolic pathway, namely 5-HIAA, showed significant positive correlations with MAO-B inhibition after ingestion in the form of blackcurrant juice (Figure 25). The same correlations for placebo intervention were not significant. With the exception of DA, these neurotransmitters are metabolites derived from MAO, suggesting downstream effects of BC-induced MAO-B inhibition on circulating neurotransmitter concentrations.

[0266] Other than DOPAC, these same neurotransmitters also showed a significant positive correlation with MAO-B inhibition after ingestion of blackcurrant powder, which was not observed with placebo. In the powder intervention format only, a significant correlation with MAO-B inhibition was also observed for XA, MN, NM, and KA, which are metabolites from the tryptophan metabolic pathway. NM is a substrate for MAO-A / B, while XA, MN, and KA are not substrates or metabolites of MAO-A / B.

[0267] The temporal changes in plasma neurotransmitter concentrations from the tyrosine metabolic pathway following BC-JC and BC-P interventions, as well as their placebos, are plotted in FIG. 26. Plasma DOPAC was significantly lower 120 min after BC-JC and BC-P ingestion compared to the placebo group at the same time points (FIG. 26A-B). Plasma VMA concentrations were significantly lower 120 and 240 min after BC-JC, but not BC-P, ingestion compared to the placebo group at these time points (FIG. 26E-F). Plasma HVA and DA concentrations tended to be lower following BC-JC and BC-P ingestion compared to their corresponding placebos, but no significant treatment differences were measured for these neurotransmitters at any time point.

[0268] Blackcurrant ingestion was also found to temporally regulate plasma concentrations of 5-HIAA, a tyrosine metabolite (Figure 27). Significantly lower plasma 5-HIAA concentrations were measured as early as 10 minutes after BC-P ingestion, and these were sustained up to 240 minutes, compared to the placebo group. This almost immediate effect was not measured in the BC-JC group, where significant decreases in plasma 5-HIAA were observed at 120 and 240 minutes, compared to the placebo group. Correlation analysis revealed significant correlations of XA, MN, NM, and KA with MAO-B, for BC-P only. Further ANOVA analysis did not reveal significant time-by-treatment interactions for these neurotransmitters after BC-P ingestion at any of the measured time points (Figure 28).

[0269] Although not significantly correlated with MAO-B inhibition, univariate ANOVA analysis revealed significant time × treatment interactions for the neurotransmitters MHPG, DHPG, and PHE (Table 17). The relative changes in concentrations of these neurotransmitters after BC-JC and BC-P ingestion are plotted in Figure 29, illustrating the variability of the effects of these neurotransmitter forms. Significantly lower plasma MHPG concentrations were measured 120 minutes after BC-JC and BC-P ingestion compared to the placebo group. However, ingestion of a single dose of BC-JC, but not BC-P, significantly reduced plasma DHPG (120-240 minutes) and PHE (20-240 minutes) compared to the placebo group at these time points.

[0270] A multivariate approach was used to further explore which neurotransmitter combinations may be driving the variability across time points across both blackcurrant formats compared to placebo. The correlation circle plot (Figure 30) represents the correlation of the top 12 variables with the strongest contributors to the principal components. Figure 30A shows the main contributor of 5-HIAA driving the variability in neurotransmitter concentrations during the time-course experiment associated with blackcurrant intervention (juice and powder combination). This further suggests a strong interaction between 5-HIAA, HVA, VMA, as well as associations with DOPAC and MHPG. These associations were not observed for the placebo intervention (Figure 30B).

[0271] Consideration Although participants in the two different BC format groups (juice concentrate and powder) received different doses of anthocyanins, the study showed that consumption of one 300 mL beverage prepared from either format resulted in equal inhibition of platelet MAO-B enzyme activity. Blackcurrant anthocyanins were detectable in plasma 10 minutes after ingestion of both formats, peaked at 120 minutes, and then gradually decreased to near baseline concentrations 480 minutes after ingestion. Higher individual and total anthocyanin concentrations were measured in plasma after ingestion of the BC powder beverage than in the BC juice concentrate beverage. In contrast, no differences in the bioavailability of sarmentosin were measured between the two blackcurrant formats at any time point. Significant changes in plasma neurotransmitter concentrations were measured after ingestion of the BC juice concentrate and BC powder beverages compared to placebo. The findings also suggest that consuming blackcurrant may be effective in regulating participants' mood or other beneficial cognitive factors, at least in part through MAO enzyme inhibition via sarmentosin activity.

