Bioactives and their uses

Abstract

This invention relates broadly towards a long-term method or use of a composition for improving or supporting cognitive outcomes, mood or treating or preventing a disease or condition in a subject.

Description

[0001] Bioactives and their uses TECHNICAL FIELD This invention relates broadly towards a long-term method or use of a composition for improving or supporting cognitive outcomes, mood or treating or preventing a disease or condition in a subject. BACKGROUND ART There is a strong need for humans to maintain, optimise, or even improve, cognitive function and mood in order to meet the demands of the ever-growing expectations of society. For example, there is increased social pressure and an expectation to deliver more efficiencies in the work environment, home environment, in the education system, and so forth. As a result, there is a huge and growing consumer demand for any medicament, functional food, herbal extract or supplement that helps individuals support their cognitive function and / or mood over the longer term (i.e. more than one week) or indefinite period, which is differentiated from a product such as coffee which provides an acute, immediate transient effect after consumption for a period of a few hours. Many products like those in the energy drink sector typically rely on high levels of caffeine or similar stimulants, and / or often have many unhealthy, or potentially harmful, ingredients in order to enhance the desired stimulatory effect or provide a beneficial taste. Many people have unfortunately taken to consuming these products over a long-term period in attempts to keep alert and engaged. Unfortunately, this can lead to serious health concerns either from the unnatural perpetual state of “fight or flight”, or from metabolic conditions like insulin resistance, glucose intolerance and diabetes from the high sugar levels which are added to enhance taste. Another problem with these energy drinks is that they rely on a caffeine which can be addictive, leading to dependence, tolerance and withdraw symptoms. Similarly, alternative, healthier, products have been developed for an immediate, temporary response, including the Applicant’s own product, shown to have an immediate temporary “mental clarity” effect shortly after consumption, i.e. within a few hours. However, not all cognitive outcomes were improved over this short-term period. It would have been impossible to predict whether long-term benefits would be evident, and if so, what those benefits would be. The short-term benefits were shown to be centred around the reduction of distracting thoughts and feelings of uncertainty, but an increased level of vigor but without the feelings of jitteriness (unlike caffeinated products as it is caffeine-free). Therefore, the product has been beneficial as a replacement for the likes of coffee or other energy drinks for short-term consumption before an important exam, interview or the like. Regardless, the long-term consumption of that product and its bioactive constituents, and its specific cognitive outcomes and uses, have not previously been investigated. There has been a growing body of research focusing on how nutrient(s), various fruits and vegetables and even whole intervention diets, when taken regularly over a long-term period, may help to improve or maintain different aspects of cognitive function. However, this area of longitudinal research (and associated outcomes from long-term consumption), is fraught with difficulty and unpredictability because of any one or more of the following issues. - The cognitive outcomes of interest typically arise through a complex network of pathways including the endocrine, immune and neural pathways – each of which may or may not be influenced by the gut microbiome. Additionally, the gut-brain axis is a very new concept and one which is still poorly understood even by the most experienced researchers in the world. - Physiological systems (including the gut microbiome) can adapt to changes in diet, environment and aging, meaning unexpected changes to the intervention. - When considering a combination of different nutrients / bioactives in a composition, the complexity is even further pronounced, as the body’s response system over the long-term may adapt to each nutrient / bioactive differently. - Cognitive effects from an acute study often do not translate to the same or even similar results in a longitudinal study (and likewise short term felt effects in consumers very rarely equate to long term felt effects), potentially arising from the body’s adaptation to the intervention over time (e.g., caffeine, likely different modes of action involved, and potential side effects observed). - Often new cognitive outcomes are surprisingly observed from a long-term study that is not observed from a short-term acute intervention, even with the same or similar composition or nutritional intervention. - Longitudinal clinical studies are very expensive and often lead to poor adherence, making their utilization very challenging investigate any long-term benefits. Even when conducted, separately run clinical trials (often with very different parameters) can prove to be contradictory in their results, further adding to the ambiguity and confusion in this space. - Any long-term effects of a nutritional intervention may often only arise after a committed, sustained period of consumption, for example weeks or months. Consumers can become disheartened or impatient before seeing the beneficial results and may stop taking the nutritional intervention as a result. - Many long-term supplements have physiological side effects, albeit even if these are relatively mild. For instance, fish oil capsules can cause uncomfortable heartburn or reflex after ongoing consumption. Other such issues might include constipation, bad breath, nausea, stomach pains and so forth. Alternatively, high costs of nutritional supplements can become a financial barrier for people to consume them long-term. Both the side effects and financial burdens can be equally off-putting and can turn consumers away from taking the nutritional intervention long term. Overall, there is a need to develop new compositions or nutritional interventions that maintain or improve cognitive function when consumed over the long-term, not only in humans but also potentially non-human animals that suffer from similar cognitive or mood related concerns or disorders. It is also an ongoing concern that a significant proportion of the population have a sub-optimal diet, which can have even more pronounced implications for various aspects of overall health, including cognition and mood compared to those with an optimal diet. For example, according to the 2018-20 New Zealand Health Survey, less than a third of adults are eating enough fruit and vegetables, and there is a significant increase in obesity. This is mirrored by an international trend of increased ultra-processed foods often void of key nutritional ingredients. These two processes have been referred to as “hidden hunger”. Therefore, new compositions or bioactives that are able to improve these effects in either (preferably both) those with sub- optimal or optimal diets are seen as desirable. It is an object of the present invention to address one or more of the foregoing problems or at least to provide the public with a useful choice. All references, including any patents or patent applications cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, 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, Australia or in any other country. Throughout this specification, the word "comprise", or variations thereof such as "comprises" or "comprising", will be understood to imply 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. Further aspects and advantages of the present invention will become apparent from the ensuing description which is given by way of example only. DISCLOSURE OF THE INVENTION In a first aspect of the present invention there is provided a method of improving, supporting and / or preventing a decline of cognitive function and / or mood in a subject (for example, any one or combination of benefit(s) as shown in Table 3) in need thereof wherein the method includes long-term consumption or long-term administration of an effective amount of polyphenol(s). Suitably, compositions containing said polyphenols and uses thereof are envisaged as discussed throughout this specification. In one embodiment, the polyphenols comprise anthocyanin(s) and / or proanthocyanidin(s), or sources thereof as further discussed throughout this specification. In a further embodiment, the polyphenol(s) are combined in a composition or kit with other bioactives such as L-theanine or sources thereof. This may provide a synergy or synergistic effect from a combination of any one or more of the bioactives or components discussed herein. In a further aspect of the present invention there is provided a use of an effective amount of polyphenol(s) in the manufacture of a composition for long-term consumption or long-term administration to a subject for improvement, support and / or prevention in a decline of cognitive function and / or mood in a subject in need thereof. In a further aspect of the present invention there is provided a composition comprising an effective amount of polyphenol(s) when used for improving, supporting and / or preventing a decline of cognitive function and / or mood in a subject in need thereof wherein the use includes long-term consumption or long-term administration. In a further aspect of the present invention there is a method of improving, supporting and / or preventing a decline of cognitive function and / or mood in a subject in need thereof wherein the method includes long-term consumption or long-term administration of a composition comprising an effective amount of polyphenols and at least one or more of a) L-theanine or a source thereof and / or b) sarmentosin or a source thereof. According to a further aspect of the present invention there is provided a use of an effective amount of polyphenols and at least one or more of a) L-theanine or a source thereof, and / or b) sarmentosin or a source thereof in the manufacture of a composition for long-term consumption or long-term administration to a subject for improvement, support and / or prevention in a decline of cognitive function and / or mood in a subject in need thereof. According to a further aspect of the present invention there is provided a composition comprising an effective amount of polyphenols and at least one or more of a) L-theanine or a source thereof, and / or b) sarmentosin or a source thereof for improving, supporting and / or preventing a decline of cognitive function and / or mood in a subject in need thereof wherein the method includes long-term consumption or long-term administration. The methods, compositions or uses as described herein utilise a source of polyphenols and preferably include: a) a berryfruit extract such as a blackcurrant extract, comprising anthocyanins as polyphenols and / or b) a pine bark extract comprising proanthocyanidins as polyphenols. Optionally, L-theanine and / or sarmentosin are included in the composition, methods or uses described herein. These options and the preferred embodiments are discussed below in further detail. According to a further aspect of the present invention there is provided a method of improving, supporting and / or preventing a decline of brain derived neurotrophic factor (BDNF), Bifidobacterium and / or Coprococcus levels in a subject in need thereof wherein the method includes long-term consumption or long-term administration of an effective amount of polyphenol(s) and / or any compositions described herein containing said polyphenol(s). According to a further aspect of the present invention there is provided an effective amount of polyphenol(s) in the manufacture of a composition for long-term consumption or long-term administration to a subject for improvement, support and / or prevention in a decline of BDNF, Bifidobacterium and / or Coprococcus in a subject in need thereof. According to a further aspect of the present invention there is provided a composition comprising an effective amount of polyphenol(s) when used for improving, supporting and / or preventing a decline of BDNF, Bifidobacterium and / or Coprococcus in a subject in need thereof wherein the use includes long-term consumption or long-term administration. The inventors of the present application have previously filed a patent application (PCT / NZ2018 / 050159 - the contents of which are incorporated in their entirety through reference) towards a composition incorporating two or more of a) a berryfruit extract containing anthocyanins b) L-theanine, and c) proanthocyanidins, and its use for temporary mental clarity effects when used over the course of 1-3 days. It was never expected by the inventors that a new use towards long term cognitive function, defined herein by the effects of working memory and / or multi-tasking, over an extended period beyond one week (7 days) could be achieved. In fact, in the previous study, it was notably observed that both working memory and multi- tasking were not improved over the short term (1-3 days) in the test results. As such, this outcome was very surprising, and a unique outcome observed when the consumption / dosage regime treatment progressed beyond one week. Advantageously, both working memory and / or multi-tasking outcomes were also substantially enhanced in subjects with a sub-optimal diet compared to those with an optimal diet. This suggests that the effects are enhanced more so in those with a sub-optimal diet, which represents a significant proportion of the global