AU2021204964A1

AU2021204964A1 - Composition for use in the treatment of cognitive disorders

Info

Publication number: AU2021204964A1
Application number: AU2021204964A
Authority: AU
Inventors: Óscar Bañuelos Hortigüela; Cristian Gaspar GRIÑAN FERRE; José Luis LÓPEZ LARRAMENDI; Mónica María OLIVARES MARTÍN; Mercè PALLÀS LLIBERIA; Luís PÉREZ MARTÍNEZ
Original assignee: Biosearch SA
Current assignee: Biosearch SA
Priority date: 2020-01-03
Filing date: 2021-01-08
Publication date: 2022-07-07
Also published as: EP4084787A1; JP2023510525A; CN115066237A; KR20220123272A; BR112022013296A2; MX2022008258A; WO2021136848A1; CO2022010461A2

Abstract

The invention relates to compositions of natural origin which are useful in the treatment of cognitive disorders which contain D-pinitol and ursolic acid or extracts from natural sources which are enriched in these components and which may additionally comprise DHA and/or a

Description

COMPOSITION FOR USE IN THE TREATMENT OF COGNITIVE

DISORDERS

FIELD OF THE INVENTION

The invention relates to a composition comprising vegetal extracts for use in the treatment of a cognitive disorder, such as Alzheimer’ s disease, mild cognitive impairment or Parkinson’s disease. BACKGROUND OF THE INVENTION

Dementia is the loss of cognitive functioning — thinking, remembering, and reasoning — and behavioral abilities to such an extent that it interferes with a person’s daily life and activities. Dementia ranges in severity from the mildest stage, when it is just beginning to affect a person’s functioning, to the most severe stage, when the person must depend completely on others for basic activities of daily living. Common types of dementia include Alzheimer's disease, vascular dementia, Lewy Body disease, Fronto-temporal dementia, or Early Onset dementia. Around 60-70% patients with dementia have Alzheimer’ s disease (AD) and about 6% of subjects aged 65 or older are diagnosed with AD.

AD is a neurodegenerative disease with a progressive pattern of cognitive and functional impairment. At the early stage of the disease, the patient simply shows a short memory loss and subtle problems with the executive functions of attentiveness, planning, flexibility, and abstract thinking, or impairments in semantic memory (memory of meanings, and concept relationships). As the disease progresses, there is an increase in memory problems, a deterioration in language, reading and writing skills, as well as in the coordination of motor sequences. In advanced stages, patients become completely dependent upon caregivers, as they might completely lose the ability to speak, and they show a deterioration in muscle mass and mobility that impedes them to feed themselves. Mild cognitive impairment (MCI) is often found to be a transitional stage between normal ageing and dementia, especially Alzheimer’ s disease. MCI affects approximately 15-20% of people aged 65 or older. It can be classified as amnestic MCI and non-amnestic MIC. Amnestic MIC is characterized by a loss of memory by the person affected, so that he/she forgets important information that he/she would previously have recalled easily, such as appointments, conversations or recent events. Non amnestic MCI is characterized by the loss of cognitive abilities by the person affected other than memory, such as the ability to make sound decisions, judge the time or sequence of steps needed to complete a complex task, or visual perception. Although MCI can revert to normal stages or remain stable, amnestic MCI is frequently seen as a prodromal stage of AD. Non-amnestic MCI patients are found to be more prone to suffer other dementias.

There is no definitive evidence supporting any particular measure for preventing AD. Additionally, no medication has been shown to delay or halt the progression of AD. Available treatments offer relatively small symptomatic benefit. No medication has been proved to be effective to cure MCI neither and no high-quality evidence has been provided for pharmaceutical drugs or dietary supplement that could improve cognitive symptoms in MCI patients.

Therefore, there is a need in the field for treatments effective for stopping, or even reverting, cognitive deterioration of patients with dementia, in particular with AD or with MCI.

SUMMARY OF THE INVENTION

The inventors have shown that a vegetal extract rich in ursolic acid (sage extract) and a vegetal extract rich in D-pinitol have a neuroprotective effect in zebra fish model organisms of neurodegenerative diseases (AB zebra fish strain treated with pentylenetetrazol (PTZ)) as well as an effect on the acetylcholinesterase activity. Studies of the development of the CNS of the zebrafish (Kimmel et ah, 1995, Developmental Dynamics 203 :255-310) show that at 24 hours the brain segmentation is already appreciable and structures, such as the neural tube, the notochord and the somites (precursors of muscle and skeleton), have formed. At five days of development, some sensorial organs, such as the eyes and ears, have formed. The heart, liver, kidneys and pancreas have also appeared, and the circulatory system, digestive system and nervous system are perfectly functional. At this stage, the fish is capable of responding to visual, olfactory and mechanical stimuli and start swimming in search of food.

AChE is an enzyme that catalyses the hydrolysis of the acetylcholine neurotransmitter in choline and an acetate group. It is mainly found in the neuromuscular junctions and the cholinergic nervous system, where its activity serves to terminate synaptic transmission. Acetylcholine is a neurotransmitter involved in controlling movement and an important modulator of the cognitive functions, such as learning and memory (Hasselmo et al, 2011, Neuropsychopharmacology, 1:52-73). Therefore, adequate levels of acetylcholinesterase reflect a healthy neuronal condition.

PTZ is a competitive stimulant of gamma-aminobutyric acid (GABA) receptors. Its activity on the receptor blocks Cl-anion conductance and the formation of inhibitory postsynaptic potentials, which increases glutamatergic excitability (McDonald et al, 1978, Science, 200:775-777). The zebrafish model exerts pro-convulsive effects, probably by blocking GABAergic inhibitory synaptic transmission (Huang et al, 2001, J. Pharmacol. Exp. Ther. 298:986-995).

Additionally, the inventors have analyzed the effect of administering a vegetal extract rich in ursolic acid together with a vegetal extract rich in D- pinitol, a Ginkgo biloba extract and a fatty acids composition rich in DHA, to a group of SAMP 8 mouse (senescence-accelerated mouse prone 8), which shows pathological similarities with AD patients (Pallas M. 2012, ISRN Cell Biology, Vol. 12, Article ID 917167). They have observed that after said treatment, the capacity of said group of mice to undertake cognitive functions deteriorated in AD patients is comparable to that of control mice (senescence-accelerated mouse resistant 1, SAMRl). They have thus shown that the administration of a composition comprising a vegetal extract rich in ursolic acid, a vegetal extract rich in D- pinitol, a Ginkgo biloba extract and a fatty acids composition rich in DHA can restore cognitive functions seriously compromised in AD patients.

The inventors have also shown that the administration of a composition comprising a fatty acid composition enriched in DHA, a Ginkgo extract enriched in flavonoids, a vegetal extract enriched in D-Pinitol and a vegetal extract enriched in ursolic acid to C.elegans results in an improved oxidative tolerance, increased lifespan, increased chemotactic activity and reduced AB aggregation when compared with control animals.

Thus, in a first aspect, the invention relates to a composition or kit-of-parts comprising D-pinitol, ursolic acid and one or more additional components selected from the group of:

(i) docosahexaenoic acid (DHA),

(ii) ginkgo flavonoids and

(iii) a mixture thereof.

In a second aspect, the invention relates to a pharmaceutical composition comprising the composition according to the first aspect and a pharmaceutically active carrier.

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

The composition or kit-of-parts according to any of claims the first aspect of the invention, or the pharmaceutical product according to the second aspect of the invention, or the food or dietary supplement according to the third aspect of the invention, for use in medicine.

DESCRIPTION OF THE FIGURES

Figure 1. SAMR1/SAMP8 preclinical study schedule.

Figure 2. Open field test (OFL) schema. Figure 3. Novel Object Recognition Test (NORT) scheme Figure 4. Object Location Test (OLT) scheme.

Figure 5. A, Animal weight gain/loss during the treatment. B, Average body weight at the beginning and the end of the study.

Figure 6. Behavioural evaluation of the groups of mice in open field (n=10 - 12). A)

Locomotor activity B) Vertical activity C) Defecations

Figure 7. Summary of the short-term NORT results for the groups (n=10-12 )

Figure 8. Summary of the long-term NORT results for the groups (n=10-12 )

Figure 9. Summary of the habituation to the three cameras (OLT) results for the groups (n=10- 12 )

Figure 10. A) Example of the physical appearance of the SAMP 8 control group mice. B) Example of the appearance of the SAMP 8 treated group mice.

Figure 11: Bar graph showing the mean ± SEM of the AChE activity of the larvae treated with PTZ, PHYS and with the different compounds combined with PTZ versus the control group, considered 100% (red broken line). The statistical method used was Dunnetfs multiple comparison test (* p <0.05; ** p <0.01 vs control group) and Bonferroni's multiple comparison test (# p <0.01; ## p <0.001 vs. PTZ).

Figure 12. Summary of oxidative stress response different C. elegans groups after treatment with each extract, mix or Vitamin C (58 pM) There is significant difference among these groups when the symbols are different (P<0.05). Data are the average of three replicates with about 120-150 worms in each group.

Figure 13. Schematic diagram of the chemotaxis assay behaviour in neuronal AB- expressing strain CL2355.

Figure 14. Chemotaxis assay results in neuronal strain CL2355. There is significant difference among these groups when the symbols are different (P<0.05). Data are the average of three replicates with about 120-150 worms in each group.

Figure 15. Quantification of ThS-positive particles in head region of CL2006 strain (A). Representative images from each group tested (B). There is significant difference among these groups when the symbols are different (P<0.05). Data are the average of three replicates with about 50-60 worms in each group.

Figure 16. Kaplan-Meier curve for the survival of C. elegans on different extracts (A). The lifespan means of C. elegans treated groups and control with DMSO 1% (B). There is significant difference among these groups when the symbols are different (P<0.05). Data are the average of three replicates with about 60-70 worms in each group.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have observed that a vegetal extract rich in ursolic acid as well as a vegetal extract rich in D-pinitol have neuroprotective effects in a zebra fish model of neurodegenerative disease. They have also observed that the administration of a composition obtained by combining a fatty acid composition rich in DHA, a vegetal extract rich in ursolic acid, a vegetal extract rich in D-pinitol and a Ginkgo biloba extract to a mouse model of accelerated senescence can restore some of the cognitive dysfunctions which are compromised in said model animals.

1. Composition or kit-of-parts of the invention.

In a first aspect, the invention relates to a composition or kit-of-parts comprising D- pinitol, ursolic acid and one or more additional components selected from the group of:

(i) docosahexaenoic acid (DHA),

(ii) ginkgo flavonoids and

(iii) a mixture thereof.

The term “composition”, as used herein, refers to a combination of compounds or ingredients. The ingredients of the composition can be supplied either separately, or in a unit dosage. Thus, if the composition is administered to a subject, the compounds of the composition might be mixed together in said unit dosage, mixed together before the administration although provided separately, be provided and administered separately but mixed once taken by the subject, i.e. inside the body of the subject. Additionally, some ingredients of the composition may be administered together and others separately, but they are all mixed once taken by the subject, i.e. inside the body of the subject. The term “kit-of-parts”, as used herein refers to a product comprising different ingredients, components, or compounds wherein said ingredients, components, or compounds are physically separated, preferably by separate packaging of each ingredient, component or compound within the kit such that it allows being transported and stored. As it will be understood, in the “kit-of-parts” according to the present invention, the individual active ingredients, components, or compounds represent therapeutic agents and, provided that the use of those compounds, either simultaneously, separately or sequentially, produces the new and unexpected joint therapeutic effect as herein described that is not attained by the compounds independently of each other. Indeed as demonstrated by the results below, the claimed combination of active ingredients did not represent a mere aggregate of known agents, but rather a new combination with the surprising, valuable property that the combined effect is much more important that the simple addition of the effects that are observed, when those active ingredients are used separately. The kit-of-parts typically comprises its components in suitable containers. Materials suitable for the packaging of the components of the kit include glass, plastic (polyethylene, polypropylene, polycarbonate, and the like), bottles, vials, paper, sachets, and the like.. For example, each container may be in the form of vials, bottles, squeeze bottles, jars, sealed sleeves, envelopes or pouches, tubes or blister packages or any other suitable form provided the container is configured so as to prevent premature mixing of components. Each of the different components may be provided separately, or some of the different components may be provided together (i.e. in the same container). Furthermore, the kits of the invention can contain instructions for the simultaneous, sequential, or separate use of the different components that are in the kit. Said instructions can be in the form of printed material or in the form of an electronic medium capable of storing instructions such that they can be read by a subject, such as electronic storage media (magnetic disks, tapes, and the like), optical media (CD-ROM, DVD), and the like. The media may additionally or alternatively contain Internet addresses providing said instructions. It will be understood that the compositions and kits-of-parts of the invention may comprise, consist essentially or consist of the ingredients mentioned above.

