AU2015101752A4

AU2015101752A4 - Composition for mood and/or cognitive function support

Info

Publication number: AU2015101752A4
Application number: AU2015101752A
Authority: AU
Inventors: Neal Mercado; Jodi Van Dyk
Original assignee: Fit Bioceuticals Ltd
Current assignee: FIT-BIOCEUTICALS Pty Ltd
Priority date: 2015-12-04
Filing date: 2015-12-04
Publication date: 2016-01-14
Anticipated expiration: 2023-12-04

Abstract

AU2015101752 Composition for Mood and/or Cognitive Function Support The invention is directed to a composition comprising an effective amount of lipoic acid (5 (1,2-dithiolan-3yl)valeric acid) or an alternative form, derivative, or salt thereof, or a combination thereof, and/or a source thereof, and at least one extract selected from the following: (i) an extract prepared from at least one Bacopa spp.; (ii) an extract prepared from Ginkgo biloba; and (iii) an extract prepared from at least one Panax spp.; to improve or maintain mood and/or cognitive function in a subject; or to slow the age-related decline in the mood and/or cognitive function in a subject..

Description

1 Composition for Mood and/or Cognitive Function Support Field of the Invention [0001] The present invention relates to compositions, methods and uses of a combination of lipoic acid and at least one extract prepared from a plant for mood and/or cognitive function support in a subject. In particular, the plant is from the Bacopa spp. and/or Ginkgo biloba and/or Panax spp. Background of the Invention [0002] Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field. [0003] Many people strive for good mental health by seeking support for enhancing cognitive function, such as memory, learning and concentration, enhancing mood and reducing anxiety in order to meet the demanding needs of society, or during times of stress. In particular, healthy school age children and young adults may need cognitive support e.g., during exams and other intellectually-demanding times. It is also desirable to increase cognitive function in slow learners e.g., a group of children having an IQ between 70-80 wherein various environment and/or genetic factors may have contributed to lower brain function, attention deficit disorders associated with hyperactivity, restlessness and lack of concentration. [0004] A decline in cognitive function is often considered a natural part of ageing. However, given the increase in general health and life expectancy of people, coupled with increasing demands in the work place, there is a desire to maintain a high degree of cognitive function and to enhance mood in addition to maintaining physical ability of the ageing population. [0005] Individuals with a decline in cognitive function can be subdivided into those with memory loss that is age-related, and in that sense benign, and those whose memory or other cognitive symptoms are the first clinical manifestations of Alzheimer's or another dementia i.e. mild cognitive impairment (MCI). [0006] Therapeutic treatments for MCI are important, since MCI is considered the prodromal phase of Alzheimer's disease. Data suggest that conversion from MCI to dementia occurs at a 2 rate of 10% to 15% per year with -80% conversion by the sixth year of follow-up; although -5% of MCI subjects remain stable or convert back to normal (Lovell MA. J Alzheimers Dis. 2010;19:219). [0007] The prevalence and incidence of Alzheimer's disease (AD) is expected to increase dramatically over the next three decades. In 2010 Alzheimer's disease affected more than five million patients in the U.S. and is predicted to rise to 16 million by 2050, costing the U.S. economy more than $140 billion/yr. Globally, an estimated 35.6 million people have dementia which is expected to reach 65.7 million in 2030 and 115.4 million in 2050 (Weiner MW et al. Alzheimers Dement. 2010;6:202-211). In Australia, there will be 75,000 so-called baby boomers with dementia by 2020 and dementia will be the third largest source of health and residential care costs by 2030; in the absence of new medications to treat dementia, it is predicted that almost 950,000 people will be living with dementia by 2050 (Access Economics report - "Dementia across Australia: 2011-2050", September 09 2011). Thus, not only is there a large economic and social burden from the current state of AD and dementia prevalence, these burdens are also expected to increase dramatically. [0008] The aetiology of cognitive decline, formation of MCI and progression to AD is complex, multifaceted, and not fully understood. This is evidenced by the numerous risk factors thought to play a role in the development of Alzheimer's (Smith AD Food Nutr Bul. 2008;29:S143-S172). [0009] It is thought that an accumulation of oxidative damage may start in pre-symptomatic phases i.e. MCI, and that progression to AD might be related to depletion of antioxidant defences (Baldeiras I et al. J Alzheimers Dis. 2010). Reactive oxygen and nitrogen species (ROS and RNS) formed during normal metabolism, and in higher fluxes under pathological conditions, can cause the oxidation of amino acid residues on proteins, forming protein carbonyls. In one study, global oxidative stress measurements revealed significantly higher levels of protein carbonyls in the MCI inferior parietal lobule (IPL) relative to PCAD (and controls), despite equal levels of neuropathology (Aluise CD, et al. J Alzheimers Dis. 2010). [0010] Despite studies attempting to identify underlying causes for the formation of MCI and progression to AD, currently there are very few treatment options available for persons with AD and the treatment options that are available are only short-lived and not overly effective. Such therapies have focused on narrow therapeutic targets such as, anti-cholinesterase inhibitors e.g., donepezil. These medications are very expensive and in Australia, available only after 3 medical approval. Thus, treating persons with AD or MCI is viewed as difficult with the current technology. [0011] Improving mood and/or cognitive function in a subject, e.g., normal healthy subjects or slow learners is desirable for increasing quality of life. Moreover, maintaining mood and/or cognitive function or slowing the decline in the mood and/or cognitive function in healthy elderly people, or ageing individuals, is not only desirable for increasing quality of life but perhaps may delay the onset of MCI and AD. [0012] There is increasing evidence that the use of nutritional/dietary supplementation e.g., vitamin or compositions prepared using plant extracts that may be taken as dietary supplements, may represent attractive cost-effective alternatives for such described uses. The use of compositions prepared from a plant extract also has the added benefit for the health conscious individual wishing to use products derived from nature. [0013] The effects of herbal extracts from a single source on mood and/or cognitive function have been previously described. For example, Bacopa spp. e.g., Bacopa monnieri is a creeper plant found throughout India, and used in Ayurvedic preparations for improving some cognitive functions. However, many extracts on the market have little or no scientific evidence to prove they are active for the purpose as intended, or lack any scientific evidence that they have any active components in them at all. This may be due to the overall mix of bioactive compounds in a chosen plant or due to the quality and method used to prepare the plant extract. Despite the recent advances of the development of standardised extracts from plants, with known bioactives, there remains a need for safe, cheap and effective therapies, e.g., effective compositions prepared from plant extracts that are efficacious in maintaining or improving mood and/or cognitive function in individuals at times of need, or for slowing the decline of cognitive function in individuals with MCI or AD, or healthy elderly individuals so that the onset of MCI or AD may be avoided. [0014] It is an objective of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art treatments, or to provide a useful alternative. Summary of the Invention [0015] In seeking alternative, cost-effective treatments with improved efficacy for cognitive support and/or mood enhancement, the inventors sought to combine one or more plant extracts, known to have an effect on cognitive function and/or mood, with other ingredients 4 e.g., antioxidants. Whilst it has been suggested to combine Bacoba with other herbal preparations thought to have an effect on cognitive function, e.g., Ginkgo biloba, until the present invention, it was not clear whether a combination of one or more herbal preparations with other ingredients e.g., antioxidants, may provide a useful product. The inventors sought to combine lipoic acid with known extracts prepared from plants to prepare a composition for affecting mood and/or cognitive function. Lipoic acid is traditionally used to treat diabetes and is known to chelate redox-active transition metals, inhibiting the formation of hydrogen peroxide and hydroxyl radicals, to scavenge reactive oxygen species (ROS), increasing the level of reduced glutathione, down-regulating inflammatory processes, to scavenge lipid peroxidation products and to induce the enzymes of glutathione synthesis and other antioxidant protective enzymes. The inventors have found that the combination of an extract from a plant known to play a role in supporting cognitive function and/or mood in a subject with lipoic acid results in a significant improvement in activity over the known plant product alone. Until the present invention, no commercial preparations have combined lipoic acid with an extract prepared from a plant for affecting mood and/or cognitive function. [0016] In one aspect, the present invention provides a composition comprising an effective amount of lipoic acid (5-(1,2-dithiolan-3-yl)valeric acid) or an alternative form, derivative, or salt thereof, or a combination thereof, and/or a source thereof, and at least one extract selected from the following: (i) an extract prepared from at least one Bacopa spp.; (ii) an extract prepared from Ginkgo biloba; and (iii) an extract prepared from at least one Panax spp., to improve or maintain mood and/or cognitive function in a subject; or to slow the age-related decline in the mood and/or cognitive function in a subject. [0017] A method for improving or maintaining mood and/or cognitive function in a subject or slowing the age-related decline in the mood and/or cognitive function in a subject comprising administering lipoic acid (5-(1,2-dithiolan-3-yl)valeric acid) or an alternative form, derivative, or salt thereof, or a combination thereof, and/or a source thereof, and at least one extract selected from the following: (i) an extract prepared from at least one Bacopa spp.; (ii) an extract prepared from Ginkgo biloba; and (iii) an extract prepared from at least one Panax spp., together, or separately in any order, in an effective amount and for a time sufficient to improve or maintain the mood and/or cognitive function in the subject, or slow the age-related decline in the mood and/or cognitive function in the subject. [0018] In another aspect, the present invention provides use of a composition comprising an effective amount of lipoic acid (5-(1,2-dithiolan-3-yl)valeric acid) or an alternative form, derivative, or salt thereof, or a combination thereof, and/or a source thereof, and at least one 5 extract selected from the following: (i) an extract prepared from at least one Bacopa spp.; (ii) an extract prepared from Ginkgo biloba; and (iii) an extract prepared from at least one Panax spp., to improve or maintain mood and/or cognitive function in a subject; or to slow the age related decline in the mood and/or cognitive function in a subject. [0019] In another aspect, the present invention provides use of a composition comprising an effective amount of lipoic acid (5-(1,2-dithiolan-3-yl)valeric acid) or an alternative form, derivative, or salt thereof, or a combination thereof, and/or a source thereof, and at least one extract selected from the following: (i) an extract prepared from at least one Bacopa spp.; (ii) an extract prepared from Ginkgo biloba; and (iii) an extract prepared from at least one Panax spp., to improve or maintain mood and/or cognitive function in a subject; or to slow the age related decline in the mood and/or cognitive function in a subject in the manufacture of a medicament, dietary/nutritional supplement, or nutraceutical for improving or maintaining mood and/or cognitive function; or for slowing the age-related decline in the mood and/or cognitive function in a subject. [0020] It will be apparent to the skilled addressee that the lipoic acid (5-(1,2-dithiolan-3 yl)valeric acid), is also known as a-lipoic acid (ALA) and thioctic acid, and includes 6,8 Dithioctanoic acid. The lipoic acid of any aspect and/or example described herein may include one or a mixture of both enantiomers (R)-(+)-lipoic acid (RLA) or (S)-(-)-lipoic acid (SLA) at various concentrations including a racemic mixture (R/S)-lipoic acid (R/S-LA). It will also be understood by a person skilled in the art that any one or more of these forms of lipoic acid includes alternative forms, derivatives or salts thereof. Any one or more of these forms of lipoic acid may be used in pure or purified form from commercially available sources and/or chemically synthesized and/or prepared from ALA rich sources e.g., kidney, heart, liver, spinach, broccoli, and yeast extract or combinations thereof, or lipoic acid may also be provided in the form of an extract or foodstuff rich in lipoic acid. Commercially available forms include, but are not limited to, dihydrolipoic acid and salts thereof. Any alternative form, derivative, or salt thereof, of lipoic acid known to the skilled artisan is also contemplated. As used herein, the alternative form, derivative, or salt thereof, or combination thereof, includes known biologically active forms and/or forms that have measurable activity as described in any known assay used in the art to measure the function of lipoic acid. Examples of biologically active forms of lipoic acid include lipoate. [0021] For example, (R)-(+)-lipoic acid (RLA) or a derivative or salt thereof is used according to the invention. In another example, (S)-(-)-lipoic acid (SLA) or a derivative or salt thereof is used according to the invention. In another example, a combination of R)-(+)-lipoic 6 acid (RLA) or a derivative or salt thereof with (S)-(-)-lipoic acid (SLA) or a derivative or salt thereof, at various concentrations of each form, is used according to the invention. In another example, a racemic mixture (R/S)-lipoic acid (R/S-LA), also known as R,S alpha lipoic acid, is used according to the invention. [0022] According to the invention any known or available species of Bacopa spp. is contemplated. In one embodiment, the Bacopa ssp. according to the invention is selected from Bacopa australis, Bacopa caroliniana, Bacopa decumbens, Bacopa crenata, Bacopa eisenii, Bacopa innominata, Bacopa madagascarienis, Bacopa monnieri, Bacopa myriophylloids, Bacopa repens, Bacopa rotundifolia and Bacopa stricta. Preferably, the Bacopa spp. is Bacopa monnieri, also known as, water hyssop, moneywort and herb of grace. [0023] According to the invention any known or available species of Panax spp. is contemplated. In one embodiment, the Panax ssp. according to the invention is selected from Panax bipinnatifidus, Panax ginseng, Panax japonicus, Panax quinquefolius, Panax vietnamensis, Panax wangianus, Panax zingiberenis, Panax pseudoginseng, Panax stipuleanatus, Panax notoginseng and Panax trifolius. Preferably, the Panax spp. is Panax ginseng, also known as, red or Korean ginseng, or Panax quinequefolius, also known as American ginseng. More preferably, the Panax spp. is Panax ginseng. [0024] Whilst the sole living species of Ginkgo spp. is Gingko biloba, any species of Ginkgo spp. later identified is also contemplated. [0025] In one or more embodiments, the effective amount of the invention comprises: (i) lipoic acid or an alternative form, derivative, or salt thereof, or a combination thereof, and/or a source thereof; and (ii) an extract prepared from Bacopa monnieri. [0026] In one or more embodiments, the effective amount of the invention comprises: (i) lipoic acid or an alternative form, derivative, or salt thereof, or a combination thereof, and/or a source thereof; and (ii) an extract prepared from Ginkgo biloba. [0027] In one or more embodiments, the effective amount of the invention comprises: (i) lipoic acid or an alternative form, derivative, or salt thereof, or a combination thereof, and/or a source thereof; and (ii) an extract prepared from at least one Panax ginseng. [0028] In one or more embodiments, the effective amount of the invention comprises: (i) lipoic acid or an alternative form, derivative, or salt thereof, or a combination thereof, and/or a 7 source thereof; and (ii) an extract prepared from Bacopa monnieri; and (iii) an extract prepared from Ginkgo biloba. [0029] In one or more embodiments, the effective amount of the invention comprises: (i) lipoic acid or an alternative form, derivative, or salt thereof, or a combination thereof, and/or a source thereof; and (ii) an extract prepared from Bacopa monnieri; and (iii) an extract prepared from at least one Panax ginseng. [0030] In one or more embodiments, the effective amount of the invention comprises: (i) lipoic acid or an alternative form, derivative, or salt thereof, or a combination thereof, and/or a source thereof; and (ii) an extract prepared from Ginkgo biloba; and (iii) an extract prepared from at least one Panax ginseng. [0031] In one or more embodiments, the effective amount of the invention comprises: (i) lipoic acid or an alternative form, derivative, or salt thereof, or a combination thereof, and/or a source thereof; and (ii) an extract prepared from Bacopa monnieri; and (iii) an extract prepared from Ginkgo biloba; and (iv) an extract prepared from at least one Panax ginseng. [0032] Any one of the extracts of the invention is prepared according to any known method of extraction in herbal and/or plant medicine or according to any example described herein, and may be prepared from a whole plant or plant part, or the extract is purchased from any commercial source or specifically manufactured by a commercial company, e.g., Lipa Pharmaceuticals Limited, provided that the extraction procedure provides a good quality extract suitable for administration to a subject. For example, any standard alcohol extraction method known in the art. Any one or more extracts may be prepared and used according to the invention, as a liquid, gel, powder, solid or any other form suitable for administration to a subject. [0033] Preferably, any one of the extracts is a standardised extract. In one or more embodiments, the extract from Bacopa monnieri is a standardised extract. In one example, the extract from Bacopa monnieri is a standardised extract which comprises active compounds including, but not limited to, Bacoside A3, Bacopaside 1l, Bacopa saponin C, Bacopaside 1, bacosine, jujubogenin isomer of bacopasaponin C, Luteolin, Apigenin, and beta-sitosterol-D glucoside, wherein the total bacosides is 40.5 to 54% w/w or wherein the total bacosides is standardised to 45%. BacoMind@ is an alcoholic extract of the whole plant Bacopa monnieri (herb extract ration, 20:1). For example, the extract from Bacopa monnieri is a standardised extract that is commercially available e.g., BacoMind@ and Bacognize@. In one example, the 8 Bacopa monnieri is a standardised extract with the same profile and amounts of active ingredients found in BacoMind@ or Bacognize@. In one example, the Bacopa monnieri is a standardised extract with a profile as shown in Figure 1 and described in Example 4. An exemplary method for preparing the standardized extract is commercially available by Natural Remedies Pty Ltd. (Bangalore India), or as described in e.g., Morgan and Stevens, J. Alternative and Complementary Medicine, 16 (7): pp753-759 (2010). [0034] In one or more embodiments, the extract from Ginkgo biloba is a standardised extract. In one example, the commercially available Indena standardised extract is used according to the invention, which is standardised to > 24% ginkoflavanoid, >/=6% ginkolides and bilogalide and </=ppm ginkgoic acids, Gingko EGB761 (Tebonin), Gingko biloba GK501 and GBE L11370. In another example, the extract from Ginkgo biloba is a standardised extract with the same profile and amounts of active ingredients found in the Indena extract. In another example, the Gingko biloba is a standardised extract with a profile as shown in Figure 2 and described in Example 5. An exemplary method for preparing the standardized extract is commercially available by Indena as Gingko select phytosome (Milan, Italy), or as described in e.g. Patent No. EP0360446. [0035] In one or more embodiments, the extract from Panax ginseng is a standardised extract. In one example, the commercially available Panax G115 is used according to the invention, which is standardised to 4.25 mg ginsenosides. In another example, the extract from Panax ginseng is a standardised extract with the same profile and amounts of active ingredients found in the Panax G115. In another example, the Panax ginseng is a standardised extract with a profile as shown in Figure 3 and described in Example 6. Preferably, the extract from Panax ginseng is prepared from the root according to standard procedures in the art, or as described in Kennedy and Scholey, Pharmacology, Biochemistry and Behaviour 75: pp 687-700 (2003). [0036] It will be understood by the skilled artisan that the composition or administered combination may comprise any one or more other constituents that are favourable for maintaining/improving mood and/or cognitive function. For example, vitamins, minerals, salts or other extracts prepared from plants. In one or more embodiments, the one or more constituents may be selected from, but not limited to, extracts prepared from Rhodiola rosea, Camel/ia sinsensis, ginger, Schizandra, Withania root, Eleuthrococcus, Hypericum perforatum, Schisandra chinesnsis and tyrosine, choline, S-Adenosyl methionine, phosphatidylcholine, phosphatidylserine, thiamine, riboflavin, nicotinamide, calcium panthenoate and pyridoxine hydrochloride.

