KR20220123272A - Composition for the treatment of cognitive impairment - Google Patents

Abstract

The present invention relates to naturally occurring compositions useful for the treatment of cognitive disorders containing D-pinitol and ursolic acid or extracts from natural sources that are rich in these components and may further include DHA and/or ginkgo extract. The present invention also relates to the use of these compositions for the treatment of cognitive disorders.

Description

Composition for treating cognitive impairment

The present invention relates to a composition comprising a botanical extract for use in the treatment of a cognitive disorder such as Alzheimer's disease, mild cognitive impairment or Parkinson's disease.

Dementia is a loss of cognitive function (thinking, memory, reasoning) and behavioral abilities that interferes with an individual's daily life and activities. The severity of dementia varies from the mildest stage, which is just beginning to affect an individual's functioning, to the most severe stage, in which the person is completely dependent on others for basic activities of daily living. Common types of dementia include Alzheimer's disease, vascular dementia, Lewy body disease, frontotemporal dementia, or early-onset dementia. About 60-70% of people with dementia have Alzheimer's disease (AD) and about 6% of subjects 65 years of age or older are diagnosed with Alzheimer's disease.

AD is a neurodegenerative disease with a progressive pattern of cognitive and functional impairment. In the early stages of the disease, patients show brief memory loss and subtle problems with attention, planning, flexibility, and executive functions of abstract thinking, or impairment of semantic memory (semantic memory and conceptual relationships). As the disease progresses, memory problems increase, language, reading and writing skills deteriorate, and coordination of motor sequences is impaired. At an advanced stage, the patient may completely lose the ability to speak, so he becomes completely dependent on the caregiver, and shows decreased muscle mass and mobility that interferes with self-eating.

Mild cognitive impairment (MCI) has often been found to be a transitional stage between normal aging and dementia, particularly Alzheimer's disease. MCI can be divided into amnestic MCI and non-amnestic MCI. Amnestic-type mild cognitive impairment (MCI) is characterized by memory loss in the affected person, forgetting important information that would have been easily remembered previously, such as appointments, conversations, or recent events. Non-amnestic MCI (non-amnestic MCI) affects a person affected other than memory, such as the ability to make sound decisions, the ability to judge the sequence or time of steps required to complete a complex task, or visual perception. It is characterized by loss of cognitive ability. While mild cognitive impairment (MCI) may return to normal or remain stable, amnestic MCI is often considered a precursor to Alzheimer's disease. Patients with non-amnestic MCI have been found to be prone to other dementias.

There is no solid evidence to support specific measures to prevent AD. Also, no drugs have been shown to delay or stop the progression of Alzheimer's disease. Available treatments provide relatively little symptomatic benefit. No drugs have been demonstrated to be effective in treating MCI, and no high-quality evidence has been provided for pharmaceuticals or dietary supplements that can improve cognitive symptoms in patients with MCI.

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

The present inventors found that a botanical extract rich in ursolic acid (sage extract) and a plant extract rich in D-pinitol have not only effects on acetylcholinesterase activity, but also a zebra fish model organism (pentylenetetrazole (pentylenetetrazole) PTZ) treated with AB zebrafish strain) showed a neuroprotective effect. Studies of CNS development in zebrafish (Kimmel et al, 1995, Developmental Dynamics 203:255-310) have shown that brain division can already be detected at 24 h and structures such as neural tube, notochord and somite (precursors of muscle and skeleton) shows that it is formed. After 5 days of development, some sensory organs, such as eyes and ears, were formed. The heart, liver, kidneys and pancreas also appeared, and the circulatory, digestive and nervous systems are fully functioning. At this stage, fish can respond to visual, olfactory and mechanical stimuli and begin swimming in search of food.

AChE is an enzyme that catalyzes the hydrolysis of the acetylcholine neurotransmitter at the choline and acetate groups. It is mainly found at neuromuscular junctions and the cholinergic nervous system, and its activity is responsible for interrupting synaptic transmission. Acetylcholine is a neurotransmitter involved in motor control and is an important regulator of cognitive functions such as learning and memory (Hasselmo et al, 2011, Neuropsychopharmacology, 1:52-73). Adequate levels of acetylcholinesterase thus reflect a healthy neurological state.

PTZ is a competitive stimulator of the gamma-aminobutyric acid (GABA) receptor. Activation on the receptor increases glutamatergic excitability by blocking Cl-anion conduction and the formation of inhibitory post-synaptic potentials (McDonald et al, 1978, Science, 200:775-777). The zebrafish model exerts an anticonvulsant effect, presumably by blocking GABAergic inhibitory synaptic transmission (Huang et al, 2001, J. Pharmacol. Exp. Ther. 298:986-995).

In addition, the present inventors combined D-pinitol-enriched vegetable extract, ginkgo biloba extract and DHA-enriched fatty acid composition along with ursolic acid-rich botanical extracts in SAMP 8 mice (aging-promoting mouse tendency 8) showing pathological similarity to AD patients. The effects of administration to groups were analyzed (Pallas M. 2012, ISRN Cell Biology, Vol. 12, Article ID 917167). They observed that after the treatment, the ability of the group of mice to perform aggravated cognitive function in AD patients was similar to that of control mice (aging-promoting mouse resistance 1, SAMR1). Therefore, they found that administration of a composition comprising a botanical extract rich in ursolic acid, a botanical extract rich in D-pinitol, a Ginkgo biloba extract and a fatty acid composition rich in DHA can restore severely impaired cognitive function in AD patients. showed

The present inventors also compared a control animal with a composition comprising a fatty acid composition rich in DHA, a ginkgo extract rich in flavonoids, a vegetable extract rich in D-pinitol and a plant extract rich in ursolic acid administered to C. elegans showed improved oxidation resistance, increased lifespan, increased chemotactic activity and decreased AB aggregation.

Accordingly, a first aspect of the present invention provides one or more selected from the group consisting of D-pinitol, ursolic acid and (i) docosahexaeoic acid (DHA) , (ii) ginkgo flavonoids and (iii) mixtures thereof. It relates to a composition or kit of parts comprising the above additional components.

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

In a third aspect, the present 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 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 is for use in a medicament.

1 relates to the schedule of the SAMR1/SAMP8 preclinical study.
2 relates to an Open Field Test (OFL) schema.
3 relates to a NORT (Novel Object Recognition Test) scheme.
4 relates to an Object Location Test (OLT) scheme.
Figure 5 relates to A: animal weight gain/loss during treatment and B: mean body weight at the start and end of the study.
Figure 6 relates to the behavioral assessment of a group of mice in an open field (n=10 - 12), showing A) motor activity, B) vertical activity, and C) defecation.
7 is a summary of short-term NORT results for groups (n=10-12).
8 relates to a summary of long-term NORT results for groups (n=10-12).
9 relates to a summary of habituation results for three cameras (OLT) for a group (n=10-12).
10 relates to A) an example of the physical appearance of a SAMP 8 control mouse and B) an example of the appearance of a SAMP 8 treated group mouse.
11 is a bar showing the mean±SEM of AChE activity of larvae treated with PTZ, larvae treated with PHYS and larvae treated with different compounds in combination with PTZ versus control (dotted line in red) considered 100%. It's about graphs. The statistical methods used were Dunnett's multiple comparison method (*p<0.05;**p<0.01 vs control) and Bonferroni's multiple comparison method (#p<0.01;##p<0.001 vs. PTZ).
12 is a summary of the oxidative stress response of various groups of C. elegans after treatment with each extract, mix or vitamin C (58 pM). There was a significant difference between these groups when the symbols were different (P<0.05). Data are averages of 3 replicates with approximately 120-150 worms in each group.
13 relates to a schematic diagram of chemotaxis assay behavior in neuronal Aβ-expressing strain CL2355.
14 relates to the chemotaxis assay in neuronal strain CL2355. There was a significant difference between these groups when the symbols were different (P<0.05). Data are averages of 3 replicates with approximately 120-150 worms in each group.
15 relates to the quantification of ThS-positive particles in the head region of the CL2006 strain (A) and representative images of each group tested (B). There was a significant difference between these groups when the symbols were different (P<0.05). Data are averages of 3 replicates with approximately 50-60 worms in each group.
16 is a Kaplan-Meier curve for the survival of C. elegans (C. elegans) for different extracts (A) and the lifespan average (B) of C. elegans treated group and DMSO 1% control group will be. There was a significant difference between these groups when the symbols were different (P<0.05). Data are averages of 3 replicates with about 60-70 worms in each group.

The present inventors observed that the vegetable extract rich in ursolic acid as well as the vegetable extract rich in D-pinitol had a neuroprotective effect in a zebrafish model of neurodegenerative disease. They also found that administration of a composition obtained by combining a fatty acid composition rich in DHA, a botanical extract rich in ursolic acid, a botanical extract rich in D-pinitol, and a ginkgo extract to an aging-promoting mouse model reduced some of the impaired cognitive function in the model animal. It was observed that recovery was possible.

1. Compositions or kits of parts of the present invention

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

(i) docosahexaenoic acid (DHA),

(ii) ginkgo flavonoids and

(iii) mixtures thereof.

As used herein, the term “composition” refers to a compound or combination of ingredients. The components of the composition may be supplied individually or in unit doses. Thus, when a composition is administered to a subject, the compounds of the composition may be mixed together in such unit doses, if provided separately, may be mixed together prior to administration, and provided and administered separately but once taken by the subject. , ie, can be mixed inside the subject's body. Also, some components of the composition may be administered together and other components may be administered separately, but once ingested by the subject, ie, all mixed in the subject's body.

As used herein, the term “kit-of-parts” refers to a product comprising different components, components or compounds, wherein the components, components or compounds are preferably each component, component or compound therein. It is a kit that can be transported and stored physically separated by its individual packaging. As will be understood, in the "kit-of-parts" according to the invention, the individual active ingredients, ingredients or compounds represent a therapeutic agent, provided that the use of these compounds simultaneously, individually or sequentially, is herein It produces novel and unexpected joint therapeutic effects as described in , which are not achieved by compounds independent of each other. Indeed, as evidenced by the results below, it is surprising and surprising that the claimed combination of active ingredients does not simply represent a collection of known agents, but that the combined effect is much more important than the mere addition of the effect observed when using each of these active ingredients. It is a new combination with valuable characteristics.

Kits of parts generally contain the components in suitable containers. Suitable ingredients for packaging the components of the kit include glass, plastic (polyethylene, polypropylene, polycarbonate, etc.), bottles, vials, paper, sachets, and the like. For example, each container may have the following form. It may be in any other suitable form if the vial, bottle, squeeze bottle, bottle, sealed sleeve, bag or pouch, tube or blister package or container is configured to prevent premature mixing of the components. Each of the different components may be provided separately or some of the different components may be provided together (ie, provided in the same container). In addition, the kits of the present invention may include instructions for the simultaneous, sequential or separate use of the different components in the kit. The instruction manual may be in the form of a printed matter or in the form of an electronic storage medium (magnetic disk, tape, etc.) or an electronic medium (CD-ROM, DVD) capable of storing the instruction so that the subject can read it, such as an optical medium. The media may additionally or alternatively include an Internet address providing the above instructions. It will be understood that the compositions and kits of parts of the present invention may comprise, consist essentially of or consist of the aforementioned components.