[0272] The concentrations of cyanidin and delphinidin derivatives measured in plasma for BC-P were approximately 7-fold higher than those for BC-JC. This difference in bioavailability is due to the different doses of anthocyanins taken by participants: on average, the dose of anthocyanins taken by participants in the BC-P group was nearly 8-fold higher than that taken by participants in the BC-JC group. Despite the difference in dose, the temporal bioavailability profiles of total and individual anthocyanins during the study days were similar for both BC-P and BC-JC, suggesting that the absorption rate of anthocyanins is similar for both formats.

[0273] Bioavailability of sarmentosin was also measured in plasma samples using standards provided by Stephen Bloor at Callaghan Innovation. Bioavailability of sarmentosin in plasma after ingestion of the two blackcurrant forms was similar and followed the same bioavailability profile as that of anthocyanins. Interestingly, the bioavailability of sarmentosin at each measured time point was similar between the forms, despite the large differences in the doses of anthocyanins ingested. It should be noted that the recovery of sarmentosin in all plasma samples was low, and the results of sarmentosin presented in this report were not corrected for recovery. Further studies are needed to refine the methodology and increase the recovery of sarmentosin in plasma for more accurate quantification of sarmentosin in plasma.

[0274] This study demonstrated a rapid, sustained, and significant decrease in peripheral platelet MAO-B activity after ingestion of a single dose of the test blackcurrant beverages (BC-JC and BC-P). The inhibition of MAO-B activity in platelets was also consistent with the bioavailability profile of anthocyanins in plasma. MAO-B activity correlated with increased bioavailability of anthocyanins in plasma, whereas a decrease in bioavailable anthocyanins was accompanied by a decrease in MAO-B inhibition 120 min after BC ingestion.

[0275] Although we were unable to eliminate the MAO-B inhibitory biological activity of anthocyanins, it is plausible that the significant platelet MAO-B inhibition observed in these clinical studies could be largely attributed to black currant components other than anthocyanins, such as sarmentosin.

[0276] Analysis of subjective data indicates the potential impact of BC beverages in mood regulation and also MAO enzyme inhibition, with increased plasma levels of sarmentosin. Ingestion of blackcurrant juice and powder variably reduced participants' stress and anxiety, participants felt less calm compared to placebo, and subjective scores of mental fatigue either maintained or significantly decreased. Greater reductions in stress, anxiety, and mental fatigue were measured in the BC-P group compared to the BC-JC group. A general increase in vigilance was measured immediately after ingestion of all beverages, which then gradually decreased over the study days.

[0277] Vigilance measured after BC-JC remained consistent across study days, whereas after PL-JC vigilance dropped below baseline values ​​at 480 min. Vigilance after BC-P remained elevated and was significantly higher than PL-P at 120 min. Taken together, these studies suggest that blackcurrant intake improved or prevented a decline in vigilance across study days.

[0278] In most mammalian tissues, including humans, MAO exists as two isoforms, namely MAO-A and MAO-B, which show regional differences in enzymatic activity, substrate specificity, and distribution in the brain and periphery (Yeung et al. 2019). 5-HT has been reported to be degraded by MAO-A, whereas MAO-B shows higher affinity for benzylamine and PEA. Catecholamines such as DA, E, NE, tryptamine, and 3-MT are substrates for both isoforms (Goldstein et al. 2021). Furthermore, plasma concentrations of DHPG and MHPG have been described as sensitive indicators of MAO-A-dependent metabolism of NE (Scheinin et al. 1991).

