JP7655920B2 - Compositions for use in treating cognitive disorders - Google Patents

Description

The present invention relates to a composition containing a plant extract for use in treating cognitive disorders such as Alzheimer's disease, mild cognitive impairment, or Parkinson's disease.

Dementia is the loss of cognitive function (thinking, remembering, and reasoning) and behavioral abilities that interferes with a person's daily life and everyday activities. Dementia can range in severity from the mildest stage, where it only just begins to affect a person's function, to the most severe stage, where a person must become completely dependent on others for basic activities of daily living. Common types of dementia include Alzheimer's disease, vascular dementia, Lewy body disease, frontotemporal dementia, or early-onset dementia. Approximately 60%-70% of dementia patients have Alzheimer's disease (AD), and approximately 6% of subjects aged 65 and older are diagnosed with AD.

AD is a neurodegenerative disease with a progressive pattern of cognitive and functional impairment. In the early stages of the disease, patients may only show short-term memory loss and minor problems with executive functions of attention, planning, flexibility, and abstract thinking, or impaired semantic memory (memory of meanings and conceptual relationships). As the disease progresses, memory impairment increases and there is a decline in language, literacy, and coordination of motor sequences. In advanced stages, patients may completely lose the ability to speak and show a decline in muscle mass and mobility that prevents them from feeding themselves, becoming completely dependent on caregivers.

Mild cognitive impairment (MCI) is often seen as a transitional stage between normal aging and dementia, especially Alzheimer's disease. MCI affects approximately 15% to 20% of people over the age of 65. MCI can be classified as amnestic and non-amnestic MCI. Amnestic MCI is characterized by a loss of memory by the affected individual, causing them to forget important information that they would previously have easily recalled, such as appointments, conversations, or recent events. Non-amnestic MCI is characterized by a loss of cognitive abilities other than memory, such as the ability to make correct decisions, determine the time or sequence required to complete a complex task, or vision. Although MCI can revert to a normal stage or remain stable, amnestic MCI is frequently seen as a prodromal stage of AD. Non-amnestic MCI patients have been found to be more susceptible to developing other dementias.

There is no conclusive evidence to support any specific measures to prevent AD. Furthermore, no drugs have been shown to slow or stop the progression of AD. Available treatments offer relatively small symptomatic benefits. No drugs have been proven effective in treating MCI, and no high-quality evidence has been provided for pharmaceutical drugs or dietary supplements that may improve cognitive symptoms in patients with MCI.

Therefore, there is a need in the art for effective treatments to halt or even reverse cognitive decline in patients with dementia, particularly AD or MCI.

The inventors have shown that plant extracts rich in ursolic acid (sage extract) and D-pinitol have neuroprotective effects as well as effects on acetylcholinesterase activity in a zebrafish model organism (the AB zebrafish line treated with pentylenetetrazole (PTZ)) for neurodegenerative diseases. Studies on the development of the zebrafish CNS (Kimmel et al., 1995, Developmental Dynamics 203:255-310) have shown that at 24 hours, the brain is already segmented, with structures such as the neural tube, notochord and somites (precursors of muscle and skeleton) forming. At the fifth day of development, several sensory organs such as the eyes and ears are formed. The heart, liver, kidneys and pancreas have also appeared, and the circulatory, digestive and nervous systems are fully functional. At this stage, the fish can respond to visual, olfactory and mechanical stimuli and begin to swim in search of food.

AChE is an enzyme that catalyzes the hydrolysis of the acetylcholine neurotransmitter to choline and acetate groups. AChE is found primarily at the neuromuscular junction and in the cholinergic nervous system, where its activity serves to terminate synaptic transmission. Acetylcholine is a neurotransmitter involved in the control of movement and is an important regulator of cognitive functions such as learning and memory (Hasselmo et al, 2011, Neuropsychopharmacology, 1:52-73). Therefore, adequate levels of acetylcholinesterase reflect a healthy neurological state.

PTZ is a competitive stimulator of gamma-aminobutyric acid (GABA) receptors. Its activity at the receptors blocks Cl- anion conductance and the formation of inhibitory postsynaptic potentials, increasing glutamatergic excitability (McDonald et al, 1978, Science, 200:775-777). In the zebrafish model, it exerts proconvulsant effects, possibly by blocking GABAergic inhibitory synaptic transmission (Huang et al, 2001, J. Pharmacol. Exp. Ther. 298:986-995).

Furthermore, the inventors analyzed the effect of administering a plant extract rich in ursolic acid together with a plant extract rich in D-pinitol, a Ginkgo biloba extract and a fatty acid composition rich in DHA to a group of SAMP8 (senescence-accelerated mouseprone 8) mice that show pathological similarities to AD patients (Pallas M. 2012, ISRN Cell Biology, Vol. 12, ArticleID 917167). They observed that after the treatment, the ability of the mice in the group to take on cognitive functions reduced in AD patients was comparable to that of control mice (senescence-accelerated mouse resistant 1, SAMR1). Thus, they demonstrated that administration of a composition containing a plant extract rich in ursolic acid, a plant extract rich in D-pinitol, a Ginkgo biloba extract and a fatty acid composition rich in DHA can restore severely impaired cognitive functions in AD patients.

The present inventors have also shown that administration of a composition comprising a DHA-rich fatty acid composition, a flavonoid-rich ginkgo extract, a D-pinitol-rich plant extract, and a ursolic acid-rich plant extract to C. elegans results in improved oxidative resistance, extended lifespan, increased chemotactic activity, and reduced Aβ aggregation when compared to control animals.

Thus, in a first aspect, the present invention provides a method for the preparation of a composition comprising D-pinitol, ursolic acid,
(i) Docosahexaenoic acid (DHA),
(ii) ginkgo flavonoids, and
(iii) mixtures thereof;
and one or more additional components selected from the group consisting of:

In a second aspect, the present invention relates to a pharmaceutical composition comprising a composition according to the first aspect and a pharma- ceutical active carrier.

In a third aspect, the present invention relates to a nutritional or dietary supplement comprising a composition according to the first aspect and a nutritionally acceptable carrier.

A composition or a kit of parts according to any one of the claims of the first aspect of the invention, or a pharmaceutical product according to the second aspect of the invention, or a dietary supplement or health supplement according to the third aspect of the invention, for use in medicine.

FIG. 1 shows the SAMR1/SAMP8 preclinical trial schedule. FIG. 1 shows an outline of the open field test (OFT). FIG. 1 shows an overview of the Novel Object Recognition Test (NORT). FIG. 1 shows an overview of the Object Location Test (OLT). A, Weight gain/loss of animals during treatment. B, Mean weights at the start and end of the study. Behavioral assessment of groups of mice (n=10-12) in an open field. A) Locomotor activity B) Vertical activity C) Defecation FIG. 1 shows a summary of short-term NORT results for each group (n=10-12). FIG. 1 shows a summary of long-term NORT results for each group (n=10-12). FIG. 1 shows a summary of the habituation (OLT) results for the three cameras for each group (n=10-12). A) Examples of the physical appearance of SAMP8 control mice and B) Examples of the physical appearance of SAMP8 treated mice. Bar graph showing the mean ± SEM of AChE activity in larvae treated with PTZ, PHYS, and various compounds in combination with PTZ, relative to the control group (dashed red line) considered as 100%. Statistical methods used were Dunnett's multiple comparison test (* p<0.05, ** p<0.01 vs. control) and Bonferroni's multiple comparison test (# p<0.01, ## p<0.001 vs. PTZ). Summary of oxidative stress response of different C. elegans groups after treatment with each extract, mixture or vitamin C (58 μM). Different symbols indicate significant differences between the groups (P<0.05). Data are the average of three replicates with approximately 120-150 worms in each group. FIG. 1 shows a schematic of chemotaxis assay behavior in the neuronal Aβ-expressing line CL2355. Chemotaxis assay with neuronal line CL2355. Different symbols indicate significant differences between groups (P<0.05). Data are the average of three replicates with approximately 120-150 worms in each group. Quantification of ThS-positive particles in the head region of strain CL2006 (A). Representative images of each group tested (B). Different symbols indicate significant differences between the groups (P<0.05). Data are the average of three replicates with approximately 50-60 worms in each group. Kaplan-Meier curves of C. elegans survival in response to various extracts (A). Mean life span of treated groups of C. elegans and DMSO 1% control (B). Different symbols indicate significant differences between the groups (P<0.05). Data are the average of three replicates with approximately 60-70 worms in each group.

The inventors have observed that plant extracts rich in ursolic acid, as well as plant extracts rich in D-pinitol, have neuroprotective effects in a zebrafish model of neurodegenerative disease. The inventors have also observed that administration of a composition obtained by combining a DHA-rich fatty acid composition, a plant extract rich in ursolic acid, a plant extract rich in D-pinitol, and a Ginkgo biloba extract to an accelerated aging mouse model can reverse some of the impaired cognitive function in the model animal.

1. Composition or kit of parts of the invention In a first aspect, the invention provides a composition comprising D-pinitol, ursolic acid,
(i) Docosahexaenoic acid (DHA),
(ii) ginkgo flavonoids, and
(iii) mixtures thereof;
and one or more additional components selected from the group consisting of:

The term "composition" as used herein refers to a combination of compounds or components. The components of a composition can be provided separately or in a unit dosage. Thus, when a composition is administered to a subject, the compounds of the composition can be mixed together in said unit dosage, provided separately but mixed together prior to administration, or provided and administered separately but mixed once ingested by the subject, i.e., within the subject's body. Additionally, some components of a composition may be administered together and other components administered separately, but they are all mixed once ingested by the subject, i.e., within the subject's body.

The term "kit of parts" as used herein refers to a product that includes various components, ingredients, or compounds, which are preferably physically separated by separate packaging of each component, ingredient, or compound in the kit to allow for transportation and storage. As will be understood, in a "kit of parts" according to the present invention, each active ingredient, ingredient, or compound represents a therapeutic agent, and when used simultaneously, separately, or sequentially, these compounds produce a novel and unexpected joint therapeutic effect described herein that is not achieved by the compounds independently of each other. Indeed, as shown by the results below, the claimed combination of active ingredients does not represent a mere collection of known drugs, but rather a novel combination that has the surprising and valuable property that the combined effect is much more significant than the simple sum of the effects observed when the active ingredients are used separately. A kit of parts typically contains its components in a suitable container. Suitable materials for packaging the components of the kit include glass, plastic (polyethylene, polypropylene, polycarbonate, etc.), bottles, vials, paper, sachets, etc. For example, each container may be a vial, bottle, squeeze bottle, jar, sealed sleeve, envelope or pouch, tube or blister package, or any other suitable form, provided that the container is configured to prevent premature mixing of the components. Each of the various components may be provided separately, or some of the various components may be provided together (i.e., in the same container). Additionally, the kits of the invention may contain instructions for the simultaneous, sequential, or separate use of the various components in the kit. The instructions may be in the form of printed matter or in the form of an electronic medium on which the instructions can be stored such that they can be read by the subject, such as an electronic storage medium (magnetic disk, tape, etc.), an optical medium (CD-ROM, DVD), etc. The medium may additionally or alternatively contain an internet address providing the instructions.

It will be understood that the compositions and kits of parts of the present invention may comprise, consist essentially of, or consist of the aforementioned components.

In this specification, the term "comprising" or "comprises" is used to indicate that the composition described must contain the recited component(s) but may optionally contain additional components. The term "consisting essentially of" or "consists essentially of" is used to indicate that the composition described must contain the recited component(s) and may also contain small amounts (e.g., up to 5 weight percent, or up to 1 weight percent or 0.1 weight percent) of other components, provided that any additional components do not affect the essential characteristics of the extract or composition. The term "consisting of" or "consists of" is used to indicate that the composition described must contain only the recited component(s).

In certain embodiments, the composition or kit of parts according to the first aspect of the invention comprises further components in addition to D-pinitol and ursolic acid. In certain embodiments, the composition or kit of parts comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 components in addition to D-pinitol and ursolic acid. In another specific embodiment, D-pinitol and ursolic acid comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 66.6%, at least 66.66%, at least 66.666%, at least 67%, at least 70%, or at least 100% of the total amount of components constituting the kit.

As used herein, the terms "D-pinitol", "pinitol", "3-O-methyl-D-chiro-inositol", and "methylinositol" refer to a compound containing the IUPAC name (1S,2S,4S,5R)-6-methoxycyclohexane-1,2,3,4,5-pentol. It can be obtained from a variety of natural sources, such as the pods of Ceratoniasilique, the leaves of Sutherlandia frutescens, or Pinus lambertiana.

The term "ursolic acid" as used herein refers to a triterpenoid with the IUPAC name (1S,2R,4aS,6aR,6aS,6bR,8aR,10S,12aR,14bS)-10-hydroxy-1,2,6a,6b,9,9,12a-heptamethyl-2,3,4,5,6,6a,7,8,8a,10,11,12,13,14b-tetradecahydro-1H-picene-4a-carboxylic acid. It is present in many plants, including fruits and medicinal herbs, used in daily life. In particular, it is found in plants of the Lamiaceae family, such as Salvia officinalis, Thymus vulgaris, Rosmarinus officinalis, and Origanum vulgare, in microalgae, and in the skin of fruits of Malus domestica, Pyrus communis, Vaccinium spp., and Prunus spp.

In certain embodiments of the composition or kit of parts of the first aspect of the invention, the ursolic acid is provided as a plant extract rich in ursolic acid and/or the D-pinitol is provided as a plant extract rich in D-pinitol.

The expression "plant extract" as used herein refers to a term commonly known by experts in the field. It usually refers to a composition containing compounds, components or substances extracted from a plant organism, or a plant product or tissue, by treating said tissue with a solvent. Non-limiting examples of solvents include water, ethanol, hydroalcohol, ethyl acetate, _CO2 , methanol, acetone, acetic acid or hexane. Methods for obtaining plant extracts from plants or plant organism products or tissues are known by experts in the field and include any of the methods for obtaining plant or natural extracts described in the examples of the present invention.