9 [0037] It will be understood by the skilled artisan that a subject according to the invention may be any age including children, school-age children, and adult subjects of any IQ range, of any cognitive ability and/or mood and not limited to any level of brain function. This includes slow learners e.g., having an IQ between 70-80. The subject may have other cognitive disorders including, but not limited to, attention deficit disorders, hyperactivity, restlessness and lack of concentration. Adult subjects may include biological adults, e.g., subjects that have completed puberty and may include any subjects over the age of about 10 e.g., about 10 to 95 years. [0038] It will also be understood by the skilled artisan that a subject according to the invention may be an ageing subject, which includes any one or more of the described subjects that increase in age. Preferably, ageing subjects includes those adult subjects that are above the age of about 30 or more. Ageing subjects also include elderly subjects. In one or more embodiments, the ageing subject is a healthy adult subject. [0039] The subject of any aspect and/or example described herein may also include patients that have Alzheimer's disease, MCI, dementia or other similar neurological impairments. The subject of any aspect and/or example described herein may also include those subjects that have a pre-disposition and/or at risk of developing to Alzheimer's disease, MCI, dementia or other similar neurological impairments. For example, subject(s) may be tested for biochemical, genetic, or other risk factors and/or markers that indicate that the subject(s) has a pre-disposition and/or at risk of developing to Alzheimer's disease, MCI, dementia or other similar neurological impairments. Such markers may be identified using any test or assay known in the art for such purposes e.g., as described in Stough et al. Nutrition Journal 2012, 11:11. Risk factors for Alzheimer's disease, MCI and/or dementia include, but are not limited to those listed in Table 1. Risk markers also include, but are not limited to measuring glycated haemoglobin, insulin levels, blood glucose levels, markers of oxidative stress, markers of inflammation, C-reactive protein, F2-Isoprostanes, inflammatory cytokines including, but not limited to, TNF-alpha, IL-2, IL-4, IL-6, IL-10, and IFN-gamma, liver function, and genetic markers including detecting the presence of Single Nucleotide Polymorphism (SNPs) in DNA samples from subjects. Any known SNP in the art associated with increased risk of AD, dementia, MCI and cognitive decline in affected and/or normal elderly may also be used. For example, this includes the SNP of APOE allele, which is associated with increased risk of AD and cognitive decline in normal elderly. SNPs associated with increased risk of AD, dementia, MCI and cognitive decline in affected and/or normal elderly may also include any number of SNPs of cytokines, e.g., inflammatory cytokines.