The terms “comprising” or “comprises” are used herein to indicate that the composition described must contain the listed ingredient(s), but may optionally contain additional ingredients. The term "consisting essentially of" or "consisting essentially of eg: up to 5 wt%, or up to 1 wt% or 0.1 wt%). The terms “consisting of” or “consisting of” are used to indicate that the composition for which the composition is described should include only the component(s) listed.

In a specific embodiment, the composition or kit of parts according to the first aspect of the present invention comprises additional components in addition to D-pinitol and ursolic acid.

In certain embodiments, the composition or kit of parts comprises at least 1, at least 2, at least 3, at least east 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least other than D-pinitol and ursolic acid. 15, at least 20 components. In another specific embodiment, D-pinitol and ursolic acid are 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% .

As used herein, the terms “D-pinitol”, “pinitol”, “3-O-methyl-D-kiro-inositol”, “methylinositol” refer to the IUPAC name (1S,2S,4S, 5R)-6-methoxy It is a compound of cyclohexane-1,2,3,4,5-pentol. It can be obtained from a variety of natural sources, such as Ceratonia silique, Sutherlandia frutescens leaves or pods of Finns lambertiana.

As used herein, the term "ursolic acid" refers to the IUPAC name (1S,2R,4aS,6aR,6aS,6bR,8aR,10S,12aR,14bS)-10-hydroxy-1,2,6a,6b,9,9 Triterpe with 12a-heptamethyl-2,3,4,5,6,6a,7,8,8a,10,11,12,13,14b-tetradecahydro-1H-picene-4a-carboxylic acid refers to the node. Ursolic acid 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 vulgare, and microalgae, or in the peels of fruits such as Malus domestica, Pyrus communis, Vaccinium, and Prunus.

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

As used herein, the expression "plant extract" refers to a term commonly known by experts in the field. means a composition comprising a compound, ingredient or substance extracted by treating a plant organism or plant product, tissue or generally said tissue with a solvent. Non-limiting examples of solvents include water, ethanol, hydroalcohol, ethyl acetate, CO2, methanol, acetone, acetic acid or hexane. Methods for obtaining a botanical extract from a product or tissue of a plant or plant organism are well known to those skilled in the art and include any of the methods for obtaining a plant or natural extract described in the Examples of the present invention.

As will be understood by one of ordinary skill in the art, "a vegetable extract rich in ursolic acid", or "a vegetable extract containing ursolic acid" is a vegetable extract obtained from a product or tissue of a plant organism rich in ursolic acid, i.e. an extract high in ursolic acid. , an extract with a high final concentration of ursolic acid. Non-limiting examples of products or tissues of plant organisms rich in ursolic acid include leaf, algae or fruit peels from plant organisms of the family Lamiacea. In particular, Salvia officinalis (Salvia officinalis), Thymus vulgaris ( Thymus vulgaris ), Rosemarinus officinalis ( Rosmarinus officinalis ), Origanum vulgare ( Origanum vulgare ), products or tissues of microalgae or Malus Mestica ( Malus domestica ), Pyrus communis ( Pyrus communis ), Vaccinium ( Vaccinium ), Prunus ( Prunus ) fruit peels are included.

The botanical extract can be obtained by a method well known to those skilled in the art, and is provided as an embodiment of the present invention, for example, as a method of obtaining an extract rich in ursolic acid, or as a method of obtaining a natural extract rich in ursolic acid. In a specific embodiment, the ursolic acid-enriched product from which the ursolic acid-enriched vegetable extract is obtained is a product from a plant organism selected from the group consisting of leaves, algae or fruit peels of plants of the family Lamiacea . In another specific embodiment, the botanical product is Salvia officinalis , Thymus vulgaris , Rosmarinus officinalis , Origanum vulgare , seaweed. , Malus domestica , Pyrus communis , Vaccinium , Prunus , and combinations thereof.

As used herein, the term "product from a plant organism" or "plant product (product)" means tissue from a plant organism, tissue from the reproductive organs of a plant organism, tissue from the non-reproductive organs of a plant organism, leaves, stems, roots , refers to any part of a vegetable organism, including a fruit, a tissue of a fruit, the outer skin of a fruit, a mesocarri of a fruit, an inner peel of a fruit, a fruit peel, a fruit pod, a fruit seed or a pod of a vegetable organism. In certain embodiments, the botanical product refers to the whole of any one of the parts of the plant organism just indicated. In another specific embodiment, said plant product refers to a portion of any part of the plant organism just indicated.

In a preferred embodiment, the product rich in ursolic acid from which a vegetable extract rich in ursolic acid is obtained is Salvia officinalis , Thymus vulgaris , Rosmarinus officinalis , Origanum. It is a leaf of a plant organism selected from the group consisting of Origanum vulgare , preferably a leaf of Salvia officinalis . In another preferred embodiment, it is a product from seaweeds, preferably selected from the group consisting of Cladophora sp , preferably Cladophora vagabunda . In another preferred embodiment, it is the fruit peel of Malus domestica , Pyrus communis , Vaccinium , Prunus . In another specific embodiment, the ursolic acid-enriched extract is obtained from one or more ursolic acid-rich products selected from any of the groups of ursolic acid-rich products indicated above. In another specific embodiment, it comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 13, at least 14, at least 15 products. In another specific embodiment, it is obtained from all products indicated in any of the ursolic acid rich product groups indicated above.

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

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

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

In another specific embodiment, the vegetable extract is obtained using an alcohol such as ethanol or methanol as the solvent. In another specific embodiment, the botanical extract is obtained using water as the solvent.

In certain embodiments, the botanical extract rich in ursolic acid is 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% ursolic acid (w/w), preferably at least 15% ursolic acid (w/w) .

In one embodiment, the extract rich in ursolic acid is a leaf of a Lamiaceae plant, such as Salvia officinalis, Thymus vulgaris, Rosmarinus officinalis, Origanum vulgare, seaweed, Malus domestica, Pyrus communis, Vaccinium, fruit skins such as Prunus, or their skins. obtained from the mixture.

In one embodiment, the selected fresh or dried raw material is extracted with a hydroalcoholic mixture having an alcohol content of 10 to 96% at a temperature of about 40 to 100° C. for 1 to 10 hours. The process may also include several stages of leaf extraction with the same or different alcohol content by combining the hydroalcoholic extracts obtained to continue the process.

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

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

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

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

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

In one embodiment, the extract has a triterpene content of 5-95% consisting of 5-90% ursolic acid. In a specific embodiment, the botanical extract rich in ursolic acid comprises 0.25% (w/w) to 100% (w/w) ursolic acid, preferably 2% (w/w) to 100% (w/w) ursolic acid. acid, more preferably 5% (w/w) to 90% (w/w) ursolic acid.

Methods for determining the percentage of ursolic acid in an extract are well known to those skilled in the art. The method includes any method capable of determining the amount of a compound in a composition, such as mass spectrometry. Preferably mass spectrometry, in particular gas chromatography coupled to mass spectrometry (GC-MS), liquid chromatography coupled to mass spectrometry (LC-MS), direct injection mass spectrometry or Fourier transform ion-cyclotron 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), Chromatography coupled to supercritical fluid mass spectrometry (SFC-MS), flow injection analysis using mass spectrometry (FIA-MS), including quadrupole mass spectrometry, sequentially coupled mass spectrometry (e.g. MS-MS or MS-MS-MS) MS-MS), inductively coupled plasma mass spectrometry (ICμmS), 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), silicon desorption /ionization (DIOS), auxiliary ion mass spectrometry (SIMS), quadrupole time-of-flight (Q-TOF), atmospheric pressure chemical ionization mass spectrometry (APCI-MS), APCI-MS/MS, APCI-(MS)<n>, Atmospheric pressure photoionization mass spectrometry (APPI-MS), APPI-MS/MS and APPI-(MS)<n>, quadrupole mass spectrometry, Fourier transform mass spectrometry (FTMS) and ion trap mass spectrometry are used, where n is an integer greater than 0. Such techniques are disclosed, for example, in Nissen, Journal of Chromatography A, 703, 1995: 37-57, US Pat. No. 4,540,884 or US Pat. No. 5,397,894. Preferably, gas chromatography combined with mass spectrometry is used as described in (Garcia A. and Barbas C. 2011, Methods Mol. Biol. (Clifton NJ) 708:191-204). Even more preferably, quadrupole time-of-flight gas chromatography combined with mass spectrometry (GC-qTOF/MS) is performed (Riera-Borrull M. et al. 2016, J. Am. Soc. Mass Spectrom. 27(1)). : 168-177, Kind T. et al., 2009, Anal. Chem. 81(24): 10038-48).

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

In certain embodiments, the extract rich in ursolic acid is obtained by a method comprising:

(i) extracting from the product rich in ursolic acid with hydroalcohol; and

(ii) removing the activated carbon after treating the hydroalcoholic extract obtained in step (i) with activated carbon.

In a specific embodiment, in the extraction step of step (i) of a method for obtaining an extract rich in ursolic acid, the ursolic acid-enriched product is selected from the aforementioned ursolic acid-rich family. In another specific embodiment, the extraction of step (i) is performed on one or more of the products rich in ursolic acid selected from any of the groups of products rich in ursolic acid indicated above. In another specific embodiment, it comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 , at least 10, at least 12, at least 13, at least 14, at least 15 products. In another specific embodiment, this is an extraction from all products indicated in any of the ursolic acid-rich product groups indicated above.

In a specific embodiment, an extract rich in ursolic acid is obtained comprising the following method:

(i) hydroalcohol extraction from leaves, seaweeds or fruit peels of plants of the family Lamiaceae; and

(ii) removing the activated carbon after treating the hydroalcoholic extract obtained in step (i) with activated carbon.

In another specific embodiment, the extraction in step (i) of the method for obtaining an extract rich in ursolic acid is any hydroalcohol indicated in the definition of hydroalcohol, preferably from 5% (v/v) to 96% (v) /v) with a hydroalcohol with an alcohol.

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

In another specific embodiment, the extraction step (i) of the method of obtaining an extract enriched in ursolic acid comprises 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 specific embodiment, the method is carried out for 0.5 to 24 hours, 1 to 12 hours, 1 to 10 hours, preferably 1 to 10 hours.