As will be understood by those skilled in the art, a "plant extract rich in ursolic acid" or a "plant extract containing ursolic acid" is a plant extract obtained from a product or tissue of a plant organism that is rich in ursolic acid, i.e. contains a large amount of ursolic acid, resulting in a high final concentration of ursolic acid in the extract. Non-limiting examples of products or tissues of plant organisms rich in ursolic acid include leaves, marine algae or fruit shells of plant organisms of the Lamiaceae family. In particular, they include products or tissues from Salvia officinalis, Thymus vulgaris, Rosmarinus officinalis, Origanum vulgare, microalgae, or the skin of fruits of Malus domestica, Pyrus communis, Vaccinium spp., Prunus spp. The plant extract can be obtained by any method known by a person skilled in the art, such as those provided in the embodiments of the method for obtaining an extract rich in ursolic acid herein or in the examples of the present invention under "Method for obtaining a natural extract rich in ursolic acid".

In a particular embodiment, the ursolic acid-rich product from which the ursolic acid-rich plant extract is obtained is a product from a botanical organism selected from the group consisting of leaves, marine algae, or fruit shells of plants of the Lamiaceae family. In another particular embodiment, the plant product is a product from a botanical organism selected from the group consisting of Salvia officinalis, Thymus vulgaris, Rosmarinus officinalis, Origanum vulgare, marine algae, Malus domestica, Pyrus communis, Vaccinium spp., Prunus spp., and combinations thereof.

As used herein, the term "product from a plant organism" or "plant product" refers to any part of a plant organism, including tissue from a plant organism, tissue from a reproductive organ of a plant organism, tissue from a non-reproductive organ of a plant organism, leaves, stems, roots, fruits, tissue from a fruit, exocarp of a fruit, mesocarp of a fruit, endocarp of a fruit, fruit shell, fruit pod, fruit seed, or pod of a plant organism. In certain embodiments, the product refers to the entirety of any of the parts of a plant organism listed above. In other specific embodiments, it refers to a portion of any of the parts of a plant organism listed above.

In a preferred embodiment, the ursolic acid-rich product from which the ursolic acid-rich plant extract is obtained is the leaves of a botanical organism selected from the group consisting of Salvia officinalis, Thymus vulgaris, Rosmarinus officinalis, Origanum vulgare, preferably the leaves of Salvia officinalis. In another preferred embodiment, it is a product from a marine alga, preferably an alga selected from the group consisting of Cladophora species, preferably Cladophora avagabunda. In another preferred embodiment, it is a fruit shell from Malus domestica, Pyrus commennis, Vaccinium spp., Prunus umbellata.

In another particular embodiment, the ursolic acid-rich extract is obtained from two or more ursolic acid-rich products selected from any of the above groups of ursolic acid-rich products. In another particular embodiment, it is obtained from at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 13, at least 14, at least 15 products selected from any of the above groups of ursolic acid-rich products. In another particular embodiment, it is obtained from all products set forth in any of the above groups of ursolic acid-rich products.

In one embodiment, the ursolic acid rich extract is obtained by extraction from a plant product rich in ursolic acid using a solvent selected from the group consisting of hydroalcohol, petroleum ether, chloroform, methanol, acetone, acetonitrile, ethyl acetate or mixtures thereof.

The product is selected from any of the above groups of ursolic acid-rich products. In another particular embodiment, the ursolic acid-rich plant extract is a hydroalcoholic extract obtained from two or more of the ursolic acid-rich products selected from any of the above groups of ursolic acid-rich products. In another particular embodiment, it is obtained from at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 13, at least 14, at least 15 products selected from any of the above groups of ursolic acid-rich products. In another particular embodiment, it is obtained from all the products indicated in any of the above groups of ursolic acid-rich products. In another embodiment, the ursolic acid-rich plant extract is a hydroalcoholic extract of a ursolic acid-rich plant product.

As will be understood by those skilled in the art, a hydroalcoholic extract of a plant product rich in ursolic acid is an extract obtained from a plant biological product rich in ursolic acid using a hydroalcohol as a solvent. As known by experts in the field, a hydroalcohol is a solvent that includes water and alcohol. Non-limiting examples of alcohols contained in a hydroalcohol are ethanol or methanol. In certain embodiments, the percentage of alcohol (volume/volume) in the hydroalcohol is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%. In certain embodiments, the hydroalcohol has 2% (v/v) to 99% (v/v) alcohol, 5% (v/v) to 96% (v/v) alcohol, 10% (v/v) to 90% (v/v) alcohol, 20% (v/v) to 80% (v/v) alcohol, 30% (v/v) to 70% (v/v), 50% (v/v) to 70% (v/v) alcohol, preferably 5% (v/v) to 96% (v/v) alcohol. As will be understood by those skilled in the art, when the hydroalcohol is defined by the percentage of alcohol present in the hydroalcohol, the percentage of water present in the hydroalcohol is the remaining percentage to reach 100%. Thus, if the hydroalcohol has 5% (v/v) alcohol, it has 95% (v/v) water, and if the hydroalcohol has 96% (v/v) alcohol, it has 4% (v/v) water. Methods for obtaining hydroalcoholic extracts of plant products rich in ursolic acid are known by those skilled in the art. Non-limiting examples of such methods are as provided in the embodiments for obtaining extracts rich in ursolic acid herein and in the examples herein under "Method for obtaining natural extracts rich in ursolic acid".

In another particular embodiment, the plant extract is obtained using an alcohol, such as ethanol or methanol, as the solvent. In another particular embodiment, the plant extract is obtained using water as the solvent.

In certain embodiments, the plant extract rich in ursolic acid has a percentage (w/w) of at least 0.1%, at least 0.2%, at least 0.25%, at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 7%, at least 10%, at least 15%, at least 17.5%, at least 20%, at least 22.5%, at least 25%, at least 27.5%, at least 30%, at least 35%, at least Each contains 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 100%, and preferably at least 15% ursolic acid.

In one embodiment, the extract rich in ursolic acid is obtained from the leaves of plants of the Lamiaceae family, such as Salvia officinalis, Thymus vulgaris, Rosmarinus officinalis, Origanum vulgare, from seaweed, from the skin of fruits of Malus domestica, Pyrus communis, Vaccinium spp., Prunus spp., or mixtures thereof.

In one embodiment, the selected raw or dried material is extracted with a hydroalcoholic mixture having an alcohol content of 10% to 96% at a temperature of about 40°C to 100°C for 1 hour to 10 hours. The method can also include several stages of extraction of leaves with the same or different alcohol contents, combining the obtained hydroalcoholic extracts and continuing the method.

In another embodiment, the hydroalcoholic extract and raw material are separated using any separation method that results in an extract that does not contain particles larger than 50 microns to 100 microns.

In one embodiment, the hydroalcoholic extract is treated with activated carbon at 40°C to 100°C for 1 hour to 4 hours. The dosage of activated carbon is 1% to 20% of the dry matter present in the extract. The hydroalcoholic extract is then separated from the activated carbon using any separation method that results in an extract that is free of activated carbon particles and particles of raw material larger than 0.45 microns to 10 microns.

In another embodiment, the aqueous extract is processed using microfiltration or heat treatment to control any possible microbial load.

In another embodiment, the resultant is concentrated to a solids content of 5% (w/w) to 50% (w/w). During this step, an insoluble precipitate is formed in the concentrated extract. The precipitate is separated from the supernatant by any method known in the art.

In another embodiment, the wet precipitate is dried using any drying method that results in a solid product having a moisture content of less than 10%-15%.

In one embodiment, the extract has a triterpene content of 5% to 95%, of which 5% to 90% is composed of ursolic acid. In a particular embodiment, the ursolic acid-rich plant extract contains 0.25% (w/w) to 100% (w/w) ursolic acid, preferably 2% (w/w) to 100% (w/w) ursolic acid, more preferably 5% (w/w) to 90% (w/w) ursolic acid.

Methods for determining the percentage of ursolic acid in an extract are known by experts in the field. Said methods include any method that allows to determine the amount of the compound in the composition, for example mass spectrometry. Preferably, mass spectrometry is used, in particular gas chromatography combined with mass spectrometry (GC-MS), liquid chromatography combined with mass spectrometry (LC-MS), direct injection mass spectrometry or Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), capillary electrophoresis combined with mass spectrometry (CE-MS), high performance liquid chromatography combined with mass spectrometry (HPLC-MS), ultra high performance liquid chromatography combined with mass spectrometry (UHPLC-MS), supercritical fluid chromatography combined with mass spectrometry (SFC-MS), flow injection analysis with mass spectrometry (FIA-MS), including quadrupole mass spectrometry, any sequentially coupled mass spectrometry such as MS-MS or MS-MS-MS, inductively coupled plasma mass spectrometry (ICPMS), pyrolysis mass spectrometry, Mass spectrometry using a photoionization technique is often used, for example, photoionization (Py-MS), ion mobility mass spectrometry or time-of-flight mass spectrometry (TOF), electrospray ionization mass spectrometry (ESIMS), ESI-MS/MS, ESI-(MS)<n>, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOFMS), desorption/ionization on silicon (DIOS), secondary ion mass spectrometry (SIMS), quadrupole time-of-flight (Q-TOF), atmospheric pressure chemical ionization mass spectrometry (APCI-MS), APCI-MS/MS, APCI-(MS)<n>, atmospheric pressure photoionization mass spectrometry (APPI-MS), APPI-MS/MS, and APPI-(MS)<n>, quadrupole mass spectrometry, Fourier transform mass spectrometry (FTMS), and ion trap mass spectrometry, where n is an integer greater than 0. The above techniques are disclosed, for example, in Nissen, Journal of Chromatography A, 703, 1995: 37-57, U.S. Pat. No. 4,540,884 or U.S. Pat. No. 5,397,894. Preferably, gas chromatography combined with mass spectrometry is used as described in (Garcia A. and Barbas C. 2011, Methods Mol. Biol. (Clifton NJ) 708:191-204). Even more preferably, quadrupole time-of-flight gas chromatography combined with mass spectrometry (GC-qTOF/MS) is used as described in (Riera-Borrull M. et al. 2016, J. Am. Soc. Mass Spectrom. 27(1): 168-177, Kind T. et al., 2009, Anal. Chem. 81(24): 10038-48).

In certain embodiments, the ursolic acid-rich plant extract contains additional triterpenes. In certain embodiments, the ursolic acid-rich plant extract contains 5% (w/w) to 95% (w/w) triterpenes. In another particular embodiment, the ursolic acid-rich plant extract contains 5% (w/w) to 95% (w/w) triterpenes, of which 5% (w/w) to 90% (w/w) is ursolic acid. Methods for determining the concentration of triterpenes in the ursolic acid-rich plant extract are known by those skilled in the art and include any of the methods provided above for determining the concentration of a compound in a composition.

In certain embodiments, the ursolic acid rich plant extract comprises:
(i) hydroalcoholic extraction of the ursolic acid-rich product;
(ii) treating the hydroalcoholic extract obtained in step (i) with activated carbon, followed by removal of the activated carbon;
The method is obtained by a method comprising the steps of:

In a particular embodiment, the extraction in step (i) of the method for obtaining an extract rich in ursolic acid is carried out on two or more of the ursolic acid-rich products selected from any of the above groups of ursolic acid-rich products. In another particular embodiment, the extraction in step (i) is carried out on two or more of the ursolic acid-rich products selected from any of the above groups of ursolic acid-rich products. In another particular embodiment, it is an extraction from at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 13, at least 14, at least 15 products selected from any of the above groups of ursolic acid-rich products. In another particular embodiment, it is an extraction from all the products shown in any of the above groups of ursolic acid-rich products.

In certain embodiments, the ursolic acid rich plant extract comprises:
(i) hydroalcoholic extraction from the leaves, marine algae, or fruit shells of plants of the Lamiaceae family;
(ii) treating the hydroalcoholic extract obtained in step (i) with activated carbon, followed by removal of the activated carbon;
The method is obtained by a method comprising the steps of:

In another particular embodiment, the extraction in step (i) of the method for obtaining an extract rich in ursolic acid is carried out using a hydroalcohol as per any of those given in the definition of hydroalcohol, preferably using a hydroalcohol having between 5% (volume/volume) and 96% (volume/volume) alcohol.

In another particular embodiment, the extraction step (i) of the method for obtaining an extract rich in ursolic acid is carried out at a temperature above 30°C, above 40°C, above 45°C, above 50°C, above 60°C, above 70°C, above 80°C, above 90°C, above 95°C, above 96°C, above 97°C, above 98°C, above 99°C, above 100°C, above 105°C, above 110°C. In a preferred embodiment, it is carried out at a temperature between about 30°C and 110°C, preferably between about 40°C and 100°C.

In another particular embodiment, the extraction step (i) of the method for obtaining an extract rich in ursolic acid is carried out for at least 0.5 hours, at least 1 hour, at least 1.5 hours, at least 2 hours, at least 3 hours, at least 3.5 hours, at least 4 hours, at least 4.5 hours, at least 5 hours, at least 5.5 hours, at least 6 hours, at least 6.5 hours, at least 7 hours, at least 7.5 hours, at least 8 hours, at least 8.5 hours, at least 9 hours, at least 9.5 hours, at least 10 hours, at least 10.5 hours, at least 11 hours, at least 12 hours, at least 15 hours, at least 18 hours, at least 20 hours, at least 24 hours. In a particular embodiment, the method is carried out for 0.5 hours to 24 hours, 1 hour to 12 hours, 1 hour to 10 hours, preferably 1 hour to 10 hours.

In another particular embodiment, step (i) of the method for obtaining an extract rich in ursolic acid is repeated at least once, at least twice, at least three times, at least four times, at least five times, and the hydroalcoholic extract used in step (ii) of the method is a combination of the hydroalcoholic extracts obtained in each repetition of step (i). In another particular embodiment, the hydroalcohol used in each repetition of step (i) of the method is the same in each repetition. In another particular embodiment, the hydroalcohol used in each repetition is not the same in each repetition. In another particular embodiment, the hydroalcohol used in each repetition has a different alcohol concentration in each repetition. In a particular embodiment, each of the hydroalcohols used in each of the above repetitions is selected from the hydroalcohols set out in the definition of hydroalcohol above.