10 [0040] Risk factors for Alzheimer's disease, MCI and/or dementia include, but are not limited to measuring cerebral energy metabolism. For example, this includes, but is not limited to measuring insulin levels, insulin function, glucose uptake and metabolism, insulin resistance and downstream effectors of the insulin resistance pathway in the brain e.g., as described in Medhi and Chakrabarty, Neurol Sci (2013) 34: 1719-1725 and/or Cholerton et al., European Journal of Pharmacology (2013) 719: 170-179. For example, the level and/or activity of P13K/Akt and/or downstream effectors, glucose uptake and metabolism in the brain including intraneuronal and/or extraneuornal glucose, UDPGLcNAc, tau-O-GIcNAc, oxidative stress, advanced glycated end products (AGE) tau phosphorylation, levels of transferrin receptors, iron influx in neurons, PgP As clearance may be measured. These parameters may be measured using any test or assay known in the art for measuring energy metabolism and/or insulin resistance e.g., as described in Medhi and Chakrabarty, Neurol Sci (2013) 34: 1719 1725 and/or Cholerton et al., European Journal of Pharmacology (2013) 719: 170-179. The levels and/or activities of each may be measured and compared to a standard known in the art. It will be apparent to a person skilled in the art whether or not a change in the level and/or activity is an indicator of risk for Alzheimer's disease, MCI and/or dementia. [0041] The subject as described according to any aspect, embodiment and/or example of the invention may be any normal human or mammal subject or any normal human or mammal subject in need administration of the composition or effective amount, as described according to the invention. Mammal subjects include, but are not limited to, apes, humans, gorillas, chimpanzees, endangered species, stock animals, e.g., cattle, pigs, horses, and companion animals, e.g., dogs and cats. The subject is preferably a human subject. [0042] Cognitive function may be measured using any test or assay known in the art for such purposes e.g., as described in Stough et al. Nutrition Journal 2012, 11:11 and/or Morgan and Stevens J Altern Complement Med 16(7): 753, 2010. Cognitive function may be assessed by the Montreal Cognitive Assessment (MCA) (Freitas S et al. Alzheimer Disease & Associated Disorders. 2013) and/or The Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) (Karantzoulis et al. Archives of Clinical Neuropsychology. 2013). Cognitive function parameters include, but are not limited to, reasoning, decision making, attention span, problem solving, spatial abilities, perceptual-motor and/or cognitive speed, and/or memory. [0043] Cerebral energy metabolism is also correlated to cognitive function. Thus, a measure of cerebral energy metabolism may also be used as a parameter to indicate changes in cognitive function. For example, measuring cerebral energy metabolism includes, but is not 11 limited to measuring insulin levels, insulin function, glucose uptake and metabolism, insulin resistance and downstream effectors of the insulin resistance pathway in the brain e.g., as described in Medhi and Chakrabarty, Neurol Sci (2013) 34: 1719-1725 and/or Cholerton et al., European Journal of Pharmacology (2013) 719: 170-179. For example, the level and/or activity of P13K/Akt and/or downstream effectors, glucose uptake and metabolism in the brain including intraneuronal and/or extraneuornal glucose, UDPGLcNAc, tau-O-GIcNAc, oxidative stress, advanced glycated end products (AGE) tau phosphorylation, levels of transferrin receptors, iron influx in neurons, PgP As clearance may be measured. These parameters may be measured using any test or assay known in the art for measuring energy metabolism and/or insulin resistance e.g., as described in Medhi and Chakrabarty, Neurol Sci (2013) 34: 1719 1725 and/or Cholerton et al., European Journal of Pharmacology (2013) 719: 170-179. The levels and/or activities of a parameter described herein may be measured at a time-frame as described according to any aspect, embodiment and/or example herein. It will be apparent to a person skilled in the art whether or not a there is a significant change in the level and/or activity of any one or more parameters described according to any aspect, embodiment and/or example herein. [0044] Positron emission tomography (PET), which is a nuclear medical imaging technique that produces a three-dimensional image or picture of functional processes in the body. PET may be used to measure cognitive function and/or a risk factor for Alzheimer's disease, MCI and/or dimentia as described according to any aspect, embodiment and/or example herein. For example, this may be achieved e.g., as described in Nasrallah and Dubroff, Semin Nucl Med 2013 November; 43(6):449-61. In one example, fluorodeoxyglucose (18-F) brain uptake is measured using PET (FDG-PET). FDG is an analogue of glucose. In FDG-PET, the concentrations of tracer imaged may be used to indicate tissue metabolic activity by virtue of the regional glucose uptake. [0045] Mood may be measured using any test or assay known in the art for such purposes e.g., as described in Stough et al. Nutrition Journal 2012, 11:11. Mood levels may be assessed by Hamilton Rating Scale for Depression (HAM-D) and the Profile of Mood States 2nd edition (POMS 2). Mood parameters include, but are not limited to, depression, anxiety and general mood assessments e.g., happiness, excitement, sadness. [0046] Cognitive function/mood is measurable over any time-frame e.g., hours, days, weeks, months or years. Cognitive function/mood which has not changed or has increased over one or more described time-frames, is considered to be maintained or improved in the subject, e.g., in any subject described herein. Cognitive function/mood may also decline with 12 increasing age in a subject, e.g., an adult subject. In this example, cognitive function/mood is measured over longer time-frames, e.g., months or years in ageing subjects as described herein. The cognitive function/mood is measured at two or more time points in an ageing subject. For example, the decline in cognitive function/mood in an ageing subject may be measured between several months or several years to establish a rate of decline. The rate of decline may be compared to a standard known in the art taking into consideration the age of the subject. The rate of decline may also be compared to cognitive function/mood that is then measured again in the same subject, after the subject has aged for a further period of months or years. Age-related decline is considered slowed if the rate of decline that is less compared to the standard or compared to a second measurement is less. [0047] It will be apparent to the skilled artisan that since cognitive function/mood may be measured, a time sufficient to improve the mood and/or cognitive function in the subject and/or maintain mood and/or cognitive function in the ageing subject or slow the age-related decline in the mood and/or cognitive function in the subject. For example, the time may be 1 month, or 2 months, or 3 months, or 4 months, or 5 months, or 6 months, or 7 months, or 8 months, or 9 months or 10 months, or 11 months, or 12 months. In another example, the time may be 1 to 2 years, or 2 to 4 years, or 4 to 6 years, or 6 to 8 years or 8 to 10 years, or greater than 10 years. It will also be apparent that the time of administration may be continued for the life of the subject. [0048] The amount of each composition or effective amount actually administered will typically be determined by a physician, nutritionist, psychologist and/or other health care practitioner in light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the combination of extract and lipoic acid according to the invention to be delivered, the age, weight, and response of the individual patient, the severity of the patient's symptoms/condition, and the like. [0049] The composition or effective amount of the invention may be formulated for administration. Preferably, the composition or combination of the invention is formulated for oral administration. Processing techniques known in the art maybe used, as well as known pharmaceutically acceptable carriers, diluents or excipients. To prepare such formulations, compositions described herein, containing active ingredient(s) are mixed with a pharmaceutically acceptable carrier or excipient for example, by mixing with physiologically acceptable carriers, excipients, or stabilizers in the form of, e.g., lyophilized powders, slurries, aqueous solutions, or suspensions (see generally Remington's Pharmaceutical Sciences and/or generally any one or more of the following list of references).

13 * Remington's Pharmaceutical Sciences (2000), Mack Publishing Company, Easton, PA., USA 20.sup.th Edition, 2000 * Hardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y. * Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y. * Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY * Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY * Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY * Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, N.Y. * British Pharmacopeia (2011), The Stationary Office on behalf of the Medicines and Healthcare products Regulatory Agency (MHRA). * Rowe, et al. (eds.) (2006) Handbook of Pharmaceutical Excipients, Buttler & Tanner, Frome Somerset., Great Britain. * United States Pharmacopeia, (2012), US Pharmacopeial Convention. * Japanese Pharmacopeia, Fifteenth Edition (2006), Evaluation and Licensing Division, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare. * Sweetman S (ed.)(2007), Martindale: The Complete Drug Reference, Pharmaceutical Press, London * Maryadele et al. (eds.)(2006), The Merck Index, Merck Research Laboratories, Merch & Co., Inc. NJ, USA.17. Katdare and Chaubal (eds.)(2006), Excipient Development for Pharmaceutical, Biotechnology, and Drug Delivery Systems, Informa Health Care, USA * Niazi (ed)(2007), Handbook of Preformulation, Chemical, biological, and Botanical Drugs, Informa Health Care, USA [0050] The composition or effective amount of the present invention can also be formulated and administered in sustained release forms or from sustained release drug delivery systems, either in separate dosage forms or in a combination dosage form. A description of representative sustained release materials can also be found in the incorporated materials in Remington's Pharmaceutical Sciences, and e.g., the above list of references). Such 14 administration can also occur via bolus administration, or via implantable devices, or patches or the like. [0051] Preferably, the composition or the effective amount according to any aspect, embodiment or example of the invention described herein is formulated in unit dosage form. A unit dosage form according to any aspect, embodiment or example described herein may be manufactured according to any standard procedure as described generally in Remington's Pharmaceutical Sciences and/or generally any one or more of the list of references above. For example, the process of manufacture of a unit dosage form of the invention is shown in Figure 4. [0052] The effective amount, or the unit dosage form according to the invention may include 100 mg to 1 g of lipoic acid according to the invention and at least one of the following: 1 to 20 g of extract from Bacopa spp., and/or 1 g to 10 g of extract from Ginkgo biloba, and/or 100 mg to 400 mg of extract from Panax spp. [0053] In one or more embodiments, the effective amount, or the unit dosage form according to the invention includes 200 mg to 1 g of lipoic acid according to the invention and at least one of the following: 2 to 10 g of extract from Bacopa spp., and/or 2 g to 5 g of extract from Ginkgo biloba, and/or 210 mg to 300 mg of extract from Panax spp. ** Add Standardised and more amounts. [0054] In one or more embodiments, the effective amount, or the unit dosage form according to the invention includes about 210 mg, or about 220 mg, or about 230 mg, or about 240 mg, or about 250 mg, or about 260 mg, or about 270 mg, or about 280 mg, or about 290 mg, or about 300 mg, or about 310 mg, or about 320 mg, or about 330 mg, or about 340 mg, or about 350 mg lipoic acid according to the invention, preferably about 300 mg of lipoic acid according to the invention, and at least one of the following: about 1 g, or about 2 g, or about 3 g, or about 4 g, or about 5 g, or about 6 g, or about 7 g, of extract from Bacopa spp., preferably, 4 g of extract from Bacopa spp and/or about 1 g, or about 2 g, or about 3 g, or about 4 g, or about 5 g of extract from Ginkgo biloba, 5 g of extract from Ginkgo biloba and/or 200 to 250 mg of extract from Panax spp., preferably about 200 mg, or about 201 mg, or about 202 mg, or about 203 mg, or about 204 mg, or about 205 mg, or about 206 mg, or about 207 mg, or about 208 mg, or about 209 mg, or about 210 mg, or about 211 mg, or about 212 mg, or about 213 mg, or about 214 mg, or about 215 mg, of extract from Panax spp., more preferably, about 212 mg of extract from Panax spp.