In this specification the term "comprising" or "comprises" is used to indicate that the composition being described must contain the listed ingredient(s) but may optionally contain additional ingredients. The term "consisting essentially of or "consists essentially of is used to indicate that composition being described must contain the listed ingredient(s) and may also contain small (for example up to 5 percent by weight, or up to 1 percent or 0.1 percent by weight) of other ingredients provided that any additional ingredients do not affect the essential properties of the extract or composition. The term "consisting of or "consists of is used to indicate that the composition being described must contain the listed ingredient(s) only.

In a particular embodiment, the composition or kit-of-parts according to the first aspect of the invention comprises further ingredients in addition to D-pinitol and ursolic acid. In a particular embodiment, it comprises at least 1, at least 2, at least 3, at east 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 ingredients in addition to D-pinitol and ursoilic acid. In another particular 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 ingredients making up the kit.

The term “D-pinitol”, “Pinitol”, “3-O-Methyl-D-chiro-inositol”, “Methylinositol” as used herein, refer to the compound with IUPAC name (lS,2S,4S,5R)-6- methoxycyclohexane-l,2,3,4,5-pentol. It can be obtained from different natural sources, such as pods of the Ceratonia silique, Sutherlandia frutescens leaves, or Finns lambertiana.

The term “ursolic acid”, as used herein, refers to a triterpenoid with IUPAC name (!S,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, 8 a, 10, 11, 12, 13,14b-tetradecahydro- 1 H-picene-4a- carboxylic acid. It is present in many plants, such as fruits and herbs used in daily life. In particular, it can be found in plants of the Lamiacea family, such as Salvia officinalis, Thymus vulgaris, Rosmarinus officinalis, Origanum vulgar e, microalgae, or in the peel of fruits such as Malus domestica, Pyrus communis, Vaccinium, Prunus.

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

The expression “vegetal extract” as used herein, refers to the term commonly known by an expert in the field. It refers to a composition comprising compounds, ingredients, or substances, that have been extracted from a product, or tissue of a vegetal organism, or a plant, usually 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 to obtain a vegetal extract from a product or tissue of a plant or vegetal organism are well-known by an expert in the field, and include any of the methods for obtaining vegetal or natural extracts described in the examples of the invention.

As understood by the skilled person, a “vegetal extract rich in ursolic acid”, or “vegetal extract containing ursolic acid”, is a vegetal extract obtained from a product or tissue of a vegetal organism rich in ursolic acid, i.e. comprising a high amount of ursolic acid, so that the final concentration of ursolic acid in the extract is high. Non-limiting examples of products or tissues of vegetal organisms rich in ursolic acid include leaves from vegetal organisms from the Lamiacea family , marine algae or fruit shells. In particular, they include products or tissues from Salvia officinalis, Thymus vulgaris, Rosmarinus officinalis, Origanum vulgare, microalgae, or the peel of fruits from Malus domestica, Pyrus communis, Vaccinium, Prunus. Said vegetal extract can be obtained by any method well-known by an expert in the field, such as that provided herein in the embodiments of the method for obtaining an extract rich in ursolic acid or in the examples of the invention in “Method for obtaining a natural extract rich in ursolic acid”.

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

The term “product from a vegetal organism” or “vegetal product”, as used herein, refers to any part of a vegetal organism, including a tissue from the vegetal organism, a tissue from the reproductive organs of the vegetal organism, a tissue from the non- reproductive organs of the vegetal organism, the leaves, the stem, the roots, the fruits, a tissue from the fruits, the exocarp of the fruit, the mesocarp of the fruit, the endocarp of the fruit, the fruit shell, the pod of the fruit, the seed of the fruit, or the pod of the vegetal organism. In a particular embodiment, the product refers to the whole of any of the parts of a vegetal organism just indicated. In another particular embodiment, it refers to a portion of any of the parts of a vegetal organism just indicated.

In a preferred embodiment, the product rich in ursolic acid from which the vegetal extract rich in ursolic acid is obtained is the leaves of a vegetal 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 from an alga selected from the group consisting of Cladophora sp preferably Cladophora vagabunda. In another preferred embodiment, it is the fruit shells from Malus domestica, Pyrus communis, Vaccinium, Prunus. In another particular embodiment, the extract rich in ursolic acid is obtained from more than one product rich in ursolic acid selected from any of the group of products rich in ursolic acid indicated above. 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 leastl3, at least 14, at least 15, products selected from any of the groups of products rich in ursolic acid indicated above. In another particular embodiment, it is obtained from all the products indicated in any of the groups of products rich in ursolic acid indicated above.

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

Said product is selected from any of the groups of products rich in ursolic acid indicated above. In another particular embodiment, the vegetal extract rich in ursolic acid is hydroalcoholic extract obtained from more than one of the products rich in ursolic acid selected from any of the groups of products rich in ursolic acid indicated above. 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 leastl3, at least 14, at least 15, products selected from any of the groups of products rich in ursolic acid indicated above. In another particular embodiment, it is obtained from all the products indicated in any of the groups of products rich in ursolic acid indicated above. In another embodiment, the vegetal extract rich in ursolic acid is a hydroalcoholic extract of a vegetal product rich in ursolic acid

As understood by a skilled person, a hydroalcoholic extract of a vegetal product rich in ursolic acid, is an extract obtained from a product of a vegetal organism rich in ursolic acid using a hydroalcohol as a solvent. As it is well-known by an expert in the field, a hydroalcohol is a solvent comprising water and an alcohol. Non-limiting examples of alcohol comprised in the hydroalcohol are ethanol or methanol. In particular embodiment, the percentage of alcohol in (v/v) in the hydroalcohol is of around 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 a particular embodiment, the hydroalcohol has between 2% (v/v) and 99% (v/v) of alcohol, between 5% (v/v) and 96% (v/v) of alcohol, between 10% (v/v) and 90% (v/v) of alcohol, between 20% (v/v) and 80% (v/v) of alcohol, between 30% (v/v) and 70% (v/v), between 50% (v/v) and 70% (v/v) of alcohol, preferably between 5% (v/v) and 96% (v/v) of alcohol. As understood by a skilled person, when defining the hydroalcohol by the percentage of alcohol present in the hydroalcohol, the percentage of water present in the hydroalcohol is the percentage remaining to arrive to 100%. Thus, when the hydroalcohol has 5% (v/v) of alcohol, it has 95% (v/v) of water and when the hydroalcohol has 96% (v/v) of alcohol, it has 4% (v/v) of water. Methods for obtaining a hydroalcoholic extract of a vegetal product rich in ursolic acid are well- known by an expert in the art. A non-limiting example of such methods are those provided herein the embodiments for obtaining an extract rich in ursolic acid and in the examples herein in “Method for obtaining a natural extract rich in ursolic acid”.

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

In a particular embodiment, a vegetal extract rich in ursolic acid comprises a % in (w/w) of ursolic acid 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 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%m, 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 15%.

In one embodiment, the extract rich in ursolic acid is obtained from the leaves from Lamiaceae plants, such as Salvia officinalis, Thymus vulgaris, Rosmarinus officinalis, Origanum vulgar e, and the like, from seaweed, from the peel of fruits from the Malus domestica, Pyrus communis, Vaccinium, Prunus, and the like or from a mixture thereof. In one embodiment, the fresh or dried raw material selected is extracted with a hydroalcoholic mixture that has an alcohol content between 10 and 96% at a temperature of approximately 40 to 100°C for 1-10 hours. The process may also include several stages of leaf extraction with the same or a different alcohol content, combining the hydroalcoholic extracts obtained to continue the process.

In another embodiment, the hydroalcoholic extract and raw material are separated using any separation methodology that results in an extract free of particles larger than 50-100 microns.

In one embodiment, the hydroalcoholic extract is treated with active carbon at 40- 100°C for 1-4 hours. The active carbon dose is between 1-20% of the dry matter present in the extract. Then, the hydroalcoholic extract is separated from the active carbon using any separation methodology that results in an extract free of active carbon particles and particles of raw material larger than 0.45-10 microns.

In another embodiment, the watery extract is treated using microfiltration or heat treatment in order to control any possible microbial load.

In another embodiment, the obtained is concentrated until the solids content is between 5 and 50% (w/w). During this phase a precipitate insoluble in the concentrated extract is generated. 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 with a moisture content of less than 10-15%.

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

Methods to determine the percentage of ursolic acid in an extract are well-known by an expert in the field. Said methods include any method allowing to determine the amount of a compound in a composition, such as mass spectrometry methods. Preferably, mass spectrometry is used, in particular gas chromatography coupled to mass spectrometry (GC-MS), liquid chromatography coupled to mass spectrometry (LC-MS), direct infusion mass spectrometry or Fourier transform ion-cyclotrone resonance mass spectrometry (FT-ICR-MS), capillary electrophoresis coupled to mass spectrometry (CE-MS), high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS), ultra-high-performance liquid chromatography coupled to mass spectrometry (UHPLC-MS), supercritical fluid chromatography coupled to 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 (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 iontrap mass spectrometry, where n is an integer greater than zero. Said techniques are disclosed in, e.g., Nissen, Journal of Chromatography A, 703, 1995: 37-57, US 4,540,884 or US 5,397,894. Preferably, Gas chromatograpy coupled to 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 coupled to 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 a particular embodiment, the vegetal extract rich in ursolic acid comprises additional triterpenes. In a particular embodiment, the vegetal extract rich in ursolic acid contains between 5% (w/w) and 95% (w/w) of triterpenes. In another particular embodiment, the vegetal extract rich in ursolic acid contains between 5% (w/w) and 95% (w/w) of triterpenes of which 5% (w/w) and 90%(w/w) is ursolic acid. Methods to determine the concentration in triterpenes in a vegetal extract rich in ursolic acid are well-known by an expert in the field and include any of the methods provided above to determine the concentration of a compound in a composition.

In a particular embodiment, the extract rich in ursolic acid is obtained by a method that comprises:

(i) the extraction from a product rich in ursolic acid with a hydroalcohol and

(ii) the treatment of the hydroalcoholic extract obtained in step (i) with activated carbon and subsequent removal of the activated carbon.

In a particular embodiment, the extraction of step (i) of the method to obtain an extract rich in ursolic acid, the product rich in ursolic acid is selected from any of the groups of products rich in ursolic acid mentioned above. In another particular embodiment, the extraction of step (i) is performed in more than one of the products rich in ursolic acid selected from any of the groups of products rich in ursolic acid indicated above. 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 leastl3, at least 14, at least 15, products selected from any of the groups of products rich in ursolic acid indicated above. In another particular embodiment, it is an extraction from all the products indicated in any of the groups of products rich in ursolic acid indicated above.

In a particular embodiment, the extract rich in ursolic acid is obtained by a method that comprises: (i) the extraction from the leaves of a plant of the Lamiacea family, from a marine algae or from fruit shells, with a hydroalcohol and

In another particular embodiment, the extraction of step (i) of the method to obtain an extract rich in ursolic acid is performed with a hydroalcohol as any of those indicated in the definition of hydroalcohol, preferably with a hydroalcohol that has between 5% (v/v) and 96% (v/v) of alcohol.

In another particular embodiment, the extraction step (i) of the method to obtain an extract rich in ursolic acid is performed at a temperature higher than 30°C, higher 40°C, higher than 45°C, higher than 50°C, higher than 60°C, higher than 70°C, higher than 80°C, higher than 90°C, higher than 95°C, higher than 96°C, higher than 97°C, higher than 98°C, higher than 99°C, higher than 100°C, higher than 105°C, higher than 110°C. In a preferred embodiment, it is performed at around 30°C-110°C, preferably at around 40-100°C.