In another specific embodiment, step (i) of the method of obtaining an extract rich in ursolic acid is repeated at least 1 time, at least 2 times, at least 3 times, at least 4 times, at least 5 times, wherein in step (ii) of the method The hydroalcoholic extract used is a combination of the hydroalcoholic extracts obtained in each iteration of step (i). In another specific embodiment, the hydroalcohol used in each iteration of step (i) of the method is the same in each iteration. In another specific embodiment, the hydroalcohol used in each iteration is not the same in each iteration. In another specific embodiment, the hydroalcohol used in each iteration has a different concentration of alcohol in each iteration. In certain embodiments, each hydroalcohol used in each of the iterations is selected from the hydroalcohols indicated in the definition of hydroalcohol above.

In another specific embodiment, the ursolic acid-enriched product used in step (i) of the method is the same in each iteration of step (i) of the method. As the skilled person will understand, in this case the same product rich in ursolic acid is extracted several times with hydroalcohol.

In a specific embodiment, the hydroalcoholic extract used in step (ii) contains 0.01 μm, 0.05 μm, 0.1 μm, 0.5 μm, 1 μm, 2 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm No particles larger than microns, 60 microns, 70 microns, 80 microns, 90 microns, 100 microns, 150 microns, 200 microns, 250 microns, 500 microns, 1 mm, 2 mm, or 5 mm. In certain embodiments, the hydroalcoholic extract is free of particles larger than 50 μm. Methods for removing particles larger than any of these sizes are well known to those skilled in the art, such as centrifugation or the use of sieves with pore sizes smaller than the particles to be removed.

As used herein, the terms “activated carbon” and “activated carbon” refer to terms known to those skilled in the art, and activated carbon is a form of carbon processed to improve adsorption properties. They are primarily treated to have small, small numbers of pores that increase the surface area available for adsorption or chemical reactions.

In certain embodiments, step (ii) of the method of obtaining an extract rich in ursolic acid is greater than 30°C, greater than 40°C, greater than 45°C, greater than 50°C, greater than 60°C, greater than 70°C, greater than 80°C, greater than 90°C , greater than 95°C, greater than 96°C, greater than 97°C, greater than 98°C, greater than 99°C, greater than 100°C, greater than 105°C, greater than 110°C. In a preferred embodiment, step (ii) is carried out at about 30° C. to 110° C., preferably about 40 to 100° C.

In certain embodiments, step (ii) of the method of obtaining an extract rich in ursolic acid comprises 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 hours, at least 12 hours, at least 15 hours, at least 18 hours, at least 20 hours, at least 24 hours. In a specific embodiment, step (ii) of the method is carried out for 0.5 to 10 hours, 1 to 5 hours, preferably 1 to 4 hours.

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

In certain embodiments, removal of the activated carbon results in at least 0.01 μm, 0.05 μm, 0.15 μm, 0.2 μm, 0.25 μm, 0.3 μm, 0.35, 0.4 μm, 0.45 μm, 0.5 μm, 0.55 μm, 0.6 μm, 0.65 μm, 0.7 μm, 0.75 μm, 0.8 μm, 0.85 μm, 0.9 μm, 0.9 μm, 0.1 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 150 μm, An extract free of raw particles and activated carbon particles larger than 200 μm, 250 μm, 500 μm, 1 mm, 2 mm, 5 mm is produced. In certain embodiments, removal of the activated carbon results in an extract free of raw particles and carbon particles larger than 0.45 μm, larger than 1 μm, larger than 5 μm, and larger than 10 μm, preferably having raw particles and carbon particles larger than 0.45 μm no extract is produced. Methods for removing particles larger than any of these sizes are well known by those skilled in the art, such as centrifugation or the use of sieves with pore sizes smaller than the particles to be removed.

In a specific embodiment, the method of obtaining an extract rich in ursolic acid comprises a further step (iii) in which the hydroalcoholic extract obtained from step (ii) is concentrated. In certain embodiments, the concentration comprises separation of the solids content of the extract from the solvent and drying of the solids content. As will be understood by those skilled in the art, the solids content is any content of the extract other than the solvent of the extract. In certain embodiments, the extract obtained in step (iii) is 50%, 45% 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10% %, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% water, preferably less than 15% water, even more preferably 10% contains less than water.

In certain embodiments, the extract obtained in step (ii) of the method and used in step (iii) is treated to remove any bacteria. Such therapies are well known by those skilled in the art. Non-limiting examples of such treatments include heat treatment or microfiltration.

As used herein, the expression "a plant extract rich in D-pinitol" or "a plant extract containing D-pinitol" refers to a product or tissue of a plant organism that is rich in D-pinitol, i.e., extracts containing large amounts of D-pinitol. The final concentration of D-pinitol is high. Non-limiting examples of products or tissues of plant organisms rich in D-pinitol include products of plant organisms selected from Ceratonia, Ceratonia siliqua, Sutherlandia frutescens or Pinus lambertiana. In certain embodiments, the botanical product from the plant is any of those indicated in the definition of "vegetable product". In a preferred embodiment, the product rich in D-pinitol is selected from the group consisting of fruits of Ceratonia, pods of Ceratonia, fruits of Ceratonia siliqua, pods of Ceratonia siliqua, leaves of Sutherlandia frutescens, or plant products of Pinus lambertiana. The botanical extract can be obtained by any method well known to those skilled in the art, for example, the method provided in the embodiment of the method for obtaining an extract enriched in D-pinitol or "Method for obtaining a natural extract enriched in D-pinitol" It is provided in the example of ". A method for obtaining an extract rich in D-pinitol is described in European Patent EP1241155-A1 using carob pods as a starting material or Gonzalez-Mauraza et al. (Natural Product Communications, 2015, 11:405-406) using the aerial portion of Retama monospema as a starting material.

In another specific embodiment, the D-pinitol-enriched extract is obtained from one or more products enriched in D-pinitol selected from any of the groups of products enriched in D-pinitol acid indicated above. In another specific embodiment it is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 13 dog, at least 14, at least 15 products selected from the D-pinitol-enriched product group indicated above. In another specific embodiment, it is obtained from all products indicated in any of the D-pinitol-rich product groups indicated above.

In certain embodiments, the botanical extract rich in D-pinitol is 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% D-pinitol (w/w), preferably at least 95% D-pinitol (w/w).

In a specific embodiment, the weight ratio of D-pinitol and ursolic acid in the composition or kit of parts of the first aspect of the invention is a:b, where a represents the amount of the composition or kit of parts, and b is ursol in the composition or kit of parts Indicates the amount of acid:

- 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 45, 50, 55, 58, 60, 61, 61.5, 62, 62.5, 63, 63.1, 63.2, 63.3, 63.4, 63.65, 63.7, 63.8, 63.9, 70, 71, 72 , 75, 80, 85 and 90 are selected from the group consisting of. In a preferred embodiment the value of a is 63.3. In preferred embodiments, the values of b are 1, 2, 3, 4, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 12, 15, 18, and 20. In a preferred embodiment, the value of b is 7.5.

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

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

(i) docosahexaeoic acid (DHA),

(ii) ginkgo flavonoids and

(iii) mixtures thereof.

As used herein, the term "docosahexaenoic acid" or "DHA" refers to (4Z, 7Z, 10Z, 13Z, 16 Z, 19Z)-docosa-4,7,10,13,16,19-hexae It means an omega-3 fatty acid, which is a major structural component with the IUPAC name of noic acid. In certain embodiments, the DHA of the composition or kit of parts of the first aspect of the invention contains or is provided as a fatty acid composition comprising DHA. In certain embodiments, a 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% DHA, preferably at least 25% DHA.

In certain embodiments, the fatty acid composition containing DHA is 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% DHA, preferably at least 25% DHA.

In another specific embodiment, the fatty acid composition is obtained from microalgae or fish. In certain embodiments, it is Schizochytrium sp , Schizochytrium , aggregates, Schizochytrium limacinum, Schizochytrium minutum , Thraustochytrium sp. Thraustochytrium sp), Thraustochytrium aureum ( Thraustochytrium aureum ), Thraustochytrium kinnei ) Nannochloropsis sp . Nannochloropsis granulata , Nannochloropsis limnetica, Nannochloropsis ocednica , Nannochloropsis ocednica , Nannochloropsis oculata , Nannochloropsis salina salina , Pinguiococcus species ( Pinguiococcus sp. ), Pinguiococcus pyrenoidosus ( Pinguiococcus pyrenoidosus ), Pavlova sp. Pavlova gyrans, Pavlova hommersandii , Pavlova pinguis , Pavlova ennorea , Pavlova lutheri , Pavlova mesolychnon , Pavlova mesolychnon ( Pavlova salina ), Pavlova virescens , Pavlova virescens ( Pavlova vi ridis ), Isocrysis sp., Isochrysis galbana , Isochrysis litoralis ( Isochrysis litoralis ) and Isochrysis maritima microalgae selected from the group consisting of is obtained from

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

As used herein, the term "Ginkgo flavonoid" refers to a flavonoid glycoside present in an extract of G. bilobd, preferably a leaf extract of G. biloba. Flavonoid glycosides are glycosides, including O-glycosides, thioglycosides, glycosylamines and C-glycosides, depending on whether the linkage between the sugar and other compounds occurs via an oxygen atom, a sulfur atom, a nitrogen atom or a carbon atom. A compound in which a sugar is conjugated to a flavone or flavonol via a seed bond. Flavonols found as glycosides in G. biloba extract include quercetin (which binds to rhamnose to form quercitrine), camperol and isorhamnetin (isorhamnetin-3-O-lutinoside-7). -O-glucoside, isorhamnetin-3-0-lutinoside-4'-O-glucoside or isorhamnetin-3-O-lutinoside (also called narcisin)). As used herein, the expression "extract of ginkgo tree" or "ginkgo extract" refers to a botanical extract obtained from a plant product of G. biloba. In a specific embodiment, the botanical product refers to the botanical product having the definition of "vegetable product" above. In a preferred embodiment, the extract of G. biloba is obtained from the leaves of G. biloba. As is well known to those skilled in the art, the extract contains flavonoids and terpene lactones. The extract may be obtained by any method well known to those skilled in the art, for example, as an example of a method for obtaining an extract of G. biloba or as a "method for obtaining a natural extract rich in ursolic acid" It is provided in an embodiment of the invention. The term "terpene lactone" is a group of compounds with a unique chemical structure first recognized in the extract of G. biloba. The main terpene lactone compounds of G. biloba include bilobalide and ginkgolide.

In a specific 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 specific embodiment, the G. biloba extract of the composition or kit of parts of the first aspect of the invention is 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%, a percentage (w/w) of ginkgo flavonoids of at least 16%, at least 18%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, preferably at least Contains a percentage (w/w) of ginkgo flavonoids of 0.6%. In another specific embodiment, the G. biloba extract of the composition or kit of parts of the first aspect of the invention is 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%, a percentage (w/w) of ginkgo flavonoids of at least 16%, at least 18%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, preferably at least Contains a percentage (w/w) of ginkgo flavonoids of 0.6%.