15 [0055] In one or more embodiments, the effective amount, or the unit dosage according to any aspect, embodiment, or example described herein includes a standardised extract of Bacopa spp., preferably Bacopa monniera, comprising 20 mg to 45 mg equivalent to bacosides A, or 45 mg to 225 mg equivalent to bacosides A, or about 50 mg, or about 55 mg, or about 60 mg, or about 65 mg, or about 70 mg, or about 75 mg, or about 80 mg, or about 85 mg, or about 90 mg, or about 95 mg, or about 100 mg, or about 105 mg, or about 110 mg, or about 120 mg, or about 125 mg, or about 130 mg, or about 135 mg or about 140 mg, or about 145 mg, or about 150 mg equivalent to bacosides A, preferably about 90 mg equivalent to bacosides A. [0056] In one or more embodiments, the effective amount, or the unit dosage according to any aspect, embodiment, or example described herein includes a standardised extract of Ginkgo biloba comprising 5 mg to 55 mg equiv. to ginkgo flavonglycosides, or 10 mg to 30 mg equiv. to ginkgo flavonglycosides, or about 10 mg, or about 11 mg, or about 12 mg, or about 13 mg, or about 14 mg, or about 15 mg, or about 16 mg, or about 17 mg, or about 18 mg, preferably about 16 mg equiv. to ginkgo flavonglycosides; and/or 1 mg to 13.5 mg equiv. to ginkgolides and bilobalide, or 2.0 mg to 6.75 mg equiv. to ginkgolides and bilobalide, or about 1.0 mg, or about 2.0 mg, or about 3.0 mg, or about 4.0 mg, or about 5.0 mg equiv. to ginkgolides and bilobalide. [0057] In one or more embodiments, the effective amount, or the unit dosage according to any aspect, embodiment, or example described herein includes a standardised extract of Panax spp., preferably Panax ginseng comprising 2.0 mg to 9.5 mg equiv. to ginkgolides and bilobalide, or 4.1 to 7.1 mg equiv. to ginkgolides and bilobalide, or about 4.0 mg, or about 4.1 mg, or about 4.2 mg or about 4.3 mg, or about 4.4 mg, or about 4.5 mg, preferably about 4.25 mg equiv. to ginkgolides and bilobalide. [0058] According to any aspect, embodiment or example of the invention described herein, the effective amount, or unit dosage form may be administered, or may be formulated for administration between 1 to 10 times per day, separately, together, or at different times throughout the day. Preferably the effective amount, or unit dosage form may be administered, or may be formulated for administration between 1 to 3 times per day separately, together, or at different times throughout the day. For example, once a day, twice a day, or three times daily. More preferably, the effective amount, or unit dosage form may be administered, or may be formulated for administration, twice daily separately, together, or at different times throughout the day.

16 [0059] Unless the context clearly requires otherwise, throughout the description and the claims, the words 'comprise', 'comprising', and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to". [0060] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter. [0061] Each aspect, embodiment and/or example of the invention described herein is to be applied mutatis mutandis to each and every aspect, embodiment and/or example unless specifically stated otherwise. [0062] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features. [0063] The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally equivalent products, compositions and methods are clearly within the scope of the invention, as described herein. Brief Description of the Drawings [0064] Figure 1 is a chromatogram of the TLC profile of Bacopa monnieriwhole plant extract of Example 4. [0065] Figure 2 is a chromatogram of the TLC profile of Ginkgo biloba leaf extract of Example 5. [0066] Figure 3 is a chromatogram of the TLC profile of Panang ginseng root extract of Example 6.

17 [0067] Figure 4 is a Flow-chart showing the process of manufacture of an exemplary unit dosage of the invention. Detailed Description of Preferred Embodiments [0068] The present invention will now be described in more detail with reference to specific but non-limiting examples describing specific compositions and methods of use. It is to be understood, however, that the detailed description of specific procedures, compositions and methods is included solely for the purpose of exemplifying the present invention. It should not be understood in any way as a restriction on the broad description of the inventive concept as set out above. [0069] The inventors sought to combine lipoic acid with a known herbal extract. The inventors have found that the combination of an extract from a plant known to play a role in supporting cognitive function and/or mood in a subject with lipoic acid results in a significant improvement in activity over the known product alone. Example 1 [0070] By way of non-limiting example only, the range of effective amounts present in an example of a suitable unit dose of the composition or administered according to the invention is outlined below. Each tablet contains: Herbal extracts standardised equiv. to dry: Bacopa monniera (bacopa) whole plant 1 g to 20 g equiv. to bacosides A 22.5 mg to 450 mg Ginkgo biloba (ginkgo) leaf 1 g to 10 g equiv. to ginkgo flavonglycosides 5 mg to 55 mg equiv. to ginkgolides and bilobalide 1 mg to 13.5 mg Panax ginseng (Korean ginseng) root 100 to 400 mg equiv. to ginsenosides Rgl,Re, Rf, Rg2, Rbl, Rb2, Rc, Rd 2.0 mg to 9.5 mg Nutrient: R,S-alpha-lipoic acid 100 mg to 1 g 18 Example 2 [0071] By way of non-limiting example only, the range of effective amounts present in an example of a suitable unit dose of the composition or administered according to the invention is outlined below. Each tablet contains: Herbal extracts standardised equiv. to dry: Bacopa monniera (bacopa) whole plant 2 g to 10 g equiv. to bacosides A 45 mg to 225 mg Ginkgo biloba (ginkgo) leaf 2 g to 5 g equiv. to ginkgo flavonglycosides 10 mg to 27.5 mg equiv. to ginkgolides and bilobalide 2.0 mg to 6.75 mg Panax ginseng (Korean ginseng) root 210 to 300 mg equiv. to ginsenosides Rgl,Re, Rf, Rg2, Rbl, Rb2, Rc, Rd 4.2 mg to 7.1 mg Nutrient: R,S-alpha-lipoic acid 200 mg to 600 mg Example 3 [0072] By way of non-limiting example only, a unit dose of the composition or administered according to the invention is outlined below. Each tablet contains: Herbal extracts standardised equiv. to dry: Bacopa monniera (bacopa) whole plant 4 g equiv. to bacosides A 90 mg Ginkgo biloba (ginkgo) leaf 3 g equiv. to ginkgo flavonglycosides 16.02 mg equiv. to ginkgolides and bilobalide 4.02 mg Panax ginseng (Korean ginseng) root 212 mg equiv. to ginsenosides Rgl,Re, Rf, Rg2, Rbl, Rb2, Rc, Rd 4.25 mg Nutrient: R,S-alpha-lipoic acid 300 mg 19 Example 4 [0073] By way of non-limiting example only, an extract of Bacopa monniera was manufactured according to standard procedures to produce an standardised extract with wherein the total bacosides is 40.5 to 54% w/w, as described herein above; Quality testing was carried out on a sample of the extract and compared to a reference standard to ensure the prepared extract was in accordance with the standardised extract. The protocol used was in accordance with the method, as described below: BACOPA MONNIERI WHOLE PLANT EXT. DRY CONC. STAND. 20:1 TESTING INSTRUCTIONS DESCRIPTION The appearance of the sample was examined under evaluation using General Testing Method GTM001 as a guide. IDENTIFICATION OF HERB (Report results as Pass/Fail) APPARATUS - A transparent Chromatographic Tank - HPTLC glass plate, Silica Gel F 254 nm 10 X 10 cm PREPARATION OF SAMPLE SOLUTION: 1.0 g of herbal material under evaluation using a mortar and pestle to get a fine powder was pulverised. The powder was transferred to a suitable screw-capped test tube and add 10 mL of methanol and cap the tube. The mixture was sonicated for 30 minutes. The extract was filtered using a Millipore Nylon Filter 0.45 pm and the particle free extract is used for HPTLC. PREPARATION OF WORKING HERBAL STANDARD SOLUTION: Pulverise 1.0 g of working herbal standard of Bacopa monnieri herb ext. dry cone. stand. 20:1 (R8139) wausing a mortar and pestle to get a fine powder. Transfer the powder to a suitable screw-capped test tube and add 10 mL of methanol and cap the tube. Sonicate the mixture for 30 minutes. Filter the extract using a Millipore Nylon Filter 0.45 pm and the particle free extract is used for HPTLC.

20 PREPARATION OF REFERENCE STANDARD Dissolve 1.8 mg of Bacoside A (Approx. 97%purity) in 1 mL of methanol (Use prepared solution, if available). PROCEDURE: Carry out the test for TLC Identification using General Testing Method GTM021 and the following conditions; Application volume: 6- 8 pL of sample and standard solutions and 8-10pL of reference solution Solvent system: Ethylacetate : Methanol : Water (7 : 2 : 1) Development path: 8 cm Before spraying: Dry in a current of cold air. Detection (1): Examine in UV 254nm Detection (2): Spray with: eerie sulfate - sulfuric acid reagent (modified) 1% Ceric sulfate in 1M Sulphuric acid. OR Spray with Vanillin-Sulfuric acid Reagent Vanillin Reagent- Carefully add, dropwise, 2 mL of cone. sulphuric acid to 100 mL of a 1% w/v solution of vanillin in ethanol (96%). Note: Use within 48 hours. After spraying: Heat at 11 0 0 cfor 5 to 10 minutes. TLC evaluation: Examine under visible light. Apply separately to the plate 6 - 8 pL of sample and standard solutions and 8 - 10 IJL of reference solution. Develop over a path of 8 cm using a mixture of 7 volumes of Ethylacetate: 2 volumes of Methanol: 1 volume of Water (7 :2 : 1). Dry the plate in a current of cold air until the solvent is removed in the fume hood. Examine the dried TLC plate in UV 254 nm. This is detection (1). Dip (or spray) the air-dried HPTLC plate in eerie sulphate-sulfuric acid reagent OR Vanillin- Sulfuric acid Reagent and heat at 110 C for 5-10 minutes. The plate is then evaluated in visible light. This is Detection (2). EVALUATION: The chromatogram obtained with the sample solution corresponds to the chromatogram obtained with the working standard solution. The chromatogram of the TLC profile is shown in Figure 1. ASSAY FOR TOTAL BACOSIDES (Report results of analysis. Carry out in duplicate) Determine the percentage content of Bacosides, following General Analytical Method GAM053 - Determination and Identification of total Bacosides by UPLC. Prepare the sample preparation as follows; 21 Sample preparation - Weigh about 150 mg of sample into a 50 mLvolumetric flask. - Add 35mL of 90%methanol and sonicate for 20-30 minutes with occasional shaking. - Cool and dilute to volume with 90% methanol. This will give a final concentration of Bacosides about 11.2mg/ml. - Filter sample solutions through 0.2pm nylon filters into the UPLC vial and cap. Note: Discard the first 1mL. - Make two 1pL injections of sample solution. MICROBIOLOGICAL TESTS: A Total Viable Count Carry out the test for Total Viable Count following Microbiological Testing method MTM006 B. Yeast and Mould Count Carry out the test for Yeast and Mould Count following Microbiological Testing method MTM006 C. Bile-tolerant Gram- Carry out the test for Bile-tolerant Gram-negative bacteria following negative bacteria Microbiological Testing method MTMO17 D. E. coli Carry out the test for E. coli following Microbiological Testing method MTM002 E. Salmonella Carry out the test for Salmonella following Microbiological Testing method F. Staphylococcus aureus MTM004 Carry out the test for Staphylococcus aureus following Microbiological Testing method MTM008 22 PARTICLE SIZE (Report results of analysis or Report results obtained) Determine the percentage of particles that passes through a 500 pm (ASTM 35 mesh) sieve using normal sieves, following General Testing Method GTMO26 or using Malvern Master sizer following Standard Operating Procedure SOP L076 Malvern Mastersizer 2000 -Operation, Calibration and Maintenance. BULK DENSITY- UNTAPPED (Report results obtained) Determine the Bulk Density- Untapped of the sample under evaluation following General Testing Method GTM023. ADDITIONAL SPECIFICATIONS TESTING INSTRUCTIONS SOLUBILITY (Report result as Passl Fail) Partially soluble in Methanol. TOTAL ASH (Report results of analysis.) Determine the Total Ash of the sample under evaluation following General Testing Method GTM022- Method II. Use 2.Og of sample. Retain the residue obtained for use in Test 3- Acid Insoluble Ash. ACID INSOLUBLE ASH (Report results of analysis.) Ash insoluble in hydrochloric acid is the residue obtained after extracting the total ash with hydrochloric acid, calculated with reference to 100 g of the sample under evaluation. To the crucible containing the residue from the determination of total ash, add 15 mlof water and 10 mol f cone. hydrochloric acid. Cover with a watch glass and boil the mixture gently for 10 minutes. Cool and filter through an ashless filter paper. Wash the residue with hot water until the filtrate is neutral.