In another particular embodiment, the ursolic acid-rich product used in step (i) of the method is the same in each repetition of step (i) of the method. As will be appreciated by those skilled in the art, in this case the same ursolic acid-rich product is subjected to several extractions with hydroalcohol.

In certain embodiments, the hydroalcoholic extracts used in step (ii) are free of particles larger than 0.01 μm, 0.05 μm, 0.1 μm, 0.5 μm, 1 μm, 2 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 150 μm, 200 μm, 250 μm, 500 μm, 1 mm, 2 mm, 5 mm. In certain embodiments, they are free of particles larger than 50 μm. Methods for removing particles larger than any of the above sizes are known by experts in the field and include centrifugation or the use of sieves with pore sizes similar to the smaller particles to be removed.

The term "active carbon" or "activated carbon" as used herein refers to a term known by those skilled in the art. It is a form of carbon that has been treated to enhance its adsorptive properties. It is primarily treated to have small, low volume pores that increase the surface area available for adsorption or chemical reactions.

In a particular embodiment, step (ii) of the method for obtaining an extract rich in ursolic acid is carried out at a temperature above 30°C, above 40°C, above 45°C, above 50°C, above 60°C, above 70°C, above 80°C, above 90°C, above 95°C, above 96°C, above 97°C, above 98°C, above 99°C, above 100°C, above 105°C, above 110°C. In a preferred embodiment, it is carried out at a temperature between about 30°C and 110°C, preferably between about 40°C and 100°C.

In certain embodiments, step (ii) of the method for obtaining an extract rich in ursolic acid is carried out for at least 0.5 hours, at least 1 hour, at least 1.5 hours, at least 2 hours, at least 3 hours, at least 3.5 hours, at least 4 hours, at least 4.5 hours, at least 5 hours, at least 5.5 hours, at least 6 hours, at least 6.5 hours, at least 7 hours, at least 7.5 hours, at least 8 hours, at least 8.5 hours, at least 9 hours, at least 9.5 hours, at least 10 hours, at least 10.5 hours, at least 11 hours, at least 12 hours, at least 15 hours, at least 18 hours, at least 20 hours, at least 24 hours. In certain embodiments, step (ii) of the method is carried out for 0.5 hours to 10 hours, 1 hour to 5 hours, preferably 1 hour to 4 hours.

In a particular embodiment, the amount of activated carbon used in step (ii) of the process for obtaining an extract rich in ursolic acid is between 0.5% (w/w) and 40% (w/w), preferably between 1% (w/w) and 20% (w/w) of the dry matter present in the extract.

In certain embodiments, removal of the activated carbon results in an extract that is free of activated carbon particles and raw material particles larger than at least 0.01 μm, 0.05 μm, 0.15 μm, 0.2 μm, 0.25 μm, 0.3 μm, 0.35 μm, 0.4 μm, 0.45 μm, 0.5 μm, 0.55 μm, 0.6 μm, 0.65 μm, 0.7 μm, 0.75 μm, 0.8 μm, 0.85 μm, 0.9 μm, 0.95 μm, 1 μm, 2 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 150 μm, 200 μm, 250 μm, 500 μm, 1 mm, 2 mm, 5 mm. In certain embodiments, they are free of carbon and feedstock particles larger than 0.45 μm, larger than 1 μm, larger than 5 μm, larger than 10 μm, preferably larger than 0.45 μm. Methods for removing particles larger than any of the above sizes are known by those skilled in the art and include centrifugation or the use of sieves with pore sizes similar to the smaller particles being removed.

In a particular embodiment, the method for obtaining an extract rich in ursolic acid comprises an additional step (iii) in which the hydroalcoholic extract obtained from step (ii) is concentrated.

In certain embodiments, concentrating includes separating the solids of the extract from the solvent and drying the solids. As will be appreciated by those skilled in the art, solids is any content of the extract other than the solvent of the extract. In certain embodiments, the extract obtained in step (iii) contains less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% water, preferably less than 15% water, even more preferably less than 10% water, in terms of % water (w/w).

In a particular embodiment, the extract obtained from step (ii) of the method and used in step (iii) is treated to remove any bacteria. Such treatments are known by experts in the field. Non-limiting examples of such treatments include heat treatment or microfiltration.

As used herein, the expression "D-pinitol-rich plant extract" or "D-pinitol-containing plant extract" refers to a plant extract obtained from a plant organism product or tissue that is rich in D-pinitol, i.e., contains a large amount of D-pinitol, resulting in a high final concentration of D-pinitol in the extract. Non-limiting examples of plant organism products or tissues that are rich in D-pinitol include products of plant organisms selected from the genus Ceratonia, Ceratoniasiliqua, Sutherlandiafrutescens, or Pinuslambertiana. In certain embodiments, the plant product of the above plants is any of those set forth in the definition of "plant product". In a preferred embodiment, the product rich in D-pinitol is selected from the group consisting of carob fruits, carob pods, Ceratonia siliqua fruits, Ceratonia siliqua pods, Sternlandia frutescens leaves, or Pinus lambertiana plant products. The plant extract can be obtained by any method known by a person skilled in the art, such as that provided in the embodiment of the method for obtaining an extract rich in D-pinitol herein or in the examples of the present invention under "Method for obtaining a natural extract rich in D-pinitol". Methods for obtaining an extract rich in D-pinitol include those as disclosed in European Patent No. 1241155 using carob pods as starting material, or those disclosed by Gonzalez-Mauraza et al. (Natural Product Communications, 2015,11:405-406) using the aerial parts of Retama monosperma as starting material.

In another particular embodiment, the D-pinitol-rich extract is obtained from two or more D-pinitol-rich products selected from any of the above groups of D-pinitol acid-rich products. In another particular embodiment, it is obtained from at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 13, at least 14, at least 15 products selected from any of the above groups of D-pinitol-rich products. In another particular embodiment, it is obtained from all products set forth in any of the above groups of D-pinitol-rich products.

In certain embodiments, the plant extract rich in D-pinitol has a % (w/w) of at least 0.1%, at least 0.2%, at least 0.25%, at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 7%, at least 10%, at least 15%, at least 17.5%, at least 20%, at least 22.5%, at least 25%, at least 27.5%, at least 30%, at least 35%, at least All contain 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 100%, preferably at least 95% D-pinitol.

In a particular embodiment, the weight ratio of D-pinitol and ursolic acid in the composition or kit of parts of the first aspect of the invention is a:b, where a represents the amount of D-pinitol in the composition or kit of parts and b represents the amount of ursolic acid in the composition or kit of parts,
the value of a is 100 to 30, 90 to 40, 80 to 50, 70 to 60, 65 to 60, preferably 70 to 60;
The value of b is 1-20, 2-18, 4-15, 5-10, 6-8, preferably 4-15.

In a preferred embodiment, the value of a is selected from the group consisting of 45, 50, 55, 58, 60, 61, 61.5, 62, 62.5, 63, 63.1, 63.2, 63.3, 63.4, 63.5, 63.6, 63.7, 63.8, 63.9, 70, 71, 72, 75, 80, 85, and 90. In a preferred embodiment, the value of a is 63.3.

In a preferred embodiment, the value of b is selected from the group consisting of 1, 2, 3, 4, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 12, 15, 18, and 20. In a preferred embodiment, the value of b is 7.5.

In a preferred embodiment, the weight ratio of D-pinitol and ursolic acid in the composition or kit of parts of the first aspect of the invention is about 63.3:7.5.

The composition or kit of parts according to the first aspect of the invention comprises:
(i) Docosahexaenoic acid (DHA),
(ii) ginkgo flavonoids, and
(iii) mixtures thereof;
The composition further comprises an additional component selected from the group consisting of:

As used herein, the term "docosahexaenoic acid" or "DHA" refers to the primary constituent omega-3 fatty acid with the IUPAC name (4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,10,13,16,19-hexaenoic acid.

In certain embodiments, the DHA of the composition or kit of parts of the first aspect of the invention is provided as a fatty acid composition that contains or comprises DHA. In certain embodiments, the composition comprising DHA comprises at least 5%, at least 10%, at least 15%, at least 18%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 32%, at least 33%, at least 35%, at least 37%, at least 40%, at least 42%, at least 45%, at least 47%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, at least 99.8%, at least 99.9%, at least 100% DHA, preferably at least 25% DHA.

In certain embodiments, the fatty acid composition containing DHA contains at least 5%, at least 10%, at least 15%, at least 18%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 32%, at least 33%, at least 35%, at least 37%, at least 40%, at least 42%, at least 45%, at least 47%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, at least 99.8%, at least 99.9%, at least 100% DHA, preferably at least 25% DHA.

In another particular embodiment, the fatty acid composition is obtained from microalgae or from fish. In a particular embodiment, it is selected from the group consisting of Schizochytrium sp., Schizochytrium, aggregatum, Schizochytriumlimacinum, Schizochytriumminutum, Thraustochytrium sp., Thraustochytrium aureum, Thraustochytriumkinnei, Nannochloropsis sp., Nannochloropsisgaditana, Nannochloropsis granulata, Nannochloropsis niger ... granulata, Nannochloropsislimnetica, Nannochloropsis oceanica, Nannochloropsis oculata, Nannochloropsis salina, Pinguiococcus sp., Pinguiococcus pyrenoidosus, Pavlova sp., Pavlova calceolate, Pavlova granifera, Pavlova gyrans, Pavlova hommersandii, Pavlova pinguis, Pavlova enolea ennorea, Pavlova lutheri, Pavlovamesolychnon, Pavlova salina, Pavlova virescens, Pavlovaviridis, Isocrysis sp., Isochrysis galbana, Isochrysis litoralis, and Isochrysis maritima.

In another particular embodiment, the DHA-rich fatty acid composition is obtained from a fish selected from the group consisting of salmon, herring, mackerel, tuna, halibut, sardine, shark, swordfish, tilefish, and albacore tuna.

The term "ginkgo flavonoids" as used herein refers to flavonoid glycosides present in extracts of Ginkgo biloba (G. biloba), preferably in extracts from the leaves of G. biloba. Flavonoid glycosides are compounds resulting from the conjugation of a flavone or flavonol with a sugar via a glycosidic bond, including O-glycosides, thioglycosides, glycosylamines, and C-glycosides, depending on whether the bond between the sugar and the other compound occurs through an oxygen, sulfur, nitrogen, or carbon atom. Flavonols found in G. biloba extracts as glycosides include quercetin (which forms quercitrin upon conjugation with rhamnose), kaempferol, and isorhamnetin (sometimes designated as isorhamnetin-3-O-rutinoside-7-O-glucoside, isorhamnetin-3-O-rutinoside-4'-O-glucoside, or isorhamnetin-3-O-rutinoside, also known as narcisin).

The expression "Ginkgo biloba extract" or "Ginkgo biloba extract" as used herein refers to a plant extract obtained from a plant product of G. biloba. In a particular embodiment, the plant product is any of the plant products set out in the definition of "plant product" above. In a preferred embodiment, the G. biloba extract is obtained from the leaves of G. biloba. As known by a person skilled in the art, the extract contains flavonoids and terpene lactones. The extract can be obtained by any method known by a person skilled in the art, such as the one provided in the embodiment of the method for obtaining a G. biloba extract herein or in the "Method for obtaining a natural extract rich in ursolic acid" in the examples of the present invention.

The term "terpene lactones" refers to a group of compounds with unique chemical structures that were first recognized in extracts of G. biloba. The major terpene lactone compounds in G. biloba include bilobalide and ginkgolide.

In certain embodiments, the ginkgo flavonoid of the composition or kit of parts of the first aspect of the invention is provided as a G. biloba extract.

In another particular embodiment, the G. biloba extract of the composition or kit of parts of the first aspect of the invention comprises a percentage (w/w) of ginkgo flavonoids of at least 0.01%, at least 0.05%, at least 0.075%, at least 0.1%, at least 0.25%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.75%, at least 1%, at least 2%, at least 5%, at least 7.5%, at least 10%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 18%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, preferably at least 0.6%. In another particular embodiment, the Ginkgo biloba extract of the composition or kit of parts of the first aspect of the invention contains a percentage (w/w) of ginkgo flavonoids of at least 0.01%, at least 0.05%, at least 0.075%, at least 0.1%, at least 0.25%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.75%, at least 1%, at least 2%, at least 5%, at least 7.5%, at least 10%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 18%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, preferably at least 0.6%.

In another specific embodiment, the G. biloba extract contains 0.01% (w/w) to 20% (w/w), 0.5% (w/w) to 15% (w/w) ginkgo flavonoids. In another specific embodiment, the G. biloba extract contains 0.01% (w/w) to 20% (w/w), 0.5% to 15% ginkgo flavonoids.

In another particular embodiment, the G. biloba extract contains less than 10%, 7%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, preferably less than 1% terpene lactones (w/w). In another particular embodiment, the G. biloba extract contains less than 10%, 7%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, preferably less than 1% terpene lactones (w/w).

In a preferred embodiment, the G. biloba extract contains ginkgo flavonoids in any of the percentages (wt/wt) described above, and terpene lactones in any of the percentages (wt/wt) described above. In a further preferred embodiment, the G. biloba extract contains 0.5% (wt/wt) to 15% (wt/wt) ginkgo flavonoids, and less than 1% (wt/wt) terpene lactones. In a preferred embodiment, the G. biloba extract contains ginkgo flavonoids in any of the percentages (wt/wt) described above, and terpene lactones in any of the percentages (wt/wt) described above. In a further preferred embodiment, the G. biloba extract contains 0.5% (wt/wt) to 15% (wt/wt) ginkgo flavonoids, and less than 1% (wt/wt) terpene lactones.