population. The 4-week trial using the composition demonstrated that long term use of the composition beneficially enhanced aspects of mood of the subjects in parallel to the enhanced cognitive outcomes noted above. Preferably, enhanced mood includes at least one or more of the following features: - a reduction in anxiety / tension, - a reduction in anger / hostility; - a reduction in depression; - a reduction in fatigue; - a reduction in confusion; and / or - a reduction in total mood disturbance. These features may be informally self-rated or simply be a felt effect of the user, or a noticeable difference identified from another individual. Alternatively, they are more formally assessed by a third party, such as a medical practitioner or psychologist. In one embodiment, the manufacturer may highlight one or more of these beneficial features (or similar / corresponding wording or connotations) on the packaging or advertisement relating to the product. These specific mood outcomes over a long-term period were also not predictable given the complex nature of nootropic effects and neurological pathways over time, compared to what has been observed in acute studies. Effects from a nootropic can quickly attenuate (become “faded out”) as the body adapts to the treatment, requiring greater dosing over time to achieve the same outcome as is the case with many psychiatric medicaments like anti-depressants but also everyday substances like nicotine, alcohol and caffeine. Furthermore, predicting responses is difficult in this field due to the wide variety of study designs used (which often don’t have acute effects nested within longer-term interventions, varying intervention periods, varying cognitive testing paradigms, and so forth). In a further embodiment, the composition may enhance, improve, or restore sleep quality as well as improved cognitive and mood outcomes. As a further embodiment, the long-term consumption of the composition may have little to no side effects or adverse effects. Definitions and Preferred Embodiments For the purposes of this specific specification, the term cognitive outcome or outcomes should be taken as meaning at least one or both of an improved working memory and / or an improved multi-tasking ability. Throughout this specification the term working memory should be taken as meaning the ability to store and recall temporary or short-term ideas, information or data. In this specification, working memory was tested employing a widely used multi-tasking framework (MTF). The memory search task presented a set of four letters which disappear after four seconds. Single probe letters are then presented, and participants have 15-seconds to indicate whether the probe letter belonged to the initial set of four letters by clicking a true or false button. However, it should be understood working memory would be understood to go significantly beyond recollection of letters, and could include for example, names and places, faces, images or memories, numbers or sequences of numbers and letters and so forth. Throughout this specification the term multi-tasking should be taken as meaning the ability of a subject to engage with, deal with, process or comprehend more than one task at a time. Throughout this specification the term mood should be taken as meaning an improved total mood disturbance (TMD) or one or more of tension, anger, depression, fatigue, confusion and / or similar subjective states. The trial has shown that each of these aspects of mood beneficially decreases in test subjects (often to statistically significant levels) compared to the placebo group. Most preferably, both the aforementioned cognitive and mood benefits are improved in the subject. Any such alternative combinations of cognitive outcomes (working memory or multi- tasking) and mood (TMD, tension, anger, depression, fatigue or confusion) are envisaged within the scope of this invention. Preferably, the compositions, methods and / or uses as described herein include improving sleep quality of the subject. Throughout this specification, the term improved sleep may be defined according to the Pittsburg Sleep Quality Index (PSQI), other such recognized tests, or even a subject’s own personal thoughts, opinions or anecdotes (or those of someone observing, such as a parent, doctor, veterinarian or owner – in the case of the subject being a non-human animal). More preferably, sleep quality is improved to below a PSQI score of 5, and / or at least an improvement of at least 0.3, 0.5, 0.75, or 1.0 points on the PSQI scale in the subject. On average in test subjects, PSQI scores improved by almost 1 point on the PSQI scale and dropped to a PSQI score of 5. A PSQI score of 5 or above represent a poor-quality sleep, which was almost achieved from the test group, but not the placebo group (PSQI score of 5.6). Throughout this specification the term subject should be taken as meaning a human or other animal, such as a companion animal or pet. Preferably, the subject is a human. More, preferably, the subject is an adult human. An adult human should be understood to be a person who is 18 years of age or older. Preferably the subject is healthy, wherein the long-term consumption or administration of the composition is primarily a means to improve or support cognitive function and / or mood. Alternatively, the subject may suffer from, or at risk of developing, one or more clinical conditions strongly related to cognitive function and / or mood. Therefore, in a broad aspect of the present invention, the long-term consumption or administration may treat, improve, reduce the risk of developing, or prevent progression of one or more of the following, provided merely for example: - Alzheimer’s Disease (or Cognitive Dysfunction syndrome, or CDS – in animals like dogs or cats) - Multiple Sclerosis - Dementia - Parkinson’s Disease - Bipolar Disorder - Schizophrenia - Depression - Anxiety - Autism or Aspergers Syndrome (now commonly referred to as “Austism Spectrum Disorder”) - Attention Deficit Disorder (ADD) or Attention Deficit Hyperactivity Disorder (ADHD) - Post traumatic stress disorder (PTSD) - Anger or behavioral disorders like intermittent explosive disorder, oppositional defiant disorder. Preferably, the subject being treated has a sub-optimal diet. The preliminary data provided in the clinical study suggested that the beneficial effects were more pronounced in subjects that had a sub-optimal diet. However, benefits were still observed in the entire test group and indeed even in subjects with an optimal diet. Throughout the specification the term “sub-optimal diet” should be taken as meaning a diet of a subject that typically lacks staple macronutrients or micronutrients intakes such as vitamin E, magnesium, zinc, fiber, and / or vitamin B6, and / or wherein the subject has a reduced biochemical status of vitamin B6, folate, and / or saturated fatty acids. In the clinical study exemplified in this specification, a diet screening tool (DST) was used to group subjects into those with a sub-optimal diet with a DST equal to or below 59, or an optimal diet with a DST equal to or greater than 60. Therefore, in one embodiment the DST test may be used to help determine if a given subject would be deemed as having a sub-optimal diet or not. It should be appreciated that other methodologies may be used to assess whether a person adheres to a sub-optimal diet or not, including the subject’s own personal thoughts, opinions or anecdotes. In the embodiment of treating the subject with a sub-optimal diet, preferably gut microbiome diversity is increased. Throughout the specification the term “long-term” should be taken as meaning consumption or administration for at least one week, typically with daily consumption of the preferred dosages described in this specification. Preferably, the long-term consumption or administration is for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, or 100 weeks, or at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 years, or otherwise indefinitely. Preferably, the long-term consumption or administration is a once-daily dosage regime. Alternatively, a larger dosage may be provided on a less frequent regime, for example every two or three days, or even a weekly or monthly dosage. In such cases, currently available slow-release formulations, coatings or devices may be employed to help deliver the composition more consistently over a longer duration to the subject, yet with the benefit of less burden for the subject in terms of daily dosing. Likewise, in some cases a dosage regime may be twice or three times daily or may fluctuate between any of the above regimes as required or desired by the subject. Throughout this specification the term “effective amount” should be taken as meaning the amount of a compound that, when administered to a human or other mammal for improving, treating, preventing or delaying a state, disorder or condition, is sufficient to affect such treatment. Of course, the “effective amount” can vary based on the compound, the particular condition and its severity as well as the age, physical state, or weight of the subject to be treated. Preferred effective amounts, including dosages, are discussed further below. Throughout the specification the term “consumption” or “administration” should be taken as meaning any means to deliver the composition to the digestive tract of the subject, typically by eating or drinking the composition orally. Of course, there may be alternative routes such as nasal delivery, sublingual, or via a gastro-tube through the abdomen wall into the stomach for those that are nil-by-mouth. Throughout the specification the term “composition” should be taken as meaning any format that is suitable for consumption or administration, such as a beverage, food, nutraceutical, pill, capsule, powder, paste, gel and so forth. Most preferably, the composition is a beverage, pill or capsule. The daily dosages described throughout the specification may be used as suitable amounts (by weight) to include in a composition, such as a single pill, capsule, or drink, or could represent a recommended serving size for example in a powder format when sold in bulk. Biomarker effects Without wishing to be bound by theory, the study results suggest at least three biomarkers (or modes of action) that may be implicated or associated with the beneficial results observed either alone or in any combination. Additionally, these biomarkers are known to be associated wide reaching health benefits beyond the downstream outcomes specifically tested and observed in this study. Therefore, in a broad aspect of the present invention, the method, use or compositions described in this specification may be used to increase, modulate, improve or maintain any one or more of the biomarkers described herein, particularly with preference to those discussed below. In a preferred embodiment, the compositions, methods and uses described in this specification upregulate BDNF. Firstly, as shown in Table 3, BDNF is positively increased in the intervention arm more than that observed in the placebo group. In support of a potential mode of action, increased or improved levels of BDNF have been shown to be associated with improved cognitive performance (Nicarstri et al., 2022), improved mood (Hashimoto et al., 2004), and improved sleep (Schmitt et al., 2016). Similarly, low levels of BDNF are associated with the development of a range of neurodegenerative disorders, including but not limited to Parkinson’s Disease, Multiple Sclerosis (MS), Alzheimer’s Disease and Huntington’s Disease (HD) (Ibrahim et al., 2022). In other non-exhaustive examples, BDNF is associated with a number of neuropsychological disorders including ADHD / ADD (Liu et al., 2015). Preferably the compositions, methods, and uses described in this specification upregulate Bifidobacteria. More preferably, the Bifidobacteria is selected from the group consisting of Bifidobacterium adolescentis, Bifidobacterium longum and Bifidobacterium bifidum. As shown in Figure 7, Bifidobacteria species (in total), and in particular Bifidobacterium longum and Bifidobacterium bifidum are increased in subjects who demonstrated the best responses with letter retrieval, amongst other potential microbial changes observed. Despite the results not showing any apparent changes in overall diversity of the gut microbiome, the data suggests that Bifidobacteria may be at least partially implicated in some or all of the beneficial outcomes relating to cognition (in particular, working memory). As a further example, Bifidobacterium adolescentis was substantially increased post intervention. Thirdly, the Applicant considers the observed upregulation of Coprococcus, and in particular the subspecies of Coprococcus eutactus to be a particularly interesting finding and is seen as an advantageous and further unexpected result of the present invention. This microbial species, when depleted in the gut, is linked to neurological outcomes relating to cognition, mood and cognitive diseases, including depression, and diseases such as Parkinson’s Disease (Notting et al., 2023). Upregulation of the species is shown to significantly improve important SCFAs including butyrate and acetate with wide ranging health benefits. Therefore, in another embodiment, the use or method comprises increasing levels of at leastone short chain fatty acid (SCFA).In a preferred embodiment, the SCFA is selected from butyrate, acetate or propionate.For instance, Bifidobacterium species are known to primarily produce acetate, which serves as a precursor for butyrate synthesis by cross-feeding interactions in the gut microbiota. Similarly, Coprococcus eutactus is recognized for its butyrogenic capabilities but may also