METHODS FOR IMPROVING CEREBROVASCULAR FUNCTION AND COGNITION IN PERI- AND POST MENOPAUSAL WOMEN

Field of the Art

[0001] The present disclosure relates generally to methods for improving cerebrovascular function and cognitive performance, and for preventing or reducing cognitive impairment or decline, in peri-menopausal and post-menopausal human subjects. The present disclosure further relates to methods for improving mood and reducing pain or pain perception in peri- menopausal and post-menopausal human subjects. The methods of the present disclosure comprise the administration of resveratrol.

Background

[0002] Menopause increases the risk of premature cognitive decline. This effect of menopause on cognition is reflected in global incidences of dementia which indicate the prevalence of dementia in women is 14% to 32% higher than that of men for those over 65 years old. Women account for 63% of dementia sufferers over 80 years old worldwide.

[0003] Severity of cognitive impairment is associated with poor cerebrovascular function, in particular the inability of cerebral vessels to dilate effectively in response to dynamic conditions. The present inventors have previously shown that cerebral artery elasticity and adequate cerebral perfusion during cognitive activities are predictive of cognitive performance in post-menopausal women; hence, maintaining cerebrovasuclar function may be key for attenuating an estrogen-related deficit in cognition in this population (Wong et al. Translational Research & Clinical Interventions, 2016, 2: 162).

[0004] There is an ongoing need in the art for means of addressing the cognitive decline associated with menopause, as well as for improving the sense of well being and for reducing pain or pain perception in post- and peri-menopausal women. Summary of the Disclosure

[0005] A first aspect of the present disclosure provides a method for improving cerebrovascular function in a peri-menopausal or post-menopausal human subject, comprising administering to the subject an effective amount of resveratrol.

[0006] The improvement in cerebrovascular function is typically observed relative to the absence of administration of the resveratrol.

[0007] The improvement in cerebrovascular function may comprise, for example, an improvement in cerebral blood flow velocity (CBFV) and/or an improvement in cerebrovascular responsiveness (CVR).

[0008] The improvement in cerebrovascular function may be associated with improvements in cognition and memory, or with the prevention or inhibition of cognitive and memory decline peri- and post-menopause.

[0009] A second aspect of the present disclosure provides a method for improving cognitive performance or cognition in a peri-menopausal or post-menopausal human subject, comprising administering to the subject an effective amount of resveratrol.

[0010] The improvement in cognitive performance or cognition is typically observed relative to the absence of administration of the resveratrol.

[0011] The improvement in cognitive performance or cognition may be associated with improvements in cerebrovascular function.

[0012] The improvement in cognitive performance or cognition may comprise improvements in memory, and executive functions. The improvements in memory may comprise improvements in verbal memory and semantic memory.

[0013] A third aspect of the present disclosure provides a method for preventing, inhibiting or reducing cognitive or memory decline in a peri-menopausal or post-menopausal human subject, comprising administering to the subject an effective amount of resveratrol.

[0014] The prevention, inhibition or reduction of cognitive or memory decline is typically observed relative to the absence of administration of the resveratrol.

[0015] The prevention, inhibition or reduction of cognitive or memory decline may be associated with improvements in cerebrovascular function. [0016] A fourth aspect of the present disclosure provides a method for improving mood in a peri-menopausal or post-menopausal human subject, comprising administering to the subject an effective amount of resveratrol.

[0017] The improvement in mood is typically observed relative to the absence of administration of the resveratrol.

[0018] The improvement in mood may comprise reducing tension and/or anxiety in the subject.

[0019] A fifth aspect of the present disclosure provides a method for treating tension or anxiety in a peri-menopausal or post-menopausal human subject, comprising administering to the subject an effective amount of resveratrol.

[0020] A sixth aspect of the present disclosure provides a method for reducing pain or pain perception in a peri-menopausal or post-menopausal human subject, comprising administering to the subject an effective amount of resveratrol.

[0021] A seventh aspect of the present disclosure provides a method for reducing pain or pain perception in a subject with osteoarthritis, comprising administering to the subject an effective amount of resveratrol.

[0022] The reduction in pain perception is typically observed relative to the absence of administration of the resveratrol.

[0023] Further aspects of the present disclosure provide the use of resveratrol for the manufacture of a medicament or supplement for improving cerebrovascular function, improving cognitive performance or cognition, preventing, inhibiting or reducing cognitive or memory decline, improving mood, treating tension or anxiety, or reducing pain or pain perception, typically in peri-menopausal or post-menopausal human subjects.

[0024] In accordance with the above aspects and embodiments, the resveratrol may be cis- resveratrol or irans-resveratrol. In an exemplary embodiment, the resveratrol is trans- resveratrol.

[0025] In accordance with the above aspects and embodiments, the resveratrol may be administered in a form suitable for oral administration. The composition may be a solid or liquid composition. The composition may be in unit dosage form. In one embodiment, the unit dosage form is a tablet, capsule, or caplet. The composition may be in the form of a beverage or food supplement. [0026] The following disclosure relates to all of the above aspects and embodiments.

Brief Description of the Drawings

[0027] Embodiments of the present disclosure are described herein, by way of non- limiting examples only, with reference to the following figures:

[0028] Figure 1. Treatment differences in group cognitive scores and overall cognitive performance. Pre-post intervention differences in cognitive performances were calculated for each group using z-scores. Significant differences between resveratrol and placebo: Verbal memory domain ^a (P = 0.037; Cohen's d = 0.47) and Overall performance ^b (P = 0.023; Cohen's d = 0.69) after adjusting for depressive symptoms (CES-D).

[0029] Figure 2. Cerebrovascular responsiveness to hypercapnia in the MCA. TCD ultrasound was used to measure blood flow velocity in the MCA in order to calculate treatment changes after 14 week in CVR to hypercapnia. Significant differences between resveratrol and placebo (P = 0.011; cohen 's d = 0.69).

[0030] Figure 3. Changes in mood scores in placebo and resveratrol treatment groups. *, P < 0.033.

[0031] Figure 4. Changes (week 14 - week 0; mean + SEM) in pain intensity scores for each pain descriptor in the McGill Pain Questionnaire in the placebo and resveratrol groups.

[0032] Figure 5. Correlation between changes (week 14 - week 0) in (A) overall pain, (B) quality of life (QoL) and (C) total well-being and the change in cerebrovascular responsiveness (CVR) to hypercapnia. MCA, middle cerebral artery.