In another particular embodiment, the Ginkgo biloba extract comprises:
(i) a water extraction from a Ginkgo biloba plant product;
(ii) treating the aqueous extract obtained in step (i) with activated carbon and then removing the activated carbon;
The compound was obtained by a method comprising the steps of:

In certain embodiments, the plant product of G. biloba is any of those set forth in the definition of "plant product," and preferably, it is G. biloba leaves.

In another particular embodiment, the extraction step (i) of the method for obtaining a G. biloba extract is carried out with water at a temperature above 30°C, above 40°C, above 45°C, above 50°C, above 60°C, above 70°C, above 80°C, above 90°C, above 95°C, above 96°C, above 97°C, above 98°C, above 99°C, above 100°C, above 105°C, above 110°C. In a preferred embodiment, it is carried out with water at a temperature between about 30°C and 110°C, preferably between about 40°C and 100°C.

In another particular embodiment, the extraction step (i) of the method for obtaining a G. biloba extract is carried out for at least 0.5 hours, at least 1 hour, at least 1.5 hours, at least 2 hours, at least 3 hours, at least 3.5 hours, at least 4 hours, at least 4.5 hours, at least 5 hours, at least 5.5 hours, at least 6 hours, at least 6.5 hours, at least 7 hours, at least 7.5 hours, at least 8 hours, at least 8.5 hours, at least 9 hours, at least 9.5 hours, at least 10 hours, at least 10.5 hours, at least 11 hours, at least 12 hours, at least 15 hours, at least 18 hours, at least 20 hours, at least 24 hours, at least 30 hours, at least 36 hours, at least 40 hours, at least 42 hours, at least 48 hours. In a particular embodiment, the method is carried out for 0.5 hours to 48 hours, 1 hour to 30 hours, 1 hour to 20 hours, preferably 1 hour to 20 hours.

In another particular embodiment, step (i) of the method for obtaining a G. biloba extract is repeated at least once, at least twice, at least three times, at least four times, at least five times. In a particular embodiment, the G. biloba plant product used in step (i) of the method is the same in each repetition of step (i) of the method. As will be appreciated by the skilled artisan, in this case the same G. biloba plant product is subjected to several aqueous extractions.

In certain embodiments, the aqueous extract used in step (ii) does not contain particles larger than 0.01 μm, 0.05 μm, 0.1 μm, 0.5 μm, 1 μm, 2 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 150 μm, 200 μm, 250 μm, 500 μm, 1 mm, 2 mm, 5 mm. In certain embodiments, they do not contain particles larger than 50 μm. Methods for removing particles larger than any of the above sizes are known by experts in the field and include centrifugation or the use of sieves with pore sizes similar to the smaller particles to be removed.

In a particular embodiment, step (ii) of the method for obtaining a G. biloba extract is carried out at a temperature above 30°C, above 40°C, above 45°C, above 50°C, above 60°C, above 70°C, above 80°C, above 90°C, above 95°C, above 96°C, above 97°C, above 98°C, above 99°C, above 100°C, above 105°C, above 110°C. In a preferred embodiment, it is carried out at a temperature between about 30°C and 110°C, preferably between about 40°C and 100°C.

In certain embodiments, step (ii) of the method for obtaining a G. biloba extract is carried out for at least 0.5 hours, at least 1 hour, at least 1.5 hours, at least 2 hours, at least 3 hours, at least 3.5 hours, at least 4 hours, at least 4.5 hours, at least 5 hours, at least 5.5 hours, at least 6 hours, at least 6.5 hours, at least 7 hours, at least 7.5 hours, at least 8 hours, at least 8.5 hours, at least 9 hours, at least 9.5 hours, at least 10 hours, at least 10.5 hours, at least 11 hours, at least 12 hours, at least 15 hours, at least 18 hours, at least 20 hours, at least 24 hours. In certain embodiments, step (ii) of the method is carried out for 0.5 hours to 10 hours, 1 hour to 5 hours, preferably 1 hour to 3 hours.

In a particular embodiment, the amount of activated carbon used in step (ii) of the method for obtaining a G. biloba extract is between 0.5% (w/w) and 40% (w/w), preferably between 1% (w/w) and 20% (w/w) of the dry matter present in the extract.

In certain embodiments, removal of the activated carbon results in an extract that is free of activated carbon particles and raw material particles larger than at least 0.01 μm, 0.05 μm, 0.15 μm, 0.2 μm, 0.25 μm, 0.3 μm, 0.35 μm, 0.4 μm, 0.45 μm, 0.5 μm, 0.55 μm, 0.6 μm, 0.65 μm, 0.7 μm, 0.75 μm, 0.8 μm, 0.85 μm, 0.9 μm, 0.95 μm, 1 μm, 2 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 150 μm, 200 μm, 250 μm, 500 μm, 1 mm, 2 mm, 5 mm. In certain embodiments, they are free of carbon and feedstock particles larger than 0.45 μm, larger than 1 μm, larger than 5 μm, larger than 10 μm, preferably larger than 0.45 μm. Methods for removing particles larger than any of the above sizes are known by those skilled in the art and include centrifugation or the use of sieves with pore sizes similar to the smaller particles being removed.

In a particular embodiment, the method for obtaining a G. biloba extract comprises an additional step (iii) in which the aqueous extract obtained from step (ii) is concentrated.

In a particular embodiment, the extract obtained from step (iii) is concentrated until the solids content of the extract is between 1% (w/w) and 90% (w/w), preferably between 5% (w/w) and 80% (w/w), more preferably between 10% (w/w) and 70% (w/w). The term "solids content" is as defined above in the embodiment of the method for obtaining an extract rich in ursolic acid.

In a particular embodiment, the extract obtained from step (iii) is a liquid extract. In another particular embodiment, the extract obtained in step (iii) of the method is dried, such that the extract obtained in step (iii) contains less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% water, preferably less than 15% water, even more preferably less than 10% water, in terms of % water (w/w).

In a particular embodiment, the extract obtained from step (ii) of the method and used in step (iii) is treated to remove any bacteria. Such treatments are known by experts in the field. Non-limiting examples of such treatments include heat treatment or microfiltration.

In certain embodiments, the G. biloba extract obtained by the method for obtaining the G. biloba extract is as described above in any of the above embodiments.

In a particular embodiment, when the composition or kit of parts according to the first aspect of the invention contains or comprises D-pinitol, ursolic acid and DHA, the weight ratio of the components is a:b:c, where a and b are as a and b of the weight ratio of D-pinitol and ursolic acid described above, and c represents the amount of DHA in the composition or kit of parts and is 10-80, 20-70, 30-60, 35-50, preferably 35-45. In a preferred embodiment, the value of c is selected from the group consisting of 5, 10, 15, 20, 25, 30, 35, 37, 40, 42, 45, 50, 55, 60, 70, 80, 90 and 100. In a preferred embodiment, the value c is 40.

In a preferred embodiment, when the composition or kit of parts according to the first aspect of the invention contains or comprises D-pinitol, ursolic acid and DHA, the weight ratio of D-pinitol, ursolic acid and DHA in the composition or kit of parts according to the first aspect of the invention is about 63.3:7.5:40.

In the case where the composition contains D-pinitol, ursolic acid, and ginkgo flavonoid, the weight ratio of the components is a:b:d, where a and b are as a and b of the weight ratio of D-pinitol and ursolic acid described above, and d represents the amount of ginkgo flavonoid in the composition or kit of parts, and is 0.01-10, 0.5-5, 0.7-2, 0.8-1.5, preferably 0.9-1.1. In a preferred embodiment, the value of d is selected from the group consisting of 0.1, 0.2, 0.4, 0.5, 0.7, 1, 1.1, 1.2, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, and 10. In a preferred embodiment, the value of d is 1.

In a preferred embodiment, when the composition or kit of parts according to the first aspect of the invention contains or comprises D-pinitol, ursolic acid and ginkgo flavonoid, the weight ratio of D-pinitol, ursolic acid and ginkgo flavonoid in the composition or kit of parts according to the first aspect of the invention is about 63.3:7.5:1.

In a particular embodiment, when the composition or kit of parts according to the first aspect of the invention contains or comprises D-pinitol, ursolic acid, DHA and ginkgo flavonoids, the weight ratio of the components is a:b:c:d, where a and b are as a and b set forth above for the weight ratio of D-pinitol and ursolic acid, c is as set forth above for the weight ratio of D-pinitol, ursolic acid and DHA, and d is as set forth above for the weight ratio of D-pinitol, ursolic acid and ginkgo flavonoids. In a preferred embodiment, when the composition or kit of parts contains D-pinitol, ursolic acid, DHA and ginkgo flavonoids, the weight ratio of the components is about 63.3:7.5:40:1.

In certain embodiments, the composition or kit of parts of the first aspect of the invention further comprises a carrier.

The term "carrier" as used herein refers to an excipient, diluent, and/or adjuvant useful in the preparation of a composition, for example by reducing viscosity, increasing solubility, or aiding in the stability of the components of the composition, for example by preventing their denaturation or aggregation over their expected shelf life. A carrier can comprise or consist of agents such as wetting or emulsifying agents, pH buffering agents, or adjuvants that enhance the effectiveness of the formulation. When the composition is administered to a subject, the carrier must be physiologically acceptable and will not normally produce an allergic reaction or similar undesirable reaction, such as stomach upset, dizziness, etc., when administered to a human or animal. Non-limiting examples of carriers include water, saline, alcohol, vegetable oils, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, flavor oils, monoglycerides and diglycerides of fatty acids, fatty acid esters petroetrals, hydroxymethylcellulose, polyvinylpyrrolidone, hydroxypropyl beta-cyclodextrin, and the like.

Although a single carrier is used in the compositions of the present invention, it will be understood that when the various components are formulated as a kit of parts, each element or part of the kit may be formulated with a carrier which may be the same or different from the carrier used for the other parts of the kit.

The concentration of the carrier in the composition is not particularly limited within the scope of the present invention. In one embodiment, the concentration of the carrier is 20% (wt/wt) to 90% (wt/wt) based on the total weight of the composition. In a preferred embodiment, the concentration of the carrier is 30% (wt/wt) to 80% (wt/wt), 35% (wt/wt) to 75% (wt/wt), 40% (wt/wt) to 70% (wt/wt), 45% (wt/wt) to 65% (wt/wt), or 50% (wt/wt) to 60% (wt/wt).

2. Pharmaceutical Compositions In another aspect, the present invention relates to a pharmaceutical product comprising a composition according to the present invention and a pharma- ceutically active carrier.

The term "pharmaceutical composition" as used herein refers to any composition that is physiologically acceptable and does not normally produce allergic reactions or similar undesirable reactions such as stomach upset, dizziness, etc., when administered to humans or animals. The composition comprises at least one pharmacoactive ingredient and one or more pharmacologic acceptable carriers. The terms "pharmaceutical acceptable carrier", "pharmaceutical acceptable excipient", "pharmaceutical acceptable diluent", or "pharmaceutical acceptable vehicle" are used interchangeably herein and refer to any conventional type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation aid. A pharmacologic acceptable carrier is essentially non-toxic to a recipient at the dosage and concentration employed and is compatible with the other ingredients of the formulation. Suitable carriers include, but are not limited to, water, dextrose, glycerol, saline, ethanol, and combinations thereof. The carrier may contain additional agents such as wetting or emulsifying agents, pH buffering agents, or adjuvants that enhance the effectiveness of the formulation. The adjuvant may be selected from the group consisting of sterile liquids such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water or aqueous saline solutions and aqueous dextrose and glycerol solutions, particularly injectable solutions, are preferably used as vehicles. Suitable pharmaceutical vehicles are described in "Remington's Pharmaceutical Sciences", 21st Edition, 2005, by E.W. Martin. Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

A pharmacologic or veterinary therapeutically effective amount of the extract of the present invention. As used herein, the term "effective amount" is synonymous with "therapeutically effective amount," "effective dose," or "therapeutically effective dose," and as used herein, refers to the minimum dose of the extract of the present invention required to obtain the desired therapeutic effect, including a dose sufficient to reduce at least one symptom of cognitive impairment. Efficacy in treating a disease or condition described herein can be determined by observing an improvement in an individual based on one or more clinical symptoms and/or physiological indicators associated with the condition. Improvement in a disease or condition described herein can also be indicated by a reduced need for concomitant therapy.

One skilled in the art can determine the therapeutically effective amount of the compositions of the present invention by determining the unit dose. As used herein, "unit dose" refers to the amount of the compositions or kit of parts of the present invention required to produce a response of 50 percent of the maximum effect (i.e., ED50). The unit dose can be estimated by extrapolation based on dose-response curves derived from in vitro or animal model test systems. The amount of the compound in the compositions of the present invention that is effective in treating a particular disorder or condition will depend on the nature of the disorder or condition and can be determined by standard clinical techniques (see, e.g., Goodman and Gilman's The Pharmacological Basis of Therapeutics, Joel G. Harman, Lee E. Limbird, Eds.; McGraw Hill, New York, 2001; The Physician's Desk Reference, Medical Economics Company, Inc., Oradell, N.J., 1995; and Drug Facts and Comparisons, Facts and Comparisons, Inc., St. Louis, Mo., 1993). The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Various administration patterns will be apparent to those skilled in the art.

Furthermore, when repeated administrations of the extract of the present invention are used, the effective amount of the extract of the present invention will further depend on factors including, but not limited to, the frequency of administration, the half-life of the extract of the present invention, or any combination thereof.

The dosage ranges for administration of the compositions of the invention are those large enough to produce the desired therapeutic effect. Preferably, the compositions according to the invention are administered one or more times daily on a regular basis. A typical dose administered to a human is from about 1 mg to about 10 g of the composition, preferably 1 mg to 1 g of the composition.

The compositions provided herein can be administered to a subject by a number of suitable methods known in the art. Examples of suitable methods include: (1) intramuscular, intradermal, intraepidermal, or subcutaneous administration; (2) oral administration; and (3) topical application, including intraocular, intranasal, and intravaginal application. However, in a preferred embodiment, the compositions are formulated for oral administration.