contribute tothe production of acetate or propionate under certain metabolic conditions.SCFAs are known to play a crucial role in maintaining gut health, regulating inflammation, and supporting metabolic functions. In addition to butyrate, other SCFAs such as acetate and propionate may also be increased, depending on the specific probiotic strains or bacterial genera utilised. Such increases in diverse SCFAs collectively support a balanced gutenvironment and systemic health benefits.In another embodiment, the use or method comprises increasing of one or moreneurotransmitters in a subject in need thereof.Preferably, the neurotransmitters are selected from serotonin, dopamine adrenalin(noradrenalin or norepinephrine) or tyramine.Without wishing to be bound by theory, it is thought that Bifidobacteria and Coprococcus genera are associated with the upregulation of key neurotransmitters in the brain, including serotonin, dopamine, and norepinephrine. This upregulation may occur through various mechanisms, including modulation of the gut-brain axis, immune system interactions, and metabolic byproducts such as short-chain fatty acids (SCFAs), including butyrate, acetate, and propionate. These SCFAs are known to influence neurotransmitter synthesis by modulating gut epithelial integrity, regulating inflammation, and affecting central nervous system function viavagal nerve stimulation and direct blood-brain barrier penetration.For example, butyrate has been shown to enhance serotonin production by stimulating enterochromaffin cells in the gut, which in turn may influence mood and cognitive function. Acetate and propionate can also contribute to neurotransmitter regulation by modulating microglial activity and neuroinflammatory pathways. Additionally, Bifidobacteria and Coprococcus may exert their effects by increasing the availability of neurotransmitter precursors, such as tryptophan for serotonin synthesis or tyrosine for dopamine and norepinephrine production. For instance, these mechanisms and their impact on brain healthare described in Grabska-Kobylecka, I. et al., 2023.The inventors also consider it may be possible that sarmentosin, a MAO (Monoamine Oxidase) inhibitor which has been identified by the Applicant in blackcurrant extracts (including the composition in the present study), may also be implicated in neurotransmitter upregulation. A key mechanism involves the inhibition of MAO, an enzyme responsible for breaking down serotonin and dopamine. By inhibiting MAO, polyphenol-derived metabolites may help maintain higher levels of these neurotransmitters, which may be beneficial for mood regulation, cognitive function, and overall mental well-being. This dynamic interaction between polyphenols, the gut microbiome, and neurotransmitter regulation underscores their potential therapeutic role in managing conditions like depression and anxiety. Polyphenols Throughout this specification the term “polyphenol(s)” should be taken as meaning a heterogenous group of bio-active compounds naturally synthesized in plants, which share a common phenolic structure of hydroxyl groups on an aromatic ring. The flavonoid class of polyphenols have been researched in relation to different aspects of brain health. However, despite various types of flavonoids having a potential to enhance microbiota-gut- brain axis function via multiple pathways, any ability to provide an impact on cognition and / or mood is misunderstood and not established, and any associated mode of action is also very poorly understood. This may be related to low bioavailability, where an estimated 90-95% of dietary polyphenols are not absorbed in the small intestine, reaching the colon where the gut microbiota transforms parent compounds into bioactive secondary metabolites. The two-way interplay between different polyphenols and the gut microbiota, including any potential prebiotic effects of polyphenols and biotransformation from gut microbiota, is thought to impact the ability of dietary polyphenols to produce their clinical effects or lack thereof. As a result, there have been very few human intervention studies that have attempted to determine any relationship between specific polyphenols and long term cognitive outcomes. Preferably, the method, compositions and / or uses of the present invention comprise at a dosage (typically administered daily) of least 100 mg total polyphenols. More preferably, the dosage is at least 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 400, 450, 500, 550, 600, 700, 800, 900, 1000, 1100, 1200, 1300 mg or more. Even more preferably, the total amount of polyphenols is between 100-500 mg, 200-400 mg, 150-350 mg, 175-325 mg, or about 300 mg. In the present study, a total dosage of 307 mg polyphenols was used to exemplify the efficacy of the composition in a long term consumption over 4 weeks. This example should not be seen as limiting. Given the nature of the longitudinal study, it is not feasible to conduct human clinical trials with different exemplary amounts of each bioactive – however, one skilled in the art would appreciate that alternative efficacious doses of polyphenols (and other bioactives) may be used. Preferably, the polyphenol is selected from at least one type of flavonoid. More preferably, the flavonoid is selected from the group consisting of anthocyanins or proanthocyanidins. These embodiments are discussed in more depth below. Anthocyanin(s) or a source thereof Throughout the specification the term anthocyanin should be taken as meaning any water- soluble flavonoid based on the general structure below: Anthocyanins are naturally present in a variety of fruit, vegetables, grains or legumes and are typically responsible for the red, purple, black or blue pigment. Anthocyanins are synthesized from the phenylpropanoid pathway and are derived from anthocyanidins. Preferably, the anthocyanin(s) are provided in the composition as a source (e.g. isolate or extract) containing said anthocyanin(s). In one embodiment the whole edible portion of a fruit or vegetable is used as a source of the anthocyanin(s) in the composition without first isolating the anthocyanin(s). Further, it should also be appreciated that any component of the fruit or vegetable that harbors the anthocyanin may be utilised, for example stems, shells, husks, roots, leaves, berries and so forth. Preferably, the anthocyanin(s) is selected from cyanidin and / or delphinidin. Preferably, the anthocyanin(s) selected from the group consisting of Delphinidin 3-O-glucoside, Delphinidin 3-O-rutinoside, Cyanidin 3-O-glucoside, Cyanidin 3-O-rutinoside, Petunidin 3-O- rutinoside, Pelargonidin 3-O-rutinoside, Peonidin 3-O-rutinoside and combinations thereof. These specific anthocyanins(s) have been shown to be the most prominent anthocyanin(s) present in blackcurrant concentrate and powder extracts. However, one skilled in the art would appreciate there are many other similar or alternative anthocyanin(s) in blackcurrant or its extracts or alternative fruit or vegetable sources of anthocyanins (such as blueberry or boysenberry). More preferably, the source of anthocyanin(s) is a berryfruit or berryfruit extract. The extract may be berryfruit juice or a more defined extract of the berryfruit containing for example, purified anthocyanin(s), sarmentosin or both. Even more preferably, the berryfruit is a blackcurrant, blueberry, boysenberry and combinations thereof. Blackcurrant, blueberry and boysenberry naturally all contain high levels of anthocyanins (albeit different types), however it is important to ensure the anthocyanins are substantially retained in any extract being prepared. Alternative sources of anthocyanin(s) may also be used without departing from the scope of the invention. These may be from a plurality of anthocyanin(s) sources, or the anthocyanins may be produced synthetically through known means, or via methodologies like precision fermentation in micro-organisms. In some types of berryfruit extracts already used or prepared, the anthocyanins may not be captured or isolated, or are purposefully removed, which is undesirable for the intent of the present invention. In the most preferred embodiment, blackcurrant fruit, polyphenols, or anthocyanins are extracted using aqueous ethanol. The resulting extract may then be passed through an adsorption resin, yielding a through-liquid, as illustrated in the flow diagram in Figure 9. The resin may then be subsequently washed with an acidic aqueous solution, and the resulting wash-through liquid may be collected. The through-liquid and wash-through liquid may then combined and mixed uniformly. The pH of the mixture may be verified to be approximately 3.7 or lower. The mixture may be dispensed without undergoing deacidification or concentration and may then be subsequently freeze-dried for further use. More preferably, the berryfruit or berryfruit extract is blackcurrant owing to its unique level and types of anthocyanins. Preferably, the daily dosage is at least 100 mg anthocyanin(s). More preferably, the daily dosage is about 50, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 300, 310, 320, 330, 340, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000, 1100, 1200, 1300 mg or more of anthocyanin(s). Most preferably, the daily dosage is between 50 – 700 mg, or 100 to 400 mg, or about 150 mg anthocyanin(s). In the present study, the intervention group received a daily dosage of 151 mg of anthocyanins. This should not be seen as limiting as discussed previously. Proanthocyanidins or a source thereof such as a pine bark extract Throughout the specification the term proanthocyanidins should be taken as meaning a flavonoid compound with the general structure as shown below, or natural or synthetic functional derivatives thereof. Preferably, the source of the proanthocyanidins is a pine bark extract. Pine bark extracts are very rich (often more than 65-75% w / w) in proanthocyanidins, and therefore are seen by the inventors as a good source of proanthocyanidins. However, this should not be viewed as an essential source, and many other sources could potentially be used as an alternative without departing from the scope of the invention. Without wishing to be bound by theory, the inventors believe proanthocyanidins to be a very beneficial flavonoid bioactive and may be responsible for at least some of the beneficial results observed when combined with the other components of the composition described herein. Therefore, any such source of proanthocyanidins may be utilised without departing from the scope of the invention. For instance, berries and fruits are very good sources of proanthocyanidins, including blackcurrants, blueberries, and pomegranate to name just a few. Therefore, the inclusion of pine bark extract or proanthocyanidins from pine bark should not be seen as an essential feature of the invention – although it is seen as very preferable. It is also possible other bioactives in the pine bark extract (either together or separately to proanthocyanins) may contribute or are fully responsible for the results observed. Therefore, according to the present invention, whilst proanthocyanidins are highly preferred, they are not seen as an essential form of polyphenol in the composition. Preferably, the pine bark extract is a Pinus radiata pine bark extract. However, as an alternative, one may use Pinus pinaster, or Maritime pine bark (found predominantly in the Mediterranean region) most likely with similar results. Commercially available pine bark extracts are Enzogenol®and Pycnogenol®which contain proanthocyanidins together with other condensed flavonoids. For instance, Enzogenol®has about 80% w / w proanthocyanidins. Therefore, a person skilled in the art would easily be able to determine how much pine bark extract would be required in order to achieve the desired levels of proanthocyanidin(s) based on the preferred dosages discussed below. Preferably, the composition includes at least 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 175 mg, 200 mg, 250 mg, 300, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg or 1000 mg proanthocyanidin(s) per dosage. More preferably, the composition includes a dosage between about 100 to 1000 mg proanthocyanidin per dosage and even more 100 – 400 mg per dosage. Most preferably the composition includes about 100-200 mg proanthocyanidin(s) per dosage. In the present study exemplified in this application, the total amount of proanthocyanidin(s) contributing to the total polyphenols is approximately 125-135 mg. This is on the basis that 150 mg of pine bark extract was used in the intervention drink, with 85% w / w proanthocyanidins in the extract. Sarmentosin or a source thereof In previously published patent (PCT / NZ2023 / 050027, the contents of which are incorporated entirely through reference), the Applicant identified a new use of sarmentosin as a MAO inhibitor, and its isolation and presence in blackcurrant, amongst other natural sources. Although not shown in the results, the intervention beverage (300 ml) used in the present studies of this application has been confirmed previously to contain approximately 29mg of sarmentosin. Throughout this specification the term “sarmentosin” should be taken as meaning a gamma hydroxy nitrile glycoside (chemical name: 4-(beta-D-glucopyranosyloxy)-2-(hydroxymethyl-2- butenenitrile)), or derivatives thereof as shown in the general chemical structure below: Alternatively the sarmentosin is a sarmentosin ester. Throughout the present invention the term “sarmentosin ester” or “ester thereof” should be taken as meaning nigrumin-p- coumarate, nigrumin caffeate and / or nigrumin ferulate or derivatives thereof as shown in the general structures below. These are phenolic acid derivatives of sarmentosin.