In another particular embodiment, the extraction step (i) of the method to obtain an extract rich in ursolic acid is performed 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 methods is performed for 0.5-24 hours, 1-12 hours, 1-10 hours, preferably for 1-10 hours.

In another particular embodiment, step (i) of the method to obtain an extract rich in ursolic acid is repeated at least once, at least twice, at least 3 times, at least 4 times, at least 5 times, wherein 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 concentration of alcohol in each repetition. In a particular embodiment, each of the hydroalcohols used in each of said repetitions are selected from the hydroalcohols indicated in the definition of hydroalcohol above.

In another particular embodiment, the product rich in ursolic acid used in step (i) of the method is the same in each repetition of step (i) of the method. As understood by a skilled person, in this case the same product rich in ursolic acid is exposed to several rounds of extraction with a hydroalcohol.

In a particular embodiment, the hydroalcoholic extracts used in step (ii) are free of particles larger than 0.01 pm, 0.05 pm, 0.1 pm, 0.5 pm, 1 pm , 2 pm, 5 pm, 10 pm, 20 pm, 30 pm, 40 pm, 50 pm, 60 pm, 70 pm, 80 pm, 90 pm, 100 pm, 150 pm, 200 pm, 250 pm, 500 pm, 1mm, 2mm, 5 mm. In a particular embodiment, they are free from particles larger than 50 pm. Methods to eliminated particles larger than any of said sizes are well known by an expert in the filed an include centrifugation or the use of a sieve having as pore size that of the smaller particle to be eliminated.

The term “active carbon”, “activated carbon”, as used herein, refers to the term well- known by an expert in the field. It is a form of carbon processed to have enhances adsorption properties. It is mainly processed to have small, low-volume pores that increase the surface area available for adsorption or chemical reaction.

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

In a particular embodiment, step (ii) of the method for obtaining an extract rich in ursolic acid is performed 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, step (ii) of the method is performed for 0.5-10 hours, 1-5 hours, preferably for 1-4 hours.

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

In a particular embodiment, the removal of the activated carbon results in an extract free of active carbon particles and of raw particles larger than at least than 0.01 pm, 0.05 pm, 0.15 pm, 0.2 pm, 0.25 pm, 0.3 pm, 0.35, 0.4 pm, 0.45 pm, 0.5 pm, 0.55 pm, 0.6 pm , 0.65 pm , 0.7 pm, 0.75 pm , 0.8 pm , 0.85 pm , 0.9 pm , 0.95 pm, 1 pm , 2 pm, 5 pm, 10 pm, 20 pm, 30 pm, 40 pm, 50 pm, 60 pm, 70 pm, 80 pm, 90 pm, 100 pm, 150 pm, 200 pm, 250 pm, 500 pm, 1 mm, 2 mm, 5 mm. In a particular embodiment, they are free from carbon and raw material particles larger than 0.45 pm, larger than 1 pm, larger than 5 pm, larger than 10 pm, preferably larger than 0.45 pm. Methods to eliminated particles larger than any of said sizes are well known by an expert in the filed an include centrifugation or the use of a sieve having as pore size that of the smaller particle to be eliminated.

In a particular embodiment, the method for obtaining an extract rich in ursolic acid comprises an additional step (iii) wherein the hydroalcoholic extract obtained from step (ii) is concentrated. In a particular embodiment, the concentration comprises the separation of the solid content of the extract from the solvent and drying the solid content. As understood by a skilled person, the solid content is any content of the extract other than the solvent of the extract. In a particular embodiment, the extract obtained in step (iii) comprises a water % in (w/w) of 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% of water, preferably less than 15%; of water, even more preferably less than 10% of water.

In a particular embodiment, the extract obtained from step (ii) of the method and used in step (iii) is treated to eliminate any bacteria. Said treatments are well-known by an expert in the field. Non-limiting examples of said treatments include heat treatment or microfiltration.

The expression “vegetal extract rich in D-pinitol”, or “vegetal extract containing D- pinitol”, as used herein, is a vegetal extract obtained from a product or tissue of a vegetal organism rich in D-pinitol, i.e. comprising a high amount of D-pinitol, so that the final concentration of D-pinitol in the extract is high. Non-limiting examples of products or tissues of vegetal organisms rich in D-pinitol include products from a vegetal organism selected from Ceratonia, Ceratonia siliqua, Sutherlandia frutescens , or Pinus lambertiana. In a particular embodiment, the vegetal products from said plants are any of those indicated in the definition of “vegetal product”. In a preferred embodiment, the product rich in D-pinitol is selected for the group consisting of fruits of Ceratonia, pods of Ceratonia, fruits of Ceratonia siliqua, pods of Ceratonia siliqua, Sutherlandia frutescens leaves, or vegetal products from Pinus lambertiana. Said vegetal extracts can be obtained by any method well-known by an expert in the field, such as that provided herein in the embodiments of the method for obtaining an extract rich in D-pinitol, or in the examples of the invention in “Method for obtaining a natural extract rich in D-pinitol”. Methods for obtaining extracts rich in D-pinitol include the method as disclosed in European patent EP1241155-A1 using carob pod as starting material or the method disclosed by Gonzalez-Mauraza et al. (Natural Product Communications, 2015, 11 :405-406) using the aerial parts of Retama monospema as starting material. In another particular embodiment, the extract rich in D-pinitol is obtained from more than one product rich in D-pinitol selected from any of the group of products rich D- pinitol acid indicated above. 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 leastl3, at least 14, at least 15, products selected from any of the groups of products rich in D-pinitol indicated above. In another particular embodiment, it is obtained from all the products indicated in any of the groups of products rich D-pinitol indicated above.

In a particular embodiment, a vegetal extract rich in D-pinitol comprises a % in (w/w) of D-pinitol 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 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%m, 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%. 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 , wherein a represent 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, and:

- the value of a is between 100 and 30, between 90 and 40, between 80 and 50, between 70 and 60, between 65 and 60, preferably between 70 and 60,

- the value of b is between 1 and 20, between 2 and 18, between 4 and 15, between 5 and 10, between 6 and 8, preferably between 4 and 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 of about 63.3:7.5.

The composition or kit of parts of the first aspect of the invention further comprises an additional component selected from the group of:

(i) docosahexaenoic acid (DHA),

(ii) Ginkgo flavonoids and

(iii) a mixture thereof.

The term “Docosahexaenoic acid”, or “DHA” as used herein, refers to is an omega-3 fatty acid that is a primary structural component with IUPAC name (4Z,7Z, 10Z, 13Z, 16Z, 19Z)-docosa-4,7, 10,13,16,19-hexaenoic acid.

In a particular embodiment, the DHA of the composition or kit-of-parts of the first aspect of the invention is provided as a fatty acid composition containing or comprising DHA. In a particular embodiment, 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 23%, 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% of DHA, preferably at least 25% of DHA.

In a particular embodiment, 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 23%, 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% of DHA, preferably at least 25% of DHA.

In another particular embodiment, the fatty acid composition has been obtained from microalgae or from fish. In a particular embodiment, it has been obtained from microalgae selected from the group consisting of Schizochytrium sp., Schizochytrium, aggregation, Schizochytrium limacinum, Schizochytrium minutum , Thraustochytrium sp., Thraustochytrium aureum , Thraustochytrium kinnei, Nannochloropsis sp., Nannochloropsis gaditana, Nannochloropsis granulata, Nannochloropsis limnetica, Nannochloropsis ocednica, Nannochloropsis oculata, Nannochloropsis salina, Pinguiococcus sp., Pinguiococcus pyrenoidosus, Pavlova sp., Pavlova calceolate, Pavlova granifera, Pavlova gyrans, Pavlova hommersandii, Pavlova pinguis, Pavlova ennorea, Pavlova lutheri, Pavlova mesolychnon, Pavlova salina, Pavlova virescens, Pavlova viridis, Isocrysis sp. Isochrysis galbana, Isochrysis litoralis and Isochrysis maritima.

In another particular embodiment, the fatty acid composition rich in DHA has been obtained from fish selected from the groups consisting of salmon, herring, mackerel, tuna, halibut, sardine, sharks, swordfish, tilefish and albacore tuna.

The term “ginkgo flavonoids”, as used herein, refer to the flavonoid glycosides present in an extract of Ginkgo biloba ( G . bilobd) G. biloba , preferably in an extract from the leaves of G. biloba. Flavonoid glycosides are compounds which result from the conjugation of a flavone or a flavonol with a sugar via a glycosidic bond including O- glycosides, thioglycosides, glycosylamines and C-glycosides depending on whether the linkage between the sugar and the other compounds occur via an oxigen atom, a sulphur atom, a nitrogen atom or a carbon atom. Flavonols found in G. biloba extracts as glycosides include quercetin (which forms quercitrin by conjugation of with rahmnnose), kaempferol, and isorhamnetin (which may appears as isorhamnetin-3-O- rutinoside-7-O-glucoside, isorhamnetin-3-0-rutinoside-4'-0-glucoside or isorhamnetin-3-O-rutinoside, also known as narcissin).

The expression “an extract of Ginkgo biloba" , or “ Ginkgo biloba extract” as used herein, refers to a vegetal extract obtained from the vegetal products of G. biloba. In a particular embodiment, said vegetal products are any of the vegetal products indicated the definition of “vegetal product” above. In a preferred embodiment, the extract of G. biloba is obtained from the leaves of G. biloba. As well-known by an expert in the field, said extract comprises flavonoids and terpene lactones. Said extract can be obtained by any method well-known by an expert in the field, such as that provided herein in the embodiments of the method for obtaining G. biloba extract or in the examples of the invention in “Method for obtaining a natural extract rich in ursolic acid”.

The term “terpene lactone” are a family of compounds with unique chemical structures, first recognized in an extract of G. biloba. Major terpene lactone compounds in G. biloba include bilobalide and ginkgolides.

In a particular embodiment, the Ginkgo flavonoids of the composition or kit-of-parts of the first aspect of the invention are 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 in (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 at least 18%, at least 20%, at least25%, 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 in (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 at least 18%, at least 20%, at least25%, at least 30%, at least 35%, at least 40%, at least 50%, preferably at least 0.6%.

In another particular embodiment the G. biloba extract comprises between 0.01% (w/w) and 20%(w/w), between 0.5% (w/w)and 15% (w/w)of ginkgo flavonoids. In another particular embodiment the G. biloba extract contains between 0.01% (w/w) and 20%(w/w), between 0.5% and 15% of ginkgo flavonoids.

In another particular embodiment, the G. biloba extract comprises a percentage in (w/w) of terpene lactones of 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%. In another particular embodiment, the G. biloba extract contains a percentage in (w/w) of terpene lactones of 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%.

In a preferred embodiment, the G. biloba extract comprises a percentage in (w/w) of ginkgo flavonoids as any of those indicated above, and a percentage in (w/w) of terpene lactones as any of those indicated above. In a further preferred embodiment, the G. biloba extract comprises between 0.5% (w/w) and 15% (w/w) of ginkgo flavonoids, and less than 1% (w/w) of terpene lactones. In a preferred embodiment, the G. biloba extract contains a percentage in (w/w) of ginkgo flavonoids as any of those indicated above, and a percentage in (w/w) of terpene lactones as any of those indicated above. In a further preferred embodiment, the G. biloba extract contains between 0.5% (w/w) and 15% (w/w) of ginkgo flavonoids, and less than 1% (w/w) of terpene lactones.

In another particular embodiment, the G. biloba extract has been obtained by a method that comprises:

(i) the aqueous extraction from a vegetal product of G. biloba and

(ii) the treatment of the aqueous extract obtained in step (i) with activated carbon and subsequent removal of the activated carbon.

In a particular embodiment, the vegetal product of the G. biloba is any of those indicated in the definition of “vegetal product”, preferably it is the leaves of G. biloba.