[0033] Figure 6. Pain perception as determined using a Short-Form McGill Pain Questionnaire. P values compared to placebo after adjusting for covariates: * significant after adjusting for multiple comparisons; ** significantly correlated with CVR to hypercapnia (r=- 0.404) and cognitive (r=-0.459) stimuli.

Detailed Description

[0034] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, typical methods and materials are described.

[0035] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article, unless the context clearly dictates otherwise. By way of example, "a strain" can mean one strain or more than one strain.

[0036] In the context of this specification, the term "about," is understood to refer to a range of numbers that a person of skill in the art would consider equivalent to the recited value in the context of achieving the same function or result.

[0037] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0038] Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a phytoestrogen (a stilbenoid) present in the skin of a range of plants including red grapes, blueberries and peanuts, and produced by the plants as part of their natural defence mechanism against pathogens or injury.

[0039] As described and exemplified herein the present inventors have demonstrated that chronic resveratrol supplementation provides significant beneficial effects on overall cognition as well as in specific memory domains and neurovascular coupling capacity in menopausal women. As exemplified herein, significant improvements were observed in cerebrovascular function, cognition, mood and pain perception.

[0040] Accordingly, one aspect of the present disclosure provides a method for improving cerebrovascular function in a peri-menopausal or post-menopausal human subject, comprising administering to the subject an effective amount of resveratrol.

[0041] Another aspect of the present disclosure provides a method for improving cognitive performance or cognition in a peri-menopausal or post-menopausal human subject, comprising administering to the subject an effective amount of resveratrol.

[0042] Another aspect of the present disclosure provides a method for preventing, inhibiting or reducing cognitive or memory decline in a peri-menopausal or post-menopausal human subject, comprising administering to the subject an effective amount of resveratrol. [0043] Another aspect of the present disclosure provides a method for improving mood in a peri-menopausal or post-menopausal human subject, comprising administering to the subject an effective amount of resveratrol.

[0044] Another aspect of the present disclosure provides a method for treating tension or anxiety in a peri-menopausal or post-menopausal human subject, comprising administering to the subject an effective amount of resveratrol.

[0045] Another aspect of the present disclosure provides a method for reducing pain perception in a peri-menopausal or post-menopausal human subject or a subject having osteoarthritis, comprising administering to the subject an effective amount of resveratrol.

[0046] Without being bound by theory, the present inventors hypothesise that resveratrol may elicit its benefits on cognition and mood through its ability to modulate cerebral perfusion during times of demand. Further, again without being bound by theory, the inventors hypothesise that the beneficial effects observed herein for resveratrol on cognition and cerebrovascular function may be due to actions of the resveratrol, either directly or indirectly, on estrogen receptors in the brain. Accordingly, the present disclosure also provides methods for modulating estrogen receptor activity in the brain, wherein the estrogen receptors are exposed to an effective amount of resveratrol. The estrogen receptors may be a- or β- receptors.

[0047] In the context of this specification, the terms "improving" and "improvement" are understood to mean that resveratrol is administered to a subject, or a method is used, for a period of time effective to improve one or more parameters or performance measures such as cerebrovascular function, cognition, memory or mood typically as determined by comparison with the same one or more parameters or performance measures in the subject absent the administration of the composition or method. Such "improvement" may comprise normalization of the parameter or performance measure, wherein normalization means restoring the parameter or performance measure to (or towards) that expected, or observed in individuals, in the absence of menopausal symptoms. Any suitable method(s) of assessing parameters and performance measures can be used to determine whether an improvement occurs, as will be readily appreciated by those skilled in the art. Improvement may be qualitative or quantitative. If quantitative, the improvement may be at least or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%. [0048] In the context of this specification, the term "cognitive performance" is understood to mean the ability or capacity of a subject in carrying out a task that involves or requires cognition, such as for example thinking, reasoning, understanding, problem solving, memory and/or decision making.

[0049] As used herein the terms "treating", "treatment", "preventing" and "prevention" refer to any and all uses which remedy a condition or symptom, prevent the establishment of a condition or symptom, otherwise prevent, hinder, retard, or reverse the progression of a condition or symptom in any way whatsoever, or improve a condition or symptom. Thus the terms "treating" and "preventing" and the like are to be considered in their broadest context. For example, treatment does not necessarily imply that the subject is treated until total recovery. In conditions which display or are characterized by multiple symptoms, the treatment or prevention need not necessarily remedy, prevent, hinder, retard, or reverse all of said symptoms, but may prevent, hinder, retard, or reverse one or more of said symptoms.

[0050] As used herein, the term "effective amount" refers to an amount of resveratrol that is sufficient to effect one or more beneficial or desired outcomes. An "effective amount" can be provided in one or more administrations. The exact amount required will vary depending on factors such as the subject being treated, the age and general health of the subject, and the form in which the composition is administered. Thus, it is not possible to specify an exact "effective amount". However, for any given case, an appropriate "effective amount" may be determined by one of ordinary skill in the art using only routine experimentation.

[0051] Embodiments of the present disclosure contemplate the use of a suitable functionally active form of resveratrol. This may comprise czs-resveratrol, zrans-resveratrol, a mixture of cis- and zrans-resveratrol, or pharmaceutically acceptable salts or derivatives thereof. Those skilled in the art will appreciate that the scope of the present disclosure is not limited by reference to any one form of resveratrol.

[0052] In order to achieve the beneficial effects the subject of the present disclosure, the resveratrol may be administered in a daily dose of, for example, between about 10 mg and about 200 mg, or between about 20 mg and about 180 mg, or between about 40 mg and about 160 mg, or between about 60 mg and about 150 mg. In an exemplary embodiment the daily dose of resveratrol is about 150 mg.

[0053] Individual doses of the resveratrol may comprise, for example, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, or about 200 mg resveratrol. In an exemplary embodiment individual doses comprise about 75 mg resveratrol.

[0054] The resveratrol may be administered as a single dose or alternatively as multiple doses sequentially, for example daily or weekly. In an exemplary embodiment, the resveratrol is administered in an amount of about 150 mg per day, in two doses of 75 mg resveratrol, one in the morning and one in the evening. The resveratrol may be administered on a daily basis for a period of at least about one week, at least about two weeks, at least about three weeks, at least about four weeks, at least about five weeks, at least about six weeks, at least about seven weeks, at least about eight weeks, at least about nine weeks, at least about 10 weeks, at least about 11 weeks, at least about 12 weeks, at least about 13 weeks, at least about 14 weeks, or more. The resveratrol may be administered on a daily basis for a period of at least about one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, 10 months, 11 months, 12 months or more. The administration of the resveratrol may be continued for as long as required by the subject, up to for example one, two, three, four, five or more years.