[0002] In preferred embodiment, the daily dosage is about 0.1 mg, 0.5 mg, 1.0 mg, 1.5 mg, 2.0 mg, 2.5 mg, 3.,0 mg, 4.0 mg, 5.0 mg, 6.0 mg, 7.0 mg, 8.0 mg, 9.0 mg, 10.0, 11.0, 12.0, 13.0, 14.0,15.0, 16.0, or 17.0, 18.0, 19.0, 20.0, 21.0, 22.0, 23.0, 24.0, 25.0, 30.0, 40.0, 50.0, 60.0, 70.0, 80.0, 90.0, 100.0 or 200.0 mg, 300.0 mg, 400.0 mg, or 500.0 mg sarmentosin (or its esters). A more preferred minimum dosage may be about 1-40 mg per day of sarmentosin. For instance, if a blackcurrant juice had about 0.3 mg / ml sarmentosin, a serving size of 86 ml of that juice would achieve a 25.8 mg dosage. A person skilled in the art would appreciate the concentration of sarmentosin may be suitably adjusted in a desired extract or composition using conventional methods and practices to achieve effective amounts / dosages and other criteria. Preferably, sarmentosin may be extracted or isolated from a number of sources without limitation. In the past, sarmentosin esters have been identified in blackcurrant seeds (Lu et al., 2002), and sarmentosin has been identified in Kalanchoe species (Fernandes et al, 2021). Preferably sarmentosin is extracted or isolated from blackcurrant fruit. The extract may also include other components from the source (such as blackcurrant) as this may provide additional benefits from bioactives or micronutrients, including the anthocyanin(s). The inventors foresee that in an alternative use embodiment, a whole fruit or plant material may be used to provide a beneficial MAO inhibitory effect from sarmentosin or its esters, so long as the material indeed has this beneficial bioactive which is the subject of this patent application. L-theanine or a source thereof Throughout the specification, the term L-theanine should be understood to mean a compound generally as shown below: Potential sources of L-theanine include but are not limited to a green tea extract (Camellia sinensis, or other species of Camellia) or the edible mushroom Xerocomus badius. Notably, the invention does not cover D-theanine. Synthetically derived L-theanine may also be used, or even L-theanine derivatives thereof, if shown to also provide the same functionality as described herein. Preferably, the composition includes a daily dosage of least 50 mg L-theanine. More preferably, the composition includes a dosage of between about 100-500 mg L-theanine. Most preferably, the composition includes about 200 mg L-theanine. The inclusion of L-theanine is seen as highly beneficial but not essential towards the long term benefits. Part of this reasoning is because it is understood that L-theanine peaks in plasma 50 minutes after ingestion and is largely cleared from plasma within 24 hours. However, the inventors cannot discount that the L-theanine may be having a misunderstood effect within the body, perhaps exerting a synergistic effect with one or more of the other polyphenols or preferred components. Further testing may be conducted. BRIEF DESCRIPTION OF THE FIGURES Further aspects of the present invention will become apparent from the ensuing description which is given by way of example only and with reference to the accompanying Figures. Figure 1 Study design schematic Figure 2 Detailed analysis of trial steps and adherence Figure 3 Screen layout of the Purple Multi-Tasking Framework (MTF) Figure 4 Participant adherence Figure 5 MTF – Letter Retrieval, Tension / Anxiety, Anger / Hostility and TMD results (from Table 3) Figure 6 Diet Group Effects Figure 7 Gut microbiome analysis Figure 8 Bray-Curtis Dissimilarity (Pre vs post intervention in optimal vs sub-optimal diet groups) Figure 9 Schematic representation of anthocyanin rich blackcurrant extraction process