In some embodiments, the preferred route of administration of the compositions provided herein is oral. In these cases, compositions for oral use are formulated, for example, as tablets, troches, lozenges, aqueous or oily suspensions, solutions, dispersible powders or granules, emulsions, hard or soft capsules, syrups or elixirs, pastes, gels, and the like. Compositions intended for oral use can be prepared according to any known method, and such compositions can contain one or more agents selected from the group consisting of sweeteners, flavoring agents, coloring agents, and preservatives to provide a pharma- ceutically elegant and palatable composition. Tablets can contain the active ingredient(s) in admixture with non-toxic pharma-ceutically acceptable excipients that are suitable for the manufacture of tablets. These excipients may be, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents such as corn starch or alginic acid; binders such as starch, gelatin or acacia; and lubricants such as magnesium stearate, stearic acid or talc. The tablets may be uncoated or may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained action over a longer period of time. For example, a time-delay material such as glyceryl monostearate or glyceryl distearate may be used. They may also be coated for controlled delivery. For example, a "delayed release" dosage form releases a product or substance at a time other than immediately after administration. Examples of delayed release systems include repeat-action tablets and capsules, and enteric-coated tablets in which the sustained release is achieved by a barrier coating.

The compositions of the present invention can also be formulated for oral use as hard gelatin capsules in which the active ingredient(s) are mixed with an inert solid diluent, such as calcium carbonate, calcium phosphate, or kaolin, or as soft gelatin capsules in which the active ingredient(s) are mixed with water or an oil medium, such as peanut oil, liquid paraffin, or olive oil.

The compositions of the invention may be formulated as aqueous suspensions in which the active ingredient(s) are in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum arabic, and the dispersing or wetting agent may be a naturally occurring phosphatide, such as lecithin, or a condensation product of an alkylene oxide with a fatty acid, such as polyoxyethylene stearate, or a condensation product of ethylene oxide with a long chain aliphatic alcohol, such as heptadecaethyl-enoxycetanol, or a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol, such as polyoxyethylene sorbitol monooleate, or a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride, such as polyethylene sorbitan monooleate. Aqueous suspensions may also contain one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

The compositions of the invention may be formulated as oily suspensions by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavouring agents may be added to provide a palatable oral composition. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.

The compositions of the invention can be formulated in the form of dispersible powders and granules suitable for constitution of an aqueous suspension by the addition of water. The active ingredient in such powders and granules is provided in admixture with a dispersing or wetting agent, a suspending agent, and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, such as sweetening, flavoring and coloring agents, may also be present.

The compositions of the invention may also be in the form of oil-in-water emulsions. The oil phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin, or a mixture thereof. Suitable emulsifiers may be naturally occurring gums, for example gum arabic or gum tragacanth, naturally occurring phosphatides, for example soya, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavoring agents.

The compositions of the present invention can also be formulated as syrups and elixirs. Syrups and elixirs can be formulated with sweetening agents, such as glycerol, propylene glycol, sorbitol, or sucrose. Such formulations can also contain demulcents, preservatives, and flavoring and coloring agents. Demulcents are protective agents used primarily to relieve inflammation, especially mucous membranes or abraded tissues. Many chemicals have demulcent properties. These substances include alginates, vegetable mucilages, gums, dextrins, starches, certain sugars, and polymeric polyhydric glycols. Others include acacia, agar, benzoin, carbomer, gelatin, glycerin, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, propylene glycol, sodium alginate, tragacanth, hydrogels, and the like.

Liquid-based oral dosage forms, like their solid counterparts, can contain, in certain embodiments, at least 0.1 mg of the provided extract. One of skill in the art will be able to appropriately formulate liquid formulations containing the appropriate amount of the provided extract per fluid ounce, depending on the excipient or carrier selected.

Formulations suitable for buccal administration include tablets and lozenges containing the extract in a flavored base such as sucrose, acacia, or tragacanth, and troches containing the extract in an inert base such as gelatin and glycerin or sucrose and acacia.

3. Nutritional and Dietary Supplements In another aspect, the present invention relates to a nutritional or dietary supplement comprising the composition of the present invention and a nutritionally acceptable carrier.

The term "nutraceutical" or "dietary supplement" as used herein refers to a concentrated source of nutrients or other substances obtained from edible products, intended to supplement the normal diet. Nutraceutical or dietary supplements according to the present invention include functional food compositions, i.e. foods, beverages, feeds or pet foods, or food, beverage, feed or pet food supplements, nutritional supplements, flavors or flavorings, oenological formulations or cosmetic formulations. In general, the term "nutraceutical" or "dietary supplement" also refers to:
(i) Any product intended to supplement the diet that has or contains one or more of the following dietary ingredients: [A] vitamins, [B] minerals, [C] herbs or other botanicals, [D] amino acids, [E] dietary substances used by humans to supplement the diet by increasing total dietary intake, or (F) any product intended to supplement the diet that has or contains one or more of the concentrates, metabolites, constituents, extracts, or combinations of any of the ingredients set forth in subparagraph (A), (B), (C), (D), or (E); or
(ii) (A) products intended for consumption; (B) products not represented as being for use as conventional foods or as a separate item in a meal or diet; and (C) products represented as dietary supplements.
It can mean:

The "nutraceuticals" or "dietary supplements" according to the invention are usually administered orally and provided with the subject's diet. They can take a wide variety of forms, including tablets, capsules, liquid suspensions, dry powders, wet compositions, dry or wet tube feedings. They can be provided as nutritional formulations, e.g. in the form of medical foods, e.g. tube feedings, or in oral nutritional forms as complete meals, as part of a meal, as food additives, as powders for dissolution, as ready-made drinks, e.g. health drinks, solutions, juices, milkshakes, yogurt drinks, smoothies or soy-based drinks, in bars or dispersed in any kind of food, such as baked products, cereal bars, dairy bars, snack foods, soups, breakfast cereals, muesli, candy, cookies, biscuits, etc.

As used herein, the term "nutritionally acceptable carrier" refers to a carrier that is suitable for consumption and intended for preparing a solution to be administered orally, as described above. Exemplary nutritionally acceptable carriers, diluents and excipients will be familiar to those skilled in the art. Non-limiting examples of such carriers are provided in U.S. Patent Nos. 6,258,846, 6,576,666 and 7,112,609.

The amount of the composition present in a dietary supplement composition, a food or food for humans or animals (e.g., a functional food composition, i.e., a food, beverage, feed or pet food or a food, beverage, feed or pet food supplement), a nutritional supplement, a flavoring or flavoring, a drug (pharmaceutical composition or formulation), a veterinary composition, a wine formulation, or a cosmetic formulation varies depending on the application. Typically, the amount of the composition present in a dietary supplement or health supplement is from about 0.001 percent to about 50 percent by weight of the dietary supplement composition, food or food, nutritional supplement, flavoring or flavoring, oenological, e.g., from about 0.01 percent to about 10 percent, or from about 0.1 percent to 1 percent.

4. Medical Uses In another aspect, the present invention relates to a composition or a kit of parts according to the invention, to a pharmaceutical product according to the invention, or to a dietary or health supplement according to the invention, for use in medicine.

In another aspect, the present invention relates to a composition or a kit of parts according to the present invention, to a pharmaceutical product according to the present invention, or to a dietary or health supplement according to the present invention, for use in the prevention and/or treatment of cognitive disorders.

As used herein, the term "cognitive disorder" or "neurocognitive disorder" refers to a disorder or disease characterized by an alteration in at least one cognitive function of a patient, resulting in a reduced ability of said patient to undertake said function. The term "cognitive function" refers to the ability developed by cognition, or mental ability. DSM-5 defines six major areas of cognitive function: executive function, learning and memory, perceptual-motor function, language, complex attention, and social cognition. Thus, cognitive disorders refer to a category of mental health disorders that primarily affect cognitive abilities, including learning, memory, perception, and problem solving. Neurocognitive disorders include delirium and mild and severe neurocognitive disorders (formerly known as dementia). Non-limiting examples of cognitive disorders include Alzheimer's disease, mild cognitive impairment (MCI), Parkinson's disease, frontotemporal degeneration, Huntington's disease, Lewy body disease, traumatic brain injury (TBI), prion disease, and dementia/neurocognitive problems due to HIV infection.

In a preferred embodiment, the cognitive disorder is selected from the group consisting of mild cognitive impairment, Alzheimer's disease, and Parkinson's disease.

The term "mild cognitive impairment (MCI)" as used herein refers to a term well known by experts in the field. It refers to a neurological disorder that occurs in older adults (approximately 15%-20% of the population aged 65 years or older) and involves cognitive impairment with minimal impairment in instrumental activities of daily living. MCI involves the onset and progression of cognitive impairment beyond that expected based on the individual's age and education, but which is not significant enough to interfere with the person's daily activities. According to the World Health Organization (WHO), MCI is diagnosed by the presence of impairment in one or more cognitive domains without meeting the diagnostic criteria for dementia. It can be classified into amnestic MCI and non-amnesic MCI. Amnesic MCI is characterized by impairment in memory-cognitive functions, such that the affected person forgets important information that the person would previously have easily recalled, such as appointments, conversations, or recent events. Non-amnesic MCI is characterized by the loss of cognitive abilities other than memory, such as the affected person's ability to make correct decisions, judge the time or sequence required to complete a complex task, or vision.

As used herein, the term "Alzheimer's disease" or "AD" refers to a disease well known by experts in the field. AD is characterized by a progressive pattern of cognitive and functional impairment. In the early stages of the disease, patients only exhibit short-term memory loss and subtle problems with attention, planning, flexibility, and executive functions of abstract thinking, or impairments in semantic memory (memory of meanings and conceptual relationships). As the disease progresses, memory impairment increases and language, literacy, and coordination of motor sequences decrease. In advanced stages, patients may completely lose the ability to speak and exhibit a decline in muscle mass and mobility that prevents them from feeding themselves, becoming completely dependent on caregivers.

The term "dementia" as used herein refers to terms commonly known by those skilled in the art. According to the World Health Organization (WHO), dementia is a syndrome, usually of a chronic or progressive nature, in which there is a decline in cognitive function beyond that which can be expected from normal aging. It affects memory, thinking, orientation, understanding, calculation, learning ability, language, and judgment. Consciousness is not affected. Impaired cognitive function is generally accompanied by, and sometimes preceded by, a decline in emotional control, social behavior, or motivation. Dementia results from a variety of diseases and injuries that primarily or secondarily affect the brain, such as Alzheimer's disease or stroke. Alzheimer's disease is the most common form, and may contribute to 60% to 70% of cases. Other major forms include a group of diseases that contribute to vascular dementia, Lewy body dementia (abnormal clumps of proteins that occur inside nerve cells), and frontotemporal dementia (degeneration of the frontal lobe of the brain).

The term "Parkinson's disease," or "PD," as used herein, refers to the term commonly known by those skilled in the art. It is a long-term degenerative disorder of the central nervous system that primarily affects the motor system. The most obvious early symptoms of the disease are tremors, stiffness, slowness of movement, and difficulty walking. Problems with thinking and behavior may also occur. Dementia becomes common in the advanced stages of the disease. Depression and anxiety are also common, occurring in more than one-third of PD patients. Other symptoms include sensory impairment, sleep disorders, and emotional disturbances.

Depending on the disorder and subject to be treated, and the route of administration, the compositions of the present invention may be administered in various doses (i.e., therapeutically effective doses administered to a patient in need thereof). In this regard, those skilled in the art will understand that the dose administered to a mammal, particularly a human, in the context of the present invention should be sufficient to affect a therapeutic response in the mammal over a reasonable time frame. Those skilled in the art will understand that the selection of the exact dose and composition, as well as the most appropriate delivery regimen, will also be influenced by, among other things, the pharmacological properties of the formulation, the nature and severity of the condition to be treated, and the health and mental acuity of the recipient, as well as the age, condition, weight, sex and response of the patient to be treated, and the stage/severity of the disease.

The subject to be treated is a mammal, preferably a human. The subject to be treated according to the present invention can be selected based on several criteria related to the neurodegenerative disease, such as imaging or behavioral tests.

In one embodiment, the composition, kit of parts, pharmaceutical product or dietary supplement for use according to the invention comprises administration of multiple doses of the composition. In yet another embodiment, the composition, kit of parts, pharmaceutical product or dietary supplement is administered for at least 2 days, at least 4 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 1 month. In some embodiments, the composition, kit of parts, pharmaceutical product or dietary supplement for use according to the invention is administered for a period of time, followed by a period of time in which the composition, kit of parts, pharmaceutical product or dietary supplement is not administered. In one embodiment, the composition, kit of parts, pharmaceutical product or dietary supplement follows a 5+2 delivery pattern, i.e., the product is delivered for 5 days, followed by no delivery for the following 2 days.

Those skilled in the art will appreciate that initial indications for appropriate therapeutic dosages of the compositions of the invention may be determined in in vitro and in vivo animal model systems, as well as in human clinical trials. Those skilled in the art will know to use animal testing and human experience to ascertain dosages that can be safely administered without causing toxicity or other side effects. For acute treatments where it is desired to substantially restore cognitive function, it is preferred that the therapeutic dosage be close to the maximum tolerated dose. For chronic prophylactic use, lower dosages may be desirable due to concerns about long-term effects.