[0055] Those skilled in the art will appreciate that single or multiple administrations of resveratrol can be carried out with dose levels and dosing regimes being determined as required depending on the circumstances and on the subject to be administered the resveratrol. The skilled addressee can readily determine suitable dosage regimes. A broad range of doses may be applicable. Dosage regimens may be adjusted to provide the optimum response. Those skilled in the art will appreciate that the exact amounts and rates of administration will depend on a number of factors such as the particular composition being administered, the age, body weight, general health, of the subject, as well as any drugs or agents used in combination or coincidental with methods of the present disclosure. Based on the teaching herein those skilled in the art will, by routine trial and experimentation, be capable of determining suitable dosage regimes on a case-by-case basis. The exemplary daily dose of resveratrol used in the studies exemplified herein has been shown by the inventors to be safe and without side effects.

[0056] Administration of the resveratrol may be initiated before, during or after menopause and may be continued for the course of the menopausal transition and post menopause. [0057] Resveratrol may be administered in accordance with the present disclosure in the form of a composition comprising one or more pharmaceutically acceptable carriers, excipients or diluents. Examples of pharmaceutically acceptable carriers, diluents and excipients include but are not limited to: demineralised or distilled water, saline solution, vegetable-based oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil and sesame oil, volatile silicones, mineral oils, cellulose derivatives such as methyl cellulose, ethyl cellulose, carboxymethylcellulose, sodium carboxymethylcellulose or hydroxypropylmethylcellulose, fatty acid esters, polyvinylpyrrolidone, carrageenan and gums. The carriers, diluents and excipients may form from 5% to 99.9% by weight of the composition.

[0058] Compositions may further comprise any suitable additives, carriers, additional therapeutic agents, bioavailability enhancers, side-effect suppressing components, diluents, buffers, flavouring agents, binders, preservatives or other ingredients that are not detrimental to the efficacy of the composition. Typically, the compositions are administered via the oral route.

[0059] Compositions suitable for oral administration may be presented as discrete units (i.e. dosage forms) such as gelatine or HPMC capsules, caplets or tablets, each containing a predetermined amount of each component of the composition as a powder, granules, as a solution or a suspension in an aqueous liquid or a non-aqueous liquid. In exemplary embodiments disclosed herein, the resveratrol is provided in capsule form, and a daily dose comprises one or two capsules.

[0060] When the composition is formulated as capsules, the components of the composition may be formulated with one or more pharmaceutically acceptable carriers such as starch, lactose, microcrystalline cellulose and/or silicon dioxide. Additional ingredients may include lubricants such as magnesium stearate and/or calcium stearate. The capsules may optionally be coated, for example, with a film coating or an enteric coating and/or may be formulated so as to provide slow or controlled release of the composition therein.

[0061] Tablets may be prepared by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the components of the composition in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant (for example magnesium stearate or calcium stearate), inert diluent or a surface active/dispersing agent. Moulded tablets may be made by moulding a mixture of the powdered composition moistened with an inert liquid diluent, in a suitable machine. The tablets may optionally be coated, for example, with a film coating or an enteric coating and/or may be formulated so as to provide slow or controlled release of the composition therein.

[0062] The compositions may be provided to the user in a powder form. For oral administration, the composition may then be mixed with a suitable volume of an aqueous medium, typically with agitation, to dissolve the components, or produce a suspension, suitable for ingestion. Thus, the compositions may be provided to a user in a powder form, which powder may then be added by the user to any type of aqueous medium (for example water or fruit juice) and consumed there after. Alternatively, the composition may be provided as a beverage, pre-mixed with an aqueous medium such as water. In another embodiment the compositions may be added in powder form by the user to any type to a food product (for example, yoghurt) and consumed there after. In another embodiment, the compositions may simply be consumed as a powder in the absence of a drink or additional food product.

[0063] Methods of the present disclosure may be employed as an adjunct to or otherwise in combination with other therapies or treatments for menopause and menopausal symptoms. Such combination treatments may involve simultaneous administration of additional active agents in the same formulation as the resveratrol or in a different formulation via the same or different routes, or sequential administration by the same or different routes. By "sequential" administration is meant a time difference of from seconds, minutes, hours or days between the administration of the agents, compositions or treatments. Sequential administration may be in any order.

[0064] The present disclosure may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification, individually or collectively, and any or all combinations of any two or more said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which this disclosure relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

[0065] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates. Examples

[0066] The following examples are illustrative of the methods disclosed herein and should not be construed as limiting in any way the general nature of the disclosure of the description throughout this specification.

Example 1 - Human study design

[0067] A 14 week randomised, double-blind, placebo-controlled (parallel comparison) dietary intervention trial was designed to evaluate the effects of resveratrol supplementation (75mg twice daily) on cognitive abilities. Assessments were carried out at baseline (week 0) and at the end of the intervention phase (week 14). The trial was conducted at the Clinical Nutrition Research Centre, University of Newcastle, in New South Wales, Australia in accordance with the Principles of Good Clinical Practice as outlined by the International Conference on Harmonisation. The protocol was approved by the University of Newcastle's Human Research Ethics Committee and registered with the ANZCTR (ANZCTR12615000291583).

[0068] Study population

[0069] To give 80% chance of detecting a statistically significant difference (P<0.05) of medium effect size {Cohen 's d= 0.67) in cognitive performance (primary outcome) between resveratrol and placebo treatments, 74 complete data sets were needed. To allow for attrition, we recruited 80 women.

[0070] Community-dwelling women residing in the Hunter region of New South Wales responded to invitations to participate via an approved media campaign. Respondents completed a health and lifestyle questionnaire to determine their potential suitability. Eligible participants were 45 - 85 years old, post-menopausal (self-reported cessation of menses for more than 6 months) and willing to maintain a stable medication regime and exercise habits. Participants were excluded if they were smokers or used nicotine, insulin, warfarin or any form of hormone replacement therapy within the last 6 months, had suspected dementia or a clinical diagnosis of depression, a history of breast or cervical cancer or had any other serious condition such as cardiovascular disease, kidney or liver disease, neurological problems as well as any other factor deemed by the investigators as likely to confound the validity of the study. Written informed consent was obtained prior to any assessments.