[0003] DETAILED DESCRIPTION The following clinical study was carried out by the Medical and Health Sciences Department at the University of Auckland. Study design The clinical study is a randomised, double-blind, placebo-controlled cross-over trial. Participants received both the intervention (referred to here as “test composition / drink) and a placebo control (taste- and colour-matched beverage). The sequence of intervention was a randomised with an equal allocation ratio, and both investigators and participants were blinded to the identify of treatments for the duration of intervention and analysis of primary outcomes. The intervention was three months in duration, with four weeks on each intervention arm, and a four-week wash-out period between arms. Participants were recruited from the Auckland area via print and social media advertising. Clinical data was collected at the University of Auckland Clinical Research Centre in Grafton, Auckland. The trial protocol has been developed in accordance with the Standard Protocol Items: Recommendations for Intervention Trials (SPIRIT) statement. The overall study is outlined Figures 1 and 2. Eligibility Criteria Key Inclusion criteria - Females aged between 18-45 years. - BMI between 18-30 kg / m2. - Not pregnant, nor intending to become pregnant during the trial. Key Exclusion criteria - Underlying medical conditions. - Medication, prebiotic / probiotic supplements, herbal extracts or antibiotics for at least 4 weeks prior to (and during) the intervention. - Self-reported alcohol intake exceeding a moderate intake (>15 standard drinks / week). - Regular use of recreational / illicit drugs. Rational for subject selection Sex hormones play an important role in the neurobiological basis of stress-related disorders, and gender differences in behavioral responses to intervention can be observed. There may also be a high level of inter-individual variation in the gut microbiota response to intervention. With complex outcomes of interest, only one gender was recruited to minimise heterogeneity in this research results. However, the results of this study in general would be expected to be applicable to the wider population, not just one gender. Also, given that epidemiological evidence has demonstrated that there may be a higher prevalence of anxiety-related disorders and functional gastrointestinal disorders (FGDs) in females compared to males, females were the population of interest in this study. Differences in gut microbiota composition across different age groups are well described, and young to middle-aged adults (18-45) was the population of interest to reduce the heterogeneity that comes with age-related gut microbiota changes. With the objective to examine the role of baseline diet and gut microbiota composition in mediating the effects of supplementation on potential outcomes, this population was balanced for ‘optimal’ and ‘suboptimal’ diets at baseline to ensure even distribution of diet quality. As it was not suitable to recruit according to baseline gut microbiota composition, diet quality served as a proxy given the close relationship between diet quality and gut microbiota composition. A modified version of the Healthy Eating Index was used at screening, showing the strongest association with gut microbiota composition. The tool was modified to provide an index of polyphenol intake using the Phenol-Explorer database, and to be compatible with the New Zealand dietary context. Intervention Subjects received the following interventions for four weeks: - Intervention composition drink (300mL) - Control composition drink (300mL). The control drink was matched for taste, colour, and macronutrient composition to the test drink. Each intervention was separated by a four-week wash-out period. Table 1. Ingredients and nutritional composition of the intervention and placebo drink Note: The Blackcurrant juice and blackcurrant extract also have about 22.7 mg non- anthocyanin polyphenols. Hence, the total polyphenols add up to approximately 307 mg. Both the intervention and control products were prepared and packaged with identical packaging except for printed batch codes and expiry dates. Batch codes and expiry dates were concealed as detailed in the blinding procedures below. Participants were directed to keep the beverages in their fridge or a cool, dark place at home to minimize polyphenol degradation, and asked to consume their 300 mL beverage daily at a similar time of day for the 4-week period. Participants were asked to maintain their normal diet and lifestyle habits for the duration of the study. Intervention compliance was assessed by weekly online questionnaires, as the return of bottles was considered impractical. Compliance questionnaires included the number of beverages the participant had consumed that week, the reason for not consuming all seven allocated beverages (if applicable), time of day the beverages were consumed, and if they had made any changes to their diet or lifestyle over the last week. Compliance to the treatment was recorded as a cumulative score (% of beverages consumed out of the total 28 provided), with minimum compliance requirements defined a priori as 80%. Trial Procedure Participants were required to visit the Clinical Research Centre (University of Auckland, New Zealand) on five separate occasions as shown in Figure 1. An enrolment / familiarization visit took place approximately two weeks before the first testing visit. Following written informed consent, participants provided health and demographic data, and then completed the 2-minute built-in familiarization session of the MTF to minimize learning effects. Participants were given a three-day food record to complete before their first testing visit, and also given a stool sample collection kit to collect a fecal sample in the 24 hours before their first testing visit. Participants were randomly allocated to a treatment order at the end of the enrolment / familiarization visit. Participants were asked to abstain from caffeine for 12 hours and alcohol for 24 hours before each testing visit. Participants attended their testing visits between 7am – 10am following an overnight fast, with the time kept consistent across the four visits for each participant. Participants completed stress reactivity and cognitive measures before providing a fasted blood sample. In-person contact time was kept minimal due to ongoing Covid-19 requirements at the time this study was conducted, and participants were instructed to complete online versions of the sleep and mood questionnaires within 24 hours (see Figure 1). Participants were provided with their 28 beverages at the end of visits 1 and 3, and were provided with another stool collection kit at the end of visits 1-3. Dietary assessment Dietary screening tool (DST) The Australian modification of the DST was used, requiring only minimal adaptation to the New Zealand context (e.g., updating the name of popular fast-food restaurants). The DST is comprised of 20 items, with 18 items related to the frequency of consumption of particular foods (e.g., “How often do you usually eat wholegrain breads or crackers?”), and two items related to the number of servings consumed (e.g., “How many different vegetable servings do you usually have at your main meal of the day?”). The DST has a maximum score of 104, with higher scores indicating better diet quality. Cut-off scores of ≤59 (sub-optimal), and ≥60 (optimal) was utilized, which have been previously validated in middle-aged adults (25), and shown to discriminate nutrient intake, blood nutrient status, mood, and cognition in middle aged adults. The intent of a priori dietary screening was to capture a wider variety of dietary intakes than typically achieved with opportunity sampling in a healthy population, countering the issue of self-selection bias. Food records Participants completed a three-day food record before their first testing visit. Instructions on how to complete the food record with as much detail as possible were provided by a Registered Dietitian at the enrolment / familiarization study visit, along with a standardized template to use. All records were checked for accuracy and completeness by a Registered Dietitian, with any concerns resolved in person. One student dietitian entered the food records into FoodWorks software (Version 10, Xyris, Australia), with 10% of the records randomly cross-checked by another student dietitian. The average daily macronutrient and micronutrient composition of participants’ diets were calculated from the nutrient composition data primarily from the New Zealand FOOD files 2016 database, or the AusBrands 2019 and AusFoods 2019 databases if a suitable item was not available in the New Zealand database. Energy over- and under-reporting was checked from the 3-day food records, and all participants had plausible energy intakes (range 5281 kJ / d – 14180 kJ / d). Nutrient intake was compared with age- and sex- specific Estimated Average Requirements (EAR) or Adequate Intakes (AI, if EAR was unavailable) according to the joint Australia and New Zealand Nutrient Reference Values. Outcomes Primary endpoint - Change in stress reactivity to a multi-tasking cognitive stressor (purple-MTF) from pre to post intervention and between intervention arms, including acute changes to subjective stress, mood, and anxiety. Secondary endpoints - Change in cognitive performance from pre to post intervention and between the intervention arms, including psychomotor function, attention, working memory, and an aggregate multi-tasking score (purple-MTF). - Change in subjective mood (POMS, WHO-5 wellbeing index) from pre to post intervention and between intervention arms. - Change in subjective sleep quality (PSQI) from pre to post intervention and between intervention arms. - Change in fecal gut microbiota markers (composition, diversity, and predictive function) from pre to post intervention and between intervention arms. - Change in serum brain-derived neurotrophic factor (BDNF) from pre to post intervention and between intervention arms - Change in serum tryptophan metabolites from pre to post intervention and between intervention arms - Change in circulating inflammatory markers from pre to post intervention and between intervention arms - Adverse events Sample size A sample size of 35 subjects per group is estimated to be adequate for a between-group difference of 20% to identify significant differences at a level of 5%. A sample size of 40 will allow for a drop-out rate not exceeding 15%. Neurocognitive responses Cognitive stressor: Multi-Tasking Framework The Purple-MTF allowed for concomitant assessment of psychomotor, memory, and attentional performances. Cognitive performance was assessed in the present study by scores on the four individual tasks and the aggregate score, which reflected the ability to multi-task. The Purple-MTF (Purple Research Solutions, Northumbria, UK) was used in this study to concurrently elicit cognitive stress through a period of multi-tasking and measure cognitive performance. The purple-MTF battery has been shown to increase subjective and physiological measures of stress, and is unique amongst lab stressors as it can repeated on several occasions with limited learned effects and suitability for use in cross-over trials.