In another particular embodiment, the extraction step (i) of the method to obtain G. biloba extract is performed with water at a temperature higher than 30°C, higher 40°C, higher than 45°C, higher than 50°C, higher than 60°C, higher than 70°C, higher than 80°C, higher than 90°C, higher than 95°C, higher than 96°C, higher than 97°C, higher than 98°C, higher than 99°C, higher than 100°C, higher than 105°C, higher than 110°C. In a preferred embodiment, it is performed with water at around 30°C-110°C, preferably at around 40-100°C.

In another particular embodiment, the extraction step (i) of the method to obtain G. biloba extract is performed 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 3.5hours at least 4hours, at least 4.5 hours, at least 5hours, 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 performed for 0.5-48 hours, 1-30 hours, 1-20 hours, preferably for 1-20 hours.

In another particular embodiment, step (i) of the method to obtain G. biloba extract is repeated at least once, at least twice, at least 3 times, at least 4 times, at least 5 times. In a particular embodiment, the vegetal product from G. biloba used in step (i) of the method is the same in each repetition of step (i) of the method. As understood by a skilled person, in this case the same vegetal product from G. biloba is exposed to several rounds of aqueous extraction.

In particular embodiment, the aqueous extracts used in step (ii) are free of particles larger than 0.01 gm, 0.05 mih, 0.1 mih, 0.5 mih, 1 mih , 2 mih, 5 mih, 10 mih, 20 mhi, 30 mih, 40 mhi, 50 mih, 60 mhi, 70 mhi, 80 mih, 90 mhi, 100 mih, 150 mhi, 200 mhi, 250 mih, 500 mhi, 1 mm, 2 mm, 5 mm. In a particular embodiment, they are free from particles larger than 50 pm. Methods to eliminated particles larger than any of said sizes are well known by an expert in the filed an include centrifugation or the use of a sieve having as pore size that of the smaller particle to be eliminated.

In a particular embodiment, step (ii) of the method for obtaining a G. biloba extract is performed at a temperature higher than 30°C, higher 40°C, higher than 45°C, higher than 50°C, higher than 60°C, higher than 70°C, higher than 80°C, higher than 90°C, higher than 95°C, higher than 96°C, higher than 97°C, higher than 98°C, higher than 99°C, higher than 100°C, higher than 105°C, higher than 110°C. In a preferred embodiment, it is performed at around 30°C-110°C, preferably at around 40-100°C.

In a particular embodiment, step (ii) of the method for obtaining a G. biloba extract is performed 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, step (ii) of the method is performed for 0.5-10 hours, 1-5 hours, preferably for 1-3 hours.

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

In a particular embodiment, the removal of the activated carbon results in an extract free of active carbon particles and of raw particles larger than at least than 0.01 pm, 0.05 pm, 0.15 pm, 0.2 pm, 0.25 pm, 0.3 pm, 0.35 pm, 0.4 pm, 0.45 pm, 0.5 pm, 0.55 pm , 0.6 pm , 0.65 pm , 0.7 pm, 0.75 pm , 0.8 pm , 0.85 pm , 0.9 pm , 0.95 pm, 1 pm , 2 pm, 5 pm, 10 pm, 20 pm, 30 pm, 40 pm, 50 pm, 60 pm, 70 pm, 80 pm, 90 pm, 100 pm, 150 pm, 200 pm, 250 pm, 500 pm, 1 mm, 2 mm, 5 mm. In a particular embodiment, they are free from carbon and raw material particles larger than 0.45 pm, larger than 1 pm, larger than 5 pm, larger than 10 pm, preferably larger than 0.45 pm. Methods to eliminated particles larger than any of said sizes are well known by an expert in the filed an include centrifugation or the use of a sieve having as pore size that of the smaller particle to be eliminated.

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

In particular embodiment, the extract obtained from step (iii) is concentrated until the solid content of the extract is between 1% (w/w) and 90% (w/w), preferably between 5% (w/w) and 08% (w/w), more preferably between 10% (w/w) and 70% (w/w). The term “solid content” is as defined above in the embodiments 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, so that the extract obtained in step (iii) comprises a water % in (w/w) of 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% of water, preferably less than 15%; of water, even more preferably less than 10% of water.

In a particular embodiment, the G. biloba extract obtained by the method for obtaining a G. biloba extract is as defined in any of the embodiments above.

In a particular embodiment, if the composition or kit-of-parts according to the first aspect of the invention contains or comprises D-pinitol, ursolic acid and DHA, then the weight ratio of the components is a:b:c, wherein a and b are as a and b of the weight ratio of D-pinitol and ursolic acid indicated above, and c represents the amount of DHA in the composition or kit of parts and is between 10 and 80, between 20 and 70, between 30 and 60, between 35 and 50, preferably between 35 and 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, if the composition or kit-of-parts according to the first aspect of the invention contains or comprises D-pinitol, ursolic acid and DHA, then the weight ratio of D-pinitol, ursolic acid and DHA in the composition or kit of parts of the first aspect of the invention is of about 63.3:7.5:40.

If the composition contains D-pinitol, ursolic acid, and ginkgo flavonoids, then the weight ratio of the components is a:b:d, wherein a and b are as a and b of the weight ratio of D-pinitol and ursolic acid indicated above, and d represents the amount of ginkgo flavonoids in the composition or kit of parts and is between 0.01 and 10, between 0.5 and 5, between 0.7 and 2, between 0.8 and 1.5, preferably between 0.9 and 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 <iis 1.

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

In a particular embodiment, if 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, then the weight ratio of the components is a:b:c:d, wherein a and b are as a and b indicated for the weight ratio of D-pinitol and ursolic acid indicated above, c is as indicated for the weight ratio of D-pinitol, ursolic acid and DHA indicated above, and d is as indicated for the weight ratio D-pinitol, ursolic acid and ginkgo flavonoids indicated just above. In a preferred embodiment, if the composition or kit-of-parts contains D-pinitol, ursolic acid, DHA and ginkgo flavonoids, then the weight ratio of the components is of about 63.3:7.5:40:1.

In a particular embodiment, 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 that is useful in preparing a composition, such as by reducing viscosity, enhancing solubility, or aiding in stability of the ingredients of the composition, such as by preventing its denaturation or aggregation over their expected shelf life. The 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. If the composition is to be administered to subject, said carriers should be physiologically tolerable and do not typically produce an allergic reaction or a similar unfavorable reaction as gastric disorders, dizziness and suchlike, when administered to a human or animal. Non-limiting examples of carriers include water, salt solutions, alcohol, vegetable oils, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oil, monoglycerides and diglycerides of fatty acids, fatty acid esters petroetrals, hydroxymethyl cellulose, polyvinylpyrrolidone, hydroxypropyl beta cyclodextrin and the like.

It will be understood that a single carrier is used in the compositions of the invention but that, if the different components are formulated as a kit-of-parts, then each element or part of the kit may be formulated with a carrier, which may be the same or different as the carriers used for the other parts of the kit.

The concentration of carrier on the composition is not particularly limitative of the scope of the invention. In one embodiment, the concentration of the carrier is of between 20% and 90% (w/w) with respect to the total weight of the composition. In preferred embodiment, the concentration of the carrier is between 30 and 80% (w/w), between 35 and 75%, between 40% and 70% (w/w), between 45% and 65% (w/w) or between 50% and 60% (w/w).

2. Pharmaceutical compositions

In another aspect, the invention relates to a pharmaceutical product comprising the composition according to the invention and a pharmaceutically active carrier.

The term “pharmaceutical composition”, as used herein, refers to any composition physiologically tolerable and do not typically produce an allergic reaction or a similar unfavourable reaction as gastric disorders, dizziness and suchlike, when administered to a human or animal. Said composition comprises at least one pharmaceutically active ingredient and one or more pharmaceutically acceptable carriers. The terms "pharmaceutically acceptable carrier," "pharmaceutically acceptable excipient", "pharmaceutically acceptable diluent", or "pharmaceutically acceptable vehicle" are used interchangeably herein, refer to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any conventional type. A pharmaceutically acceptable carrier is essentially non-toxic to recipients at the dosages and concentrations employed, and is compatible with other ingredients of the formulation. Suitable carriers include, but are not limited to water, dextrose, glycerol, saline, ethanol, and combinations thereof. The carrier can contain additional agents such as wetting or emulsifying agents, pH buffering agents, or adjuvants that enhance the effectiveness of the formulation. Adjuvants could be selected from the group consisting of sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water or saline aqueous solutions and aqueous dextrose and glycerol solutions, particularly for injectable solutions, are preferably used as vehicles. Suitable pharmaceutical vehicles are described in "Remington's Pharmaceutical Sciences" by E.W. Martin, 21st Edition, 2005. Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated. The pharmaceutical or veterinary comprise an extract of the invention in a therapeutically effective amount. As used herein, the term "effective amount" is synonymous with "therapeutically effective amount", "effective dose", or "therapeutically effective dose" and when used in the present invention refers to the minimum dose of the extract of the invention necessary to achieve the desired therapeutic effect and includes a dose sufficient to reduce at least one symptom of the cognitive disorder. Effectiveness in treating the diseases or conditions described herein can be determined by observing an improvement in an individual based upon one or more clinical symptoms, and/or physiological indicators associated with the condition. An improvement in the diseases or conditions described herein also can be indicated by a reduced need for a concurrent therapy.

A skilled artisan can determine a therapeutically effective amount of the inventive compositions by determining the unit dose. As used herein, a "unit dose" refers to the amount of inventive composition or kit-of-parts required to produce a response of 50 percent of maximal effect (i.e. ED50). The unit dose can be assessed by extrapolating from dose-response curves derived from in vitro or animal model test systems. The amount of compounds in the compositions of the present invention which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. (See, for example, 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.

Additionally, where repeated administration of the extract of the invention is used, an effective amount of the extract of the invention will further depend upon factors, including, without limitation, the frequency of administration, the half-life of the extract of the invention, or any combination thereof.

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

Compositions provided herein may 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 (such as ocular, 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 compositions provided herein is oral. In those cases, the composition for oral use is 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 or the like. Compositions intended for oral use may be prepared according to any known method, and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents, and preserving agents in order to provide pharmaceutically elegant and palatable compositions. Tablets may contain the active ingredient(s) in admixture with non-toxic pharmaceutically-acceptable excipients which 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, for example, corn starch or alginic acid; binding agents, for example, starch, gelatin or acacia; and lubricating agents, for example, magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They also may be coated for controlled delivery. For example, a "delayed release" dosage form releases a product or substance at a time other than promptly after administration. Examples of delayed-release systems include repeat- action tablets and capsules, and enteric-coated tablets where timed release is achieved by a barrier coating.

Compositions of the present invention also may be formulated for oral use as hard gelatin capsules, where the active ingredient(s) is(are) mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or soft gelatin capsules wherein the active ingredient(s) is (are) mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil.

The compositions of the present invention may be formulated as aqueous suspensions wherein the active ingredient(s) is (are) in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example, sodium carboxymethylcellulose, methylcellulose, hydroxy- propylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide such as lecithin, or condensation products of an alkylene oxide with fatty acids, for example, polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example, heptadecaethyl-eneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions also may contain one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin. Compositions of the present 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 flavoring 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.

Compositions of the present invention may be formulated in the form of dispersible powders and granules suitable for composition 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, suspending agent, and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, or example, sweetening, flavoring and coloring agents also may be present.

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

The compositions of the invention also may be formulated as syrups and elixirs. Syrups and elixirs may be formulated with sweetening agents, for example, glycerol, propylene glycol, sorbitol or sucrose. Such formulations also may contain a demulcent, a preservative, and flavoring and coloring agents. Demulcents are protective agents employed primarily to alleviate irritation, particularly mucous membranes or abraded tissues. A number of chemical substances possess demulcent properties. These substances include the alginates, 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, in certain embodiments contain at least 0.1 mg of a provided extract. One skilled in the art will be able to properly formulate a liquid formulation containing an appropriate amount of a provided extract per fluidic ounce, depending on the additive or carrier selected.

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

3. Food and dietary supplements

In another aspect, the invention relates to a food or dietary supplement comprising the composition of the invention and a nutritionally acceptable carrier.