[0279] Initial multivariate analysis identified five neurotransmitters that showed significant correlation with MAO-B inhibition with both juice and powder format interventions, and four more that showed significant correlation with the powder format only. Three (5-HIAA, DOPAC, and VMA) showed consistent treatment x time effects that were statistically significant with the juice format only. Further analysis identified strong interactions between 5-HIAA, HVA, VMA, and associations with DOPAC and MHPG. Our findings also confirmed significant treatment x time effects for MHPG and its precursor, DHPG, although only the latter reached significance levels for the juice format. The treatment x time effect for HVA was not statistically significant (p=0.024), but the changes in concentration profile were very similar to those observed with VMA, especially with the juice intervention format. The significant changes in concentration for each of these analytes were maximal at 120 min after ingestion of the treatment, with lower plasma concentrations in the blackcurrant group compared to placebo. Notably, all of the neurotransmitters highlighted by our analysis are end products of MAO-metabolized neurotransmitters as part of the tyrosine and tryptophan metabolic pathways.

[0280] Interestingly, the decrease in circulating concentrations of 5-HIAA and MHPG / DHPG after blackcurrant ingestion suggests an inhibitory effect of blackcurrant on both MAO-A and MAO-B activity.

[0281] Although both blackcurrant juice and powder interventions inhibited platelet MAO-B activity to a similar extent, differences in the regulation of some neurotransmitters were observed between the two formats. Even when a higher dose of anthocyanins was ingested in participants in the BC-P intervention, the neurotransmitters VMA, DHPG, and PHE were significantly reduced after ingestion of BC-JC but not BC-P. It is possible that these differences could be minimized by testing the efficacy of these formats with a larger sample size. It is also plausible that the differences in these formats could be due to variations in bioactive substances other than anthocyanins between the juice and powder formats.

[0282] The small sample size of participants enrolled in each BC format is an important limitation that must be considered when interpreting the mood and neurotransmitter data. The number of participants recruited in this study was based on the detection of differences in MAO-B inhibition as the primary outcome, and mood and neurotransmitter data as secondary outcomes. To account for this limitation, a stringent approach was taken to determine whether any treatment effects or interactions were statistically significant (p<0.01) in the final analysis of neurotransmitter results. Another limitation is the uncontrolled environmental effects that may have affected participants' mood during the study day, as participants could choose to leave or enter the facility after the 20-minute collection time point.

[0283] The significant difference in blood glucose concentrations measured between BC-JC and PL-JC may be due to the difference in sugar content between the two interventions. To equalize the sugar content between the two doses, BC-JC and PL-JC were formulated with the same soluble solids content (measured as °Brix). However, it should be noted that Brix is ​​only an approximate measurement of sugar content and may be affected by other solids present in the solution. Anthocyanins are also known to control blood glucose concentrations (Kim et al. 2016), which may partially explain the lower blood glucose concentrations observed with the anthocyanin-rich BC-JC intervention compared to the PL-JC intervention.

[0284] Conclusions and future research Results showed very similar, albeit to different degrees, bioavailability profiles for the blackcurrant juice concentrate and powdered intervention.Temporal bioavailability of the specific metabolite of interest in this study, sarmentosin, also followed a very similar bioavailability profile between the two blackcurrant formats up to 480 minutes after ingestion.

[0285] The results showed that consumption of beverages prepared from blackcurrant powder and juice concentrate reduced platelet MAO-B enzyme activity to the same extent and at the same rate, despite the different formats and administration approaches used for each format. To the applicant's knowledge, this is the first study to demonstrate the effectiveness of blackcurrant powder in inhibiting platelet MAO-B to the same extent as blackcurrant juice. The MAO-B activity data combined with the bioavailability data indicate that a relatively low dose of blackcurrant anthocyanins in a beverage formulated from juice concentrate causes MAO-B inhibition equivalent to the higher doses of blackcurrant anthocyanins in a beverage formulated from blackcurrant powder.

[0286] Inhibition of platelet MAO-B activity after ingestion of a single dose of BC juice or powder was also accompanied by a transient significant decrease in the circulating monoamine neurotransmitters DOPAC, 5-HIAA, and MHPG. A format effect on monoamine neurotransmitters was also observed, with a statistically significant decrease in circulating VMA and DHPG after ingestion of BC juice, but not BC powder. The reasons behind these format effects are unclear, but may be due to the small sample size of the study or compositional differences between the two formats.

[0287] Subjective data collected during this study suggest benefits of consuming blackcurrant for reducing resting stress, anxiety, and mental fatigue, as well as improving vigilance.