[0004] During the task, four concurrent cognitive and psychomotor tasks are presented in quadrants of a split screen as per Figure 3 and as discussed in Table 2 below. A 20-minute version of the platform was used, with ‘Mental Arithmetic’, ‘Stroop Colour-Word’, ‘Memory Search’ and ‘Visual Monitoring’ tasks included (See Figure 3). Mathematical processing required participants to complete addition of the numbers on screen, entering their response using the number pad displayed. The Stroop task presented colour words (“blue”, “yellow”, “red”, or “green”) in text which were displayed in one of the four corresponding colours, and participants indicated the font colour by clicking on the correct colour displayed in the panel within 20-seconds. The memory search task presented a set of four letters which disappear after four seconds. Single probe letters were then presented, and participants had 15-seconds to indicate whether the probe letter belonged to the initial set of four letters by clicking a true or false button. The visual monitoring task presented a red dot which moves outwards through a series of concentric circles. Participants needed to press a “reset” button before the red dot moves beyond the outermost circle. Participants were instructed to simultaneously attend to the four tasks, whilst also monitoring the central counter which displayed their aggregate score. The score was determined by accuracy and speed of responses across the four tasks. Throughout the battery, research staff were positioned within participant’s peripheral vision to seemingly monitor performance and increased performance anxiety. Assessment of stress reactivity to a cognitive stressor: Stress, mood, and anxiety Stress reactivity was assessed through questionnaires immediately before and after completing the Purple-MTF cognitive stressor. Stress reactivity was comprised of stress, mood, and anxiety measures, including Visual Analogue Mood Scales (VAMS), and State-Trait Anxiety Inventory: State Subscale (STAI-S). Visual Analogue Mood Scales Bond-Lader Visual Analogue Mood Scales The Bond-Lader VAMS included 16 visual analogue items with antonyms at either end separated by a 100mm line, for example “alert” and “drowsy”. Scores were combined to form three mood dimensions, including ‘alert’, ‘calm’, and ‘contented’. For each item, participants marked on the line how they feel at that time. The distance from the negative antonym will be measured in millimeters, and the average score from the combined scales will be used to compute a score for the three mood dimensions. The VAMS have been used extensively in research on psychopharmacological interventions, and changes following completion of the Purple-MTF have been reported. Stress and Fatigue Visual Analogue Mood Scales An additional two measures of subjective stress and fatigue were included in the same form of VAMS. Each item of stress and fatigue had the words “extremely” and “not at all” at either end of a 100mm line. As for the Bond-Lader scales, items were scored as the distance from the low end. State-Trait Anxiety Inventory: State Subscale The STAI-S is a widely used tool which measures anxiety in the present emotional state, and shows sensitivity across a range of experimental paradigms. The STAI-S is comprised of 20 items, each scored on a 4-point Likert scale (1=not at all, 4=very much so). The STAI-S instructs participants to respond with the statement which describes their present feelings best. Scores were calculated on a scale from 20-80, with higher scores reflecting higher present-state anxiety. Mood Profile of Mood States (POMS) The POMS questionnaire consists of 65 mood-related adjectives. On a 5-point Likert scale (0 = Not at all, 4 = Extremely), participants indicated the degree to which they identified with each of the 65 items over the past week. The 65 items were summed into six mood dimensions; tension / anxiety, confusion / bewilderment, anger / hostility, depression / dejection, fatigue / inertia, and vigor / activity. A total mood disturbance score was calculated as the sum of the first five dimensions minus the vigor / activity score). WHO-5 wellbeing index The WHO-5 wellbeing index consists of 5 positive statements. On a 6-point Likert scale (0 = At no time, 5 = All of the time), participants indicated the degree to which they have identified with each statement over the past week. A total score was calculated as the sum of the five items, ranging from 0-25 with 25 representing the best possible quality of life. Sleep quality – Pittsburgh Sleep Quality Index The PSQI questionnaires consisted of 19 items and assessed sleep quality during the previous month. These items were grouped into seven component scores, each weighed on a 0-3 scale. Components included sleep quality, sleep latency, sleep duration, habitual sleep efficiency, sleep disturbance, use of sleeping medications, and daytime dysfunction. The component score was then summed to calculate a global PSQI score ranging from 0-21, with higher scores indicating worse sleep quality. Faecal microbiota Stool samples were self-collected by participants 24 hours before their study visits. Participants were instructed to store samples within their home freezer and transport them to their study visit with the provided ice packs. Upon receipt, samples were thawed and aliquoted, combining 100 mg of stool in 900 µl of DNA / RNA Shield (Zymo Research, R1100) by vortex. Samples were subsequently stored at -80°C until DNA extraction was performed at the completion of the study. In total, DNA was extracted from 156 study samples, 3 ZymoBIOMICS Gut Microbiome Standards (Zymo Research, D6331), and 3 extraction blanks using the standard protocol for ZymoBIOMICS 96 MagBead DNA Kit (Zymo esearch, D4308). Mechanical bead beating was performed on a 1600 MiniG (SPEX SamplePrep), shaking at full speed (1500 rpm) for 5 minutes. Extract purity was assessed by spectrophotometry. DNA concentration was measured using the Qubit dsDNA Broad Range Assay Kit (Invitrogen, Q33266). DNA extracts were sent to a commercial provider (Novogene, China) for shotgun metagenomic sequencing on an Illumina NovaSeq6000 platform, generating an average sequencing depth of 21.8 ± 1.4 million paired-end reads / sample (range 17.4 – 29.4 million). Metagenomic sequencing data were processed by KneadData (version 0.10.0) to trim and remove poor quality reads and those that mapped to the human genome (hg19). Taxonomic profiling was subsequently performed with MetaPhlAn3 (version 3.1). Functional profiling was performed with HUMAnN3 (version 3.6) with resulting gene families and pathway abundance tables renormalized to copies per million (CPM). Downstream analysis and plot generation were conducted in R statistical software (v 4.2.1). The vegan package was used to calculate α-diversity (Shannon index) and β-diversity (Bray- Curtis dissimilarity) based on species-level composition. Ordinations were performed by non- metric multidimensional scaling and differences in microbiome structure between intervention arms were assessed by PERMANOVA. The Maaslin2 package was used to test for differentially abundant species, genera, and pathways utilizing both general linear models and linear mixed effects modeling designs. All models included a log transformation, minimum prevalence was set to 0.1, and the default significance threshold following FDR correction was used (q < 0.25). To identify microbiome features that changed after consumption of the active beverage, models were fitted with fixed effects for intervention group (0 = placebo, 1 = active), time (0 = baseline, 1 = 4-weeks), and their interaction (1 = active & 4-weeks, 0 = all other combinations), and subject ID was added as a random effect. Inflammatory markers Circulating IL-6 was measured by ELISA assay according to manufacturer’s instructions. Brain derived neurotrophic factor Circulating BDNF was measured by ELISA assay (Biosensis, USA) according to manufacturer’s instructions. Tryptophan metabolites Serum tryptophan and kynurenine were measured by ELISA assay (ImmuSmol, Talence, France) according to manufacturer’s instructions. Physical activity Usual physical activity was assessed using the IPAQ-SF. The IPAQ-SF is comprised of seven items, providing information on the time spent walking, in vigorous- and moderate-intensity activity, and in sedentary activity in the last seven days. Adverse events Adverse events were collected by observing and interviewing the subject during the study, and for up to 30d following the study (for serious adverse events). Psychological assessments were checked weekly by the research team. Statistical analyses Statistical analysis was conducted on an intention to treat basis, including all participants who received at least one treatment dose and satisfied the inclusion criteria at enrolment into the study. Statistical analyses were analysed using R 4.1.2 by a researcher blinded to group allocation. Before analysis, multiple imputations of missing data was conducted using chained equations, and distribution of continuous variables was graphically assessed for normality and outliers to ensure their appropriateness. Linear mixed effect models were used to examine changes in stress reactivity, cognitive, mood, sleep, and blood measures from baseline to follow-up within and between intervention groups. The models included time (baseline, 4-weeks), intervention group (active, placebo), their interaction (time x intervention group) and sequence allocation as fixed factors, and subject ID as a random factor to account for repeated measures. Significant interaction effects were followed by multiple pairwise comparisons with Tukey’s adjustment. Associations between microbiome features and clinical