[0071] Investigational product and allocation

[0072] The resveratrol and placebo capsules were of identical appearance and supplied by DSM Nutritional Products Ltd, Switzerland. The active capsule comprised of 75mg of 99% pure synthetic trans-resveratrol (ResVida™). The placebo comprised several inert excipients (calcium hydrogen phosphate, microcrystalline cellulose, prosolv 50 and hydrated magnesium silicate). Capsules were identifiable only by code numbers; an independent investigator who held the code allocated participants to each treatment group based on the minimisation method using years since ceasing menses (Altman et al., Br Med J, 2005, 330:843). The first participant was allocated to a group randomly. Blinding was maintained until all data analysis had been completed.

[0073] Screening and baseline assessments

[0074] Detailed protocols of each outcome assessment were as described in Evans et al. (Nutrients, 2016, 8, 150).

[0075] Potentially eligible volunteers attended the screening/baseline visit (week 0) having refrained for at least an hour from medication, food or beverages other than water. Height, weight and waist circumference were measured followed by seated blood pressure (BP) readings to determine BP eligibility. Participants undertook the Australian version of the Modified Mini-Mental State Examination (3MS) to exclude those with suspected dementia (score of <78/100) (Bravo and Hebert, International Journal of Geriatric Psychiatry, 1991, 12, 1008-1018).

[0076] Those eligible were then fitted with a transcranial Doppler (TCD) ultrasound headpiece to assess basal cerebral haemodynamics and cerebrovascular responsiveness (CVR) to hypercapnia. CVR is a measure of the ability to increase cerebral blood flow in response to a physiological or mental demand. Measurements were performed bilaterally in the middle cerebral artery (MCA). The hypercapnic challenge required participants to inhale a carbogen gas mixture (5% C02, 95% 02) for 180s. The headpiece then remained in place for the neuropsychological test battery. The test battery consisted of four validated tests, namely The Rey Auditory Verbal Learning Test (RAVLT) (Schmidt and Rey, Auditory and Verbal Learning Test: A handbook. Western Psychological Services. Los Angeles, CA. 1996), the Cambridge Semantic Memory Battery (Adlam et al., Neurocase, 2010, 16, 193-207), the Double Span Task (Lovato et al., Perceptual and Motor Skills, 2013, 116, 368-381) and the Trail Making Task (TMT) (Sanchez-Cubillo et al., Journal of the International Neuropsychological Society, 2009, 15, 438-450). Cognitive tasks were performed in the same order for both resveratrol and placebo groups and additionally between visits to negate order of effect variability.

[0077] At the conclusion of the cognitive test battery, participants then completed two paper-based questionnaires pertaining to their mood, the Profile of Mood States (POMS) and the Centre for Epidemiological Studies Depression scale (CES-D).

[0078] Intervention

[0079] Participants were instructed to take two capsules daily, one in the morning and one in the evening, and record each intake in the assigned supplement diary in order to assist with treatment compliance. Participants were encouraged to maintain their habitual diet throughout the intervention; however, any changes in dietary supplement and/or medication intake during the intervention were to be recorded in the supplement diary.

[0080] A follow-up phone call was made at mid-intervention to enquire about the participants' welfare such as well-being, alterations to their habitual diet, physical activity or medication use. This call also served to encourage compliance. At the end of the trial, all remaining capsules were counted and tallied with the corresponding diary records to calculate overall compliance.

[0081] Participants returned at the end of the 14-week intervention, fasted for at least one- hour and instructed to refrain from consuming their supplement on the day of their visit; Participants underwent further TCD measurements of CVR to hypercapnia and cognitive stimuli, while performances on the test battery were scored against baseline results in order to assess changes in cognitive performance. Mood questionnaires were then completed to conclude the visit.

[0082] Statistical analysis

[0083] Using an intention to treat analysis, treatment by time effects were determined by analysis of covariance (ANCOVA) using SPSS version 21.0 (SPSS by IBM inc. Chicago, IL, USA) with significance for the primary outcome set at P<0.05. The primary outcome was the effect of resveratrol supplementation versus placebo on overall cognitive performance, determined by summing the Z-scores for each cognitive test; Z-scores at the end of the 14- week intervention were derived from the cohort's mean scores obtained at week 0 (baseline). Where cognitive tests had more than one scoring profile, they were each converted to Z- scores and averaged. Secondary outcomes were treatment changes in haemodynamic parameters (basal mean cerebral blood flow velocity - basal mean cerebral blood flow velocity (CBFV), pulsatility index (PI) and CVR to both hypercapnic and cognitive stimuli) as well as changes in mood. Correlational analysis was then performed between primary and secondary outcomes; the treatment change in CES-D scores was used as a covariate in ANCOVA analysis and further linear correlations involving cognitive performance as they significantly (r=-0.272; P=0.022) interacted. Effect sizes were calculated using Cohen 's d. Bonferroni post-hoc analysis of false discovery rates was used to adjust for multiple comparisons; the resultant significance for secondary outcomes was set at P<0.025.

[0084] Participant disposition

[0085] Eighty participants were recruited (eight withdrawals - 72 participants completing both the baseline and final 14 week intervention assessments. As all eighty participants completed their baseline visit, they were used in determining treatment differences according to our analysis method. One participant was identified as a statistical outlier (> 3 standard deviations from the mean for the majority of outcome measures); from the resveratrol group which was associated with a major life event and her data was excluded from the final analysis. Thus 38 participants in the resveratrol group and 41 in the placebo group were included in group-wise comparisons. Compliance with capsule consumption was 92% for both groups. All withdrawals were unrelated to the study; this coupled with the fact that no adverse events were reported in the resveratrol group and that there was a lower withdrawal rate than those on placebo indicates the resveratrol dose was safe and tolerable.

[0086] Participant baseline characteristics are shown in Table 1. Participants were normotensive though slightly overweight (25<BMI<30). Post-menopausal duration averaged 11.56 + 1.02 years; the range was broad (38years) but normally distributed. Both 3MS and self -reported cognitive impairment scores were similar between groups indicating no cognition bias after randomisation. There were no significant differences in other baseline measures between those randomised initially to the resveratrol or placebo treatment arms. Table 1. Participant baseline characteristics.

Participants characteristics mean ± standard error of the mean (SEM)

Age (years) 61.5 d b 0.9

Years since cessation of menses 11.6 d b 1.0

Years of formal education 15.5 d b 0.5

3MS score (%) 96.0 d b 0.4

BMI (kg/m ² ) 26.7 d b 0.6

Waist circumference (cm) 87.0 d b 1.4

Systolic blood pressure (mmHg) 125.3 d t 2.1

Diastolic blood pressure (mmHg) 70.9 d b 1.4

Basal mean blood flow velocity (cm/s) 50.1 d b 2.0

Pulsatility index 0.8 ή : 0.1

Example 2 - Effect of trans-resveratrol on cognitive performance, cerebrovascular function and mood

[0087] Participants in the study described in Example 1 were assessed for effects of 14 week irans-resveratrol supplementation (75 mg twice daily) on aspects of cognitive performance, cerebrovascular function and mood.