The term “food supplement”, or “dietary supplement”, as used herein, refers to concentrated sources of nutrients or other substances obtained from edible products, whose purpose is to supplement the normal diet. Food supplement or dietary supplements according to the present invention include functional food compositions, i.e. food, drink, feed or pet food or a food, drink, feed or pet food supplements), nutritional supplements, fragrances or flavourings, oenological or cosmetic formulations. In general, The terms “food supplement”, or “dietary supplement”, can also mean:

(i) A product intended to supplement the diet that bears or contains one or more of the following dietary ingredients: [A] a vitamin, [B] a mineral, [C] an herb or other botanical, [D] an amino acid, [E] a dietary substance for use by man to supplement the diet by increasing the total dietary intake; or (F) a concentrate, metabolite, constituent, extract, or combination of any ingredient described in clause (A), (B), (C), (D), or (E); or (ii) Aa product that (A) is intended for ingestion; (B) is not represented for use as a conventional food or as a sole item of a meal or the diet; and (C) is labelled as a dietary supplement.

“Food supplement” or “dietary supplement” according to the present invention are usually administered orally and provided together with the diet of a subject. They can take very different forms, including tablets, capsules, liquid suspensions, dried powder, wet composition, a dry tube feeding or a wet tube feeding. They may be provided as a nutritional formulation, e.g. medical food, e.g. in form of a tube feeding, or in oral nutritional form as a complete meal, as part of a meal, as food additive, as a powder for dissolution, e.g. health drinks, as a solution, as a ready-made drink, including juices, milk-shake, yogurt drink, smoothie or soy-based drink, in a bar, or dispersed in foods of any sort, such as baked products, cereal bars, dairy bars, snack- foods, soups, breakfast cereals, miiesli, candies, cookies, biscuits.

The term “nutritionally acceptable carrier”, as used herein, refers to a carrier, as defined above, which is eatable and is aimed at preparing solutions to be administered orally. Typical nutritionally acceptable carriers, diluents and excipients will be familiar to the skilled person in the art. Non-limiting examples of said carriers are provided in U.S. Pat. Nos. 6,258,846, 6,576,666 and 7,112,609.

The amount of the composition present in nutraceutical compositions, dietary or food products for humans or animals (such as functional food compositions, i.e. food, drink, feed or pet food or a food, drink, feed or pet food supplements), nutritional supplements, fragrances or flavourings, pharmaceuticals (pharmaceutical compositions or formulations), veterinary compositions, oenological or cosmetic formulations will vary depending on the application. Typically, the amount of composition present in the food supplement or dietary supplement will be from about 0.001 to about 50 percent by weight of the nutraceutical compositions, dietary or food product, nutritional supplement, fragrance or flavouring, oenological such as from about 0.01 to about 10 percent, or from about 0.1 to 1 percent.

4. Medical uses

In another aspect, the invention relates to the composition or kit-of-parts according to the invention, to the pharmaceutical product of the invention or to the food or dietary supplement according to the invention for use in medicine.

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

The term “cognitive disorder”, or “neurocognitive disorder” as used herein, refers to a disorder or disease characterized in that at least one cognitive function of a patient is altered, so that said patient has a reduced capacity to undertake said function. The term “cognitive function” refers to abilities developed by cognition, or to mental abilities. The DSM-5 defines six key domains 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 major neurocognitive disorder (previously known as dementia). Non-limiting examples of cognitive disorders include Alzheimer 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 issues 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 the term well- known by an expert in the field. It refers to a neurological disorder that occurs in older adults (approximately 15-20% of people aged 65 or older) which involves cognitive impairments with minimal impairment in instrumental activities of daily living. MCI involves the onset and evolution of cognitive impairments beyond those expected based on an individual's age and education, but which are not significant enough to interfere with his or her daily activities. According to the World Health Organization (WHO), MCI is diagnosed by the presence of impairment in one or more cognitive domains without fulfilling the diagnostic criteria for dementia. It can be classified as amnestic MCI and non-amnesic MCI. Amnestic MCI is characterized by an impairment in the memory cognitive function, so the person affected forgets important information that he/she would previously have recalled easily, such as appointments, conversations or recent events. Non-amnestic MCI is characterized by the loss of cognitive abilities by the person affected other than memory, such as the ability to make sound decisions, judge the time or sequence of steps needed to complete a complex task, or visual perception.

The term “Alzheimer’s disease”, “Alzheimer’s” or “AD”, as used herein, refers to the disease well-known by an expert in the field. AD is characterized by a progressive pattern of cognitive and functional impairment. At the early stage of the disease, the patient simply shows a short memory loss and subtle problems with the executive functions of attentiveness, planning, flexibility, and abstract thinking, or impairments in semantic memory (memory of meanings, and concept relationships). As the disease progresses, there is an increase in memory problems, a deterioration in language, reading and writing skills, as well as in the coordination of motor sequences. In advanced stages, patients become completely dependent upon caregivers, as they might completely lose the ability to speak, and they show a deterioration in muscle mass and mobility that impedes them to feed themselves.

The term “dementia”, as used herein, refers to the term commonly known by a person 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 deterioration in cognitive function beyond what might be expected from normal ageing. It affects memory, thinking, orientation, comprehension, calculation, learning capacity, language, and judgement. Consciousness is not affected. The impairment in cognitive function is commonly accompanied, and occasionally preceded, by deterioration 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 disease is the most common form and may contribute to 60-70% of cases. Other major forms include vascular dementia, dementia with Lewy bodies (abnormal aggregates of protein that develop inside nerve cells), and a group of diseases that contribute to 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 a person skilled in the art. It is a long-term degenerative disorder of the central nervous system that mainly affects the motor system. The most obvious early symptoms of the disease are shaking, rigidity, slowness of movement, and difficulty with walking. Thinking and behavioral problems may also occur. Dementia becomes common in the advanced stages of the disease. Depression and anxiety are also common, occurring in more than a third of people with PD. Other symptoms include sensory, sleep, and emotional problems.

Depending on the disorder, and the subject, to be treated, as well as the route of administration, the compositions of the invention may be administered at varying doses (i.e. therapeutically effective doses, as administered to a patient in need thereof). In this regard, the skilled person will appreciate 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 timeframe. One skilled in the art will recognize that the selection of the exact dose and composition and the most appropriate delivery regimen will also be influenced by inter alia the pharmacological properties of the formulation, the nature and severity of the condition being treated, and the physical condition and mental acuity of the recipient, as well as the age, condition, body 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 invention can be selected on the basis of several criteria associated to the neurodegenerative diseases such as imaging methods or behavioural tests.

In one embodiment, the composition, kit-of-parts, pharmaceutical product or the food or dietary supplement for use according to the present invention comprises the administration of multiple doses of the composition. In yet another embodiment, the composition, kit-of-parts, pharmaceutical product or the food or dietary supplement are administered during at least 2 days, at least 4 days, at least 6 days, at least one week, at least two weeks, at least three weeks or at least 1 month. In some embodiments, the composition, kit-of-parts, pharmaceutical product or the food or dietary supplement for use according to the present invention are administered during a period of time followed by a period of time wherein the composition, kit-of-parts, pharmaceutical product or the food or dietary supplement is not administered. In one embodiment, the composition, kit-of-parts, pharmaceutical product or the food or dietary supplement follows a 5+2 delivery pattern; that is, delivering the product for five days, followed by two days without delivery.

Those skilled in the art will recognize that initial indications of the appropriate therapeutic dosage of the compositions of the invention can be determined in in vitro and in vivo animal model systems, and in human clinical trials. One of skill in the art would know to use animal studies and human experience to identify a dosage that can safely be administered without generating toxicity or other side effects. For acute treatment where it is desirable to substantially restore cognitive function, it is preferred that the therapeutic dosage be close to the maximum tolerated dose. For chronic preventive use, lower dosages may be desirable because of concerns about long term effects.

Alternatively, the composition of the present invention may be administered at least once per day in combination with a drug which is prescribed for the indication that is to be treated. For example, when the composition or kit-of-parts according to the present invention is for use in the treatment of Alzheimer’s disease, then the composition or kit-of-parts is administered together with one Alzheimer's disease medication. Suitable Alzheimer's disease medications include acetylcholinesterase inhibitors (e.g. Tacrine, also known as tetrahydroaminoacridine (THA); Rivastigmine; Donepezil; Galantamine; Carbamates; Physostigmine; Neostigmine; Pyridostigmine; Ambenonium; Demecarium; Phenanthrene derivatives; Caffeine - non-competitive (also an Adenosine receptor antagonist); Rosmarinic acid - ester of Caffeic acid found in plants species of Lamiaceae family; Alpha-Pinene - noncompetitive reversible; Piperidines; Edrophonium; Huperzine A; Ladostigil; Ungeremine; Lactucopicrin), NMDA receptor antagonists (e.g. memantine), gamma secretase inhibitors (e.g. semagacestat, ELND006, avagacestat, begacestat, NIC5-15 and CHF-5074, MK- 8931, LY2886721, AZD3293, LY3314814, E2609), antibodies directed against Abeta (bapineuzumab, solanezumab, gantenerumab, crenezumab, aducanumab, crenezumab, ponezumab, GSK933776 and BAN-2401, a human polyclonal anti- Abeta antibody or immunoglobulin therapies (such as, e.g., Gammagard(R), Flebogamma(R)), agents directed against the tau protein (e.g. inhibitor of tau hyperphosphorylation (such as LMTX), epothilone D, TPI-287, an anti-tau vaccine (sucha as AADvacl or ACI-35), and a GSK-3beta inhibitor (such as Tideglusib, intranasal Humulin R or intranasal Glulizine).

For example, when the composition or kit-of-parts according to the present invention is for use in the treatment of Parkinson’s disease, then the composition or kit-of-parts is administered together with one Parkinson’s disease medication. Suitable Parkinson’s disease medications include synucleinopathy therapeutic agents such as glucosylceramide synthase inhibitors (e.g., GZ667161), iron chelation agents, epigallocatechin gallate (EGCG), myeloperodixase inhibitors (e.g., AZD3241), affitopes (e.g., AFFITOPE PD01A, AFFITOPE PD03A), and anti-synuclein antibodies (e.g., PRX002, BIIB054). Other suitable Parkinson’s disease medications include levodopa, carbidopa, entacapone, ropinirole, rotigotine, pramipexole, bromocriptine, rasagiline, selegiline, amantadine and trihexphenidyl. For example, when the composition or kit-of-parts according to the present invention is for use in the treatment of MCI, then the composition or kit-of-parts is administered together with one MCI medication, with a non-pharmacological treatment or with a therapy suitable for a disease which is known to affect mental function. Suitable MCI medications include cholinesterase inhibitors. Suitable non-pharmacological treatments for mci include regular exercise and cognitive training .Suitable therapies for diseases which are known to affect mental function include therapies for high blood pressure, therapies for depression and/or therapies for sleep apnea.

While the compositions of the invention may be administered as such, the invention also contemplates that possibility that at least one of the components of the composition, kit- of-parts, pharmaceutical composition or food or dietary supplement is administered separately from the rest of components of the composition, of the kit-of-parts, of the pharmaceutical product, or of the food or dietary supplement. In one embodiment, the D- pinitol or the vegetal extract enriched in D-pinitol is administered separately from the other components of the composition or of the kit-of-parts. In another embodiment, the ursolic acid of the hydroalcoholic extract of a vegetal product rich in ursolic acid is administered separately from the other components of the composition or of the kit-of- parts. In another embodiment, the DHA or fatty acid composition comprising DHA is administered separately from the other components of the composition or of the kit-of- parts. In another embodiment, the ginkgo flavonoids or the Ginkgo biloba extract is administered separately from the other components of the composition or of the kit-of- parts.

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

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

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

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

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

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

In the case that the inventive composition is provided as a kit-of-parts, then the different parts of the kit may be separately administered or, alternatively, they may be combined prior to the administration. In another embodiment, the composition, kit-of-parts, pharmaceutical product, or the food or dietary supplement for use according to the present invention is administered at the following daily dosages:

(i) about 150 mg/day of the vegetal extract rich in ursolic acid (ii) about 480 mg/day of fatty acids rich in DHA;

(iii) about 500 mg/day of Ginkgo Biloba extract; and/or

(iv) about 200 mg/day of D-pinitol.