23 ACID INSOLUBLE ASH (Report results of analysis.) Dry the filter paper and residue at 1050C. Ignite to constant weight at 6000C, cooling in a desiccator between weighings. Note: Constant weight is where the difference between 2 consecutive weighings is not more than 1 mg. Acid Insoluble Ash WResidue x 100 Sample Where; WResidue Weight of Residue obtained in step 3.7 Wsample = Weight of sample used in step 2.2 HEAVY METALS (Report result as Pass / Fail) Carry out the Limit of Lead, Arsenic and Cadmium following General Analytical Method GAMO45 - Metal analysis using ICP -OES. Record the limit of Mercury result from the manufacturer's Certificate of Analysis.

24 RESULTS: STANDARD NAME: BACOPA MONNIERI WHOLE PLANT EXT. DRY J ITEM NO CONG. STAND. 20.1 _ ______ I SPPERTRADE NAME:. Bacopa nne whole pMnt ext dy conc. sMAJd. R8139q 20:~~~~ _(RMEO/Bcpa m-on ed 4501 Racosides / BcNni ______ TEST SECWMATJON RESULT 2 Identification TLC - oihe, 3 Los cm n 0yinq NO momtM SIV :4 Assay for TOWtac waos' es Nci 4 les toan 40 S> A. Total Vle AerobW MM>:r No> man: aMn LOX 1 (Jdu/q Gmimmnhrt r--cahve No nmmcs t IC 11 cbio ----- .. ...... ...... ...... .. ... .. ... . .. ........ .... ... .......... . -------- ------------- c~----------~4 25 STANDARD NAME: BACOPA MONNIERf WHOLE PLANT EXT, DRY I ITEM NO CONC. STAND, 20:1 SUPPLIER TRADE NAME. Baco monnien whole plant ext dry conc. stand. RB139 20:1 (NRBME40) / Bacopa monnir 45% Bcosides fBacoM nd@ TEST SPECFATIONRESULT 2 TotaI AhRo 3 A00 d se ub As h Ntmr hn3 4 Heavy Matals Lead NMT 0 ppm SArsenicNMT 2 ppm Cadi NMIT I ppm M NMT 0 1 ppm TLC Evaluation The chromatogram obtained with the sample solution corresponds to the chromatogram obtained with the working standard solution, as shown in Figure 1.

26 Example 5 [0074] By way of non-limiting example only, an extract of Gingko Biloba was manufactured according to standard procedures to produce an standardised extract which is standardised to > 24% ginkoflavanoid, >/=6% ginkolides and bilogalide and </=ppm ginkgoic acids, as described herein above; Quality testing was carried out on a sample of the extract and compared to a reference standard to ensure the prepared extract was in accordance with the standardised extract. The protocol used was in accordance with the method, as described below: TESTING INSTRUCTIONS DESCRI PTION (Report result as Pass / Fail) Examine the appearance of the sample under evaluation using General Testing Method GTM001 as a guide. Check the colour of the sample against pantone colours. Evaluation criteria Bright yellow- brown (matches with pantone colour 1245 U - 1255 U) fine powder with a characteristic odour. IDENTIFICATION A (Report results as Pass / Fail) APPARATUS A transparent Chromatographic Tank HPTLC plate - Silica Gel 60 F 254 10 X 10 cm SAMPLE SOLUTION /TEST SOLUTION: Pulverise 0.200 g of herbal sample under evaluation using a mortar and pestle to get a fine powder and transfer to a suitable screw-capped test tube. Add 10 mL of a mixture of 2 volumes of water and 8 volumes of Methanol (2: 8) and cap the tube. Warm the mixture on a water bath for 10 minutes. Cool the extract to room temperature and filter using a Millipore Nylon Filter 0.45pm and the particle free extract to be used for HPTLC. PRERARATION OF WORKING STANDARD SOLUTION: Pulverise 0.200 g of working standard of GINKGO BILOBA LEAF EXT. DRY CONC. STAND. 50:1 (RG0607) using a mortar and pestle to get a fine powder and transfer to a suitable screw-capped test tube.

27 Add 10 mL of a mixture of 2 volumes of water and 8 volumes of Methanol (2: 8) and cap the tube. Warm the mixture on a water bath for 10 minutes. Cool the extract to room temperature and filter using a Millipore Nylon Filter 0.45pm and the particle free extract to be used for HPTLC. REFERENCE SOLUTIONS: Dissolve 3 mg of Rutin and 1 mg of Chlorogenic Acid in 5 mL methanol. Procedure: Carry out the test for TLC Identification using General Testing Method GTM021 and the following conditions; Application volume: 2- 4pL Sample solution, Standard solution and Reference solution Solvent system: Ethyl Acetate: Anhydrous Formic acid: Glacial Acetic acid: Water( 1 00: 11 : 11 : 26) Before spraying: Dry in a current of cold air. Spray reagent: A 1% w/v solution of Diphenylboric acid 2-Aminoethyl Ester in Methanol (NP Reagent), followed by separate application of a 5% w/v solution of Polyethylene glycol 4000 in 96% Ethanol (PEG Reagent). After spraying: Dry in a current of cold air. TLC evaluation: Examine under UV366nm. CAUTION: The entire process to be completed in a Fume Hood Apply separately to the plate 2- 4pL of sample solution, standard solution and reference solution. Develop over a path of 8 em using a mixture of 100 volumes of Ethyl Acetate : 11 volumes of Anhydrous Formic acid: 11 volumes of Glacial Acetic acid : 26 volumes of (1 00: 11 : 11 : 26). Dry the plate in a current of cold air until the solvent is removed in the fume hood. Dip the air-dried HPTLC plate in NP Reagent, followed immediately with PEG reagent. Air-dry the plate in a fume hood. Examine under UV366nm.

28 RESULTS AND TLC PROFILE: The chromatogram obtained with the sample solution corresponded to the chromatogram obtained with the working herbal standard solution. The TLC profile obtained is shown in Figure 2. The chromatogram of the reference solution exhibits a yellow-brown fluorescent zone due to rutin in the lower section and a light blue fluorescent zone due to chlorogenic acid in the middle section. See below the sequence of zones present in the chromatograms obtained with the reference solution and the test solution. Furthermore, other, weaker fluorescent zones may be present in the chromatogram obtained with the test solution. I D EN T IFICAT TION B (Report result as Pass /Fail) Examine the chromatograms obtained in the assay for Assay Ginkgoflavonglycosides. See below. The retention times of the peaks for quercetin, isorhamnetin, and kaempferol in the chromatogram of the sample solution correspond to those in the chromatogram of the standard solution. The peak for kaempferol is not less than 0. 7 times the size of the quercetin peak and the peak for isorhamnetin is not less than 0. 1 times the size of the quercetin peak. W AT ER (Report results of analysis. Carry out in duplicate) Determine the% content of water in the sample under evaluation using Karl Fischer Titration following SOP L-083- Metrohm Auto-Titration and Karl Fischer- Operation, Calibration and Maintenance. Use 100 mg of sample.

29 ASSAY FOR GIN KGOFLAVONGLYCOSI DES (Report results of the analysis / Perform in duplicate) Determine the % content of Ginkgoflavonglycosides (On the anhydrous basis) in the sample under evaluation by following General Analytical Method GAM054 Prepare the Sample solution as follows; Sample preparation: * Accurately weigh about 220 mg finely ground sample, equivalent to about 50mg of Ginkgoflavonglucosides into a 50 ml volumetric flask. * Add 30 ml of Methanol (80% v/v), shake for 5 minutes and sonicate for 20 minutes or until dissolved. * Cool, dilute to volume with Methanol (80%v/v) and mix. * Centrifuge a portion of sample solution (- 10 ml) for 20 minutes or until clear. * Pipette 5.0 ml of the clear supernatant and 15.0 ml of acidic methanol solution into a 25 mL pressureproof screw-cap test tube (with a red PTFE rubber septa) and close the cap tightly. * Put the tube into a thermo stated water-bath or block heater at 85 0 C for 2 hours. Remove the test tube from the water-bath and allow to cool to room temperature. * Filter through the nylon 0.22pm filter, (discarding the first 2 mL - Note: this step is critical), into the UPLC vial and cap. * Make duplicate 21pL injections of each sample solution. ASSAY FOR TERPENE LACTONES (GINKGOLIDES AND BILOBALIDE) (Report the result from the manufacturer's Certificate of Analysis) Record the result from the manufacturer's Certificate of Analysis. Note: USP 36/NF31- "Powdered Ginkgo Extract monograph" -Content of terpene lactones method is given below. Solution preparation: Solvent- Prepare a mixture of methanol and water (9: 1). Buffer Dissolve 1.19 g of dibasic sodium phosphate and 8.25 g of solution - monobasic potassium phosphate in 1000 ml of water, and adjust to a pH of about 5.8. Diluent - Prepare a mixture of methanol and water (1 : 1). Solution A - Use filtered and degassed water. Solution B - Use filtered and degassed methanol. Mobile Use variable mixtures of Solution A and Solution Bas directed for phase - Chromatographic system . Make adjustments if necessary. Standard solutions: Using the labeled content of the individual terpene lactones, prepare five solutions of the USP Ginkgo Terpene Lactone reference standard in Diluent, within the range of 5-500 pg per mL for each of the relevant terpene lactones. Use sonication to dissolve the analytes if necessary. Pass through a filter of 0.45pm or finer porosity. Sample solution: 30 Transfer about 120 mg of sample under evaluation, accurately weighed, to a 25-mL beaker. Add 10 mL of Buffer solution to the residue, and sonicate for 5 minutes. Quantitatively transfer the solution to a glass chromatographic tube filled with chromatographic siliceous earth capable of holding 20 mL of aqueous phase. Rinse the beaker with two 5-mL portions of Buffer solution, and transfer the washings to the column. [NOTE- Do not exceed 20 mL of total aqueous phase or the holding capacity of the chromatographic tube.) Allow the Buffer solution to be absorbed into the column. After 15 minutes, elute the column with 100 mL of ethyl acetate, collect the ethyl acetate solution, and evaporate dryness under vacuum on a water bath maintained at 500C Dissolve the residue in 20.0 mL of Diluent. Chromatographic system Analyse the sample and standard solutions by High Performance Liquid Chromatography following SOPO30 as a guide and with the following conditions: Column: C18 (4.6 x 250 mm) 5.pm or equivalent (Octadecyl silane chemically bonded to porous silica or ceramic micro-particles,1.5 to 10 pm in diameter, or a monolithic sil ica rod.) Column temperature: 25 ± 1 Flow rate: 1.OmL Iminute Detector: ELSD (Evaporative light-scattering detector) Note: The parameters of the detector are adjusted to achieve the best signal-to-noise ratio, according to manufacturer recommendations. The chromatograph is programmed as follows. 23-28 5248is rac 354010+7 9096 fiear gra'die 40-50 75~ 25iocaA 31 System suitability: Chromatograph the Standard solutions (about 15 pL) and record the peak responses. The chromatograms obtained are similar to the Reference Chromatogram provided with USP Ginkgo Terpene Lactones reference standard and the relative standard deviation determined from the bilobalide peak for replicate injections is not more than 2.0%. Procedure Separately inject equal volumes (about 15 pL) of each of the Standard solutions and the sample solution into the chromatograph, record the chromatograms, and identify the peaks of the relevant analytes in the chromatogram of the Standard solution by comparison with the Reference Chromatogram. Measure the areas of the analyte peaks. Plot the logarithms of the relevant peak responses versus logarithms of concentrations, in mg per ml, of each analyte obtained from the Standard solutions, and determine the regression line using a leastsquares analysis. The correlation coefficient for the regression line is not less than 0.995. From the graphs so obtained, determine the concentration, C, in mg per mL, of the relevant analyte in the sample solution. Calculation: Separately calculate the percentages of bilobalide (C 1 5