[0088] Cognitive performance

[0089] Raw scores (presented as a percentage) and treatment differences for individual cognitive test and their memory domains are presented in Table 2; when analysing z-scores, the resveratrol group scored better than the placebo group in all individual cognitive tasks (Figure 1). Significant improvements were observed in overall cognitive performance (P = 0.003; Cohen 's d = 0.69), in addition to the domains of semantic (P = 0.043; Cohen's d = 0.48) and verbal memory (P = 0.043; Cohen's d = 0.48). However, after controlling for depressive symptoms analysis, only the domain of verbal memory (P = 0.037) and overall cognitive performance (P = 0.023) remained significantly improved by resveratrol (Figure 1). Table 2. Participants' raw performance data (percentage) for each cognitive test and its component. Data are presented as mean + SEM

Week 0 (VI) Week 14 (V2) Δ (V2 -VI) Placebo vs. Resveratrol

Memory domain Placebo Resveratrol Placebo Resveratrol Placebo Resveratrol P-value P-value

Cognitive task (unadjusted) (CES-D covariate)

Components (n=41) (n=38) (n=41) (n=38) (n=41) (n=38)

Verbal Memory 50.7 + 1.2 51.3 + 1.1 53.2+1.0 56.5 + 1.1 2.5 + 0.8 5.2 + 0.9 0.024* 0.021*

RAVLT immediate 52.0 + 0.8 52.3 + 0.9 53.9 + 0.7 55.5 + 0.8 1.8 + 0.6 3.2 + 0.7 0.136 0.135

Learning 66.3 + 1.7 71.0 + 2.0 73.6+1.8 78.0+1.9 7.3 + 1.0 7.0 + 1.2 0.837 0.830

Proactive memory 46.1 + 1.2 43.1 + 1.3 43.8 + 1.2 44.5 + 1.5 -2.3 + 1.6 1.4 + 1.4 0.160 0.155

Retroactive interference 43.8 + 1.1 42.7 + 1.1 44.2+1.2 44.0+1.0 0.4+1.3 1.3 + 1.4 0.630 0.637

RAVLT delayed 49.3 + 1.6 50.3 + 1.6 52.5 + 1.4 57.5 + 1.7 3.2+1.2 7.2 + 1.4 0.035* 0.029*

Delayed recall 42.3 + 1.3 41.4+1.5 43.0+1.2 45.4+1.2 0.7 + 1.1 3.9 + 1.8 0.116 0.110

Delayed recognition 56.3 + 2.3 59.1+2.3 62.1+2.0 69.6 + 2.6 5.8 + 2.0 10.5 + 2.0 0.097 0.073

Semantic Memory 75.6 + 0.7 75.6 + 0.8 75.9 + 0.7 76.9 + 0.8 0.3 + 0.3 1.3 + 0.4 0.032* 0.150

Category fluency 48.7 + 1.3 48.3 + 2.0 48.0+1.5 49.2+1.8 -0.7 + 0.7 0.9 + 1.0 0.239 0.293

Letter fluency 45.2 + 2.3 46.4+1.8 45.1+2.0 48.0 + 2.0 -0.1 + 1.0 1.6 + 1.4 0.320 0.348

Naming 97.0 + 0.3 95.9 + 0.5 97.6 + 0.3 97.1+0.4 0.6 + 0.3 1.2 + 0.4 0.292 0.290

Category comprehension 99.0 + 0.2 99.0 + 0.2 99.4 + 0.2 99.5 + 0.1 0.4 + 0.2 0.6 + 0.2 0.475 0.587

Camel and cactus 88.0 + 0.8 87.6+1.1 89.1+0.7 89.9 + 0.7 1.8 + 0.6 2.3 + 1.0 0.353 0.410

Visuospatial working memory

Double Span 85.9 + 0.8 86.0+1.3 87.4+1.0 87.5 + 1.2 1.5 + 0.7 1.6 + 1.1 0.965 0.987

Executive Function

Trail Making Task ¹

Time taken A(s) 34.9 + 1.2 36.0 + 0.9 33.1 + 1.2 33.1 + 1.1 -1.9 + 1.1 -2.9 + 1.0 0.512 0.498

Number of Errors A 0.2 + 0.1 0.1+0.1 0.2 + 0.1 0.1+0.1 -0.1+0.1 -0.1+0.1 0.973 0.959

Time taken B (s) 72.7 + 2.9 75.4 + 3.8 70.5 + 2.8 68.6 + 3.1 -2.2 + 2.9 -6.9 + 2.6 0.236 0.215

Number of Errors B 0.5 + 0.1 0.7 + 0.1 0.5 + 0.1 0.4 + 0.1 0.1+0.1 -0.4 + 0.2 0.043* 0.084

Interference (ratio) 2.1+0.1 2.1+0.1 2.2 + 0.1 2.0 + 0.1 0.1+0.1 -0.1+0.1 0.578 0.602

Overall cognitive performance 70.6 + 0.6 70.8 + 0.7 71.6 + 0.6 73.1 +0.7 1.0 + 0.3 2.3 + 0.3 0.004* 0.018*

* Independent t-test, P < 0.05.

1

Not presented as a percentage; higher values = worse performance.

[0090] Cerebrovascular function

[0091] Basal mean CBFV (treatment difference; placebo - 1.1 + 1.5, resveratrol - 0.2 + 1.3) and PI (treatment difference; placebo - 0.1 + 0, resveratrol - 0 + 0) were unaffected by intervention. After adjusting for multiple comparisons, CVR to hypercapnia (Figure 2) was significantly improved, by a magnitude of 7%, with resveratrol compared to placebo (P = 0.011; Cohen 's d = 0.69). CVR to the cognitive test battery (Table 3) was also enhanced by resveratrol (P = 0.008; Cohen 's d = 0.71). This enhancement of CVR by resveratrol was also observed during individual tasks: RAVLT immediate, Category fluency and Camel and Cactus (Table 3). Moreover, treatment differences in CVR to the cognitive test battery correlated significantly with the differences in overall cognitive performance (unadjusted r = 0.328, P = 0.032; adjusted for depressive symptoms r = 0.327; P = 0.048).