Alternatively, the compositions of the invention may be administered at least once daily in combination with a drug prescribed for the indication being treated. For example, when a composition or kit of parts according to the invention is used in the treatment of Alzheimer's disease, the composition or kit of parts is administered with one Alzheimer's disease therapeutic. Suitable Alzheimer's therapeutic agents include acetylcholinesterase inhibitors (e.g., tacrine, also known as tetrahydroaminoacridine (THA), rivastigmine, donepezil, galantamine, carbamates, physostigmine, neostigmine, pyridostigmine, ambenonium, demecarium, phenanthrene derivatives, caffeine - noncompetitive (also an adenosine receptor antagonist), rosmarinic acid - an ester of caffeic acid found in species of plants in the Lamiaceae family, α-pinene - noncompetitive reversible, piperidine, edrophonium, huperzine A, ladostigil, ungeremine, lactucopicrin), NMDA receptor antagonists (e.g., memantine), gamma secretase inhibitors (e.g., semagacestat, ELND006, avagacestat, , vegacestat, NIC5-15 and CHF-5074, MK-8931, LY2886721, AZD3293, LY3314814, E2609), antibodies against Aβ (bapineuzumab, solanezumab, gantenerumab, crenezumab, aducanumab, crenezumab, ponezumab, GSK933776 and BAN-2401, human polyclonal anti-Aβ antibodies or immunoglobulin therapy (e.g., GammaGuard™, Flebogamma™), drugs against tau protein (e.g., tau hyperphosphorylation inhibitors (LMTX, etc.)), epothilone D, TPI-287, anti-tau vaccines (e.g., AADvacl or ACI-35), and GSK-3β inhibitors (e.g., tideglusib, intranasal humulin R or intranasal glulisine, etc.).

For example, when the composition or kit of parts according to the invention is used in the treatment of Parkinson's disease, the composition or kit of parts is administered with one Parkinson's disease therapeutic agent. Suitable Parkinson's disease therapeutic agents include synucleinopathies such as glucosylceramide synthase inhibitors (e.g., GZ667161), iron chelators, epigallocatechin gallate (EGCG), myeloperoxidase inhibitors (e.g., AZD3241), affitopes (e.g., AFFITOPE PD01A, AFFITOPE PD03A), and anti-synuclein antibodies (e.g., PRX002, BIIB054). Other suitable Parkinson's disease therapeutic agents include levodopa, carbidopa, entacapone, ropinirole, rotigotine, pramipexole, bromocriptine, rasagiline, selegiline, amantadine, and trihexyphenidyl.

For example, when a composition or kit of parts according to the invention is used in the treatment of MCI, the composition or kit of parts is administered with one MCI treatment, with a non-pharmacological treatment, or with a suitable therapy for a disease known to affect mental function. Suitable MCI treatments include cholinesterase inhibitors. Suitable non-pharmacological treatments for MCI include regular exercise and cognitive training. Suitable therapies for diseases known to affect mental function include therapy for hypertension, therapy for depression, and/or therapy for sleep apnea.

While the composition of the invention can be administered by itself, the invention also contemplates the possibility that at least one of the components of the composition, kit of parts, pharmaceutical composition, or dietary or dietary supplement may be administered separately from the remaining components of the composition, kit of parts, pharmaceutical product, or dietary or dietary supplement. In one embodiment, D-pinitol or a plant extract rich in D-pinitol is administered separately from the other components of the composition or kit of parts. In another embodiment, ursolic acid of a hydroalcoholic extract of a plant product rich in ursolic acid is administered separately from the other components of the composition or kit of parts. In another embodiment, DHA or a fatty acid composition comprising DHA is administered separately from the other components of the composition or kit of parts. In another embodiment, ginkgo flavonoids or Ginkgo biloba extract are administered separately from the other components of the composition or kit of parts.

In another embodiment, D-pinitol or a plant extract rich in D-pinitol and ursolic acid or a hydroalcoholic extract of a plant product rich in ursolic acid are administered separately from other components of the composition or kit of parts.

In another embodiment, the D-pinitol or plant extract rich in D-pinitol and the DHA or fatty acid composition comprising DHA are administered separately from other components of the composition or kit of parts.

In another embodiment, the D-pinitol or D-pinitol rich plant extract and the ginkgo flavonoids or Ginkgo biloba extract are administered separately from other components of the composition or kit of parts.

In another embodiment, the ursolic acid or hydroalcoholic extract of a plant product rich in ursolic acid and the DHA or a fatty acid composition comprising DHA are administered separately from other components of the composition or kit of parts.

In another embodiment, the DHA or a fatty acid composition comprising DHA and ginkgo flavonoids or Ginkgo biloba extract are administered separately from other components of the composition or kit of parts.

In another embodiment, the D-pinitol or plant extract rich in D-pinitol, the ursolic acid or hydroalcoholic extract of a plant product rich in ursolic acid, and the DHA or a fatty acid composition comprising DHA are administered separately from the other components of the composition or kit of parts.

In another embodiment, the ursolic acid or hydroalcoholic extract of a plant product rich in ursolic acid, the DHA or a fatty acid composition comprising DHA, and the ginkgo flavonoids or Ginkgo biloba extract are administered separately from the other components of the composition or kit of parts.

When the composition of the invention is provided as a kit of parts, the various parts of the kit may be administered separately or, alternatively, they may be combined prior to administration.

In another embodiment, the composition, kit of parts, pharmaceutical product, or dietary or dietary supplement for use according to the invention comprises the following daily dosage:
(i) about 150 mg/day of a plant extract rich in ursolic acid;
(ii) about 480 mg/day of DHA-rich fatty acids;
(iii) about 500 mg/day of Ginkgo biloba extract, and/or
(iv) about 200 mg/day of D-pinitol;
It is administered at .

In another embodiment, the composition, kit of parts, pharmaceutical product, or dietary or dietary supplement for use according to the invention is administered at the following dosages per kg of patient:
(i) about 2.5 mg/kg of a plant extract rich in ursolic acid;
(ii) about 8 mg/kg of a DHA-rich fatty acid composition;
(iii) about 8.33 mg/kg of Ginkgo biloba extract, and/or
(iv) about 3.33 mg/kg D-pinitol;
It is administered at .

The present invention is illustrated in the following examples, which are merely illustrative and are not intended to limit the scope of the invention.

Preparation of Biological Extracts Preparation of Ursolic Acid-Rich Natural Extracts Preparation of ursolic acid-rich natural extracts is carried out by a method comprising the following:
(i) Providing the raw material, leaves of plants of the Lamiaceae family (Salvia officinalis, Thymus vulgaris, Rosmarinus officinalis, Origanum vulgare, etc.) In addition, other sources rich in ursolic acid, such as seaweed or fruit peels of Malus domestica, Pyrus communis, Vaccinium spp., Prunus spp., etc., can also be used.
(ii) Extraction of the raw or dried material with a hydroalcoholic mixture having an alcohol content of 10% to 96% for 1 to 10 hours at a temperature of about 40° C. to 100° C. The process may also include several stages of extraction of the leaves with the same or different alcohol contents, combining the hydroalcoholic extracts obtained and continuing the process.
(iii) The hydroalcoholic extract is separated from the raw material using any separation method that results in an extract that is free of particles larger than 50 microns to 100 microns.
(iv) The hydroalcoholic extract is treated with activated charcoal for 1 to 4 hours at 40° C. to 100° C. The dosage of activated charcoal is 1% (w/w) to 20% (w/w) of the dry matter present in the extract.
(v) The hydroalcoholic extract is separated from the activated carbon using any separation method that results in an extract that is free of activated carbon particles and particles of raw material larger than 0.45 microns to 10 microns.
(vi) The aqueous extract is treated using microfiltration or heat treatment to control any possible microbial load.
(vii) The resulting aqueous extract is concentrated to a solids content of 5% (w/v) to 50% (w/v). During this step, an insoluble precipitate is formed in the concentrated extract.
(viii) The precipitate is separated by a separation method that separates the resulting precipitate from the supernatant.
(ix) Drying the wet precipitate using any drying method that results in a solid product having a moisture content of less than 10% (w/w) to 15% (w/w).
(x) the extract obtained using this method has a triterpene content of between 5% (wt/wt) and 95% (wt/wt), of which between 5% (wt/wt) and 90% (wt/wt) is composed of ursolic acid.

This method stands out as being simple, affordable, and capable of obtaining the desired product with high ursolic acid content and a ratio of 5 to 40 to the raw material.

Preparation of Ginkgo Biloba Extract Ginkgo biloba leaf extract is prepared by a method comprising the following steps:
(a) Extraction of fresh green or dried Ginkgo biloba leaves with water for 1 hour to 20 hours at a temperature of about 40° C. to 100° C. The process may also include several stages of extraction of the leaves, combining the resulting aqueous extracts and continuing the process.
(b) The aqueous extract and ginkgo biloba leaves are separated by any separation method that results in an aqueous extract that is free of leaf particles larger than 50 microns to 100 microns.
(c) The aqueous extract is treated with activated charcoal for 1 to 3 hours at 40° C. to 100° C. The dosage of activated charcoal is 1% (w/w) to 20% (w/w) of the dry matter present in the extract.
(d) The aqueous extract is separated from the activated charcoal using any separation method that results in an aqueous extract that is free of activated charcoal particles and leaf particles larger than 0.45 microns to 10 microns.
(e) The aqueous extract is treated by microfiltration or heat treatment to control any possible microbial load.
(f) Concentrate the resulting extract until the solid content is between 10% and 70%.
(g) Optionally, the concentrated aqueous extract is dried using any drying method that results in a solid product having a moisture content of less than 10% (w/w) to 15% (w/w).

The above process results in an extract containing a ginkgo flavonoid content of 0.5% (w/w) to 15% (w/w) and a terpene lactone content of 0.01% (w/w) to less than 1% (w/w). This process is distinguished by using water as the only extraction solvent. It is inexpensive and gives the desired product in a ratio of 2 to 8 with respect to the raw material.

Preparation of D-pinitol-rich extract
The preparation of a natural extract rich in D-pinitol can be carried out essentially according to the method as described in European Patent Application No. 1241155. This method comprises:
(i) Providing the raw material, dried carob pods.
(ii) Carob pods are crushed to obtain pulp.
(iii) Extraction of the carob pulp with water at slightly acidic pH and at 10°C to 70°C until an extract of 30°Brix to 50°Brix is obtained.
(iv) Pressing the pulp to remove retained water.
(v) filtering the raw juice and passing the filtrate through a strong cation resin to remove most of the calcium and magnesium ions;
(vi) The aqueous extract is treated using microfiltration or ultrafiltration to control any possible microbial load.
(vii) Concentrate the syrup to about 60°Brix.
(viii) The sucrose in the syrup is converted either by an enzymatic process or by an acid process using a cationic resin such as RPI resin.
(ix) A syrup having a concentration between 20°Brix and 30°Brix is successively desalted and decolorized by passing it through a strong cation (H) and then a strong anion resin (OH) until a composition is obtained having a conductivity of less than 10 μΩ and a color of less than 25 Icumsa at 420 nm.
(x) The desalted and decolorized syrup is fractionated by strong cation exchange resin, preferably using ISMB technology (sequential chromatographic separation). The separation is typically carried out starting from carob syrup of about 60°Brix, using 4 ^m3 of UBK 530 resin, at a feed rate of 0.038 L/h, with a W/F of 3.750 (vol/vol) and a P/R of 2.166 (vol/vol), at a temperature of 65°C, and with a feed volume of 2.07 T/D and a pinitol fraction volume of 0.36 T/D.
(xi) The pinitol fraction thus obtained, containing 44.2% salt, is desalted and decolorized.

The resulting composition contains 90% pinitol, 5% glucose and 5% fructose and is concentrated and crystallized by the addition of ethanol.

The extract obtained using this method has a pinitol content of 85% (w/w) with a specific rotation of (+)64 and a water content of 2%.

Alternatively, pinitol can be extracted from the converted, desalted and decolourised carob syrup using a strong anionic resin, in which case the process entails following steps (i) to (ix) above:
(x) The desalted and decolorized syrup, having a Brix of about 25° and a composition of 35% pinitol, 27% glucose, 37% fructose and 0.4% non-sugars, is fractionated on 250 ml or resin SA 11A contained in a column of diameter 2 cm and height 100 cm at a rate of 10 ml/min and eluted with demineralized water at a temperature of 6° C.
(xi) The fractions having a positive °Brix are mixed and the mixture is concentrated.
(xii) The concentrated mixture is crystallized from ethanol.

The extract obtained using this method has a pinitol content of 91% (w/w) with a specific rotation of (+)63 and a water content of 2.5%.

Methods Test Subjects and Compositions for the Zebrafish Model The species selected for testing was the AB wild type strain of zebrafish (Danio rerio). The fish were contacted with five compositions (C1, C2, C3, C4 and C5) at a single dose each. The compositions are as follows:
Compound C1
Compound C2
Compound C3: D-pinitol, RS=98.58, pinitol=93.29% wt/wt Compound C4: Sage extract Compound C5:

The following table summarizes the experimental conditions.
Table 1. Table showing compound code, weight of compound and volume of DMSO required for preparation of stock solution, solution state and final concentrations tested.

All compounds were initially tested at a concentration of 100 mg/l. However, in the first test, compound C4 at this concentration caused 100% mortality of the larvae. Therefore, two new tests were performed in which all compounds were tested at 100 mg/l, except for C4, which was tested at a concentration of 10 mg/l.

Once the embryos were obtained, they were seeded in Petri dishes containing 50 ml of diluent (DS) and kept for 5 days post-fertilization (5 DPF), which is considered the larval stage. Only larvae that did not show any external abnormalities were used for the study. Then, using a Pasteur pipette, the larvae were transferred to 24-well microplates, with 5 larvae in each well, with 10 replicas per condition. First, pretreatment was performed on the 5 DPF larvae. To do this, the larvae were incubated in a volume of 2 ml of physostigmine (PHYS, a commercially available AChE enzyme inhibitor) and the test compound for 1 hour at 26 ± 1 °C. Next, the medium of the larvae was replaced and they were incubated with PHYS, PTZ, and other compounds in combination with PTZ for 6 hours at 26 ± 1 °C. After this incubation period, all the larvae were examined and it was determined that the general condition of the larvae was completely normal and free of any visible abnormalities or abnormal behavior. Finally, the larvae were processed to analyze AChE activity. A control group in which the embryos were seeded without any treatment was run in parallel.

Determining AChE levels:
At the end of the experimental period, the larvae were processed to determine AChE levels: they were mechanically homogenized and samples were centrifuged to obtain supernatants that were used to determine AChE enzyme levels according to the treatments administered.

In addition, the total protein in each experimental group was determined according to the normalization process.

Finally, the AChE levels determined in the control group were considered as 100% and taken as the baseline measurement.