H

1 8 0 8 ), ginkgolide A (C 20

H

24 0 9 ), ginkgolide B (C 20

H

24 0 10 ), and ginkgolide C (C 20

H

24 0 1 1 ) in the portion of sample taken by the formula: 2000(C/W) Where; C = Concentration, in mg per mL, of the relevant analyte in the sample solution W = Weight, in mg, of the sample taken to prepare the sample solution. Calculate the total percentage of ginkgolides and bilobalide (On the Anhydrous Basis) in the sample taken by adding the percentages calculated for each analyte. Acceptance criteria: * Total terpene /actones: NL T 6.0% (On the Anhydrous Basis) * Bilobalide: 2.6% - 3.2% (On the Anhydrous Basis) LIMIT OF GINKGOLIC ACIDS (Report results of the analysis / Perform in duplicate) Perform the limit of Ginkgolic acids either by method given below (Ref: USP 36/NF31 "Powdered Ginkgo Extract monograph'? or by method given in BP 2013 "Refined and Quantified Ginkgo Dry Extract" monograph. Standard solution: 32 Dissolve an accurately weighed quantity of USP Ginkgolic Acids Reference Standard in methanol, and dilute quantitatively, and stepwise if necessary, with water to obtain a solution having a known concentration of about 0.25pg per ml of ginkgolic acids. Sample solution: Transfer about 0.5 g of the sam pie under evaluation, accurately weighed, to a 10-mL volumetric flask, add 8 ml of methanol to dissolve, and dilute with water to volume. Chromatographic system Column: 4.6-mm x 5-cm column that contains base-deactivated packing Octylsilane chemically bonded to totally porous silica particles, 1.5 to 10 pm in diameter) or equivalent Column temperature: 350C Flow rate: 1.0mL /minute Detector: UV 210nm Mobile phase: Solution A - Prepare a solution of 0.01% phosphoric acid in water. Solution B - Prepare a solution of 0.01% phosphoric acid in acetonitrile. Use variable mixtures of Solution A and Solution Bas directed below The chromatograph is programmed as follows. Time Solution A Solution B noites) (%~) (%)____ B4ut on 0-6 25 >10 7590 ar gradient 7 10 90 scai 7-8 -10 >25 90*1-,75 line,-r gaim 8-0 25 73 socrafic System suitability Chromatograph the Standard solution (about 100 IJL), and record the peak responses. The chromatogram obtained is similar to the Reference Chromatogram provided with USP Ginkgolic Acids Reference Standard. The tailing factor is not more than 2.0 for Ginkgolic Acid C 15:1 peak. Relative standard deviation for replicate injections is not more than 5.0% for Ginkgolic Acid C 15:1 peak. Analysis: Standard solution and sample solution Separately inject equal volumes (about 100 IJL) of the sample solution into the chromatograph, record the chromatograms, identify the peaks of the relevant analytes by comparison with the Reference Chromatogram, and measure the areas for the major peaks.

33 [NOTE- Identify the peaks of the relevant analytes by comparison with the reference chromatogram of the USP Ginkgolic Acids reference standard lot being used. If deterioration of peak shapes is observed, wash the column using a mixture of methanol and water (9:1) for 30 minutes]. TLC profile as shown in Figure 2. Calculation: Calculate the concentration, in pg per g (ppm), of each ginkgolic acid in the portion of sample taken by the formula: 10 P ( C/W) (r u I r s) Where; P = Content, in pg per g, of the relevant ginkgolic acid in USP Ginkgolic Acids Reference Standard C = Concentration, in mg per mL, of USP Ginkgolic Acids Reference Standard in the Standard solution W = Weight, in mg, of sample taken to prepare the sample solution r- Peak areas for the relevant analyte obtained from the sample solution rs Peak areas for the relevant analyte obtained from the standard solution Calculate the total amount of ginkgolic acids by adding the individual contents. Limit of Ginkgolic acids is not more than 5 pg per g (NMT 5 ppm). MICROBIOLOGICAL TESTS: A. Total Viable Count Carry out the test for Total Viable Count following Microbiological Testing method MTM006 B. Yeast and Mould Count Carry out the test for Yeast and Mould Count following Microbiological Testing method MTM006 C. Bile-tolerant Gram Carry out the test for Enterobacteriaceae following negative bacteria Microbiological Testing method MTMO17 D. E. coli Carry out the test for E coli following Microbiological Testing method MTM002 E. Salmonella spp Carry out the test for Salmonella spp following Microbiological Testing method MTM004 F. Staphylococcus aureus Carry out the test for Staphylococcus aureus following Microbiological Testing method MTM008 Note: To be tested only for Total Viable Count (TVA C). If TVA C 210 cfulg, proceed for the other micro testing. (Refer to Change Request Number CI-057). PARTICLE SIZE (Report results obtained or Report results of analysis) Determine the percentage of particles that passes through a 250 pm (60 mesh) and passes through a 180 pm (80 mesh) using Malvern Mastersizer following Standard Operating Procedure SOP L076- Malvern Mastersizer 2000- Operation, Calibration and Maintenance or using normal sieves following General Testing MethodGTM026.

34 ADDITIONAL SPECIFICATIONS TESTING INSTRUCTIONS SULPHATED ASH (Report results of analysis.) Determine the Total Ash of the sample under evaluation following General Testing Method GTMO13- Test B. Use 2.0 g of sample. Retain the residue obtained for use in Test 2- Heavy Metals. HEAVY METALS (Report result as Pass / Fail) Carry out the Limit of Lead, Arsenic and Cadmium following General Analytical Method GAMO45 - Metal analysis using ICP - OES. Record the limit of Mercury from the manufacturer's Certificate of Analysis. RESULTS OF TESTS: STANDARD NAME:GINKGO BILOBA LEAF EXT. DRY CONC. STAND. 50:1 ITEM NO SUPPLER T RADE NAME: Ginkgo Biloba Dry Extract / Ginkgoselect (R) RG0607 _____Product Code 36GK60090 TEST [SPECFlCATION RESULT 1 De--r-pt-on Bg---w - brown (ma.ch s wth p ntone c-Oiour 12415 U - 1255 U) flne powder with a characieristic odour Z dentification A -TLC Pao i v 3 dent cation BI The retentin tr es of the peaks for quMcethA iorhamneiin and kampfeo( n the chrlornatogrr of thesmp solution corrcnd~ to thosefln th chromtogamof th standard solutio The pemk k ermpftEi beiwon u 8 and 12 tmres the stze of the qu e' peak; ardthe peak for netn unless han 0A .mes the size of he qe ctn ea :4, Water o ~ot ~~ 5. Assay for Nti tess tha~n 24 0% (On the Anhydrous Ginkgoflavonglycosides Bss fAssay for Terpenei LwAcones Ginkgods and Nct ess tan 60% (On the Anhydrous B' obalide 2.6 -2% (On the Anhydrus BFas e 7. Ltn4 of GifktofkAdd P18ht mcwE th 5 in'i 35 TEST SPECW1CATMO R ESU T A Tota bM e AVmh e C 0or thN l .0 X 10-u Yeast and MouNm a X Byerr G HeNoW mo-ir thani 10 X 10 -c No etedg SEm, ea p Not d lftd 0g SCphylacccud s au No0 td Pat lesh aRecord tepercennge ha passes- throug-h 250TLC sieve mRcord ithe percentage aplesesothot chomt gam obtine wit thmorkng stnadsouin5shw niue2 Tecr NMT U pp OfC NRSL Lnedic NMT 3 -ppm TLC EVALUATION (see also above) The chromatogram obtained with the sample solution corresponds to the chromatogram obtained with the working standard solution, as shown in Figure 2.

36 Example 6 By way of non-limiting example only, an extract of Panax Ginseng was manufactured according to standard procedures to produce an standardised extract which is standardised to 4.25 mg ginsenosides, as described herein above; Quality testing was carried out on a sample of the extract and compared to a reference standard to ensure the prepared extract was in accordance with the standardised extract. The protocol used was in accordance with the method, as described below: TESTING INSTRUCTIONS DESCRIPTION (Report result as Pass IFail) Examine the appearance of the sample under evaluation using General Testing Method GTM001 as a guide. Check the colour of the sample against pantone colour. Evaluation criteria Pale yellowish tan (matches with pantone colour 467 U - 468 U) fine powder with characteristic odour. IDENTIFICATION A (Report results as Pass /Fail) APPARATUS A transparent Chromatographic Tank HPTLC glass plate, Silica Gel, 10 X 10 em (TLC silica gel plate 5-40 pm or TLC silica gel plate 2-10 pm). PREPARATION OF SAMPLE SOLUTION (TEST SOLUTION): Pulverise 1.0 g of herbal material using a mortar and pestle to get a fine powder. Transfer the powder to a suitable screw-capped test tube and add 10 mL of a mixture of 70 : 30 Methanol :Water and cap the tube. Reflux the mixture for 15 minutes followed by sonicating for 15 minutes. Filter the extract using a Millipore Nylon Filter 0.45 11 m and the particle free extract is used for HPTLC. PREPARATION OF WORKING HERBAL STANDARD SOLUTION: Pulverise 1.0 g of working standard of PANAX GINSENG ROOT EXT. DRY CONC. STAND. 3:1 (RP1546) using a mortar and pestle to get a fine powder. Transfer the powder to a suitable screw-capped test tube and add 10 mL of a mixture of 70 : 30 Methanol:Water and cap the tube. Reflux the mixture for 15 minutes followed by sonicating for 15 minutes.

37 Filter the extract using a Millipore Nylon Filter 0.45 pm, and the particle free extract to be used for HPTLC. PREPARATION OF REFERENCE STANDARD SOLUTION Dissolve 5.0 mg of aescin and 5.0 mg of Arbutin in 1 mL of methanol. IDENTIFICATION A Procedure: Carry out the test for TLC Identification using General Testing Method GTM021 and the following conditions; Application volume: 5-6 pL sample and standard solutions and 2-4pL reference solution. Solvent system: Ethyl acetate : Water: Butanol (25 : 50 : 100 VNN); allow to stand for 10 minutes and use the upper layer. Before spraying: Dry in a current of cold air Spray reagent: Anisaldehyde - Sulphuric acid reagent. Prepare by mixing 0.5mL of Anisaldehyde with 10mL of Glacial Acetic acid, followed by 85ml of Methanol and 5mol f Cone. Sulphuric acid, in that order. After spraying: Heat at 105 0 C for 5 to 10 minutes. Examine in visible light. CAUTION: The entire process to be completed in a Fume Hood Apply separately to the plate 5-6 pL of sample solution and standard solution and 2- 4 pL of reference solution. Develop over a path of 8 em using the upper layer of 25 volumes of Ethyl acetate : 50 volumes of Water: 100 volumes of Butanol (25: 50 : 100 VNN) in a saturated chromatographic chamber. Air-dry the plate in a fume hood. Spray the air-dried HPTLC plate in Anisaldehyde- Sulphuric acid Reagent. Heat at 105 0 C for 5 - 10 minutes and examine in visible light.