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

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

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

In another specific example, the G. biloba extract was obtained by a method comprising:

(i) aqueous extraction from botanical products of G. biloba and

(ii) treating the aqueous extract obtained in step (i) with activated carbon and subsequently removing the activated carbon.

In a specific embodiment, the botanical product of G. biloba is any of those indicated in the definition of "vegetable product", preferably the leaves of G. biloba.

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

In another specific embodiment, step (i) extraction of the method for obtaining a G. biloba extract comprises 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 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 is carried out In a specific embodiment, the method is carried out for 0.5-48 hours, 1-30 hours, 1-20 hours, preferably 1-20 hours.

In another specific embodiment, step (i) of the method of obtaining the G. biloba extract is repeated at least 1 time, at least 2 times, at least 3 times, at least 4 times, at least 5 times. In certain embodiments, the vegetable product from G. biloba used in step (i) of the method is the same in each iteration of step (i) of the method. As will be appreciated by those skilled in the art, in this case the same botanical product of G. biloba is exposed to several aqueous extractions.

In certain embodiments, the aqueous extract used in step (ii) includes 0.01 μm, 0.05 μm, 0.1 μm, 0.5 μm, 1 μm, 2 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm , 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 150 μm, 200 μm, 250 μm, 500 μm, 1 mm, 2 mm, 5 mm. In certain embodiments, they are free of particles larger than 50 μm. Methods for removing particles larger than any of these sizes are well known by those skilled in the art, including centrifugation or the use of sieves with pore sizes smaller than the particles to be removed.

In certain embodiments, step (ii) of the method of obtaining an extract of G. biloba is greater than 30°C, greater than 40°C, greater than 45°C, greater than 50°C, greater than 60°C, greater than 70°C, greater than 80°C. , greater than 90°C, greater than 95°C, greater than 96°C, greater than 97°C, greater than 98°C, greater than 99°C, greater than 100°C, greater than 105°C, greater than 110°C. In a preferred embodiment, the above step is carried out at about 30°C to 110°C, preferably about 40 to 100°C.

In certain embodiments, step (ii) of the method of obtaining an extract of G. biloba comprises 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 specific embodiment, step (ii) of the method is performed for 0.5 to 10 hours, 1 to 5 hours, preferably 1 to 3 hours.

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

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

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

In a specific embodiment, the extract obtained in step (iii) has a solids content of 1% (w/w) to 90% (w/w), preferably 5% (w/w) to 08% (w) of the extract. /w), more preferably from 10% (w/w) to 70% (w/w). The term "solids content" is as defined above in the examples of the method for obtaining an extract rich in ursolic acid.

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

In certain embodiments, the extract obtained in step (ii) of the method and used in step (iii) is treated to remove any bacteria. Such therapies are well known by those skilled in the art. Non-limiting examples of such treatments include heat treatment or microfiltration.

In a specific embodiment, the G. biloba extract obtained by the method for obtaining the G. biloba extract is as defined in any of the preceding Examples.

In a specific embodiment, when the composition or kit of parts according to the first aspect of the present invention contains or comprises D-pinitol, ursolic acid and DHA, the weight ratio of the components is a:b:c, wherein a and b are above a and b of the indicated weight ratios of D-pinitol and ursolic acid, c denotes the amount of DHA in the composition or kit of parts and is 10 to 80, 20 to 70, 30 to 60, 35 to 50, preferably 35 to 45 . In a preferred embodiment, the values of c are 5, 10, 15, 20, 25, 30, 35, 37, 40, 42, 45, 50, 55, 60, 70, 80, 90 and 100. In a preferred embodiment, the value c is 40.

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

When the composition contains D-pinitol, ursolic acid and ginkgo flavonoid, the weight ratio of the components is a:b:d, where a and b are a and b of the weight ratio of D-pinitol and ursolic acid indicated above, and d is The amount of ginkgo flavonoid in the composition or kit of parts is 0.01 to 10, 0.5 to 5, 0.7 to 2, 0.8 to 1.5, preferably 0.9 to 1.1. In a preferred embodiment, the values of d are 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 to be. In a preferred embodiment, the value of d is 1.

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

In a specific embodiment, when the composition or kit of parts according to the first aspect of the present invention contains or comprises D-pinitol, ursolic acid, DHA and ginkgo flavonoid, the weight ratio of the components is a:b:c:d, wherein a and b are 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 the D- indicated immediately above As indicated for the weight ratio of pinitol, ursolic acid and ginkgo flavonoid. In a preferred embodiment, when the composition or kit of parts contains D-pinitol, ursolic acid, DHA and ginkgo flavonoid, the weight ratio of the components is about 63.3:7.5:40:1.

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

As used herein, the term “carrier” refers to excipients, diluents and/or adjuvants useful in preparing compositions, for example, by reducing viscosity, enhancing solubility, or aiding in the stability of composition components. For example, to prevent denaturation or agglomeration during the expected shelf life. The carrier may comprise or consist of agents such as wetting or emulsifying agents, pH buffering agents, or adjuvants that enhance the effectiveness of the agent. When the composition is administered to a subject, the carrier must be physiologically acceptable and should not generally cause allergic reactions or similar adverse reactions such as gastrointestinal disturbances, dizziness, etc. when administered to humans or animals. Non-limiting examples of carriers include water, salt solutions, alcohols, vegetable oils, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oils, monoglycerides and diglycerides of fatty acids, fatty acid esters petroleum, hydroxymethyl cellulose, polyvinylpyrrolidone, hydroxypropyl beta cyclodextrin, and the like.

Where a single carrier is used in the compositions of the present invention, but the different components are formulated into a kit of parts, each element or part of the kit may be formulated with a carrier, which may be the same as the carrier used in the other parts of the kit or can be different.

The concentration of the carrier on the composition does not specifically limit the scope of the present invention. In one embodiment, the concentration of carrier is from 20% to 90% (w/w) by weight of the composition. In a preferred embodiment, the concentration of carrier is from 30 to 80% (w/w), from 35 to 75%, from 40% to 70% (w/w), from 45% to 65% (w/w), or from 50% to 60% (w/w).

2. Pharmaceutical composition

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

As used herein, the term "pharmaceutical composition" refers to any composition that is physiologically acceptable and does not typically cause allergic reactions or similar adverse reactions such as gastric disorders, dizziness, etc. when administered to humans or animals. The composition comprises one or more pharmaceutically active ingredients 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, and include a non-toxic solid, semi-solid or liquid filler; refers to a diluent, encapsulating material or agent. All existing types of auxiliary. Pharmaceutically acceptable carriers are essentially nontoxic to recipients at the dosages and concentrations employed and are compatible with the other ingredients of the formulation. Suitable carriers include, but are not limited to, water, dextrose, glycerol, saline, ethanol, and combinations thereof. The carrier may contain additional agents such as wetting or emulsifying agents, pH buffering agents or adjuvants that enhance the effectiveness of the formulation.

The adjuvant may be selected from the group consisting of sterile liquids such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, or sterile liquids such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Particularly in the case of injectable solutions, water or aqueous saline and aqueous dextrose and glycerol solutions are preferably used as vehicles. Suitable pharmaceutical vehicles are described in "Remington's Pharmaceutical Sciences" (EW Martin, 21st Edition, 2005). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in therapeutic or pharmaceutical compositions is contemplated.

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

One of ordinary skill in the art can determine a therapeutically effective amount of a composition of the present invention by determining the unit dose. As used herein, “unit dose” refers to the amount of a composition or kit of parts of the invention required to produce a response of 50% of maximal effect (ie, ED50). Unit doses can be assessed by extrapolation from dose-response curves derived from in vitro or animal model test systems. The amount of compound in the compositions of the present invention that will be effective for 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, e.g., Goodman and Gilman's The Pharmacological Basis of Therapeutics, Joel G. Harman, Lee E. Limbird, Eds.; McGraw Hill, New York, 2001; The Physician's Desk Reference, Medical Economics Company, Inc., Oradell, NJ, 1995 and Drug Facts and Comparisons, Facts and Comparisons, Inc., St. Louis, MO, 1993). The exact dosage used in the formulation also depends on the route of administration and the severity of the disease or disorder, and should be determined according to the judgment of the physician and each patient's circumstances. Various dosing patterns will be apparent to those skilled in the art.

Further, when repeated administrations of the extracts of the present invention are used, the effective amount of the extracts of the present invention is further dependent on factors including, but not limited to, the frequency of administration, the half-life of the extract, the present invention, or any combination thereof. something to do.

Dosage ranges for administration of the compositions of the present invention are large enough to produce the desired therapeutic effect. Preferably, the composition according to the present invention is regularly administered at least once a day. A typical dose administered to a human is from about 1 mg to about 10 g of the composition, preferably from 1 mg to 1 g of the composition.

The compositions provided herein can be administered to a subject by a number of suitable methods known in the art. Examples of suitable methods include (1) intramuscular, intradermal, intradermal or subcutaneous administration, (2) oral administration, and (3) topical application (eg, ocular, nasal and vaginal application). However, in a preferred embodiment, the composition is formulated for oral administration.

In some embodiments, the preferred route of administration for a composition provided herein is oral. In this case, compositions for oral use are, for example, tablets, troches, lozenges, aqueous or oily suspensions, solutions, dispersible powders or granules, emulsions, hard or soft capsules, syrups or elixirs, pastes, gels. . Compositions 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 preservatives to provide a pharmaceutically elegant and palatable composition. have. Tablets may contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients suitable for the manufacture of tablets. Such excipients include, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents such as corn starch or alginic acid; binders such as starch, gelatin or acacia; and lubricants such as magnesium stearate, stearic acid or talc. Tablets may be uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained action over an extended period of time. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be used. It can also be coated for controlled delivery. For example, a “delayed release” dosage form releases the product or substance at a time other than immediately after administration. Examples of delayed release systems include repeat acting tablets and capsules, and enteric coated tablets in which timely release is achieved by a barrier coating.

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

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

The compositions of the present invention may be formulated as oily suspensions by suspending the active ingredient in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil, such as liquid paraffin. Oily suspensions may contain thickening agents, for example beeswax, hard paraffin or cetyl alcohol. Sweetening and flavoring agents such as those described above may be added to provide a palatable oral composition. Such compositions can be preserved by the addition of antioxidants such as ascorbic acid.

The composition of the present invention may be formulated in the form of dispersible powders and granules suitable for the composition of an aqueous suspension by adding water. The active ingredients of such powders and granules are provided in admixture with dispersing or wetting agents, suspending agents and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, or for example sweetening, flavoring and coloring agents, may also be present.

The composition of the present invention may also be in the form of an oil-in-water emulsion. The oily phase may be a vegetable oil, such as olive oil or arachis oil, or a mineral oil, such as liquid paraffin, or mixtures thereof. Suitable emulsifiers include natural gums, such as gum acacia or gum tragacanth, natural phosphatides, such as soybeans, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monool rates, and condensation products of partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. Emulsions may also contain sweetening and flavoring agents.