parameters were restricted to those clinical parameters shown to improve in the analyses above. This included analyses to identify differences in baseline microbiome features of clinical ‘responders’, using maaslin2’s linear mixed effect models fitted with a binary response category (0 = non-responder, 1 = responder). Responders were defined as those participants with changes to clinical scores in the top quartile. Safety considerations This study was conducted in accordance with International Conference on Harmonisation (ICH) Guidelines for Good Clinical Practice (GCP). Results Adherence As shown in Figure 4, there was excellent adherence to the intervention overall, with all participants meeting the minimum requirements of 80%. Additionally, no side / adverse effects were observed or reported during the study. The clinical parameter results are summarised in Table 3 below. Abbreviations: BDNF, brain derived neurotrophic factor; LMM, linear mixed effect model; PSQI, Pittsburgh sleep quality index; STAI-S, State trait anxiety inventory – Stress subscale; VAMS, Visual analogue mood scale; VAS, Visual analogue scale. Estimated marginal means and p-values are derived from linear mixed effect models. MTF – Cognitive Results As shown in Figure 5a, letter retrieval (working memory) improved with 4-weeks of daily intervention consumption (with similar findings in sensitivity analysis). As shown in Table 3, total MTF (multi-tasking) scores were also improved more so in the intervention compared to the placebo. Secondary analyses – Diet Group Effects on Cognitive Results As shown in Figure 6 (data not shown in Table 3), the beneficial effects of the intervention drink on letter search (working memory) and total MTF (multi-tasking) were stronger in those with a sub-optimal diet compared to those with an optimal diet, although beneficial results were observed in both groups, and in total. Sleep quality (PSQI) As shown in Table 3, sleep quality scores improved by almost 1 point on the PSQI scale after 4 weeks of daily consumption of the intervention drink. The PSQI score also dropped to 5 in the intervention (active) group, but not in the placebo (control) group. A PSQI score of 5 or greater indicates a gradient of poorer sleep quality. Mood (POMS) As shown in Figure 5 (and in more depth in Table 3), post-hoc analyses of significant time x intervention group interactions revealed significant improvements following 4-weeks of the active intervention for A) Letter retrieval scores: Δ = 1026 point increase, p<0.001; B but not placebo (p>0.05); B) Tension / anxiety: Δ = 1.7 point reduction, p=0.023; C) Anger / hostility: Δ = 1.9 point reduction, p=0.013; D) Total mood disturbance: Δ = 8.9 point reduction, p=0.013, though note a near-significant interaction effect for this domain (p=0.052). Therefore, it can be concluded that the anger, tension, fatigue, depression, confusion domains as well as Total Mood Disturbance (TMD) all improved in the active (intervention) group after 4 weeks and to a greater degree compared to the placebo (control) group. Blood markers As shown in Table 3, no statistically significant changes to blood markers (BDNF, tryptophan, kynurenine, tryptophan / kynurenine) were seen in either the active (intervention) or placebo (control) groups, although there were a wide variety of individual responses. Regardless, there was still a notable improvement in BDNF in the test group compared to the placebo group. This was seen as a beneficial effect. Without wishing to be bound by theory, this may be a potential mechanism contributing to the cognitive and / or mood outcomes observed. Gut microbiome - Richness Figure 7 (A) shows the Shannon diversity index based on species composition at baseline and 4-weeks after supplementation with active and placebo beverages. Figure 7 (B) shows Bray- Curtis dissimilarity in species composition between participant’s baseline and 4-week sample for both intervention arms. Figure (C) shows non-metric multi-dimensional scaling (MDS) plot showing that variability in species composition was largely driven by subject rather than by intervention. Therefore, from these results, no statistical difference in microbiome richness metrics were observed in either group. Furthermore, there were no noticeable shifts in overall species composition post-intervention, and there was also no apparent shifts in abundance of specific species (not shown) in either group. However, looking deeper at the species and genera (see Tables 4a-b below), the inventors did identify some potentially beneficial effects in terms of upregulation of some microbes, and downregulation of other microbes, following the study with the intervention drink. Any one or combination of these are envisaged as a potential beneficial result and may be linked to one or more of the advantageous effects observed in the study and as described herein. Table 4a: Species (those shaded in grey have greater significance (p value < 0.05), but other selected results with less significance are shown below). __ _ _ _ _ __ _ _ Table 4b: (those shaded in grey have greater statistical significance (p value < 0.05), but other selected results with lower statistical significance are shown below). __ _ In relation to Tables 4a-b: o “Feature” – refers to the name of the species or genera. o “Metadata” – refers to what is being compared, namely “post-active” refers to comparing post intervention (i.e. post-active) data compared to pre intervention (i.e. pre-active) data. o “Coefficient” and “standard error” – refers to the model coefficient or contrast between two categories, in this case between post-active and pre-active data. The actual value itself is not used in interpretation. A positive coefficient = increase in level of the species or genera, whereas a negative coefficient = decrease in level of species or genera. Therefore, a positive coefficient is seen as the relative level of upregulation of that species / genera after the intervention compared to before the intervention, and vice versa. o “N” = refers to the total number of samples used in the model. This model included pre / post data for both active / placebo, but has been specifically set for this output summary to compare pre / post for the active. o “N.not.0” = refers to the number of samples in which the feature is non-zero. Not everyone possesses each species. o P value – nominal significance. Needs to be <0.050 to be considered nominally significant. Additionally, and turning now to Figures 7 (D-F), relative abundance of the genus Bifidobacterium (D) and species Bifidobacterium longum (E) and Bifidobacterium bifidum (F) was higher at baseline in the top quartile of participants whose letter retrieval scores improved (i.e. the “Top responders”). Therefore, participants with the greatest cognitive responses had a greater abundance of Bifidobacteria species at baseline which may implicate the gut microbiome, and perhaps specifically Bifidobacteria species in mediating some or all of the benefits observed in the present application. Additionally, as shown in Figure 8, a considerable subset of participants in the sub-optimal group showed a greater shift in microbiome composition with the active (intervention) drink, suggesting greater microbiome diversity changes in those with a sub-optimal diet. This is seen as a very advantageous and beneficial result and may be linked to the improved responses in this group vs the optimal diet (see Figure 6). Discussion The study surprisingly showed that long term consumption of the intervention composition had long term benefits towards multi-tasking and working memory, as well as a number of mood characteristics including a reduction of anger, tension, fatigue, depression, confusion and TMD. The beneficial improvements in cognitive function and mood do not appear to associated with any widespread gut microbiome alterations such as improved diversity (although it may play a role for those with a sub-optimal diet where increased diversity was observed in the intervention group). Yet, the study supported that Bifidobacterium and / or Coprococcus levels are improved in the gut microbiome after intervention, and these are seen as very positive and unexpected outcomes for reasons previously discussed. Yet, to the best of the Applicant’s knowledge, this is also the first human intervention trial which has shown differences in baseline gut microbiome composition in cognitive ‘responders’. Specifically, the inventors found that the clinical responder group (in the top quartile of response) for letter recall scores had nominally higher concentrations of Bifidobacterium spp., at baseline, including B. longum and B. bifidum. These findings may implicate a role for Bifidobacterium spp., in moderating how participants responded to the polyphenol rich intervention. Other than BDNF, there were no other differences in the blood markers which could help to elucidate how the surprising cognitive or mood benefits were seen over the long-term consumption period. The results reconfirm the very difficult and complex nature of predicting cognitive, mood, and sleep outcomes from a given nutritional intervention, especially the outcomes relating to long term consumption and effectiveness. It was also unexpected that the beneficial effects did not taper off or reduce over the trial period and supports that long term consumption effects may continue longer term, and potentially even improve over time in a compounding fashion. More-so, it was unexpected to have such a high adherence during the trial, which supports there were no negative side effects with long term consumption. This is an important outcome, as many cognitive supplements or therapeutic products do have poor adherence due to side effects.