In another embodiment, the composition, kit-of-parts, pharmaceutical product, or the food or dietary supplement for use according to the present invention is administered at the following dosages per kg of the patient.

(i) about 2,5 mg/kg of the vegetal extract rich in ursolic acid,

(ii) about 8 mg/kg of the fatty acid composition rich in DHA,

(iii) about 8, 33 mg/kg of Ginkgo biloba extract; and/or (iv) about 3,33 mg/kg of D-pinitol.

* * *

The invention is shown in the examples below, which are merely illustrative and not limitative of the scope of the invention. EXAMPLES

Preparation of biological extracts

Preparation of a natural extract rich in ursolic acid A natural extract high in ursolic acid is carried out by a process that comprises:

(i) Providing the source material which is leaves from plants of the Lamiaceae family (e.g. Salvia officinalis, Thymus vulgaris, Rosmarinus officinalis, Origanum vulgare, etc. In addition, other sources that are high in ursolic acid can also be used, such as seaweed or the peel of fruits from the Malus domestica, Pyrus communis, Vaccinium, Prunus, etc.

(ii) Extraction of the fresh or dried raw material with a hydroalcoholic mixture that has an alcohol content between 10 and 96% at a temperature of approximately 40 to 100°C for 1-10 hours. The process can also include several stages of leaf extraction with the same or a different alcohol content, combining the hydroalcoholic extracts obtained to continue the process.

(iii) Separation of the hydroalcoholic extract from the raw material using any separation methodology that results in an extract free of particles larger than 50-100 microns.

(iv) Treatment of the hydroalcoholic extract with active carbon at 40-100°C for 1-4 hours. The active carbon dose is between 1-20% (w/w) of the dry matter present in the extract.

(v) Separation of the hydroalcoholic extract from the active carbon using any separation methodology that results in an extract free of active carbon particles and particles of raw material larger than 0.45-10 microns.

(vi) Treatment of the watery extract using microfiltration or heat treatment in order to control any possible microbial load.

(vii) Concentration of the watery extract obtained until the solids content is between 5 and 50% (w/v). During this phase a precipitate insoluble in the concentrated extract is generated.

(viii) Separation of the precipitate by a separation method that separates the precipitate generated from the supernatant.

(ix) The wet precipitate is dried using any drying method that results in a solid product with a moisture content of less than 10-15% (w/w).

(x) The extract obtained using the methodology has a triterpene content of 5- 95% (w/w) of which 5-90% (w/w) is composed of ursolic acid.

This methodology stands out for being simple, affordable and for obtaining the target product high in ursolic acid and a ratio of between 5 and 40 with respect to the raw material.

Preparation of a Ginkgo Biloba extract

The Ginkgo biloba leaf extracts are prepared by a process comprising the following steps:

(a) Extraction of fresh green or dried Ginkgo biloba leaves with water at a temperature of approximately 40-100°C for 1-20 hours. The process can also include several stages of leaf extraction, combining the watery extracts obtained to continue the process.

(b) Separation of the watery extract and ginkgo leaves by any separation methodology that results in a watery extract free of leaf particles larger than 50- 100 microns.

(c) Tratment of the watery extract with active carbon at 40-100°C for 1-3 hours. The active carbon dose is between 1-20% (w/w)of the dry matter present in the extract.

(d) Separation of the watery extract from the active carbon using any separation methodology that results in a watery extract free of active carbon particles and leaf particles larger than 0.45-10 microns.

(e) Treatment of the watery extract with microfiltration or heat treatment in order to control any possible microbial load.

(f) Concentration of the extract obtained is concentrated until the solids content is between 10 and 70%,

(g) Optionally drying the concentrated watery extract using any drying method that results in a solid product with a moisture content of less than 10-15% (w/w).

The above method results in an extract that contains a ginkgo flavonoid content of 0.5- 15% (w/w) and a terpene lactone content of less than 0.01-1% (w/w). This methodology stands out for using water as the only extraction solvent. It is cheap and obtains the target product with a ratio of between 2 and 8 with respect to the raw material.

Preparation of an extract rich in D-pinitol

A natural extract high in D-pinitol may be carried out by a process essentially as described in European patent application EP1241155-A1. The process comprises:

(i) Providing the source material which is dry carob pod.

(ii) Grinding the carob pods to obtain a pulp

(iii) Extraction of the carob pulp with water between 10°C and 70^,oC at a slightly acidic pH until an extract between 30° and 50° Brix is obtained,

(iv) Pressing the pulp to remove retained water (v) Filtering the raw juice and passing the filtrate through a strong cationic resin so as to remove most of the calcium and magnesium ions.

(vi) Treatment of the watery extract using microfiltration or ultrafiltration in order to control any possible microbial load.

(vii) Concentrating the syrup to approximate 60° Brix.

(viii) Inverting the saccharose in the syrup by either enzymatic method or by an acid method using a cationic resin, such as RPI resin

(ix) The syrup at a concentration of 20-30° Brix is demineralized and decolorized by passing it successively through strong cationic resins (H) and then through strong anionic resins (OH), until a composition is obtained having a conductivity of less than 10 mW and a color of less than 25 Icumsa at 420 nm.

(x) Fractionation of the demineralized and decolorized syrup by strong cationic exchange resin, preferably using the ISMB technique (continuous chromatographic separation). The separation is typically carried out starting from a carob syrup of approximately 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 v/v and a P/R of 2.166 v/v at a temperature of 65°C and at a feed capacity of 2.07 T/D and a capacity of the pinitol fraction of 0.36 T/D.

(xi) Demineralized and decolorization of the pinitol fraction thus obtained which contains 44.2% of salts.

The resulting composition contains 90% pinitol, 5% glucose and 5% fructose is concentrated and crystallized by the addition of ethanol. The extract obtained using the methodology has a pinitol content of 85% (w/w) with a specific rotatory power of (+) 64 and a moisture content of 2 %.

Alternatively, pinitol can be extracted from inverted, demineralized and decolorized carob syrup using a strong anionic resin. In this case, the method requires steps (i) to (ix) as described above followed by:

(x) Fractionation of the demineralized and decolorized syrup of approximately 25° Brix and a composition of 35% pinitol, 27% glucose, 37% fructose and 0,4% non sugars through a 250 ml or resin SA 11 A, contained in a column with a diameter of 2 cm and a height of 100 cm at a rate of 10 ml/min. and eluting with demineralized water at a temperature of 6°C.

(xi) Mixing the fractions with positive °Brix and concentrating the mixture (xii) Crystallization of the concentrated mixture with ethanol.

The extract obtained using the methodology has a pinitol content of 91% (w/w) with a specific rotatory power of (+) 63 and a moisture content of 2,5 %.

Methods relating to the zebra fish model

Test subjects and compositions

The species chosen for the trial was the AB wild-type strain of zebrafish ( Danio rerio). Fish were contacted with 5 compositions (Cl, C2, C3, C4 and C5), each at a single dose, said compositions being.

• Compound Cl

• Compound C2

• Compound C3: D-pinitol; RS=98,58; pinitol=93,29% w/w

• Compound C4: sage extract

• Compound C5:

Below is a table summarising the conditions of the experiment:

Table 1. Table showing the compound code, the compound weight anc the volume of

DMSO required to prepare the stock solution, the condition of the solution and the final concentration tested.

All the compounds were initially tested at a concentration of 100 mg/1. However, in the first test, with this concentration the C4 compound induced a mortality rate of 100% of the larvae. Therefore, two new tests were carried out in which all compounds were tested at 100 mg/1, except for C4, which was tested at a concentration of 10 mg/1. Once obtained, the embryos were seeded in a petri dish with 50 ml of dilution solution (DS) and raised for 5 days post-fertilization (DPF), considered larval stage. Only the larvae that did not show any external anomaly were used for the test. Then, using a Pasteur pipette, the larvae were transferred to a 24-well microplate, so that each well contained five larvae, making ten replicas per condition. First, the pre-treatment was carried out on the 5 DPF larvae. To do this, the larvae were incubated in a volume of 2 ml of physostigmine (PHYS, commercial AChE enzyme inhibitor) and of the test compounds at 26 ±1°C for 1 hour. Then, the larvae medium was changed and they were incubated with PHYS, PTZ and the other compounds combined with PTZ for 6 hours at 26±1°C. After this incubation time, all the larvae were examined and it was determined that the general condition of the larvae was completely normal, without any visible anomalies or abnormal behaviour. Finally, the larvae were processed in order to analyse the AChE activity. Control groups that were seeded in embryos without treatment were used in parallel.

Determination of the AChE levels: Once the experimental period was over, the larvae were processed to determine the AChE levels. The larvae were mechanically homogenised and the samples were centrifuged to obtain the supernatant, which were used to determine the AChE enzyme levels according to the treatments administered.

In addition, the total protein of each experimental group was determined according to the normalisation process. Finally, the AChE levels determined in the control group were taken as a reference measurement, considering them 100%.

Methods relating to the mouse SAMP8 model

Experimental subjects

The experimental procedure described here was approved by the Comiti elico para la experimentacion animal (Ethics committee for animal experimentation) of the University of Barcelona and the Generalitat (no. 222/18).

Females of the SAMR1 and SAMP8 strains of mouse, bom between December 2018 and January 2019 in the facilities of the Faculty of Pharmacy at the University of Barcelona, were used.

The animals were divided into 3 experimentation groups, the treatment being started at 5 months old; the behavioural evaluation was carried out using an open field test (OFT), object recognition test (NORT) and object location test (OLT) after 8 weeks of treatment. Subsequently, the animals were euthanised to collect samples.

SAMR1 Control Group (n=10). SAMP8 Control Group (n=10).

SAMP8 Treated Group (n=12).

Treatment with Ginkgo biloba extract, vegetal extract rich in ursolic acid (such as Ursolia®) , D-pinitol and a fatty acid composition with 25% ofDHA

According to the protocol, female mice were treated with a combination of extracts and DHA (product) from the company Biosearch S.A. orally via gastric tube. The extracts were dissolved in 40% hydroxypropyl beta cyclodextrin. The concentration of extracts was calculated according to the weight of the animals in order to reach the specified dose, shown in Table 1.

Table 1. Dose and concentration ratio for each extract

The treatment with the product lasted 8 weeks with a 5+2 delivery pattern; that is, delivering the product for five days, followed by two days without delivery. The product was delivered during the behavioural test days and they were euthanised three days after the last behavioural test. They were given two deliveries through a daily probe, one containing DHA and the second containing Gingko biloba extract, Ursolia- and pinitol (Fig. 1).

Open field test (OFT) The Open Field Test (Fig.2) is a classic test of behavioural patterns in which the animal is exposed to a new, open and brightly lit environment (Seibenhener ML and Wooten MC, 2015 J Vis Exp. 96:e52434). Its behaviour in this space is determined by the balance between animals’ natural interest in the new space (exploratory behaviour) and fear of unknown, open and brightly lit spaces (fear/anxiety). The apparatus comprised a white wooden box (50X50X25 cm high). The brightness of the light in the centre of the field was 30 lx. The mouse was placed in the centre of the apparatus and its behaviour was evaluated for 5 minutes. The variables measured were the total horizontal (total distance, number of line crossings) and vertical (rearing or number of times the rodent stands on its hind legs) locomotor activity and variables associated to emotionality; the time spent in the centre, on the edge, distance in the central area, distance in the peripheral area, time spent in the centre as a (%), time at the edge as a (%), the number of defecations and number of urinations. The purpose of the tasks was to obtain a video for subsequent analysis by recording it using SMART® ver. 3.0 (PanLab, SLU, Spain).