[0092] The significant improvements in CVR during both hypercapnic provocation and neuronal load suggest resveratrol is also able to modulate CBFV; an important finding when it is noted that reductions of CVR in response to hypercapnia of just 10% are related to a 64% increase in stroke susceptibility and that mild cognitive impairment and Alzheimer's disease patients have lesser CVR responses to hypercapnia as measured by Blood Oxygen Level- Dependant (BOLD) spectroscopy.

[0093] Correlations between cerebrovascular function and cognitive performance

[0094] Differences in CVR to hypercapnia pre-post intervention for all participants correlated with differences in CVR to cognitive stimuli in the left MCA (r=0.359; P=0.018) and combined MCA (r=0.337; P=0.016). Likewise changes in overall cognitive performance for all participants correlated significantly with pre-post intervention differences in CVR to cognitive stimuli in the left (r=0.315; P=0.032), right (r=0.331; P=0.022) and combined MCA (r=0.328, P=0.016); after correcting for CES-D only the right (r=0.350; P=0.017) and combined MCA (r=0.327; P=0.024) remained significant.

[0095] Mood

[0096] Following resveratrol supplementation, anxiety (a component of the POMS) was significantly reduced (P = 0.025; Cohen 's d = 0.50) (Table 4) as was tension (P < 0.033; Figure 3). No significant changes were observed in other components of the POMS or in depressive symptoms, although participants tended to score better in the resveratrol group (Table 4; Figure 3). Table 3. Cerebrovascular responsiveness (percentage) for individual cognitive tasks and overall cognitive CVR. Data are presented as mean SEM.

Week 0 (VI) Week 14 (V2) Δ (V2-V1) Placebo vs. Resveratrol

Outcome measure Placebo Resveratrol Placebo Resveratrol Placebo Resveratrol P-value

(n=33) (n=28) (n=33) (n=28) (n=33) (n=28)

RAVLT immediate 16.8 ± 1.6 17.4 ± 2.3 16.4 ± 1.5 21.9 ± 2.2 -0.4 ± 1.6 4.5 ± 1.3 0.020*

RAVLT delayed 12.9 ± 1.5 15.2 ± 1.4 13.5 ± 1.3 16.5 ± 1.7 0.6 ± 1.0 1.3 ± 1.2 0.686

Category fluency 6.1 ± 1.6 6.9 ± 1.9 4.6 ± 0.9 10.1 ± 1.7 -1.4 ± 0.9 3.2 ± 1.7 0.016*

Letter fluency 9.4 ± 1.9 9.6 ± 1.8 7.4 ± 1.4 9.3 ± 1.7 -1.9 ± 1.9 -0.3 ± 1.7 0.449

Naming 7.4 ± 1.8 9.7 ± 1.7 8.9 ± 1.4 7.3 ± 1.5 1.5 ± 1.6 -2.4 ± 1.6 0.097

Category comprehension 11.4 ± 1.6 9.1 ± 1.5 12.2 ± 1.7 9.3 ± 1.4 0.8 ± 1.6 0.2 ± 1.3 0.761

Camel and cactus 7.7 ± 1.5 5.1 ± 1.2 4.8 ± 1.0 8.0 ± 1.5 -2.9 ± 1.5 2.8 ± 1.2 0.006*

Double Span 5.3 ± 1.1 9.5 ± 1.5 6.3 ± 1.3 9.6 ± 1.4 1.0 ± 1.1 0.1 ± 1.4 0.606

TMT 14.0 ± 2.0 15.8 ± 1.8 11.8 ± 1.9 17.9 ± 2.1 -2.2 ± 1.3 2.1 ± 1.7 0.047

Overall cognitive CVR 9.6 ± 0.9 10.8 ± 0.8 9.2 ± 0.8 12.6 ± 1.0 -0.4 ± 0.5 1.7 ± 0.5 0.008*

* Independent t-test (Bonferroni adjusted), P < 0.025.

Table 4. Participants' mood profiles and individual measures. Data are presented in mean + SEM.

Placebo vs.

Week 0 (VI) Week 14 (V2) Δ (V2-V1) Resveratrol

Mood questionnaire Placebo Resveratrol Placebo Resveratrol Placebo Resveratrol P-value

(n=41) (n=38) (n=41) (n=38) (n=41) (n=38)

CES-D ¹ 8.18 + 1.18 8.50 + 1.18 9.74 + 1.42 7.74 + 1.17 1.56 + 1.42 -0.76 + 0.89 0.104

POMS

Tension and anxiety ¹ 6.43 + 0.77 7.37 + 1.00 6.15 + 0.77 5.21 + 0.82 -0.28 + 0.60 -2.16 + 0.57 0.025*

Depression ¹ 4.20 + 0.85 5.63 + 1.09 4.05 + 1.23 4.26 + 0.92 -0.15 + 0.89 -1.37 + 0.70 0.290

Anger ¹ 4.98 + 0.96 5.45 + 1.00 4.23 + 0.99 3.97 + 0.85 -0.75 + 0.65 -1.48 +0.73 0.458

Fatigue ¹ 6.63 + 0.97 5.87 +0.78 6.73 + 1.13 4.55 + 0.77 0.10 + 0.72 -1.32 + 9.68 0.159

Confusion ¹ 5.40 + 0.59 5.13 + 0.59 5.23 + 0.64 4.47 + 0.51 -0.18 + 0.43 -0.66 + 0.38 0.404

Vigour 16.98 ± 1.01 17.97 + 0.95 17.75 + 1.05 19.50 + 1.01 0.78 + 0.73 1.53 +0.79 0.486

Total mood score ¹ 10.65 + 3.87 11.47 + 4.41 8.63 + 4.76 2.97 + 4.03 -2.03 + 2.63 -8.50 + 2.54 0.085

* Independent t-test (Bonferroni adjusted), P < 0.025

1 A more negative value indicates better outcome

[0097] Conclusions

[0098] This is the first study to demonstrate benefits of chronic resveratrol supplementation (75mg twice daily for 14 weeks) on overall cognition as well as in specific memory domains and neurovascular coupling capacity. From the results of the present study, it can be seen that chronic resveratrol supplementation is able to influence cognitive processes while also modulating the responsiveness of cerebral vessels. 14 weeks of resveratrol consumption had a beneficial effect in post-menopausal women improving both cognition and cerebral blood flow velocity.

[0099] As participants were instructed to refrain from consuming their supplement 24 hours before their final visit, these improvements represent a sustained effect of resveratrol rather than an acute effect, as resveratrol would not have been present in the circulation at the time of testing. The half-life of resveratrol in the circulation is ~9 hours.