The compositions of the present invention may also be formulated as syrups and elixirs. Syrups and elixirs may be formulated with sweetening agents such as glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain emollients, preservatives, flavoring and coloring agents. Emollients are protective agents primarily used to relieve irritation, particularly mucous membranes or worn tissue. Many chemicals have mitigating properties. These substances include alginates, mucilages, gums, dextrins, starches, certain sugars and high molecular weight 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 may contain at least 0.1 mg of a provided extract in certain embodiments, such as its solid counterpart. One of ordinary skill in the art will be able to properly formulate liquid formulations containing appropriate amounts of a given extract per ounce of fluid, depending on the selected additive or carrier.

Formulations suitable for buccal administration include tablets and lozenges comprising the extract in a flavored base such as sucrose, acacia or tragacanth; and tablets 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 present invention relates to a food or dietary supplement comprising the composition of the present invention and a nutritionally acceptable carrier.

As used herein, the term "food supplement" or "dietary supplement" refers to a source of concentrated nutrients or other substances obtained from edible products intended to supplement the general diet. A food supplement or dietary supplement according to the present invention comprises a functional food composition, i.e. a food, beverage, feed or pet food or food, beverage, feed or pet food supplement, nutritional supplement, fragrance or flavoring agent, brewing or cosmetic preparation. do. In general, the term "food supplement" or "dietary supplement" may also mean:

(i) Products that contain or are intended to supplement a diet containing one or more of the following dietary ingredients: [A] vitamins, [B] minerals, [C] herbs or other plants, [D] amino acids, [E] total dietary intake dietary substances used by humans to supplement their diet by increasing or (F) a concentrate, metabolite, component, extract or combination of components described in clauses (A), (B), (C), (D), or (E); or

(ii) (A) a product intended for ingestion; (B) When used as a conventional food or not used as a sole item in a meal or diet, (C) It is labeled as a dietary supplement.

A "food supplement" or "dietary supplement" according to the present invention is generally administered orally and given with the subject's diet. They can take a wide variety of forms, including tablets, capsules, liquid suspensions, dry powders, wet compositions, dry tube feeds or wet tube feeds. They may be provided as a nutritional preparation, for example in the form of a medical food, for example a tube supply, or as a complete meal in the form of oral nutrition, as part of a meal, as a food additive, as a powder for dissolution, for example as a health drink. can For example, ready-made beverages, including juices, milkshakes, yogurt drinks, smoothies or soy-based beverages, dispersed at the bar or in any kind of food such as baked goods, cereal bars, dairy bars, snack foods, soups, breakfasts It may be cereal, misesley, candy, cookie, biscuit.

As used herein, the term "nutritionally acceptable carrier" means a carrier as defined above for the purpose of preparing a solution which is edible and to be administered orally. Typical nutritionally acceptable carriers, diluents and excipients will be familiar to those skilled in the art. Non-limiting examples of such carriers are described in US Pat. 6,258,846; 6,576,666; and 7,112,609.

Nutraceutical compositions, diets or foods for humans or animals (e.g., nutraceutical compositions, i.e. food, beverage, feed or pet food or food, beverage, feed or pet food supplement), nutritional supplements, fragrances or flavorings, pharmaceuticals ( The amount of pharmaceutical composition or formulation), veterinary composition, brewing or cosmetic formulation will vary depending on the application. Generally, the amount of composition present in a food supplement or dietary supplement will be from about 0.001 to about 50% by weight by weight of the nutraceutical composition, diet or food, nutritional supplement, flavor or flavoring, brewed food, for example about 0.01 to about 10% by weight, or from about 0.1 to 1% by weight.

4. Medical use

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

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

As used herein, the term "cognitive impairment", or "neurocognitive disorder" refers to a disorder or disease characterized by an alteration in at least one cognitive function of a patient, thereby reducing the patient's ability to perform said function. . The term “cognitive function” refers to cognitively developed or mental abilities. DSM-5 defines six major domains of cognitive function: executive function, learning and memory, perceptual motor function, language, complex attention, and social cognition. Thus, cognitive impairment refers 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 disorders (formerly known as dementia). Non-limiting examples of cognitive impairment include Alzheimer's disease, mild cognitive impairment (MCI), Parkinson's disease, frontotemporal degeneration, Huntington's disease, Lewy body disease, traumatic brain injury (TBI), prion disease, and dementia/neurocognitive problems due to HIV infection includes

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

As used herein, the term “mild cognitive impairment (MCI)” refers to a term well known to those skilled in the art. Neurological disorders that occur in the elderly (approximately 15-20% of the population aged 65 years and older), including cognitive impairments that cause minimal impairment in instrumental activities of daily living. MCI involves the onset and development of cognitive impairments beyond those expected based on an individual's age and education, but not severe enough to interfere with daily activities. According to the World Health Organization (WHO), MCI does not meet the criteria for diagnosis of dementia and is diagnosed with impairment in one or more cognitive areas. It can be divided into amnestic MCI and non-amnestic MCI. Amnesia MCI is characterized by impairment of memory-cognitive functioning, so that affected individuals forget important information that could have been easily remembered before, such as appointments, conversations, or recent events. Nonamnestic MCI is characterized by loss of cognitive abilities by the affected person other than memory, such as the ability to make sound decisions, the ability to judge the sequence or time of steps required to complete a complex task, or visual perception.

As used herein, the terms "Alzheimer's disease", "Alzheimer's disease" or "AD" refer to diseases well known by those skilled in the art. Alzheimer's disease is characterized by a progressive pattern of cognitive and functional impairment. In the early stages of the disease, patients show brief amnesia and subtle problems with attention, planning, flexibility, and executive functions of abstract thinking, or impairment of semantic memory (semantic memory and conceptual relationships). As the disease progresses, memory problems increase, language, reading and writing skills deteriorate, and coordination of motor sequences is impaired. At an advanced stage, the patient may completely lose the ability to speak, so he becomes completely dependent on the caregiver, and shows decreased muscle mass and mobility that interferes with self-eating.

As used herein, the term “dementia” refers to a term commonly known to one of ordinary skill in the art. According to the World Health Organization (WHO), dementia is a syndrome that is usually chronic or progressive, a syndrome in which cognitive function declines beyond what would be expected from normal aging. It affects memory, thinking, orientation, comprehension, calculation, learning ability, language and judgment. Consciousness is not affected. Impairment of cognitive function usually accompanies and sometimes precedes deterioration of emotional regulation, social behavior, or motivation. Dementia is caused by a variety of diseases and injuries that primarily or secondary affect the brain, such as Alzheimer's disease or stroke. Alzheimer's disease is the most common form and can account for 60-70% of cases. Other major forms include vascular dementia, Lewy body dementia (abnormal protein aggregates that develop inside nerve cells), and a group of diseases that contribute to frontotemporal dementia (frontal lobe degeneration of the brain).

As used herein, the term "Parkinson's disease" or "PD" refers to a term generally known by one of ordinary skill 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 tremors, stiffness, slow movements, and difficulty walking. Thinking and behavioral problems may also arise. Dementia becomes common in advanced stages of the disease. Depression and anxiety are also common and occur in more than one-third of people with PD. Other symptoms include sensory, sleep, and emotional problems.

Depending on the disorder, the subject being treated as well as the route of administration, the compositions of the present invention may be administered in various doses (ie, a therapeutically effective dose as administered to a patient in need thereof). In this regard, the person skilled in the art will understand that in the context of the present invention the dose administered to a mammal, in particular a human, should be sufficient to affect the therapeutic response of the mammal over a reasonable period of time. One of ordinary skill in the art will know that the exact dosage and composition and selection of the most appropriate delivery regimen will depend, inter alia, on the pharmacological properties of the formulation, the nature and severity of the condition being treated, and the physical condition and mental power of the recipient, age, condition, weight, sex and response of the patient to be treated; It will be appreciated that will be affected by the stage/severity of the disease.

The subject of treatment is a mammal, preferably a human. Subjects to be treated in accordance with the present invention may be selected based on several criteria related to neurodegenerative diseases, such as imaging methods or behavioral tests.

In one embodiment, a composition, kit of parts, pharmaceutical product or food or dietary supplement for use in accordance with the present invention comprises administration of multiple doses of the composition. In another embodiment, the composition, kit of parts, pharmaceutical product or food or dietary supplement is administered for at least 2 days, at least 4 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month. In some embodiments, the composition, kit of parts, pharmaceutical product or food or dietary supplement for use according to the present invention is administered for a period followed by a period during which the composition, kit of parts, pharmaceutical product or food or dietary supplement is not administered. In one embodiment, the composition, kit of parts, pharmaceutical product or food or dietary supplement follows a 5+2 delivery pattern; That is, we ship the product for 5 days and not for 2 days.

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

Alternatively, the composition of the present invention may be administered one or more times per day in combination with a drug prescribed for the indication to be treated. For example, when a composition or kit of parts according to the invention is for use in the treatment of Alzheimer's disease, the composition or kit of parts is administered together with one Alzheimer's disease drug. Suitable Alzheimer's disease drugs include acetylcholinesterase inhibitors such as tacrine, also known as tetrahydroaminoacridine (THA); rivastigmine; donepezil; galantamine; carbamate; physostigmine; neostigmine; pyridostigmine; ambenonium; demecarium; phenanthrene derivatives; caffeine - non-competitive (also an adenosine receptor antagonist); rosmarinic acid - ester of caffeic acid found in Lamiaceae plant species; alpha Alpha-Pinene - non-competitive reversibility; Piperidines; Edrophonium; Huperzine A; Ladostigil; Ungeremine; Lactuco picrin (Lactucopicrin), NMDA receptor antagonists (eg memantine), gamma secretase inhibitors (eg semagacestat, ELND006, avagacestat, begacestat, NIC5-15 and CHF-5074, MK-8931, LY2886721, AZD3293, LY3314814, E2609), antibodies to Abeta (bapineuzumab, solanezumab, gantenerumab, crenezumab) , aducanumab, crenezumab, ponezumab, GSK933776 and BAN-2401, human polyclonal anti-Abeta antibody or immunoglobulin therapy (e.g., gammagard(R), plevo Gamma(R)), agents against tau protein (e.g. tau hyperphosphorylation inhibitors (e.g. LMTX), epothilone D, TPI-287, anti-tau vaccines (e.g. AADvacl or ACI-35) and GSK-3beta inhibitors (eg, Tideglusib, intranasal Humulin R, or intranasal Glulizine).