Methods for the Murine SAMP8 Model Experimental Subjects The experimental procedures described herein were approved by the Comitetíco para la experimentacion animal (Committee on Ethics of Animal Experimentation) of the University of Barcelona and the Provincial Government (No. 222/18).

Female mice of the SAMR1 and SAMP8 strains born between December 2018 and January 2019 at the University of Barcelona Faculty of Pharmacy were used.

Animals were divided into three experimental groups, treatment was initiated at 5 months of age, and behavioral assessments were performed after 8 weeks of treatment using the open field test (OFT), object recognition test (NORT), and object location test (OLT), after which the animals were euthanized and samples were collected.
SAMR1 control group (n=10)
SAMP8 control group (n=10)
SAMP8 treated group (n=12)

Treatment with Ginkgo biloba extract, plant extract rich in ursolic acid (such as Ursolia™), D-pinitol and fatty acid composition containing 25% DHA According to the protocol, female mice were treated orally via stomach tube with a combination of extracts and DHA (product) from Biosearch S.A. The extracts were dissolved in 40% hydroxypropyl beta-cyclodextrin. The concentration of the extracts was calculated according to the weight of the animals to reach the specified doses shown in Table 1.

Table 3. Dosage and concentration ratios for each extract

Treatment with the products lasted for 8 weeks in a 5+2 delivery pattern, i.e., the products were delivered for 5 days followed by 2 days without delivery. The products were delivered across the behavioral testing days and the mice were euthanized 3 days after the final behavioral testing. Mice received two deliveries via dailyprobe, one containing DHA and the other containing Ginkgo biloba extract, Ursolia™ and pinitol (Figure 1).

Open Field Test (OFT)
The open field test (Figure 2) is a classic test of behavioral patterns in which animals are exposed to a novel, open, brightly lit environment (SeibenhenerML and WootenMC, 2015 J Vis Exp.;96:e52434). Behavior in this space is determined by the balance between the animal's natural interest in new spaces (exploratory behavior) and its fear of unknown, open, brightly lit spaces (fear/anxiety). The apparatus consists of a white wooden box (50cm x 50cm x 25cm height). The luminance of the light in the center of the field was 30 lx. Mice were placed in the center of the apparatus and their behavior was evaluated for 5 min. The variables measured were overall horizontal (total distance traveled, number of linecrossings) and vertical (number of rearing or rearing of the rodent) locomotor activity as well as variables related to emotionality, time spent in the center, time spent at the edge, distance traveled in the center area, distance traveled in the peripheral area, % time spent in the center, % time at the edge, number of defecations and number of urinations. The aim of the task was to obtain a video for subsequent analysis by recording it using SMART™ ver.3.0 (PanLab, SLU, Spain).

Novel Object Recognition Test (NORT)
The NORT (Figure 3) paradigm is a widely used explicit memory test. This behavioral test exploits two rodent characteristics: the natural tendency of these animals to explore novel objects that are not particularly important to the animals and are not associated with reinforcement, and the innate preference to explore novel objects over familiar objects (EnnaceurA et al., 1988, Behav Brain Res. 31:47-59). In the NORT test, mice were placed in a black maze consisting of two L-shaped branches measuring 25 cm in length, 20 cm in height, and 5 cm in width. The luminance of the light in the center of the field was 30 lx. Different plastic objects were used. For the first three days, mice were allowed to grasp the maze for 10 min. On the fourth day, mice were allowed to explore identical objects at both ends of one of the maze branches. A 10-min retention test was performed 2 h later for short-term memory tests or 24 h later for long-term memory tests. During the second test, a novel object was placed at one end of a branch of the maze that the mice had not previously explored, and the time that the animals spent investigating the novel object (TN) and the old object (TO) was recorded. This was done to calculate the discrimination index (DI), defined as (TN-TO)/(TN+TO). To avoid object preferences, objects A and B were counterbalanced, with half of the mice in each experimental group exposed first to object A and then to object B, and the other half exposed first to object B and then to object A. To eliminate olfactory cues, the maze and objects were cleaned with 70% ethanol after each test. NORT testing began on the first Friday after completing the treatment week from 09:30 to 15:30.

Object Location Test (OLT)
The Object Location Test (OLT) specifically assesses cognitive deficits in spatial memory and spatial discrimination (HattiangadyB et al., 2014, Front Behav Neurosci. 8:78). This task involves utilizing the rodent's ability to recognize when an object has been relocated and is essentially non-stressful to the rodent. Testing was performed for 4 days in a wooden box (50cm x 50cm x 25cm) with four walls marked with white and one with a black and white square pattern. On day 1, the box was empty and the animal was allowed to habituate to the OFT cage for 10 min. On day 2, two objects were placed on the patterned wall at equal distances from each other and from the wall. These objects were 10cm high and replicas of each other. The animal was placed in the center of the cage and allowed to explore the objects and surroundings for 10 min. The mouse was then returned to the cage and the OLT apparatus was cleaned with 70% ethanol. On day 3, the object was moved in front of the opposite white wall to test spatial memory (Figure 4). The test was recorded using a camera mounted in the working area, and the total investigation time was determined by recording the time (seconds) spent sniffing the object in the new location (novel) and the object in the old location (old). To analyze the cognitive performance, the location index (%) was calculated using the following method: (Tnovel x 100)/(Tnovel + Told), where Tnovel is the time spent sniffing the object in the new location and Told is the time spent investigating the object in the old location.

Three days after the last behavioral test, animals were euthanized by cervical dislocation. Brains were dissected, immediately frozen on dry ice, stored at −80°C and made available to Biosearch S.A.

Analysis of the results of the open field test, the object recognition test and the object location test To analyze the tests performed, the activity of the mice during all familiarization, retention and memory tests, as well as the open field test, was recorded and then analyzed manually according to the SOPs for each procedure, which are included as an appendix to this report.

Statistical analysis To evaluate possible significant differences between the study groups, a one-way ANOVA statistical test with post-hoc Tukey's multiple comparison test was performed to compare the three groups involved in the treatment. Furthermore, the t-Student statistical tool was used to compare the SAMP8 control and SAMP8 treated groups when there was a clear trend and it was not significant using Dunnett's test. Prior to statistical analysis, Grubbs' test was performed for abnormal data at the 90% confidence level. For graphical presentation, the obtained data are expressed as mean ± standard error of the mean (mean ± SEM). Differences in t-Student were also considered significant at p<0.05 and are indicated by a single (*) with one, two or three more stars being added depending on the value of p (* p<0.01, ** p<0.001 and *** p<0.0001). For statistical analysis and graphs, the statistical analysis program GraphPad 8.0 was used.

Methods for the C. elegans model
1.1. Compounds Test doses were established according to previous studies of Biosearch SA extract in SAMP8 (Senescence accelerated mice prone 8), a mouse model of late-onset AD (LOAD), maintaining and adjusting the range of test doses found in the literature review (1-10). For each compound, concentrations lower than the documented effective concentration were selected. Higher concentrations closer to the published effective dose were also tested when studying drug responses in the oxidative resistance assay. Regarding the composition of the mixture, the proportions of each compound were established in light of previous studies reporting synergistic effects in mice.

Stock concentrations of Biosearch SA extract were prepared in 100% dimethyl sulfoxide (DMSO). Stock solutions were then diluted with MiliQ ddH20 to obtain test dilutions with a maximum concentration of 1% DMSO and stored at -20°C.

1.2. Maintenance and Treatment of C. elegans Wild-type Caenorhabditis elegans (C. elegans) strain N2, transgenic strain CL2006, transgenic strain CL2355 and control strain CL2122 were used. Standard methods were used for the cultivation and observation of C. elegans. N2 was grown at 20°C and CL2006, CL2355 and CL2122 were maintained at 16°C on solid nematode growth medium (NGM) seeded with the OP50 strain of Escherichia coli (E. coli) as a food source in a temperature-controlled incubator. To obtain eggs of age-synchronized populations, egg-bearing adults were treated with an alkaline hypochlorite solution (0.5 M NaOH, approximately 2.6% NaClO) for 5-7 min. Fertilized eggs were suspended in S medium for 12 h and L1 larvae were allowed to hatch overnight in the absence of food.

In most cases, drug assays were performed in 96-well plate format, liquid culture, and treated for 4 days at 20°C. Each well contained a final volume of 60 μl and contained 25-30 animals at L1 stage, the appropriate dose of Biosearch SA extract, and OP50 inactivated by freeze-thaw cycles and completely suspended in S medium until a final OD ₅₉₅ of 0.9-0.8 was measured with a microplate reader. For chemotaxis assays, synchronized CL2355 and its control CL2122 were treated with Biosearch SA products starting from the L1 stage on fresh NGM plates seeded with inactivated E. coli. They were cultivated at 16°C for 36 hours, then at 23°C for another 36 hours.

1.3. Oxidative resistance assay
To investigate the susceptibility to oxidative stress after treatment with Biosearch SA products, treated adults of N2 were transferred to plates containing 6.2 mM t-butyl hydroperoxide (Sigma) in NGM agar. Worms were incubated on these plates for 2 hours at 20°C. Worms were then transferred to new NGM plates seeded with OP50 and lacking t-butyl hydroperoxide. Worms were observed 2, 24, and 48 hours after intervention and scored as dead if they did not respond to repeated prodding with a pick.

1.4. Chemotaxis assay After each treatment, nematodes were collected and washed with M9. Briefly, assays were performed in 100 mm NGM plates, and 10 μL of odorant (0.5% benzaldehyde in 96% ethanol) was added to the "attractant" spot along with 1 M sodium azide. On the opposite side, 10 μL of control odorant (96% ethanol) was added along with 1 M sodium azide. Immediately after, 50-60 worms were placed in the center of the plate. The assay plates were incubated for 1 h at 23°C, and the chemotaxis index (CI) was scored as follows: CI = (number of worms in attractant - number of worms in control)/total number of worms. At least 60 worms from each group were analyzed in each experiment.

1.5. Thioflavin S stained Aβ aggregates Age-synchronized CL2006 worms were then fixed in 4% paraformaldehyde/PBS, pH 7.5, 4°C for 24 h and permeabilized in 5% fresh β-mercaptoethanol, 1% TritonX-100, 125 mm Tris, pH 7.5, 37°C for another 24 h. Worms were stained with 0.125% Thioflavin S (Sigma) in 50% ethanol for 2 min, destained in 50% EtOH for 2 min, washed three times with PBS, and transferred to a drop of Fluoromount G on a microscope slide in a volume of approximately 10 μL. Fluorescent images were acquired using a 20 Å-objective on a fluorescence microscope (Olympus BX51, Germany). Amyloid deposition in the head region of the worm was quantified by counting the number of thioflavin S (ThS)-positive spots using ImageJ and expressed as Aβ deposits/frontal area.

1.6. Life span assay Worms were treated as above in liquid culture for 4 days starting at the L1 stage. However, 1 μL of 5'-fluorodeoxyuridine (FUdR) was added at 4 days of age to a final concentration of 120 μM to prevent the production of progeny. After treatment, approximately 30 young adult worms were placed on three different NGM plates per condition, transferred to a new seeding plate every 3 days, and scored for dead animals. Animals were considered dead if no mechanical response was elicited by tapping the head three times with a platinum wire.

1.7. Statistics Data analysis was performed using GraphPad Prism ver. 9 statistical software. Data are expressed as the mean ± standard error of the mean (SEM) of at least three experiments. Means were compared by one-way analysis of variance (ANOVA) followed by Tukey's posthoc test. Comparisons between groups were also performed by two-tailed Student's t-test for independent repeats, when appropriate. p values <0.05 were considered statistically significant. Statistical outliers were determined by Grubs' test and excluded from the analysis.

Example 1
Effect of different compositions in the mouse SAMP8 model
1. Weight Control of Plant Extract and Lipid Treatment All animals in each treatment group were weighed. All animals were weighed on the Monday before treatment began and every Monday throughout the treatment period until euthanasia. Table 5 shows the weights of each group at the beginning and end of treatment. Improvements in physical appearance were observed as a result of plant extract and lipid treatment.
Table 5. Weights of animals during treatment

Figure 5A shows the weight gain or loss during treatment of the mouse groups. The results show that at the end of treatment, all groups recorded weight gain, with an increase of 2% for the SAMR1 control group and 4% for the two SAMP8 groups. Figure 5B shows a significant difference in the weight of the mice at the end of treatment as well as at the start of treatment for the SAMR1 and SAMP8 control groups. However, the treatment has no effect on weight gain in the SAMP8 group compared to the control group.

2. Behavioral Results Herein, we provide an overview of the cognitive profile, emotional changes and behavioral characterization results induced by treatment with the products after 8 weeks of treatment.

2.1. Summary of Open Field Test (OFT) Results Figures 6A-C show the behavioral results in the open field test for the groups after 8 weeks of treatment.

SAMR1 control vs. SAMP8 control vs. SAMP8 treated:
ANOVA tests showed significant differences in vertical locomotor variables (p<0.001) and defecation variables (p<0.0001), but not in locomotor variables (p>0.05). Post-hoc analyses showed significant and similar changes in vertical locomotor variables for SAMP8 control and treated animals (p<0.05), and between SAMR1 controls and SAMP8 treated (p<0.01).

Similarly, post-hoc analyses also showed significant and similar changes in defecation variables for control animals (p<0.01) and between SAMR1 controls and SAMP8-treated (p<0.001). A clear trend towards a return to the SAMR1 control group by the SAMP8-treated group was observed for locomotor variables.

2.2. Summary of Object Location Test (NORT) Results Below is a summary of the results of product treatment characterizing short-term and long-term working memory cognitive profiles using the Object Recognition Test after 8 weeks of treatment.

2.2.1. Short-Term Memory FIG. 7 shows the results of an object recognition test after 8 weeks of treatment to assess the short-term working memory cognitive function of the mice groups.