Novel ob ject recognition test (NORT)

The NORT (Fig.3) paradigm is a widely-used explicit memory test. This behavioural test takes advantage of two characteristics of rodents: the natural tendency of these animals to explore a novel object, which has no particular significance for the animal and which has never been associated with reinforcement, and their innate preference to investigate a novel object over a familiar object (Ennaceur A et ak, 1988, Behav Brain Res. 31:47-59). In the NORT test, the mice were placed in a black maze comprising two L-shaped branches, measuring 25 cm long, 20 cm high and 5 cm wide. The brightness of the light in the centre of the field was 30 lx. Various plastic objects were used. For the first 3 days, the mice got to know the maze for 10 minutes. On the fourth day, the mice were subjected to the investigation of a duplicate object located at either end of one branch of the maze. Two hours later for the short-term memory test or 24 h later for the long-term memory test, a 10-minute retention test was performed. During the second test, a novel object was placed at one end of the branch of maze that the mice had not previously explored and the times the animal investigated the new object (TN) and the old object (TO) were recorded. This was done in order to calculate the discrimination index (DI), which was defined as (TN- TO)/(TN+TO). In order to avoid preference for the object, objects A and B were counterbalanced so that half of the mice in each experiment group were first exposed to object A and then to B, whilst the other half were first exposed to object B and then to A. The maze and objects were cleaned with 70% ethanol after each test in order to eliminate olfactory cues. The NORT test began on the first Friday after completing the first week of treatment at 9:30-15:30.

Object location test (OLT)

The Object Location Test (OLT) evaluates the cognitive deficiencies, specifically of the spatial memory and discrimination (Hattiangady B et al, 2014, Front Behav Neurosci. 8:78). This task involves exploiting the ability of rodents to recognise when an object has been relocated and they are inherently stress-free. The test was carried out for 4 days in a wooden box (50 x 50 x 25 cm), in which four walls were white and one was marked with a pattern of black and white squares. On day one, the box was empty and the animals got used to the OFT cage for 10 minutes. On day two, two objects were placed on the patterned wall, equidistant from each other and the wall. The objects were 10 cm high and a replica of each other. The animals were placed in the centre of the cage and were allowed to investigate the objects and surroundings for 10 minutes. Then the mice were returned to their cages and the OLT apparatus was cleaned with 70% ethanol. On day three, an object was moved in front of the opposite white wall to test spatial memory (Fig 4.) The tests were recorded using a camera mounted on the work area, and the total investigation time was determined by recording the amount of time (seconds) spent sniffing the object at the new location (novel) and the object at the previous location (old). In order to analyse the cognitive performance, a 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 position and Told is the time spent investigating the object in the previous location.

Sample collection procedure

Three days after the last behavioural test, the animals were euthanised by cervical vertebrae dislocation. The brain was dissected and immediately frozen in dry ice and stored at - 80°C, leaving them available to Biosearch S.A.

Analysis of the open field test, object recognition test and object location test results

To analyse the tests performed, all of the familiarisation, retention and memory tests and the activity of the mice during the open field test were recorded. Subsequently, they were analysed manually according to the SOP of each procedure, which are included as an annex to this report.

Statistical analysis

To evaluate the possible significant differences between the groups studied, the one way ANOVA statistical test with post-hoc Tukey multiple comparison test was performed to compare the three groups involved in the treatment. In addition, the t- Student statistical tool was used to compare the SAMP8 Control group with the SAMP8 Treated group when there was a clear trend and it was not significant using the Dunnetf s test. Before the statistical analysis, the Grubbs’ test was carried out for anomalous data with 90% confidence. Regarding the graphical representation, the data obtained is expressed as the mean ± standard error of mean (mean ± SEM). The differences in t-Student were also considered significant with p <0.05 and are represented with one (*) and one, two or three more stars will be added depending on the value of p (* p <0.01, ** p <0.001 and *** p <0.0001). The statistical analysis program GraphPad 8.0 was used for the statistical analysis and graphs.

Methods relating to the Qelegans model 1.1. Compounds

The tested doses were established according to the previous study of Biosearch S.A. extracts in Senescence accelerated mice prone 8 (SAMP8), a mice model of late-onset AD (LOAD), maintaining and arranging the range tested dosed found in the literature review (1-10). A concentration lower than the demonstrated effective concentration was chosen for each compound. In the case of studying drug-response in the oxidative tolerance assay, a higher concentration, close to the published effective dose, was also tested. Regarding the composition of the Mix, the proportions of each compound were established in light of an earlier work that reported the synergic effect in mouse.

Stock concentrations of Biosearch S.A. extracts were prepared in 100% dimethyl sulphoxide (DMSO). Then, stock solutions were diluted in MiliQ ddH20, obtaining the tested dilutions at a maximum concentration of DMSO 1%, and stored at -20°C.

1.2. C. elegans maintenance and treatment The wild-type Caenorhabditis elegans (C. elegans) strain N2, the transgenic strain CL2006, the transgenic strain CL2355 and the control strain CL2122 were used. Standard methods were used for culturing and observing C. elegans. N2 were propagated at 20°C, while CL2006, CL2355, and CL2122 were maintained at 16°C in a temperature-controlled incubator on solid nematode growth medium (NGM) seeded with Escherichia coli ( E . coli ) OP50 strain as food source. To obtain the age synchronized population of eggs, gravid adults were treated with alkaline hypochlorite solution (0.5 M NaOH, ~2.6% NaCIO) for 5-7 min. Fertilized eggs were suspended in S-medium for 12 hours and LI larvae were allowed to hatch overnight in the absence of food.

In most cases, drug assays were performed in 96-well plate format, in liquid culture, and treated for 4 days at 20°C. Each well contained a final volume of 60 mΐ, comprising 25-30 animals in LI stage, Biosearch S.A. extracts at the appropriate doses and OP50 inactivated by freeze-thaw cycles and suspended in S-medium complete to a final OD595 of 0.9-0.8 measured in the microplate reader. For chemotaxis assay, synchronized CL2355 and its control CL2122 were treated with Biosearch S.A. products on fresh NGM plates seeded with E. coli inactivated, starting from the LI stage. They were cultured in 16°C for 36 hours, and then in 23°C for another 36 hours.

1.3. Oxidative Tolerance assay

To investigate sensitivity to oxidative stress after Biosearch S.A. products treatment, N2 treated adults were transferred onto plates that included 6.2 mM t-butyl hydroperoxide (Sigma) in NGM agar. Worms were incubated on these plates at 20°C during 2h. Then, worms were transferred to new NGM plates seeded with OP50, and without t-butyl hydroperoxide. Worms were observed 2h, 24h, and 48h after intervention and scored as dead when they did not respond to repeated prodding with a pick.

1.4. Chemotaxis assay

Nematodes were collected after their respective treatments and washed with M9. Briefly, the assay was performed in 100 mm NGM plates, 10 pL of odorant (0.5% benzaldehyde in 96% ethanol) along with 1M of sodium azide were added to the “attractant” spot. On the opposite side, 10 pL of control odorant (96% ethanol) along with 1M of sodium azide were added. Immediately after, 50-60 worms were placed to the center of the plate. Assay plates were incubated at 23°C for 1 hour and chemotaxis index (Cl) was score as follows: Cl = (number of worms at attractant - number of worms at control)/total number of worms. In each experiment, at least 60 worms from each group were analyzed.

1.5. Thioflavin-S staining AB aggregation

Age- synchronized CL2006 worms after were fixed in 4 % paraformaldehyde/PBS, pH

7.5, for 24 hours at 4°C, and permeabilized in 5 % fresh b-mercaptoethanol, 1 % Triton X-100, 125 mm Tris, pH 7.5, at 37°C for another 24 hours. The nematodes were stained with 0.125% thioflavin S (Sigma) in 50 % ethanol for 2 min, destained in 50 % EtOH for 2 min, washed 3 times with PBS and transferred in approximately 10 pL volume on a droplet of Fluoromount G on a glass slide for microscopy. Fluorescence images were acquired using a 20 A~ objective of a fluorescence microscope (Olympus BX51, Germany). Amyloid deposits in the head region of worms were quantified by counting the number of Thioflavin S (ThS) positive spots using ImageJ, and were expressed as AB deposits/anterior area.

1.6. Lifespan assay

Worms were treated as described above in liquid culture for 4 days, starting at LI stage. However, to prevent the progeny production, lpL of 5’-fluorodeoxyuridine (FUdR), for a final concentration of 120 pM, was added at 4 day of age. After treatment, approximately 30 young adult worms were placed on 3 different NGM plates per condition and transferred to fresh seeded plates every 3 days, scoring dead animals. An animal was considered dead if no mechanical response was elicited upon 3 light touches on the head with a platinum wire. 1.7. Statistics

Data analysis was conducted using GraphPad Prism ver. 9 statistical software. Data are expressed as the mean ± Standard error of the mean (SEM) of at least 3 experiments. Means were compared with One-way Analysis of variance (ANOVA), followed by the Tukey post hoc test. Comparison between groups was also performed by two-tailed Student’s t-test for independent replicates when it was necessary. Statistical significance was considered when p values were <0.05. The statistical outliers were determined with Grubs' test and removed from the analysis.

EXAMPLE 1

Effects of the different compositions in the mouse SAMP8 model 1. Weight control of the treatment with plant extract and lipids

Every animal in each treatment group was weighed. They were weighed on the Monday before starting the treatment and on every Monday throughout the treatment until they were euthanised. Table 2 shows the weight of each group at the beginning and end of the treatment. An improvement in the physical appearance was observed as a result of the treatment with the plant extract and lipids.

Table 2. Weights of the animals during treatment Figure 5A shows the weight gain or loss during the treatment for the groups of mice. The results show that at the end of the treatment all groups recorded a weight gain, the gain being 2% in the SAMRl Control group and 4% in the two SAMP8 groups. Figure 5B shows a significant difference in the weight of the mice at the end of the treatment for the SAMR1 Control and SAMP8 Control groups; the same as at the start of the treatment. However, the treatment has no effect on weight gain for the SAMP8 groups compared to the control group.

2. Behavioural results

A summary is herein provided of the results of the cognitive profile, emotional alterations and behaviour characterization induced by treatment with the product after 8 weeks of treatment.

2.1. Summary of the Open Field Test (OFT) results

Figures 6A-6C show the behaviour results in the open field test for the groups after 8 weeks of treatment.

SAMRl Control vs. SAMP8 Control vs. SAMP8 Treated:

The ANOVA test showed significant differences in the vertical activity variables (p<0.001) and the defecations variable (p<0.,0001) but not in the locomotor activity variable (p>0.05). The post-hoc analysis showed significant and similar changes in the vertical activity variable for the SAMP8 control and treated animals (p<0.05) and between the SAMRl control and SAMP8 treated (p<0.01).

Similarly, the post-hoc analysis also showed significant and similar changes in the defecations variable for the control animals (p<0.01) and between the SAMRl control and SAMP8 treated (p<0.001). A clear trend to revert to the SAMRl control group by the SMAP8 treated group was observed for the locomotor activity variable.

2.2. Summary of the Object Location Test (NORT) results

Below is a summary of the results of the short-term and long-term working memory cognitive profile characterization through the object recognition test for treatment with the product after 8 weeks of treatment.

2.2.1. Short-term memory

Figure 7 shows the results of the object recognition test after 8 weeks of treatment in order to evaluate the short-term working memory cognitive function of the groups of mice.

SAMR1 Control vs. SAMP8 Control vs. SAMP8 Treated The ANOVA test showed significant differences in the discrimination index variable (p<0.0001). The post-hoc analysis showed significant and similar changes in the discrimination index variable for the SAMR1 and SAMP8 control animals (p<0.0001). In addition, the post-hoc analysis also showed significant and similar changes in the discrimination index variable between the SAMP8 control and SAMP8 treated groups (p<0.0001). 2.2.2. Long-term memory

Figure 8 shows the results of the object recognition test after 8 weeks of treatment in order to evaluate the long-term working memory cognitive function of the groups of mice. SAMRl Control vs. SAMP8 Control vs. SAMP8 Treated The ANOVA test showed significant differences in the discrimination index variable (p<0.01). The post-hoc analysis showed significant and similar changes in the discrimination index variable for the SAMRl and SAMP8 control animals (p<0.001). In addition the post-hoc analysis also showed significant and similar changes in the discrimination index variable between the SAMP8 control and SAMP8 treated groups (p<0.01).

2.3. Summary of the Object Location Test results

Below is a summary of the results of the spatial memory cognitive profile characterization through the object location test for treatment with plant extract and lipids carried out on females 8 weeks after starting the treatment. 2.3.1. 2 3.1.- Spatial memory

Figure 9 shows the results of the object location test after 8 weeks of treatment in order to evaluate the spatial memory cognitive function of the groups of mice.