38 TESTING INSTRUCTIONS IDENTIFICATION OF PANAX NOTOGINSENG RESULTS AND TLC PROFILE: The chromatogram obtained with the sample solution corresponds to the chromatogram obtained with the working herbal standard solution, as shown in Figure 3. See below the sequence of zones present in the chromatograms obtained with the reference solution, the working standard solution and the sample solution. filtr sampl A red-brown color develops at the zone of contact. ID ENTIFICATION B (Report results as Pass /Fail) Examine the sample chromatograms obtained in the assay for Total Ginsenosides See below. The retention times of the peaks for ginsenosides Rg 1 Re 1 Rf, Rb 1 , Rb 2 , Rc 1 and Rd in the chromatogram of the sample solution correspond to those in the chromatogram of the standard solution, as obtained in the test for total ginsenosides.

39 LOSS ON DRYING (Report results of analysis. Perform in duplicate) Determine the Loss on Drying of the sample under evaluation following General Testing Method GTM006. Use 1.000g of sample and dry at 1050C in an oven for 3 hours. ASSAY FOR TOTAL GINSENOSI DES (Report results of analysis. Perform in duplicate) Note: Determine the % content of Total Ginsenosides on the dried basis by UPLC following General Analytical Method GAM076. Prepare the sample solution in duplicate as follows; Sample Solution: - Accurately weigh about 0.500g finely ground sample under evaluation into a 1OOmL low actinic volumetric flask. - Add about 70mL of diluent and shake manually to disperse the sample. Place in an ultrasonic bath for - 15 minutes. Diluent- Ethanol: Acetonitrile: Water (20: 20: 60). - Cool, dilute to volume with diluent and mix well. This would give the final concentration of total - Ginsenosides of about target of 0.3 mg/mL in the solution. - Filter through the nylon 0.22 pm filter, discarding the first few drops, into the UPLC vial and cap. - Inject 5 pL of sample solution into the UPLC system twice. Note: The final concentration of total Ginsenosides standard/sample solution should be in the range of 0.15 - 0.45 mg/mL. SUBSTITUTION OF PANAX QUINQUEFOLIUM (Report results as Pass /Fail) Examine the sample chromatograms obtained in Total Ginsenosides assay (See Test 6). The chromatogram obtained with the sample solution shows a peak due to ginsenoside Rf. In the case of a substitution by Panax quinquefolium no peak due to ginsenoside Rf is present.

40 MICROBIOLOGICAL TESTS: A. Total Viable Carry out the test for Total Viable Count following Microbiological Testing Count method MTM006 B. Yeast and Mould Carry out the test for Yeast and Mould Count following Microbiological Count Testing method MTM006 C. Bile-tolerant Carry out the test for Bile-tolerant Gram-negative bacteria following Gram- negative Microbiological Testing method MTM017 bacteria Carry out the test for E. coli following Microbiological Testing method D. E. coli MTM002 E. Salmonella spp Carry out the test for Salmonella spp following Microbiological Testing method MTM004 F. Staphylococcus Carry out the test for Staphylococcus aureus following Microbiological aureus Testing method MTM008 41 PARTICLE SIZE (Report results obtained) Determine the percentage of particles that passes through a 250 pm (60 mesh) sieve and passes through a 180 pm (80 mesh) sieve using Malvern Master sizer following Standard Operating Procedure SOP L076- Malvern Mastersizer 2000 -Operation, Calibration and Maintenance or using normal sieves following General Testing Method GTM026. ADDITIONAL SPECIFICATIONS TESTING INSTRUCTIONS SULPHATED ASH (Report result of analysis) Carry out the test for Sulphated Ash following General testing Method GTMO13- Method II. Use 1.300 g of sample. HEAVY METALS (Report result as Pass /Fail) Dissolve the ash obtained from Sulphated ash Test using two 5mL quantities of 2M Hydrochloric acid and add 0.1mL of Phenolphthalein solution and 13.5M Ammonia drop-wise until a pink colour is produced. Cool and add glacial Acetic acid until the solution is decolourised, then add a further 0.5mL of glacial Acetic acid. Filter if necessary and dilute the solution to 20mL with water. This is the sample stock solution. Carry out the Heavy Metals Limit Test (A) following General Testing Method GTMO19. Use 10 mL of 2ppm Lead Standard solution as the standard solution and 12 mL of sample stock solution as the sample solution. Any brown colour produced in the Sample solution is not more intense than that obtained in the Standard solution. RESULTS: TLC EVALUATION (see also above) The chromatogram obtained with the sample solution corresponds to the chromatogram obtained with the working standard solution, as shown in Figure 3 42 STANDARD NAME: PANAX GINSENG ROOT EXT. DRY CONC. iTEM NO STAND. 31 SUPPLY ER TRADE NAME Gnse PE 3: 6% (GP3) 1RP546 TEST SPECIFICATION RESULT Desception Pale yellowish ean ( e wit parno coour 467 U - 468 U) fine powder wilh characeristio odour 2 Identification A TLC - Posive 3 dentf cation B Red - brown coomr develops ldent=fcation C The retention times of the peaks for insenosides Rg , Re, Rf Rb, Rb, eand 4 Rd in the chromatogr of the sample sokinn correspond to those in the shrornalogrem of the standard solution, as obtabned in the test for totaldcnsndes s LOsS on Drying No more han 7,0% w/w g Assay for Total Ginenosides NLT do % wi (On the dried basi) Substitution of Panax The sample shou d not he subtitled by quinquefoium Rena>:uirquefiurm (Ginsenoside Rf is present) . 8 Microbioogica Tests: A ftl Viable Aerobc Cun No ore than X 10 ug Yeast and Moud Not mm% M ha M X 1 e u g B e ient Grmnegiative Not momr tan 0 X 1 W a g heterm DE cl COONot detected/g Sarmosa $PC NOt devcted Cg St apyiococaus aureus No: dletecoted/ Pafce a;le Recoat he percentage that passes though 250pm. (60 mesh) sieve. Reoor the perconrage that pas-ss lough a1lp (80 IS rneshi) seve (lfformaton COnly) TEST SPECIFfCATION RESULT 1 Suphated Ash Record 2 Heavy metls Not more than 3 ppr.

43 Example 7 [0075] By way of non-limiting example only, a clinical trial is performed to determine whether 12 months, 18 months and/or 24 months administration of the composition of the invention or effective amount according to any aspect or example described herein, improves cognitive function in a subject. The subject is selected from any group of subjects described herein and the composition of the invention is administered for the described period. One arm of the clinical trial comprises subjects that receive placebo. One or more further clinical trials may be performed by administering the composition of the invention for any length of time and in any subject as described according to any aspect or example herein. [0076] The any one of or more of the following cognitive batteries and tasks are used as primary outcome measures of the clinical trials. These measures are administered at all time points (e.g., baseline, 3, 6, 9, 12, 18 and/or 24 months). -Cognitive Drug Research Computerized Battery -Swinburne University Computerized Cognitive Assessment Battery -Inspection Time Task -Cognitive Demand Battery -Mini Mental State Examination - Hick Reaction Time Paradigm Cognitive Drug Research Computerised Battery [0077] The CDR Computerized Cognitive Assessment System is used to measure the cognitive effects of the treatments. A tailored version of the battery is used, similar to that which has previously been found to be sensitive to improved cognitive function as a consequence of ingestion of numerous nutraceuticals. The selection of computer controlled tasks from the system is administered with parallel forms of the tests being presented at each testing session. Presentation is via colour monitors on laptops, and, with the exception of written word recall tasks all responses are recorded via two-button (YES/NO) response boxes. The word recall tasks are performed with pen and paper, and the Tracking task is performed with a joystick. The entire selection of tasks takes approximately 25-30 minutes. [0078] The tasks are presented in the following order: Immediate Word Recall, Picture Presentation, Simple Reaction Time, Digit Vigilance, Choice Reaction Time, Spatial Working Memory, Numeric Working Memory, Delayed Word Recall, Word Recognition, Picture Recognition, Rapid Visual Information Processing and the Bond-Lader VAS of Mood and Alertness (Bond and Lader, 1974).