SAMR1 Control vs. SAMP8 Control vs. SAMP8 Treated
ANOVA test showed significant differences in the discrimination index variables (p<0.0001). Post-hoc analysis showed significant and similar changes in the discrimination index variables for SAMR1 and SAMP8 control animals (p<0.0001). Furthermore, post-hoc analysis also showed significant and similar changes in the discrimination index variables between the SAMP8 control and SAMP8 treated groups (p<0.0001).

2.2.2. Long-term Memory FIG. 8 shows the results of an object recognition test after 8 weeks of treatment to assess the long-term working memory cognitive function of the mice groups.

SAMR1 Control vs. SAMP8 Control vs. SAMP8 Treated
ANOVA test showed significant differences in the discrimination index variables (p<0.01). Post-hoc analysis showed significant and similar changes in the discrimination index variables for SAMR1 and SAMP8 control animals (p<0.001). Furthermore, post-hoc analysis also showed significant and similar changes in the discrimination index variables between the SAMP8 control and SAMP8 treated groups (p<0.01).

2.3. Summary of Object Location Test (OLT) Results Below is a summary of the results of the characterization of the spatial memory cognitive profile by the Object Location Test performed on females treated with plant extracts and lipids, 8 weeks after the start of treatment.

2.3.1.- Spatial Memory FIG. 9 shows the results of the object location test after 8 weeks of treatment, to assess the spatial memory cognitive function of the mice groups.

SAMR1 Control vs. SAMP8 Control vs. SAMP8 Treated
ANOVA test showed significant differences in the discrimination index variables (p<0.001). Post-hoc analysis showed significant and similar changes in the discrimination index variables for SAMR1 and SAMP8 control animals (p<0.0001). Furthermore, post-hoc analysis also showed significant and similar changes in the discrimination index variables between the SAMP8 control and SAMP8 treated groups (p<0.01).

3. Events and Observations One SAMP8 mouse in the control group died during treatment. No internal damage was observed and the death was determined to be natural. Improvements were observed in the physical appearance of the mice treated with the product (Figure 10).

Example 2
Effects of different compositions on zebrafish treated with PTZ
Neuroprotective effects of compounds C1-C5 in a zebrafish model of PTZ-induced neurotoxicity
Table 6: Percentage of AChE enzyme activity (mu) by total protein (μg) relative to the control group (considered as 100%) in 5 DPF larvae treated for 6 h with PTZ, PHYS, and various compounds (C1-C5) in combination with PTZ. The table shows the mean, standard deviation (SD), standard error of the mean (SEM), and statistical analysis using Dunnett's multiple comparison test (relative to the control group).

As shown in Table 6, treatment of larvae with 5 mm PTZ induced a significant decrease in AChE activity of about 21% compared to the control group (p<0.01). Moreover, physostigmine, a strong inhibitor of AChE activity, reduced AChE activity by 76% compared to the control group, thus confirming the validity of the test (p<0.01). Among all problem groups, only PTZ treatment in combination with up to 100 mg/l C1 showed significantly lower activity than the control group, but not more than the decrease caused by the effect of PTZ itself.

Once it was determined that PTZ reduced AChE activity in zebrafish larvae at 5 DPF, the neuroprotective effects of the compound against neurotoxin-induced toxicity were analyzed.

As shown in Figure 11, two of the tested compounds induced a significant increase in AChE activity compared to PTZ-treated larvae. Treatment of larvae with 10 mg/l C4 inhibited the PTZ-induced effect on AChE activity, reaching 103% activity (#p<0.01). Finally, 100 mg/l C3 not only significantly prevented the PTZ-induced decrease in AChE activity, but also slightly increased AChE activity (108%) compared to the control group, although this increase was not significant.

This study demonstrates that PTZ, a convulsant antagonist of GABA receptors in the CNS, induces strong toxic effects by acutely decreasing AChE activity in zebrafish larvae. It has been shown that the zebrafish genome does not encode the butyrylcholinesterase (BCHE) enzyme, which is also the enzyme that degrades acetylcholine in humans. However, the AChE enzyme is encoded by a single gene in zebrafish and has been functionally detected in the brain. Therefore, the variations in AChE activity recorded in zebrafish larvae reflect only the function of the cholinergic system in the CNS.

The aim of this study was to analyze the potential neuroprotective effects of treatment with five compounds provided by the client and coded as C1, C2, C3, C4, and C5.

First, it is important to note that none of the groups treated with the compounds tested in combination with PTZ had lower AChE activity than that induced by PTZ itself, indicating that there was no additive neurotoxic effect induced by the compounds.

AChE activity assay showed that treatment with C3 (D-pinitol) and C4 (sage extract) compounds inhibited PTZ-induced neurotoxicity in 5 DPF zebrafish larvae, restoring AChE activity by 29% and 23%, respectively, compared with convulsant-treated larvae. These results indicate that both compounds have neuroprotective capabilities against PTZ-induced neurotoxicity.

Therefore, the compounds D-pinitol at 100 mg/l and sage extract at 10 mg/l were shown to significantly inhibit the PTZ-induced decrease in AChE activity in 5DPF larvae and exhibit neuroprotective effects.

Example 3
Effect of different compositions in the C. elegans model
1. Reduction of oxidative stress in C. elegans by the mixture To investigate whether the extracts and mixtures have any beneficial effect on oxidative stress, they were tested after tert-butyl (6.2 mM). Treatment began when synchronized worms reached the L4 stage and were maintained at 20 °C until the end of the experiment. First, it was found that each extract group did not reach protection against oxidative stress compared to the untreated control, and in addition, a significant decrease in survival rate was obtained compared to the vitamin C group (Figure 12). On the other hand, worms preincubated in the mixed extract (dosage) were significantly protected against tert-butyl (6.2 mM)-induced oxidative stress compared to the untreated group, reaching a survival rate of worms close to that of vitamin C (58 μM), indicating a synergistic effect of the combined administration of the extracts (Figure 12).

2. Suppression of neural Aβ expression-induced impairment in chemotaxis behavior in transgenic C. elegans (CL2355) by the mixed extract To investigate the synergistic effect of the mixed extract on the ability of chemotaxis behavior, benzaldehyde was applied as an attractant and ethanol as a control, both containing sodium azide that paralyzes the insects on contact (Figure 13). Chemotaxis index was scored for all groups at 5 days of age. Figure 14 shows that the CL2355 strain shows a significant reduction in CI compared to the control strain CL2122. Moreover, each extract separately shows a slight tendency to increase CI compared to the CL2355 strain, but not significantly (Figure 14). Interestingly, the CI of the mixed extract was significantly increased compared to the CL2355 group, showing a synergistic effect and restoring the chemotaxis behavior to the control strain CL2122 (Figure 14).

3. Improvement of Aβ burden in transgenic C. elegans (CL2006) by the mixed extract To investigate whether the mixture affects Aβ aggregation, the CL2006 worm strain, which constitutively overexpresses human Aβ _1-42 in muscle cells, was used. It was found that the mixture had a significant effect on the deposition of Aβ _1-42 peptide, suggesting that the mixture can synergistically reduce the aggregation of Aβ species to a great extent (Figure 15). As expected, the different extracts (DHA, Ginkgo, Pinitol, and Ursolia™) had no effect on the deposition of Aβ _1-42 , indicating that the synergistic effect obtained in the mixed group was not evident (Figure 15).

4. Average life span extension by mixed extract in C. elegans To investigate the effect on aging, we investigated the change in life span in C. elegans after treatment with different extracts and mixtures. First, Kaplan-Meier curves showed no significant changes between groups (Figure 16A). However, the average life span was extended by up to 15% by mixed extract treatment compared to the DHA, pinitol and Ursolia™ groups, showing a synergistic effect (Figure 16B), but there was no change between the mixture and ginkgo groups due to the well-described effect of ginkgo on life span (REF).

5. Conclusion In summary, the superior neuroprotective effects of the mixed extract compared to each individual extract on behavior, Aβ pathology, oxidative stress resistance, as well as life expectancy were demonstrated, clearly indicating that the synergistic effect of the mixed extract was not evident.

Drawing translation 1
Control
Treated
Start treatment
5 months
8 weeks
Behavioral tests
7 months
1 week
2-24 h 2 hours to 24 hours
Finish treatment
Euthanasia Euthanasia Figure 3
Habituation phase
Test phase
Familiarization phase (Day 4)
Short-term memory test (Day 4)
Long-term memory test (Day 5)
Object Figure 4
First day
Second day
Third day Figure 5
Weight evolution
Weeks
Average animal weight
Group
Initial
Final Figure 6
Locomotor Activity
Distance travelled
Control
Treated
Rearings
Shits Defecation diagram 7
Summary of NORT short-term memory
Discrimination Index
Control
Treated Figure 8
Summary NORT long-term memory
Discrimination Index
Control
Treated Figure 9
Discrimination Index
Control
TreatedFig.11
Normalized (% vs control) AChE activity
(mU/ug protein)
Figure 12
Oxidative Tolerance Assay
Percent survival
Vitamin C
Mix
Ginkgo
Pinitol Figure 13
1 hour
Attractant spot
Control spot Fig. 14
Chemotaxis index (neuronal Aβ strain CL2355) Chemotaxis index (neuronal Aβ strain CL2355)
Chemotaxis index Chemotaxis index
Mix
Ginkgo
Pinitol Figure 15
Number of Aβ aggregates
Mix
Ginkgo
Pinitol
Untreated CL2006 Untreated CL2006
Figure 16
Percent survival
Time (days)
Pinitol
Ginkgo
Mix
Mean Lifespan
Days

Claims (22)

A composition or kit of parts for use in the prevention and/or treatment of cognitive impairment, comprising the components D-pinitol, ursolic acid , docosahexaenoic acid (DHA) and ginkgo flavonoids, wherein the weight ratio of said components is about 63.3:7.5:40:1. The composition or kit of parts of claim 1, wherein the D-pinitol is provided as a D-pinitol-rich plant extract. 2. The composition or kit of parts of claim 1 , wherein the ursolic acid is provided as a ursolic acid-rich plant extract, the ursolic acid-rich plant extract being a hydroalcoholic extract of a plant product rich in ursolic acid. 4. The composition or kit of parts according to claim 3, wherein the plant extract rich in ursolic acid is selected from leaves of plants of the Lamiaceae family, marine algae or fruit shells. 5. The composition or kit of parts according to claim 3 or 4, wherein said ursolic acid rich plant extract contains between 5% and 90% ursolic acid. The ursolic acid-rich plant extract is
(i) hydroalcoholic extraction from the leaves, marine algae, or fruit shells of plants of the Lamiaceae family;
(ii) treating the hydroalcoholic extract obtained in step (i) with activated carbon, followed by removal of the activated carbon;
A composition or kit of parts according to any one of claims 3 to 5, obtained by a process comprising the steps of: The composition or kit of parts according to any one of claims 2 to 6, wherein the plant extract rich in D-pinitol is an extract from the fruit of a plant of the genus Carob. The composition or kit of parts according to any one of claims 1 to 7, wherein the DHA is provided as a fatty acid composition containing at least 25% DHA. The composition or kit of parts of claim 8, wherein the fatty acid composition is obtained from fish or from microalgae. The composition or kit of parts according to any one of claims 1 to 9, wherein the ginkgo flavonoid is provided as a Ginkgo biloba extract. The composition or kit of parts of claim 10, wherein the Ginkgo biloba extract contains 0.5% (w/w) to 15% (w/w) ginkgo flavonoids and less than 1% (w/w) terpene lactones. The ginkgo biloba extract is
(i) an aqueous extract from Ginkgo biloba leaves;
(ii) treating the aqueous extract obtained in step (i) with activated carbon and then removing the activated carbon;
12. A composition or kit of parts according to claim 10 or 11, obtained by a process comprising: A composition or kit of parts according to any one of claims 1 to 12, further comprising a carrier. The composition according to claim 13, wherein the concentration of the carrier is 20% (w/w) to 90% (w/w). A pharmaceutical product for use in the prevention and/or treatment of cognitive impairment, comprising a composition according to any one of claims 1 to 14 and a pharma- ceutical active carrier. A nutritional or dietary supplement for use in the prevention and/or treatment of cognitive impairment, comprising a composition according to any one of claims 1 to 14 and a nutritionally acceptable carrier. The composition or kit of parts of any one of claims 1 to 14, the pharmaceutical product of claim 15, or the dietary or health supplement of claim 16, wherein the cognitive disorder is selected from the list consisting of mild cognitive impairment, Alzheimer's disease, and Parkinson's disease. 18. The composition or kit of parts, pharmaceutical product, or dietary supplement or dietary supplement of claim 17, wherein at least one of the components of the composition or kit of parts, pharmaceutical composition, or dietary supplement or dietary supplement is administered separately from the remaining components of the composition or kit of parts, pharmaceutical product, or dietary supplement or dietary supplement. said administration of said components contained in said composition or kit of parts, said pharmaceutical product, or said nutritional or dietary supplement,
(i) about 150 mg/day of a plant extract rich in ursolic acid;
(ii) about 480 mg/day of DHA-rich fatty acids;
(iii) about 500 mg/day of Ginkgo biloba, and/or
(iv) about 200 mg/day of D-pinitol;
20. The composition, kit of parts, pharmaceutical product, or dietary or health supplement of claim 18, comprising administering said administration of said components contained in said composition or kit of parts, said pharmaceutical product, or said nutritional or dietary supplement,
(i) about 2.5 mg/kg of a plant extract rich in ursolic acid;
(ii) about 8 mg/kg of a DHA-rich fatty acid composition;
(iii) about 8.33 mg/kg of Ginkgo biloba extract, and/or
(iv) about 3.33 mg/kg D-pinitol;
20. The composition, kit of parts, pharmaceutical product, or dietary or health supplement of claim 18, comprising administering The composition or kit of parts, pharmaceutical product, or dietary or dietary supplement of any one of claims 18 to 20, wherein the administration comprises administration of multiple doses of the components contained in the composition or kit of parts, pharmaceutical product, or dietary or dietary supplement for at least one month. The kit of parts according to any one of claims 18 to 21, wherein the parts of the kit of parts are combined prior to said administration.