[00100] Overall cognitive performance was significantly better in those taking resveratrol and this trend was reflected in each specific task. For example, those in the resveratrol group performed significantly better in the domains of verbal and semantic memory, demonstrating the potential for resveratrol to improve all memory functions in peri- and post-menopausal women. Additionally, improvements in mood states were observed, indicating that resveratrol supplementation can provide an overall benefit to quality of life in the years following menopause.

[00101] The inventors hypothesise that resveratrol may elicit its benefits on cognition and also mood through its ability to modulate cerebral perfusion during times of demand. Resveratrol may be acting through its own mechanisms, such as NAD+-dependent, class III histone deacetylase sirtuin 1 (SIRTl) and AMP-activated protein kinase (AMPK) and nuclear factor-erythroid-derived 2-related factor-2 (Nrf2), to modulate nitric oxide production and vasomotor tone in order to influence cognition while also complementing this with ER modulated activation of endothelial nitric oxide synthase resulting in increased cerebral perfusion.

Example 3 - Effect of trans-resveratrol on pain perception

[00102] Following assessments of cerebrovascular responsiveness (CVR) to a hypercapnic challenge, participants concluded the visit by completing six paper-based questionnaires pertaining to overall well-being (i.e pain, sleep, mood, perception of physical and psychological well-being and menopausal symptoms) that were designed to assess varying aspects of one's general living.

[00103] Participants' pain symptomatology was assessed through the Short-form McGill Pain questionnaire, containing 15 descriptors of sensory pain where the women had to assign an intensity of either none, mild, moderate or severe to each descriptor (Dworkin et al., Pain, 2009, 144, 35-42). A numerical value was assigned to each intensity scale with a maximum score of 45, indicating most pain experienced. Participants were also told to mark on the 10- cm Visual Analogue Scale (VAS) and quantify their Present Pain Intensity (range from '0' for no pain to '5' for excruciating) for an estimate of their overall pain intensity. Each subscale score was expressed as a percentage (i.e. a 3cm mark on the VAS would be 30%) and averaged to give a composite score for pain.

[00104] Sleep was assessed through the Pittsburgh Sleep Quality Index where the maximal score of 21 indicated worst quality (Buysse et al., Psychiatry Res, 1989, 28, 193-213). Menopausal symptoms were assessed using the Menopausal Rating Scale (MRS) as this has been shown to be effective in measuring treatment effects on quality of life across the full range of severity of complaints in ageing women (Heinemann et al., Health Qual Life Outcomes, 2004, 2, 67). The MRS has 11 questions with an option to check one of five ratings of the severity of symptoms (range from 'none'=0 to 'very severe'=4). Subscales pertaining to somatic, psychological and urogenital symptoms were also examined. A composite for the severity of menopausal symptoms was expressed as a percentage of the sum of scores from a maximum score of 44.

[00105] The participants' own perceptions of their physical and mental health were also recorded using the Short-form 36 (SF-36) Health Survey, a validated questionnaire in menopausal women. It consists of eight subscales of physical functioning, general health perceptions, vitality, bodily pain, mental health and physical role, emotional role and social role functioning. A maximum score of 100, equivalent to no disability, was assigned to each scale (Mishra et al., Qual Life Res, 1998, 7, 215-220). Overall perception of quality of life (QoL) was calculated by averaging the scores on the eight subscales.

[00106] Mood states and depressive symptoms were assessed by Profile of Mood States (POMS) and Centre for Epidemiologic Studies Depression Scale (CES-D) questionnaires respectively. Six subscales of tension, depression, anger, fatigue, confusion and vigour in the POMS were expressed as percentages of their maximum score for each subscale. Overall mood was then calculated by summing the percentages for all subscales (except vigour) and subtracting the percentage obtained for vigour. The CES-D composed of 20 questions which participants rated the frequency of their experience during the last week for each question. A numerical score of up to three for 'most days (5-7 days)' was assigned to each question. A maximum score of 60 indicated most depressive symptoms experienced.

[00107] Measures of QoL, pain, sleep quality, menopausal symptoms, mood states and depressive symptoms were averaged to give a composite score for total well-being, as the combination and severity of symptoms were likely to differ between individuals.

[00108] During the 14- week intervention, participants were instructed to consume one capsule containing 75mg of 99% pure synthetic trans-resveratrol (ResVida™, DSM Nutritional Products Ltd, Switzerland) or matching placebo twice daily. They were also told to maintain their habitual dietary and medication regime. The first participant was allocated to a group randomly using years since onset of menopause. The placebo comprised several inert excipients (calcium hydrogen phosphate, microcrystalline cellulose, prosolv 50 and hydrated magnesium silicate). Compliance was facilitated by a follow-up phone call at mid-intervention to enquire about the participants' well-being and participants were told to record the time of capsule consumption in their supplement diary each day. At the end of the trial, all remaining capsules were counted and tallied with the corresponding diary records to calculate overall compliance. The participants returned at the end of the 14-week intervention for reassessment of outcome measures. Blinding was maintained until all data analysis had been completed.

[00109] Using a per protocol (pp) analysis, treatment differences by time effects were determined by Generalised Linear Modelling, if the assumption of equal variances was met (Levene's test of homogeneity)(SPSS version 21.0, IBM Inc. Chicago, IL, USA). An intention-to-treat (ITT) analysis was also performed on the outcomes by multiple imputation for missing values. Treatment differences for all measures except QoL were reversed to reflect improvements following 14 weeks of supplementation. Pearson's correlational analysis was used to determine whether the improvements in each measure of well-being and total well- being were related to enhancements of CVR to hypercapnia, hereby used as a surrogate marker of circulatory function. The adjusted level of significance was set at P<0.017 to account for multiple comparisons in the secondary outcomes. Data are presented as mean + SEM.

[00110] As shown in Table 5, 60% of participants from both resveratrol and placebo group reported 'aching' pain as the most common pain complaint at baseline. Table 6 depicts the individual measures and their subscale percentages of outcome measures, overall well-being at baseline and at the end of the intervention. Both ΓΓΤ and PP analyses showed that regular resveratrol supplementation significantly reduced overall pain by 10% compared to placebo, with reductions in all three subscales of pain.