For example, when a composition or kit of parts according to the invention is for use in the treatment of Parkinson's disease, the composition or kit of parts is administered together with one Parkinson's disease drug. Suitable Parkinson's disease drugs include glucosylceramide synthase inhibitors (eg GZ667161), iron chelators, epigallocatechin gallate (EGCG), myeloperodixase inhibitors (eg AZD3241), apitopes (eg AFFITOPE PD01A, AFFITOPE PD003A), and anti-synuclein antibodies (eg, PRX002, BIIB054). Other suitable Parkinson's disease drugs include levodopa, carbidopa, entacapone, ropinirole, rotigotine, pramipexole, bromocriptine, rasagiline, selegiline, amantadine and trihexphenidyl.

For example, if a composition or kit of parts according to the present invention is for use in the treatment of MCI, the composition or kit of parts may be combined with one MCI drug for non-pharmacological treatment or treatment suitable for diseases known to affect mental function. use Suitable MCI drugs include cholinesterase inhibitors. Appropriate non-pharmacological treatment for MI includes regular exercise and cognitive training. Appropriate treatments for conditions known to affect mental function include treatment of hypertension, treatment of depression, and/or treatment of sleep apnea.

The composition of the present invention may be administered as is, but the present invention also provides that at least one of the components of the composition, kit of parts, pharmaceutical composition or food or dietary supplement comprises the other of the components of the composition, kit of parts, drug or food or dietary supplement. Consider the possibility of administration separately from other ingredients. In one embodiment, D-pinitol or a D-pinitol-enriched botanical extract is administered separately from the other components of the composition or kit of parts. In another embodiment, ursolic acid of a hydroalcoholic extract of a botanical product rich in ursolic acid is administered separately from the other components of the composition or kit of parts. In another embodiment, DHA or a fatty acid composition comprising DHA is administered separately from the other components of the composition or kit of parts. In another embodiment, the ginkgo flavonoid or ginkgo extract is administered separately from the other components of the composition or kit of parts.

In another embodiment, D-pinitol or D-pinitol-enriched botanical extracts and ursolic acid or hydroalcoholic extracts of ursolic acid-enriched botanical products are administered separately from the other components of the composition or kit of parts.

In another embodiment, D-pinitol or a D-pinitol-enriched vegetable extract and DHA or a fatty acid composition comprising DHA is administered separately from the other components of the composition or kit of parts.

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

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

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

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

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

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

In another embodiment, a composition, kit of parts, pharmaceutical product, or food or dietary supplement for use in accordance with the present invention is administered in the following daily dosages:

(i) about 150 mg/day of a vegetable 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 food or dietary supplement for use in accordance with the present invention is administered in the following dosages per kg of patient.

(i) about 2.5 mg/kg of a vegetable extract rich in ursolic acid,

(ii) about 8 mg/kg of a 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 following examples, which are illustrative only and do not limit the scope of the invention.

EXAMPLES

Preparation of biological extracts

Preparation of natural extracts rich in ursolic acid

The natural extract rich in ursolic acid is carried out by a process comprising:

(i) providing leaves of Lamiaceae plants (eg, Salvia officinalis, Thymus vulgaris, Rosmarinus officinalis, Origanum vulgare, etc.) as a raw material; You can also use other sources of ursolic acid, such as seaweed or the skins of fruits such as Malus domestica, Pyrus communis, Vaccinium, and Prunus.

(ii) extracting the fresh or dried raw material with a hydroalcoholic mixture having an alcohol content of 10 to 96% at a temperature of about 40 to 100° C. for 1 to 10 hours; The process may also include several stages of leaf extraction with the same or different alcohol content by combining the hydroalcoholic extracts obtained to continue the process.

(iii) isolating the hydroalcoholic extract from the raw material using any separation method that produces an extract free of particles larger than 50-100 microns;

(iv) treating the hydroalcoholic extract with activated charcoal at 40-100° C. for 1-4 hours; Activated carbon dosage is 1-20% (w/w) of the dry matter present in the extract.

(v) isolating the hydroalcoholic extract from the activated carbon using any separation method that produces an extract free of raw particles and activated carbon particles larger than 0.45-10 microns;

(vi) treating the water extract using microfiltration or heat treatment to control possible microbial load;

(vii) concentrating the obtained water extract until the solids content is 5 to 50% (w/v); In this step, an insoluble precipitate is formed in the concentrated extract.

(viii) separation of the precipitate by a separation method of separating the precipitate produced from the supernatant;

(ix) the wet precipitate is dried using any drying method that yields a solid product having 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) consists of ursolic acid.

This method stands out in that it is simple, inexpensive, and can obtain a target product with a high ursolic acid content and a ratio to raw material between 5 and 40.

Preparation of Ginkgo Biloba Extract

Ginkgo biloba leaf extract is prepared by a process comprising the following steps:

(a) Fresh green or dried Ginkgo biloba leaves are extracted with water at a temperature of about 40-100° C. for 1-20 hours. The process may also include several stages of leaf extraction combining the water extract obtained to continue the process.

(b) Separate the water extract and ginkgo leaves by any separation method that produces a water extract free of leaf particles larger than 50-100 microns.

(c) Treating the water extract with activated charcoal at 40-100° C. for 1-3 hours. Activated carbon dosage is 1-20% (w/w) of the dry matter present in the extract.

(d) Separation of the water extract from the activated carbon using any separation method that produces a water extract free of activated carbon particles and leaf particles larger than 0.45-10 microns.

(e) Treat the water extract by microfiltration or heat treatment to control possible microbial load.

(f) concentrated until the concentration of the obtained extract is 10 to 70% of the solid content,

(g) optionally drying the concentrated water extract using any drying method to produce a solid product having a water content of less than 10-15% (w/w);

The method produces an extract containing a ginkgo flavonoid content of 0.5-15% (w/w) and a terpene lactone content of less than 0.01-1% (w/w). This method stands out in that it uses water as the only extraction solvent. The price is low and the target product is obtained in a ratio of 2 to 8 with respect to the raw material.

Preparation of Extracts Rich in D-Pinitol

A natural extract with a high content of D-pinitol can be carried out essentially by a process as described in European patent application EP1241155-A1. The process consists of:

(i) Provide raw materials that are dried carob pods.

(ii) grinding carob pods to obtain pulp.

(iii) Extract carob pulp with water at 10°C to 70°C at slightly acidic pH until an extract of 30° to 50° Brix is obtained.

(iv) Press the pulp to remove the residue.

(v) Filter the raw juice and pass the filtrate through a strong cationic resin to remove most of the calcium and magnesium ions.

(vi) Treat the water extract using microfiltration or ultrafiltration to control possible microbial load.

(vii) Concentrate the syrup to approximately 60°Brix.

(viii) inverting the sugars in the syrup by an enzymatic method or a method using a cationic resin such as an RPI resin.

(ix) 20-30 brix concentration syrup is passed through a strong cationic resin (H) until a conductive composition is obtained and then continuously passed through a strong anionic resin (OH) to obtain 10 microomega It is desalted and bleached to a color of less than and less than 25 Icumsa.

(x) Desalting by strong cation exchange resin and separating the decolorizing syrup, preferably using ISMB technology (continuous chromatography separation). Separation is usually starting from carob syrup at about 60° Brix using 4 m3 of UBK 530 resin, at a feed rate of 0.038 L/h at a temperature of 65° C. feed volume 2.07 T/D, pinitol fractionation volume 0.36 T/D at W/ F 3.750v/v and P/R 2.166v/v.

(xi) desalting and decolorizing the pinitol fraction thus obtained containing 44.2% salt.

The extract obtained using the method has a pinitol content of 91% (w/w), a specific torque of (+) 63, and a water content of 2.5%.

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

Alternatively, pinitol can be extracted from inverted, desalted and bleached carob syrup using strong anionic resins. In this case, this method requires steps (i) to (ix) described above and the following.

(x) 250 ml or about 25° Brix desalted and decolorized syrup through resin SA 11A and a composition of 35% pinitol, 27% glucose, 37% fructose and 0.4% sugar-free, 10 ml/min in a column with a diameter of 2 cm and a height of 100 cm. and eluted with demineralized water at a temperature of 6° C.

(xi) Mix fractions with positive °Brix and concentrate the mixture.

(xii) Crystallization of the concentrated mixture with ethanol.

The extract obtained using the method has a pinitol content of 91% (w/w), a specific torque of (+) 63, and a water content of 2.5%.

How about the zebrafish model

Test Subjects and Compositions

The species selected for testing was the AB wild-type strain of zebrafish (Danio rerio).

Fish were contacted with five compositions (Cl, C2, C3, C4 and C5), each in a single dose, the composition being as follows.

- compound C1

- compound C2

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

- Compound C4: Sage Extract

- compound C5

Below is a table summarizing the experimental conditions:

Table 1. Table showing the compound code, the compound weight and volume of DMSO required to prepare the stock solution, the state of the solution, and the final concentration tested.

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

Once obtained, embryos were seeded in Petri dishes with 50 ml of dilute solution (DS) and grown for 5 days post-fertilization (DPF), which is considered the larval stage. Only larvae showing no external abnormalities were used in the test. The larvae were then transferred to a 24-well microplate using a Pasteur pipette, containing 5 larvae in each well, making 10 replicates per condition. First, pretreatment was performed on 5 DPF larvae. For this, the larvae were incubated for 1 hour at 26±1° C. in 2 ml of physostigmine (PHYS, a commercial AChE enzyme inhibitor) and the test compound. Then, the larval medium was replaced and incubated with PHYS, PTZ and other compounds bound to PTZ at 26±1° C. for 6 hours. After this incubation time, all larvae were examined and the general condition of the larvae was determined to be completely normal with no noticeable abnormalities or abnormal behavior. Finally, larvae were treated to assay AChE activity. Controls inoculated into untreated embryos were used in parallel.

Determination of AChE Levels

At the end of the experimental period, larvae were treated to determine AChE levels. The larvae were mechanically homogenized and the samples centrifuged to obtain a supernatant, which was used to determine AChE enzyme levels according to the treatment administered.

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

Finally, the AChE level determined in the control group was regarded as 100% and used as a reference measurement value.

Methods for the mouse SAMP8 model

test subject

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

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

Animals were divided into 3 experimental groups, and treatment was started at 5 months; Behavioral assessments were performed using the open field test (OFT), object recognition test (NORT) and object position test (OLT) after 8 weeks of treatment. Afterwards, the animals were euthanized and samples were collected.

SAMR1 control (n=10).

SAMP8 control (n=10).

SAMP8 treatment group (n=12).

Ginkgo biloba extract, plant extract rich in ursolic acid (e.g. Ursolia®D-Pinitol and treated with a fatty acid composition containing 25% DHA)

According to the protocol, female mice were treated orally via gavage with a combination of extracts from Biosearch SA and DHA (product). The extract was dissolved in 40% hydroxypropyl beta cyclodextrin. The concentration of the extract was calculated according to the body weight of the animal to reach the indicated dose as shown in Table 1 below.