Claims

What we claim is:

1. A method of improving, supporting and / or preventing a decline of cognitive function and / or mood in a subject in need thereof wherein the method includes long-term consumption or long-term administration of an effective amount of polyphenol(s).

2. A use of an effective amount of polyphenol(s) in the manufacture of a composition for long- term consumption or long-term administration to a subject for improvement, support and / or prevention in a decline of cognitive function and / or mood in a subject in need thereof.

3. A composition comprising an effective amount of polyphenol(s) when used for improving, supporting and / or preventing a decline of cognitive function and / or mood in a subject in need thereof wherein the use includes long-term consumption or long-term administration.

4. The method of claim 1, the use of claim 2, or the composition of claim 3, wherein the polyphenol is a flavonoid, and preferably wherein the flavonoid is selected from either anthocyanin(s) and / or proanthocyanidin(s).

5. The method of claim 1, the use of claim 2, or the composition of claim 3, wherein the method, use or composition also comprises at least one of: a. L-theanine or a source thereof, and b. sarmentosin or a source thereof.

6. The method, use or composition of any one of the above claims wherein at least two of the following are used: a. a berryfruit or berryfruit extract, comprising anthocyanin(s) and / or sarmentosin; b. a pine bark extract comprising proanthocyanidin(s); c. L-theanine or a source thereof.

7. The method, use or composition of any one of the above claims wherein the cognitive function comprises working memory and / or multi-tasking.

8. The method, use or composition of any one of the above claims wherein mood is selected from the group consisting of total mood disturbance (TMD), tension, anger, confusion, fatigue, depression and combinations thereof.

9. The method, use or composition of any one of the above claims wherein sleep quality is improved in the subject.

10. The method, use or composition as claimed in claim 8 wherein the sleep quality is improvedbelow a PSQI score of at 5 or below and / or an improvement of at least 0.3, 0.5, 0.75 or 1.0 points on the PSQI scale in the subject.

11. The method, use or composition of any one of the above claims wherein levels of BDNF are increased in the subject.

12. The method, use or composition of any one of the above claims wherein levels of Bifidobacteria, preferably Bifidobacterium longum and / or Bifidobacterium bifidum, and / or Coprococcus, preferably Coprococcus eutactus are increased in the subject.

13. The method, use or composition of any one of the above claims wherein the subject is healthy.

14. The method, use or composition of any one of the above claims wherein the subject has a sub-optimal diet.

15. The method, use or composition of claim 13 wherein the sub-optimal diet is defined equal to or below 59 on the diet screening tool (DST).

16. The method, use or composition of any one of the above claims wherein the long-term consumption or administration of the composition is for at least 1 to 100 weeks.

17. The method, use or composition of any one of the above claims for the treatment or prevention of one or more clinical conditions related to cognitive function and / or mood selected from the group consisting of Alzheimer’s disease, Multiple Sclerosis, cognitive dysfunction syndrome (in companion pets), dementia, Parkinson’s Disease, bipolar disorder, schizophrenia, depression, anxiety, autism or Aspergers syndrome, attention deficit disorder (ADD) or attention deficit hyperactivity disorder (ADHD), post traumatic stress disorder (PTSD), anger or behavioral disorders, and combinations thereof.

18. The method, use or composition of any one of claims 4-17 wherein the daily dosage of polyphenol(s) is at least 100 mg.

19. The method, use or composition of any one of claims 4-17 wherein the daily dosage of anthocyanin(s) is at least 100 mg.

20. The method, use or composition of any one of claims 4-17 wherein the daily dosage of proanthocyanidin(s) is at least 50 mg.

21. The method, use or composition of any one of claims 6-19 wherein the daily dosage of sarmentosin is at least 1 mg.

22. The method, use or composition of any one of claim 5-20 wherein the daily dosage of L-theanine is at least 50 mg.

23. A method of improving, supporting and / or preventing a decline of BDNF, Bifidobacterium and / or Coprococcus levels in a subject in need thereof wherein the method includes long- term consumption or long-term administration of an effective amount of polyphenol(s).

24. A use of an effective amount of polyphenol(s) in the manufacture of a composition for long- term consumption or long-term administration to a subject for improvement, support and / or prevention in a decline of BDNF, Bifidobacterium and / or Coprococcus in a subject in need thereof.

25. A composition comprising an effective amount of polyphenol(s) when used for improving, supporting and / or preventing a decline of BDNF, Bifidobacterium and / or Coprococcus in a subject in need thereof wherein the use includes long-term consumption or long-term administration.

Images