44 Swinburne University Computerised Cognitive Assessment Battery (SUCCAB) [0079] The SUCCAB is a battery of screen-based cognitive tasks used to measure cognitive processes that have been found to most likely decline with age. As some cognitive tasks have a motor or decision time component, simple and complex reaction time tasks are also included in the test battery to control for such effects. The tasks forming the battery are described below: Stroop Colour-Word [0080] The test consists of two congruent and two incongruent trials, presented alternately. Stimulus words are randomly presented (RED, BLUE, GREEN, and YELLOW) in either congruent or incongruent colours for 1.7-seconds, with an ISI of 0.5-seconds. Participants respond by pressing one of four buttons corresponding to the colour of the word, irrespective of what the word reads. This task is used as a measure of executive function and more specifically inhibition; participants are required to inhibit the automatic reading response. Spatial Working Memory [0081] In each trial participants are presented with a 4 x 4 white grid on a black background, with six grid positions containing white squares. Participants are given 3 seconds to remember where the white squares are located. The grid becomes blank and a series of four white squares are sequentially displayed in various grid positions for 2 seconds each. Participants respond with a yes/no response to indicate whether each square matches a position that is originally filled. In total, participants complete 14 trials, each of which is separated by a blank screen displayed for 2-seconds. Each trial is set such that two out of the four locations in the response series correspond to the original grid locations, and two do not. The task requires participants to hold spatial information in a store that has previously been described as working memory. Contextual Memory [0082] A series of 20 everyday images are presented at the top/bottom/left/right of the screen for 3-seconds each with no ISI. On completion of the series the same images are displayed again in randomised order in the centre of the screen for 2-seconds each with no ISI. Participants respond with a top/bottom/left /right button press to indicate the original 45 location of each image. This task requires participants to recall the spatial context of the original presentation and is used as a measure of episodic memory. Immediate/Delayed Recognition [0083] Participants are asked to study a series of 40 abstract images presented serially in the centre of the screen for 3-seconds each with no ISI. On completion, another series of images is presented, half of which are from the studied series and half are new (Immediate condition). Participants indicate with a right (yes) or left (no) button press whether or not they recognise the image from the studied series. This task is repeated at the end of the testing session with the remaining 20 images from the studied series and another 20 new images (Delayed condition). Because abstract patterns are difficult to verbalise, the task can be described as a measure of non-verbal recognition memory. Inspection Time Task [0084] In addition, the Inspection Time task is administered to assess speed of information processing. This task assesses the presentation time that a subject requires to discriminate between two possible stimuli. The task consists of a stimulus with two vertical parallel lines joined at the top by a horizontal line. There are two versions of the stimulus; either the left line is shorter than the right or the right line is shorter than the left. Stimuli are flashed on a computer screen and the participant is instructed to press a key corresponding to the side of the symbol that is shorter. Each stimulus presentation is followed by the presentation of a backward visual mask. This prevents further processing of the stimulus in iconic memory. The speed of stimulus presentation is varied according to the accuracy of the participants' responses. The length of presentation of the backward visual mask also varies to determine the optimal visual encoding time. The objective is to respond as accurately, rather than as quickly, as possible. The duration of stimulus presentation is varied until an 80% accuracy level is obtained by the participant. This is taken as the outcome measure for speed of visual information processing speed. Cognitive Demand Battery Mini-Mental State Examination (MMSE) [0085] The Mini-Mental State Examination (MMSE) (Folstein et al., 1975) is a brief 30 point test that is commonly used to screen for dementia. The MMSE evaluates six areas of cognitive function: orientation, attention, immediate recall, short-term recall, language, and 46 the ability to follow simple verbal and written commands. The MMSE is divided into two sections. The first part requires vocal responses to the examiner's questions. The participant is asked to repeat a short phrase after the examiner; to count backward from 100 by 7s; to name the current season and similar brief items. It tests the participants' orientation, memory, and attention. The maximum score for this section is 21. In the second part of the examination, the participant is asked to follow verbal and written instructions, write a sentence spontaneously, and copy a geometric figure. The test is not timed but usually takes less than 10 minutes to complete. Hick Reaction Time Paradigm [0086] The Hick Reaction Time Paradigm (Jensen's Box) is administered to measure choice reaction time. The standard box has a sloping face on which 8 buttons are arrayed in a semicircle, with a 'home' key in the lower center. Above each response button lies a small led which can be illuminated, and the box contains a speaker to play alerting sounds. Several parameters can be extracted: The mean 1-choice reaction time (RT) gives simple reaction time. The slope of the function across 1, 2, 4, and 8 lights gives the rate of information processing; a variance or standard deviation in RTs can be extracted to give a measure of response variability within subjects. Mood and health assessment [0087] Mood and health is assessed using any one or more of the below questionnaires at all time points (e.g., baseline, 3, 6, 9, 12, 18and 24 months). - Bond and Lader Visual Analogue Scales -Profile of Mood Scales -Short Form 36 - Beck Depression Inventory II -The Spielberger State-Trait Anxiety Inventory - Food Frequency Questionnaire - Community Healthy Activities Model Program for Seniors (CHAMPS) questionnaire - Chalder Fatigue Scale - Leeds Sleep Evaluation Questionnaire Bond and Lader Visual Analogue Scales [0088] Visual analogue scales is administered as part of the CDR system to assess self reported mood. In total, 16 dimensions of mood are administered. The participant is required to mark, on a 100 mm line to what extent the described state is appropriate to him/her either 47 at that moment in time ("right now") or over the past week. The Bond and Lader VAS discriminates three affective dimensions: alertness, contentment, and calmness. Profile of Mood States (POMS; McNair, Lorr, & Droppleman, 1992) [0089] The POMS is a self-report questionnaire designed to measure six dimensions of mood: tension-anxiety; depression-dejection; anger-hostility; vigour-activity; fatigue-inertia; and confusion-bewilderment. The POMS consists of 65 adjectives describing feeling and mood which is answered on a five-point Likert-type scale ranging from not at all to extremely. Respondents are asked to indicate mood reactions for the "past week including today". SF-36 (version 2) [0090] The SF-36 is a short-form health survey containing 36 questions. It yields an 8 scale profile of functional health and well-being scores as well as psychometrically-based physical and mental health summary measures and a preference-based health utility index. It will be used to assess changes in health and general well-being throughout the trial. Beck depression Inventory-Il (BDI; Beck et al 1996) [0091] The BDI-Il is a 21-item; self-report inventory designed to measure the severity of depressive symptoms. The BDI-Il is one of the most widely used depression inventories in both clinical and research settings. The BDI-Il asks participants to rate how they have felt over the past 2 weeks on a scale of 0 (no symptoms) to 3 (severe symptoms). Higher scores n the BDI-Il indicates more severe depressive symptoms, with the maximum total score being 63. The BDI-Il has adequate test-retest reliability and high internal consistency. Furthermore, the BDI-Il has been demonstrated to have strong psychometric properties as an assessment tool among older adults in the general population (Segal et al 2008). The Spielberger State-Trait Anxiety Inventory (STAI; Spielberger et al 1969) [0092] The STAI consists of two 20-item questionnaires, designed to measure general anxiety (Trait portion) and anxiety at the time of testing (State portion). EBm consumption has previously lead to a significant decrease in state anxiety using this measure (Stough et al 2001).

48 Chalder Fatigue Scale [0093] The Chalder fatigue Scale (Chalder et al., 1993) is completed by participants to assess physical and mental fatigue. The questionnaire consists of 14 items each rated on a 4-point likert scale. The Chalder Fatigue Sclae has good internal consistency. Leeds Sleep Evaluation Questionnaire [0094] The Leeds Sleep Evaluation Questionnaire (Parrot & Hindmarch, 1978) is completed by all participants to retrospectively assess aspects of sleep including getting to sleep, quality of sleep, awake following sleep and behaviour following wakening. These four dimensions of sleep are assessed through 10 visual analogue scales. Screening and baseline assessment [0095] Although not necessarily clinical endpoints, the following tests are used as screening instruments or to assess the participant's baseline characteristics or function: Mini-Mental State Examination (MMSE) [0096] The MMSE (Folstein et al., 1975) is a brief 30-point test that is used to screen for dementia. The MMSE evaluates six areas of cognitive function: orientation, attention, immediate recall, short-term recall, language, and the ability to follow simple verbal and written commands. The MMSE is divided into two sections. The first part requires vocal responses to the examiner's questions. The participant is asked to repeat a short phrase after the examiner; to count backward from 100 by 7s; to name the current season and similar brief items. It tests the participants' orientation, memory, and attention. The maximum score for this section is 21. In the second part of the examination, the participant is asked to follow verbal and written instructions, write a sentence spontaneously, and copy a geometric figure. The test is not timed but usually takes less than 10 minutes to complete. Dementia Rating Scale II (DRS-II) [0097] The DRS-Il is designed to assess the presence of or the progression towards dementia. Through the administration of 36 tasks and 32-stimulus cards, the DRS-Il is a sensitive in differentiating levels of cognitive deficit. Consequently, the DRS-Il is used to assess cognitive impairment and or dementia. The rater's cognitive performance is evaluated with respect to scaled scores corrected for age and education as outlined in the 49 manual. The DRS-Il is a highly reliable and valid psychometric instrument. The DRS-Il will be used to exclude participants with probable dementia. Geriatric Depression Scale (GDS) [0098] The GDS (Yesavage et al. 1982) is a basic screening measure for depression suitable for elderly populations. Although the GDS cannot be used to diagnose clinical depression it provides insight into the severity of depressive symptoms experienced by the rater. The GDS sums 30 questions each of which are answered "yes" or "no". A total score of 0-9 is considered normal, a score of 10-19 indicates mild depressive symptoms and a score of 20-30 indicates severe depressive symptoms. The GDS is a reliable and valid self rating scale for assessing depressive symptoms in the elderly. The GDS will be used to exclude participants with severe depression. General intelligence (IQ), Wechsler abbreviated scale of intelligence (WASI) [0099] Participants complete the vocabulary and matrix reasoning subsets of the Wechsler Abbreviated Scale of Intelligence (WASI). The vocabulary subtest is a 42-itemtask, which requires the examinee to orally define words that are presented visually and orally. The matrix reasoning subtest shows a series of 35 incomplete grid patterns, which the examinee is asked to complete by pointing to, or stating, the correct pattern from five possible choices. The WASI is a reliable measure of intelligence for use in clinical and research settings. The Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) [00100] The RBANS is used as a neuropsychological get battery for the clinical diagnosis/tracking of dementia. The RBANS is a series of 12 subtest that are conducted by a trained physician. The RBANS generates index scores for 5 neurocognitive domains as well as a total scale index. (Karantzoulis S et al. Archives of Clinical Neuropsychology. 2013). Montreal Cognitive Assessment (MCA) [00101] The MCA is a cognitive screening test for the diagnosis and tacking of mild cognitive impairment. The MCA consists of 30 point questionnaire that is delivered by trained physicians. (Freitas et al. Alzheimer Disease & Associated Disorders. 2013).

50 Analysis [0100] The primary analysis investigates the effects of treatment on all cognitive outcomes over the course of the trial using Analysis of Variance (ANOVA) techniques. Other techniques may also be used, e.g., linear mixed modelling and intention to treat analysis. Similarly, effects of treatment on secondary outcomes are analysed. Pearson's correlation coefficients are used to investigate whether any improvements in cognition are related to improvements in other variables e.g., biochemical, cardiovascular or mood/health factors and/or cerebral energy including glucose uptake. Correlations and regression models are used to examine baseline associations between variables. Results are considered statistically significant at p<0.05 corrected for multiple cognitive factors (primary outcome variables) or p<.05 for secondary outcome variables. [0101] Covariates such as age, gender and baseline cognitive screening measures are adjusted for in the analyses. Results are presented as appropriate effect sizes with a measure of precision e.g., 95% confidence intervals. Compliance to treatment are determined by counting each of the trial subject's remaining supplements after completion of the trial.

Claims

1. A composition comprising an effective amount of lipoic acid (5-(1,2-dithiolan-3 yl)valeric acid) or an alternative form, derivative, or salt thereof, or a combination thereof, and/or a source thereof, and at least one extract selected from the following: (i) an extract prepared from at least one Bacopa spp.; (ii) an extract prepared from Ginkgo biloba; and (iii) an extract prepared from at least one Panax spp., to improve or maintain mood and/or cognitive function in a subject; or to slow the age-related decline in the mood and/or cognitive function in a subject.

2. A method for improving or maintaining mood and/or cognitive function in a subject or slowing the age-related decline in the mood and/or cognitive function in a subject comprising administering lipoic acid (5-(1,2-dithiolan-3-yl)valeric acid) or an alternative form, derivative, or salt thereof, or a combination thereof, and/or a source thereof, and at least one extract selected from the following: (i) an extract prepared from at least one Bacopa spp.; (ii) an extract prepared from Ginkgo biloba; and (iii) an extract prepared from at least one Panax spp., together, or separately in any order, in an effective amount and for a time sufficient to improve or maintain the mood and/or cognitive function in the subject, or slow the age related decline in the mood and/or cognitive function in the subject.

3. The composition or the method of claim 1 or 2, wherein the Bacopa spp. is Bacopa monnieri and/or the Panax spp. is Panax ginseng.

4. The composition or method according to any one of claims 1 to 3, wherein the lipoic acid (5-(1,2-dithiolan-3-yl)valeric acid) is selected from alpha-lipoic acid (ALA), thioctic acid and 6,8-Dithioctanoic acid, salts thereof and combinations thereof; or the lipoic acid (5-(1,2 dithiolan-3-yl)valeric acid) is alpha-lipoic acid (ALA), including one or a mixture of both enantiomers (R)-(+)-alpha-lipoic acid (RLA), or (S)-(-)-alpha-lipoic acid (SLA), at various concentrations or preferably, a racemic mixture (R/S)-alpha-lipoic acid (R/S-LA), or salts thereof or combinations thereof.

5. The composition or the method according to any one of claims 1 to 4, wherein the effective amount comprises a standardised Bacopa monnieri extract in an amount from 2 g to 10 g, preferably about 4 g of whole leaf extract standardised equivalent to about 90 mg 52 bacosides A; and/or a standardised Ginkgo biloba extract in an amount from 2 g to 5 g, preferably about 4 g of whole leaf extract standardised equivalent to about 16 mg ginkgo flavonglycosides, and about 4 mg ginkgolides and bilobalides; and/or a standardised Panax gingseng extract in an amount from 210 mg to 300 mg, preferably about 212 mg of ginseng root extract standardised equivalent to about 4 mg ginsenosides Rgl,Re, Rf, Rg2, Rbl, Rb2, Rc, Rd; and (R/S)-alpha-lipoic acid an amount from 200 mg to 600 mg, preferably about 300 mg.