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

3. Incidents and observations One SMAP8 mouse from the control group died during the treatment. No internal damage was observed, concluding death due to natural causes. An improvement was observed in the physical appearance of the mice treated with the product (Fig. 10).

EXAMPLE 2 Effects of the different compositions in zebra fish treated with PTZ

Neuroprotective effect of compounds Cl to C5 in the PTZ-induced neurotoxicity zebrafish model

Table 3: Percentage of AChE enzyme activity (mu) by total protein (pg) vs. control group

(considered 100%) in the 5 DPF larvae treated with PTZ, PHYS and with the different compounds (Cl - C5) combined with PTZ for 6 hours. The table shows the mean, the standard deviation (SD), the standard error of the mean (SEM) and statistical analysis using Dunnetf s multiple comparison test (vs. control group).

As Table 3 shows, treating the larvae with PTZ at 5 mm induced a pronounced decrease in AChE activity, approximately 21% compared to the control group (p <0.01). In addition, physostigmine, a powerful inhibitor of AChE activity, reduced AChE activity by 76% compared to the control group, thus validating the test (p <0.01). Of all the problem groups, only the PTZ combined with Cl to 100 mg/1 treatment showed significantly less activity than the control group but no less than the decrease caused by the effect of PTZ per se. Once it was determined that PTZ decreased AChE activity in the 5 DPF zebrafish larvae, the neuroprotection effect of the compounds against neurotoxin-induced toxicity was analysed.

As shown in Fig. 11, two of the compounds studied induced a significant increase in AChE activity compared to larvae treated with PTZ. Treating the larvae with C4 at 10 mg/1 prevented the effect induced by PTZ on the AChE activity, reaching an activity of 103% (# p <0.01). Finally, C3 at 100 mg/1 not only significantly prevented a decrease in the AChE activity induced by PTZ but also slightly increased it compared to the control group (108%), this increase not being significant. This study demonstrates that PTZ, a convulsant antagonist of GABA receptors in the CNS, induces a strong toxic effect by sharply lowering the AChE activity of zebrafish larvae. It has shown that the zebrafish genome does not encode the butyrylcholinesterase (BCHE) enzyme; an enzyme that also degrades acetylcholine in humans. However, the AChE enzyme is encoded by a single gene in the zebrafish and has been functionally detected in the brain. Therefore, the variations in AChE activity recorded in zebrafish larvae only reflect the functioning of the cholinergic system of the CNS.

The purpose of the trial was to analyse the possible neuroprotection effect of the treatment with five compounds provided by the client and coded as Cl, C2, C3, C4 and C5.

Firstly, it is important to note that none of the groups treated with the compounds studied in combination with PTZ had lower AChE activity than that induced by PTZ per se, indicating the absence of an additive neurotoxic effect induced by the compounds.

The AChE activity analysis showed that treatments with the C3 (D-pinitol) and C4 (sage extract) compounds prevented the neurotoxic effect induced by PTZ in the 5 DPF zebrafish larvae, recovering the AChE activity by 29% and 23%, respectively, compared to the larvae treated with the convulsant. These results show that both compounds have a neuroprotection capacity against the neurotoxic effect induced by PTZ. Therefore, it has been shown that the D-pinitol at 100 mg/1 and sage extract at 10 mg/1 compounds significantly prevent a reduction in the AChE activity in 5DPF larvae induced by PTZ, showing a neuroprotective effect. EXAMPLE 3

Effects of the different compositions in the C.elegans model 1. Mix attenuates oxidative stress in C. elegans

To investigate if the extracts and Mix have any beneficial effects on oxidative stress, we tested them after tert-butyl (6.2 mM). The treatment was started when synchronized worms reached the L4 stage, and maintained at 20°C until the experiment was finished. Firstly, we found that each extract group did not reach protection against oxidative stress in comparison with the untreated control as well as we obtained a significant reduction in the percent survival in comparison with the vitamin C group (Fig. 12). On the other hand, the worms pre-incubated in Mix extract (dose) were significantly protected against tert-butyl (6.2 mM) induced oxidative stress in comparison with the untreated group, and reaching the percent of survival worms close to the vitamin C (58 mM), demonstrating the synergistic effect of combined extract administration (Fig. 12).

2. Mix extracts suppresses neuronal Ab expression-induced defect in chemotaxis behaviour in transgenic C. elegans (CL2355)

To investigate the synergistic effects of the Mix extract on the performance of chemotaxis behaviour, we applied benzaldehyde as an attractant and ethanol as a control, both containing sodium azide, which paralyzes the worms on contact (Fig. 13). The chemotaxis index was scored for all groups at day 5 of age. Figure 14 shows that the CL2355 strain exhibits a significant reduction in the Cl compared to the control strain CL2122. Moreover, each extract per separated show slight tendency to increase the Cl compared to the CL2355 strain, but not significantly (Fig. 14). Interestingly, a significant increase Cl in the Mix extract compared to the CL2355 group, demonstrating the synergistic effect, and restoring the chemotaxis behaviour to the control strain CL2122 (Fig. 14). 3. Mix extracts improved amyloid fi burden in transgenic C. elegans ( CL2006 )

To explore whether the Mix affects the aggregation of the amyloid B, CL2006 worm strain that constituently over expresses human ABi-42 in muscle cells was used. We found the Mix group had remarkable effect on the deposits of the AB_I-42 peptide, suggesting that the Mix could decrease the aggregation of the AB species significantly in a synergistic way (Fig. 15). As expected, the different extract groups (DHA, Ginkgo, Pinitol, and Ursolia^®) did not reach any effect on the deposits of the AB_I-42 , demonstrating that the synergistic effect obtained in the Mix group is not obvious (Fig. 15).

4. Mean lifespan extension by Mix extract in C. elegans

To investigate the effect on aging, we examined changes in lifespan in C. elegans after different extract treatments and Mix. Firstly, we did not find any significant change among groups by Kaplan-Meier curve (Fig. 16A). However, the mean lifespan was extended by up to 15% with Mix extract treatment in comparison with the DHA, Pinitol and Ursolia^® groups, demonstrating the synergistic effect (Fig. 16B), whereas there was no changes between the Mix and Ginkgo group due to the well described effects of Ginkgo in the lifespan (REF).

5. Conclusions

Together, we demonstrated greater neuroprotective effects of the Mix extract in comparison with each extract per separated on behaviour, AB pathology, oxidative stress tolerance as well as mean lifespan. This clearly shows that the synergistic effect of the Mix extract was not obvious.

Claims

1. A composition or kit-of-parts comprising D-pinitol, ursolic acid and one or more additional components selected from the group of: (i) docosahexaenoic acid (DHA),

(ii) ginkgo flavonoids and

(iii) a mixture thereof.

2. The composition or kit-of-parts according to claim 1 wherein the ursolic acid is provided as a vegetal extract rich in ursolic acid and/or wherein the D-pinitol is provided as a vegetal extract rich in D-pinitol.

3. The composition or kit-of-parts according to claim 2 wherein the vegetal extract rich in ursolic acid is a hydroalcoholic extract of a vegetal product rich in ursolic acid.

4. The composition or kit-of-parts according to claim 3 wherein the vegetal product rich in ursolic acid is selected from the leaves of a plant of the Lamiaceae family, a marine algae or fruit shells.

5. The composition or kit-of-parts according to claims 2 to 4 wherein the vegetal extract rich in ursolic acid contains between 5 and 90% of ursolic acid.

6 The composition or kit of parts according to any of claims 2 to 5, wherein the vegetable extract rich in ursolic acid has been obtained by a process which comprises:

(i) the extraction from the leaves of a plant of the Lamiaceae family, from a marine algae or from fruit shells, with a hydroalcohol and

7. The composition or kit-of-parts according to any of claims 2 to 6 wherein the extract containing D-pinitol is an extract from the fruit of a plant of the genus Ceratonia.

8. The composition or kit-of-parts according to claims 1 to 7 wherein the weight ratio of D-pinitol and ursolic acid is of about 63.3:7.5:

9. The composition or kit-of-parts according to any of claims 1 to 8 wherein the DHA is provided as a fatty acid composition that contains at least 25% DHA.

10. The composition or kit-of-parts according to claim 9 wherein the fatty acid composition has been obtained from fish or from microalgae.

11 The composition or kit-of-parts according to any of claims 1 to 10 wherein the ginkgo flavonoids are provided as a Ginkgo biloba extract.

12. The composition or kit of parts, according to claim 11, wherein the Ginkgo biloba extract contains between 0,5% (w/w) and 15% (w/w) of ginkgo flavonoids and less than 1% (w/w) of terpene lactones.

13. The composition or kit-of-parts according to any of claims 11 or 12 wherein the Ginkgo biloba extract has been obtained by a process which comprises:

(i) the aqueous extraction from Ginkgo biloba leaves and

14. The composition or kit-of-parts according to any of claims 1 to 13 wherein:

(i) If the composition or kit-of-parts contains D-pinitol, ursolic acid and a DHA, then the weight ratio of the components is of about 63.3:7.5:40,

(ii) If the composition or kit-of-parts contains D-pinitol, ursolic acid and Ginkgo flavonoids, then the weight ratio of the components is of about 63.3:7.5:1. (iii) If the composition or kit-of-parts contains D-pinitol, ursolic acid, DHA and Ginkgo flavonoids, then the weight ratio of the components is of about 63.3:7.5:40:1.

15. The composition or kit-of-parts according to any of claims 1-14, further comprising a carrier.

16. The composition according to claim 15 wherein the concentration of the carrier is of between 20% and 90% (w/w).

17. A pharmaceutical product comprising the composition according to any of claims 1 to 16 and a pharmaceutically active carrier.

18. A food or dietary supplement comprising the composition according to any of claims 1 to 16 and a nutritionally acceptable carrier.

19. The composition or kit-of-parts according to any of claims 1-16, the pharmaceutical product according to claim 17, or the food or dietary supplement according to claim 18 for use in medicine.

20. The composition or kit-of-parts according to any of claims 1-16, the pharmaceutical product according to claims 17, or the food or dietary supplement according to claim 18 for use in the prevention and/or treatment of a cognitive disorder.

21. The composition, kit-of-parts, the pharmaceutical product or the food or dietary supplement for use according to claim 20, wherein the cognitive disorder is selected from the list consisting of mild cognitive impairment, Alzheimer’s disease and Parkinson’s disease.

22. The composition, kit-of-parts, the pharmaceutical product, or the food or dietary supplement for use according to claims 20 or 21, wherein at least one of the components of the composition, kit-of-parts, pharmaceutical composition or food or dietary supplement is administered separately from the rest of components of the composition, of the kit-of-parts, of the pharmaceutical product, or of the food or dietary supplement.

23. The composition, kit-of-parts, pharmaceutical product, or the food or dietary supplement for use according to any of claims 20-22, wherein the administration of the composition, of the components comprised in the kit of parts, of the pharmaceutical product, or the food or dietary supplement comprises administering:

(i) about 150 mg/day of the vegetal extract rich in ursolic acid

(ii) about 480 mg/day of fatty acids rich in DHA;

(iii) about 500 mg/day of Ginkgo Biloba extract; and/or

(iv) about 200 mg/day of D-pinitol.

24. The composition, kit-of-parts, pharmaceutical product, or the food or dietary supplement for use according to any of claims 20-22, wherein the administration of the composition, of the components comprised in the kit of parts, of the pharmaceutical product, or the food or dietary supplement comprises administering

(i) about 2,5 mg/kg of the vegetal extract rich in ursolic acid,

(ii) about 8 mg/kg of the fatty acid composition rich in DHA,

(iii) about 8,33 mg/kg of Ginkgo biloba extract; and/or

(iv) about 3,33 mg/kg of D-pinitol.

25. The composition, kit-of-parts, pharmaceutical product, or the food or dietary supplement for use according to any of claims 20-24, wherein the treatment comprises the administration of multiple doses of the composition, the components comprised in the kit of parts, the pharmaceutical product, or the food or dietary supplement during at least 1 month.

26. The kit-of-parts for use according to any of claims 20-25, wherein the parts of the kit-of-parts are combined prior to the administration.