Table 1. Dosage and concentration ratio for each extract

Treatment with the product lasted 8 weeks with a 5+2 delivery pattern. That is, the product was delivered for 5 days and not delivered for 2 days. The product was delivered during the behavioral testing period and was euthanized 3 days after the last behavioral test. Delivered twice via daily probes, one containing DHA and the other containing Gingko biloba extract, Ursolia® and pinitol (Figure 1).

Open Field Testing (OFT)

The open field test ( FIG. 2 ) is a classic test of behavioral patterns in which animals are exposed to new, brightly lit environments (Seibenhener ML and Wooten MC, 2015 J Vis Experiment 96:e52434). The animal's behavior in this space is determined by the balance between the animal's natural interest in a new space (exploration behavior) and the fear of the unknown open bright space (fear/anxiety). The device consisted of a white wooden box (50X50X25cm high). The brightness of the light in the center of the field was 30 lx. Mice were placed in the center of the device and their behavior was evaluated for 5 minutes. The variables measured were total horizontal (total distance, number of line crossings) and vertical (nurture or number of times the rodent stood on hind legs) motor activity and variables related to emotion; Time spent in the center, time spent at the edge, distance from the central area, distance from the peripheral area, time spent in the center (%), time spent at the edge (%), number of bowel movements and frequency of urination. The purpose of the assignment is SMART®ver. To get video for analysis by 3.0 (PanLab, SLU, Spain).

New Object Recognition Test (NORT)

The NORT (Figure 3) paradigm is a widely used explicit memory test. This behavioral test takes advantage of two characteristics of rodents. It is the animal's natural tendency to search for new objects, which has no special meaning and has not been reinforced, and the innate preference to explore new objects over familiar objects (Ennaceur A et ak, 1988, Behav Brain Res. 31:47). -59). In the NORT test, mice were placed in a black maze containing two L-shaped branches that were 25 cm long, 20 cm high and 5 cm wide. The brightness of the light in the center of the field was 30 lx. Various plastic objects were used. During the first 3 days, mice were acquainted with the maze for 10 minutes. On day 4, mice were irradiated with replicas at one end of the maze. The short-term memory test was performed 2 hours later, and the long-term memory test was performed 24 hours later, followed by a 10-minute memory test. In the second experiment, mice placed a new object at one end of a previously unexplored branch of the maze and recorded the time the animals examined the new object (TN) and the old object (TO). This was done to calculate the differential index (DI) defined as (TN-TO)/(TN+TO). To avoid subject preference, subjects A and B were balanced so that half of the mice in each experimental group were first exposed to subject A and then to subject B, and the other half were exposed first to subject B and then to subject A. The maze and objects were cleaned with 70% ethanol after each test to remove olfactory cues. The NORT test started at 9:30-15:30 on the first Friday after completing the first week of treatment.

Object Position Test (OLT)

The Object Position Test (OLT) specifically assesses cognitive deficits in spatial memory and identification (Hattiangady B et al, 2014, Front Behav Neurosci. 8:78). This task involves exploiting the rodent's ability to recognize when an individual is relocated and is essentially stress-free. The test was conducted in a wooden box (50 x 50 x 25 cm) for 4 days, the walls are white and one is marked with a black and white square pattern. On the first day, the box was empty and the animals acclimatized to the OFT cage for 10 minutes. On the second day, two objects were placed on the wall and equidistant from each other on the patterned wall. The objects are 10 cm high and replicated with each other. The animal was placed in the center of the cage and allowed to examine the object and its surroundings for 10 minutes. The mice were then returned to the cage and the OLT device was washed with 70% ethanol. On the third day, the object was moved in front of the opposite white wall to test spatial memory. (seconds) spent sniffing objects in new locations (novel) and objects in old locations (old). To analyze cognitive performance, the position index (%) was calculated using the following method. (Tnovel x 100) / (Tnovel + Told), where Tnovel is the time spent sniffing the object at the new location and Told is the time spent examining the object at the old location.

Sample Collection Procedure

Three days after the last behavioral test, animals were euthanized by cervical dislocation. Brains were dissected and immediately frozen on dry ice and stored at -80°C for use in Biosearch SA.

Open field test, object recognition test and object position test result analysis

To analyze the tests performed, the activity of mice was recorded during all familiarization, retention and memory tests and open field tests. After that, manual analysis was performed according to the SOP for each procedure, and it was included as an appendix to this report.

statistical analysis

To assess possible significant differences between the studied groups, a one-way ANOVA statistical test using the post-hoc Tukey multiple comparison test was performed to compare the three groups involved in 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 important to use the Dunnetf test. Prior to statistical analysis, the Grubbs' test was performed on the anomalous data with 90% confidence. Data obtained with respect to graphical representations are presented as mean ± standard error of the mean (mean ± SEM). Differences in t-Student are also considered significant with p<0.05 and marked with one (*), with one, two or three stars added depending on the value of p (*p<0.01, **p<0.001 and ** *p <0.0001). Statistical analysis and graphs were performed using GraphPad 8.0, a statistical analysis program.

How to model C. elegans

1.1. compound

Test doses were established according to a previous study of Biosearch SA extracts in the accelerated senescence mouse trend 8 (SAMP8), a mouse model of late-onset AD (LOAD), maintaining and aligning the test dose ranges found in the review (1-10) literature. do. Concentrations lower than the demonstrated effective concentration were chosen for each compound. When studying drug response in an oxidation resistance assay, higher concentrations close to the published effective dose were also tested. The proportions of each compound with respect to the composition of the mix were established in light of earlier studies reporting synergistic effects in mice.

Stock concentrations of Biosearch SA extracts were prepared in 100% dimethyl sulfoxide (DMSO). The stock solution was then diluted in MiliQ ddH20 to obtain a test dilution at a maximum concentration of 1% DMSO and stored at -20°C.

1.2. Maintenance and treatment of C. elegans

Wild-type Caenorhabditis elegans (C. elegans) strain N2, transformed strain CL2006, transformed strain CL2355 and control strain CL2122 were used. Standard methods were used for culture and observation of C. elegans. N2 was grown at 20 °C, whereas CL2006, CL2355 and CL2122 were maintained at 16 °C in a temperature controlled incubator of solid nematode growth medium (NGM) inoculated with E. coli OP50 strain as a food source. To obtain an age-synchronized population of eggs, pregnant adults were treated with an 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 incubated overnight in the absence of food.

In most cases, drug assays were performed in 96-well plate format in liquid culture and treated at 20°C for 4 days. Each well contains 25-30 animals in LI stage, appropriate dose of Biosearch SA extract and OP50 inactivated by freeze-thaw cycle and suspended in complete S-medium to a final OD595 of 0.9-0.8 to a final volume of 60mL. contained For chemotactic analysis, synchronized CL2355 and its control CL2122 were obtained from Biosearch S.A. in fresh NGM plates seeded with inactivated E. coli starting at the LI stage. treated as a product. Incubated at 16°C for 36 hours and then incubated at 23°C for 36 hours.

1.3. Oxidation Resistance Analysis

To investigate the sensitivity to oxidative stress after Biosearch SA product treatment, N2-treated adults were transferred to plates containing 6.2 mM t-butyl hydroperoxide (Sigma) on NGM agar. Worms were incubated on these plates at 20°C for 2 hours. The worms were then transferred to a new NGM plate without OP50 and t-butyl hydroperoxide. Bugs were observed 2, 24 and 48 hours after intervention and were scored as dead if they did not respond to repeated forceps stings.

1.4. chemotaxis analysis

After each treatment the nematodes were collected and washed with M9. Briefly, assays were performed in 100 mm NGM plates and 10 μl of odorant (0.5% benzaldehyde in 96% ethanol) along with 1M sodium azide was added at the “attractant” point. To the opposite side, 10 μl of control odor (96% ethanol) was added with 1 M sodium azide. Immediately thereafter, 50-60 worms were placed in the center of the plate. Assay plates were incubated at 23° C. for 1 h and the chemotaxis index (Cl) was scored as follows: Cl = (number of worms in decoy minus number of worms in control)/total number of worms. In each experiment, more than 60 worms from each group were analyzed.

1.5. Thioflavin-S staining AB aggregation

Age-synchronized CL2006 worms were fixed in 4% paraformaldehyde/PBS, pH 7.5 for 24 h at 4°C, 5% fresh b-mercaptoethanol, 1% Triton X-100, 125mm Tris, pH 7.5, 37°C was fixed for an additional 24 hours. Nematodes were stained with 0.125% Thioflavin S (Sigma) for 2 min in 50% ethanol, destained in 50% EtOH for 2 min, washed 3 times with PBS and in a volume of about 10 μl in Fluoromount G drops on glass. moved to slides for microscopes. Fluorescence images were acquired using a 20A objective of a fluorescence microscope (Olympus BX51, Germany). Amyloid deposits on the head of the worms were quantified by counting the number of Thioflavin S (ThS) positive spots using ImageJ and marked as AB deposits/anterior area.

1.6. Lifetime analysis

Worms were treated as described above in liquid culture for 4 days starting at the LI stage. However, 1 μl of 5'-fluorodeoxyuridine (FUdR) was added at 4 days of age for a final concentration of 120 micromolar to prevent progeny generation. After treatment, approximately 30 young adult worms were placed on 3 different NGM plates per condition and transferred to a new seed plate every 3 days to score dead animals. Animals were considered dead if there was no mechanical response when the head was lightly touched with a platinum wire three times.

1.7. statistics

Data analysis was performed using GraphPad Prism ver. 9 is conducted by 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 Tukey's post hoc test. Comparisons between groups were performed by two-tailed Student's t-test for independent replicates when needed. Statistical significance was considered when the p value was <0.05. Statistical outliers were determined by Grubs' test and removed from the analysis.

Example 1 (EXA MPLE 1)

Effect of different compositions in the mouse SAMP8 model

1. Weight Control of Plant Extracts and Lipid Treatment

All animals in each treatment group were weighed. Weights were measured on the Monday prior to initiation of treatment and on Mondays until euthanasia. Table 2 shows the body weights of each group at the start and end of treatment. Improvements in appearance were observed as a result of treatment with plant extracts and lipids.

2. Animal weight during treatment

5A shows body weight gain or loss during treatment for groups of mice. Results show that all groups recorded weight gain at the end of treatment, the increase was 2% in the SAMR1 control group and 4% in the two SAMP8 groups. 5B shows a significant difference in mouse body weight at the end of treatment for the SAMR1 control group and the SAMP8 control group: the same as at the beginning of the treatment. However, the SAMP8 group had no effect on weight gain compared to the control group.

2. Behavioral Consequences

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

2.1. Summary of Open Field Test (OFT) Results

6A-6C show behavioral results in open field testing for groups after 8 weeks of treatment.

SAMR1 control group vs. SAMP8 control group vs. SAMP8 treated group:

The ANOVA test showed a significant difference in the vertical activity variable (p<0.001) and defecation variable (p<0.0001), but not in the motor activity variable (p>0.05). Post hoc analysis showed significant and similar changes in vertical activity parameters for SAMP8 control and treated animals (p<0.05) and for SAMR1 control and SAMP8 treated animals (p<0.01).