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Claims

1. A composition comprising an effective amount of sarmentosin or an ester thereof for maintaining or improving a non-clinical cognitive condition or pathology associated with monoamine oxidase enzyme A (MAO-A) or monoamine oxidase enzyme B (MAO-B) enzyme activity.

2. The composition of claim 1, wherein the non-clinical cognitive condition or pathology is selected from the group consisting of mood, anxiety, social anxiety, fatigue, cognitive ability or function, motor performance, attention / vigilance, calmness, mental clarity, executive function, working memory, secondary memory, mood, stress, stress reactivity, nootropic effects, and combinations thereof.

3. The composition of claim 1, wherein the composition comprises a plant, or part of a plant material, or an extract containing sarmentosin or an ester thereof.

4. 4. The composition of claim 3, wherein the plant or plant material is blackcurrant (Ribes nigrum).

5. The composition of claim 3, wherein the composition is formulated into or is part of a food or beverage product.

6. The composition described in claim 1, wherein the effective amount of sarmentosin or its ester partially or completely inhibits monoamine oxidase enzyme A (MAO-A) and / or monoamine oxidase enzyme B (MAO-B) in a subject.

7. The composition described in claim 6, wherein the inhibition of MAO-A and / or MAO-B is reversible inhibition.

8. The composition of claim 1, wherein the ester is selected from the group consisting of niglumine-p-coumarate, caffeic acid niglumine and / or ferulic acid niglumine, and derivatives thereof.

9. 10. The composition of claim 1, wherein the use further comprises increasing the level of a neurotransmitter selected from the group consisting of dopamine, serotonin, adrenaline, and tyramine.

10. The composition of claim 1, wherein the composition contains 0.05 to 200 mg / g of sarmentosin or its ester, or at least 2.0 mg / g of sarmentosin or its ester, or at least 3.0 mg / g of sarmentosin or its ester, or at least 4.0 mg / g of sarmentosin or its ester, or at least 5.0 mg / g of sarmentosin or its ester.

11. The composition of claim 1, wherein the composition is formulated for a dose of sarmentosin or its ester of at least 1 mg per day, or at least 20 mg per day, or 1 mg to 200 mg per day.

12. A composition comprising an effective amount of sarmentosin or an ester thereof for treating or preventing a neurological or psychiatric disease or condition associated with monoamine oxidase enzyme A (MAO-A) or monoamine oxidase enzyme B (MAO-B) enzyme activity.

13. 13. The composition of claim 12, wherein the neurological or psychiatric disease or condition is selected from the group consisting of depression, atypical depression, panic disorder, social anxiety disorder, bipolar disorder, post-traumatic stress disorder (PTSD), obsessive-compulsive disorder (OCD), adult ADHD, Alzheimer's disease, dementia, Parkinson's disease, and Huntington's disease.

14. The composition of claim 12, wherein the composition comprises a plant, or part of a plant material, or an extract containing sarmentosin or an ester thereof.

15. 15. The composition of claim 14, wherein the plant or plant material is blackcurrant (Ribes nigrum).

16. The composition of claim 12, wherein the composition is formulated into or is part of a food or beverage product.

17. The composition of claim 12, wherein the effective amount of sarmentosin or its ester partially or completely inhibits monoamine oxidase enzyme A (MAO-A) and / or monoamine oxidase enzyme B (MAO-B) in a subject.

18. The composition described in claim 12, wherein the inhibition of MAO-A and / or MAO-B is reversible inhibition.

19. The composition of claim 12, wherein the ester is selected from the group consisting of niglumine-p-coumarate, niglumine caffeate and / or niglumine ferulate, and derivatives thereof.

20. 13. The composition of claim 12, wherein the use further comprises increasing the level of a neurotransmitter selected from the group consisting of dopamine, serotonin, adrenaline, and tyramine.

21. The composition of claim 12, wherein the composition contains 0.05 to 200 mg / g of sarmentosin or its ester, or at least 2.0 mg / g of sarmentosin or its ester, or at least 3.0 mg / g of sarmentosin or its ester, or at least 4.0 mg / g of sarmentosin or its ester, or at least 5.0 mg / g of sarmentosin or its ester.

22. The composition of claim 12, wherein the composition is formulated for a dose of sarmentosin or its ester of at least 1 mg per day, or at least 20 mg per day, or 1 mg to 200 mg per day.

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