Table 5

Week O Week 14 Δ (week 14 - week 0) ^a

Levene's P-value P-value

Placebo Resveratrol Placebo Resveratrol Placebo Resveratrol

sig. PP ITT

Overall pain 16.5+2.4 15.9+2.2 20.3+2.5 10.1+2.4 -3.8+2.0 5.8+1.5 0.785 <0.001 * 0.007*

■ Pain score 8.2+1.6 10.7+1.5 9.6+1.5 6.1+1.5 -1.4+1.1 4.6+1.2 0.135 0.001 * 0.004*

■ VAS 19.6+3.1 17.4+2.9 23.8+3.1 11.2+2.9 -4.2+2.4 6.2+1.7 0.159 <0.001 * <0.001 *

■ PPI 20.6+2.9 19.4+2.8 23.0+2.9 13.1+2.8 -2.4+2.8 6.3+2.5 0.288 0.011 * 0.019

Total sleep index 35.9+2.9 30.1+2.9 30.7+2.8 24.9+2.8 5.2+2.3 5.2+1.8 0.145 0.230 0.484

■ Sleep duration 34.3+5.7 19.0+4.6 28.3+5.3 14.3+3.7 6.0+3.4 4.8+4.6 0.461 0.799 0.817

■ Sleep disturbances 59.6+3.2 52.4+3.7 50.5+2.9 48.6+3.4 9.1+3.6 3.8+3.8 0.419 0.302 0.990

■ Sleep latency 48.5+5.4 41.0+6.1 35.4+5.4 32.4+5.2 13.1+5.6 8.6+5.0 0.349 0.515 0.651

■ Daytime sleepiness 21.2+3.5 27.6+2.2 21.2+4.1 17.1+3.2 0.0+4.6 10.5+3.0 0.599 0.131 0.585

■ Sleep efficiency 43.4+6.2 26.7+5.1 41.4+6.2 27.6+5.2 2.0+6.3 -1.0+5.2 0.556 0.397 0.874

■ Medication use 15.2+5.4 10.5+4.3 11.1+5.2 12.4+4.5 4.0+4.0 -1.9+3.9 0.925 0.288 0.875

■ Sleep quality 49.5+5.3 42.9+3.8 35.4+4.8 27.6+3.5 14.1+5.6 15.2+3.7 0.047 0.644 ^b 0.710"

Menopausal symptoms 23.9+2.1 20.5+2.1 18.0+1.8 14.1+1.8 5.9+1.7 6.4+1.6 0.504 0.547 0.580

■ Somatic 35.3+2.6 25.4+2.6 26.7+2.4 17.7+2.4 8.6+2.2 7.7+2.2 0.066 0.756 0.760

■ Psychological 16.5+2.7 16.2+2.6 13.5+2.4 12.1+2.4 3.0+1.9 4.1+1.8 0.856 0.701 0.688

■ Urogenital 19.2+3.5 22.1+3.4 12.6+2.2 13.1+2.1 6.6+2.8 9.0+2.7 0.851 0.315 0.808

QoL 77.3+2.3 81.6+2.3 79.7+2.1 84.4+2.1 2.4+2.3 2.8+2.3 0.233 0.804 0.761

Depressive symptoms 12.7+2.9 14.1+1.8 15.3+2.3 12.8+1.3 -2.6+1.9 1.3+1.9 0.238 0.099 0.103

Overall mood -35.8+14.0 -35.6+13.8 -26.4+13.5 -6.6+13.3 9.4+7.4 29.0+7.2 0.465 0.079 0.434

Total well-being 35.3+3.0 32.1+3.0 31.8+3.1 24.7+3.1 3.5+1.6 7.4+1.6 0.179 0.008* 0.046

Table 6

Placebo Resveratrol

Improvement No change Worsen Improvement No change Worsen

Pain descriptors -3 -2 -1 0 +1 +2 +3 -3 -2 -1 0 +1 +2 +3 Chi ²

Throbbing 2.9 2.9 2.9 74.3 14.3 2.9 0 2.9 8.6 17.1 68.6 2.9 0 0 0.140

Shooting 0 2.9 11.4 65.7 17.1 2.9 0 0 5.7 14.3 77.1 2.9 0 0 0.255

Stabbing 0 2.9 5.7 80.0 5.7 5.7 0 0 5.7 5.7 82.9 5.7 0 0 0.672

Sharp 0 5.7 17.1 60.0 8.6 8.6 0 0 11.4 8.6 68.6 11.4 0 0 0.286

Cramping 2.9 0 11.4 65.7 11.4 5.7 2.9 0 5.7 17.1 74.3 2.9 0 0 0.211

Gnawing 0 0 11.4 80.0 2.0 9.0 5.7 0 2.9 8.6 88.6 0 0 0 0.368

Hot-burning 0 0 8.6 77.1 5.7 8.6 0 0 5.7 17.1 65.7 11.4 0 0 0.137

Aching 0 5.7 11.4 54.3 22.9 5.7 0 0 5.7 20 65.7 8.6 0 0 0.242

Heavy 0 5.7 2.9 80.0 8.6 2.9 0 0 2.9 5.7 82.9 5.7 2.9 0 0.927

Tender 0 2.9 20 68.6 5.7 2.9 0 0 0 14.3 65.7 17.1 2.9 0 0.500

Splitting 0 2.9 2.9 85.7 5.9 2.9 0 0 2.9 0 94.3 2.9 0 0 0.483

Tiring 2.9 5.7 0 71.4 14.3 2.9 2.9 0 2.9 25.7 62.9 5.7 2.9 0 0.046

Sickening 0 0 3.0 91.4 5.7 2.9 0 0 5.7 5.7 85.7 0 2.9 0 0.194

Fearful 0 0 2.9 94.3 2.9 0 0 0 0 8.6 88.6 2.9 0 0 0.588

Punishing 0 0 0 97.1 0 2.9 0 0 2.9 0 91.4 5.7 0 0 0.255

[00111] The resveratrol-treated group showed reductions in pain intensity to most pain descriptors (Figure 4). The treatment-induced improvement in overall pain correlated with the treatment change in CVR to hypercapnia (pain: r=0.405, P=0.004) (Figure 5). Improvements in QoL and total well-being significantly correlated with treatment changes in CVR to hypercapnia (r=0.382, P=0.007 and r=0.453, P=0.002 respectively) (Figure 5). Moreover, when considering overall pain scores, and the Visual Analogue Scale for pain (VAS), after adjusting for covariates, pain perception was significantly reduced in the resveratrol group compared to the placebo group (Figure 6).

[00112] Results from this study indicate that resveratrol may be effective for reducing pain and improving total well-being in postmenopausal women through improvements in circulatory function. Given the systemic inflammation milieu of age-related osteoarthritis, the results point to the potential for resveratrol alone, or in combination with other polyphenols such as curcumin, to decrease pro-inflammatory mediators and reduce pain associated with age-related osteoarthritis.