Similarly, post hoc analysis also showed significant and similar changes in bowel parameters in control animals (p<0.01) and SAMR1 control and SAMP8 treated (p<0.001) stool variables. A clear trend back to the SAMR1 control group by the SMAP8 treatment group was observed for the locomotor activity parameters.

2.2. Summary of Object Position Test (NORT) Results

The following is a summary of the results of the characterization of long and short working memory cognitive profiles through the subject recognition test for product treatment after 8 weeks of treatment.

2.2.1. short term memory

7 shows the results of an object recognition test after 8 weeks of treatment to evaluate the short-term working memory cognitive function of a group of mice.

SAMR1 control group vs. SAMP8 control group vs. SAMP8 treatment group

ANOVA test showed significant differences in discriminant index variables (p<0.0001). Post hoc analysis showed significant and similar changes in discriminant index variables for SAMR1 and SAMP8 control animals (p<0.0001). Also, post-hoc analysis showed significant and similar changes in the discriminant index variables between the SAMP8 control group and the SAMP8 treatment group (p<0.0001).

2.2.2. long-term memory

8 shows the results of an object recognition test after 8 weeks of treatment to evaluate the long-term working memory cognitive function of a group of mice.

SAMR1 control group vs. SAMP8 control group vs. SAMP8 treatment group

The ANOVA test showed significant differences in the discriminant index variables (p<0.01). Post hoc analysis showed significant and similar changes in discriminative index variables for SAMR1 and SAMP8 control animals (p<0.001). In the post-hoc analysis, significant and similar changes were observed in the discrimination index variables between the SAMP8 control group and the SAMP8 treatment group (p<0.01).

2.3. Summary of Object Position Test Results (OLT)

The following is a summary of the results of spatial memory cognitive profile characterization through plant extract and lipid treatment target location test in women 8 weeks after the start of treatment.

2 3.1.- Spatial memory

9 shows the results of an object position test after 8 weeks of treatment to evaluate the spatial memory cognitive function of a group of mice.

SAMR1 control group vs. SAMP8 control group vs. SAMP8 treatment group

The ANOVA test showed significant differences in the discriminant index variables (p<0.001). Post hoc analysis showed significant and similar changes in discriminant index variables for SAMR1 and SAMP8 control animals (p<0.0001). In the post-hoc analysis, significant and similar changes were observed in the discrimination index variables between the SAMP8 control group and the SAMP8 treatment group (p<0.01).

3. Events and observations

One SMAP8 mouse in the control group died during treatment. No internal damage was observed and death resulted from natural causes. An improvement was observed in the physical appearance of the mice treated with the product ( FIG. 10 ).

Example 2 (EXAMPLE 2)

Effect of different compositions in zebrafish treated with PTZ

Neuroprotective effect of compounds C1 to C5 in PTZ-induced neurotoxicity zebrafish model

Table 3. Percentage of AChE enzyme activity (mu) by total protein (pg) versus control (considered 100%) in 5 DPF larvae treated with PTZ, PHYS and other compounds (Cl-C5) bound to PTZ for 6 h.

This table shows the statistical analysis using the mean, standard deviation (SD), standard error of the mean (SEM), and Dunnetf's multiple comparison test (control).

As shown in Table 3, treatment of larvae with PTZ at 5 mm induced a significant decrease in AChE activity by about 21% compared to the control group (p <0.01). In addition, physostigmine, a potent inhibitor of AChE activity, reduced the AChE activity by 76% compared to the control group (p<0.01). Among all the problem groups, only PTZ treated with 100 mg/l of Cl had significantly lower activity than the control group, but not as much as the decrease due to the effect of PTZ itself.

When PTZ was determined to reduce AChE activity in 5 DPF zebrafish larvae, the neuroprotective effect of the compound against neurotoxin-induced toxicity was analyzed.

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

This study shows that PTZ, an anticonvulsant antagonist of GABA receptors in the CNS, induces potent toxic effects by dramatically lowering AChE activity in zebrafish larvae. The zebrafish genome contains butyrylcholinesterase (BCHE) enzymes; It has also been shown that human acetylcholine does not encode an enzyme that breaks it down either. However, the AChE enzyme is encoded by a single gene in zebrafish and has been functionally detected in the brain. Thus, changes in AChE activity recorded in zebrafish larvae reflect only the function of the cholinergic system of the CNS.

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

First, none of the groups treated with the compounds studied in combination with PTZ had AChE activity lower than that induced by PTZ itself, indicating no additive neurotoxic effects induced by the compounds.

As a result of the AChE activity assay, treatment with C3 (D-pinitol) and C4 (sage extract) compounds prevented the neurotoxic effect induced by PTZ in 5 DPF zebrafish larvae, thereby restoring AChE activity by 29% and 23%, respectively, compared to larvae treated with anticonvulsants. These results show that both compounds have neuroprotective ability against the neurotoxic effects induced by PTZ.

Therefore, it was shown that D-pinitol 100mg/l and sage extract 10mg/l compound significantly prevented the decrease in AChE activity of 5DPF larvae induced by PTZ, thereby exhibiting neuroprotective effects.

Example 3 (EXAMPLE 3)

Effect of various compositions in C. elegans model.

1. A mixture that attenuates the oxidative stress of C. elegans.

Extracts and mixtures were tested after tert-butyl (6.2 mM) to investigate whether they had a beneficial effect on oxidative stress. Treatment was started when the synchronized worms reached the L4 stage and kept at 20°C until the end of the experiment. First, we found that each extract group not only did not reach protection against oxidative stress compared to the untreated control group, but also obtained a significant reduction in survival compared to the vitamin C group (Figure 12). On the other hand, worms pre-incubated in the mixed extract (dose) compared to the untreated group were significantly protected against tert-butyl (6.2 mM) induced oxidative stress and reached a percentage of viable worms close to vitamin C (58 μM). , showing a synergistic effect of administration of the complex extract ( FIG. 12 ).

2. Mixed extract inhibits neuronal Ab expression-induced defects of chemotactic behavior in transgenic C. elegans (CL2355).

To investigate the synergistic effect of the mixed extracts on the performance of chemotactic behavior, benzaldehyde as an attractant containing sodium azide which paralyzes the worms upon contact and ethanol as a control were applied ( FIG. 13 ). Chemotaxis indices were scored for all groups on day 5 of age. 14 shows that the CL2355 strain shows a significant reduction in Cl compared to the control strain CL2122. Moreover, each of the isolated extracts showed a tendency to slightly increase Cl compared to the CL2355 strain, but it was not significant ( FIG. 14 ). Interestingly, compared to the CL2355 group, Cl was significantly increased in the mixed extract, showing a synergistic effect, and the chemotactic behavior was restored for the control CL2122 strain (Fig. 14).

3. The mixed extract improved the amyloid fiber burden in transgenic C. elegans (CL2006).

To investigate whether Mix affects the aggregation of amyloid B, the CL2006 worm strain constitutively overexpressing human ABi-42 in muscle cells was used. We found that the Mix group had a significant effect on AB _I-42 peptide deposition, suggesting that Mix could significantly reduce the aggregation of AB species in a synergistic manner (Figure 15). As expected, the other extract groups (DHA, Ginkgo, Pinitol and Ursilia ^® ) did not have any effect on the AB _I-42 precipitate, indicating that the synergistic effect obtained with the Mix group was not evident (Fig. 15).

4. Life expectancy extension by mixed extract from C. elegans

To investigate the effect on aging, the lifespan changes of C. elegans were investigated after treatment and mixing of various extracts. First, no significant change was found between groups in the Kaplan-Meier curve (Fig. 16A). However, life expectancy was extended by up to 15% with Mix extract treatment compared to DHA, Pinitol and Ursilia ^® groups. While demonstrating a synergistic effect (Figure 16B), there was no change between the Mix and Ginkgo groups because of the well-described effect (REF) of ginkgo on lifespan.

5. Conclusion

As such, we demonstrated a greater neuroprotective effect of the mixed extract compared to each isolated extract on behavior, AB pathology, oxidative stress tolerance and life expectancy. This clearly shows that the synergistic effect of the mixed extracts is not self-evident.

Claims (26)

A composition or kit of parts comprising D-pinitol, ursolic acid and one or more additional components selected from the group:
(i) docosahexaenoic acid (DHA),
(ii) ginkgo flavonoids, and
(iii) mixtures thereof. The composition or kit of parts according to claim 1 , wherein the ursolic acid is provided as a vegetable extract rich in ursolic acid and/or the D-pinitol is provided as a D-pinitol rich vegetable extract. The composition or kit of parts according to claim 2, wherein the ursolic acid-enriched vegetable extract is a hydroalcoholic extract of a ursolic acid-enriched vegetable product. The composition or kit of parts according to claim 3 , wherein the plant product rich in ursolic acid is selected from leaves, algae or fruit peels of plants of the family Lamiacea. 5. The composition or kit of parts according to any one of claims 2 to 4, wherein the vegetable extract rich in ursolic acid contains 5 to 90% ursolic acid. 6. The method according to any one of claims 2 to 5,
The vegetable extract rich in ursolic acid,
(i) extracting with hydroalcohol from leaves, algae or fruit peels of Lamiacea and plants; and
(ii) removing the activated carbon after treating the hydroalcoholic extract obtained in step (i) with activated carbon;
A composition or kit of parts obtained by a method comprising: The composition or kit of parts according to any one 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 any one of claims 1 to 7, wherein the weight ratio of D-pinitol and ursolic acid is about 63.3:7.5. 9. The composition or kit of parts according to any one of claims 1 to 8, wherein the DHA is provided as a fatty acid composition containing at least 25% DHA. 10. The method of claim 9,
Wherein the fatty acid composition is obtained from fish or microalgae, the composition or kit of parts. 11. The method according to any one of claims 1 to 10,
The composition or kit of parts, wherein the ginkgo flavonoid is provided as a Ginkgo biloba extract. 12. The method of claim 11, wherein the Ginkgo biloba extract contains 0.5% (w/w) to 15% (w/w) ginkgo flavonoids and less than 1% (w/w) terpene lactones. A composition or kit of parts containing The method according to claim 11 or 12, wherein the extract of Ginkgo biloba,
(i) aqueous extraction from Ginkgo biloba leaves; and
(ii) treating the aqueous extract obtained in step (i) with activated carbon and then removing the activated carbon;
A composition or kit of parts obtained by a method comprising: 14. The method according to any one of claims 1 to 13,
(i) when the composition or kit of parts contains D-pinitol, ursolic acid and DHA, the weight ratio of the components is about 63.3:7.5:40;
(ii) when the composition or kit of parts comprises D-pinitol, ursolic acid and ginkgo flavonoid, the weight ratio of the components is about 63.3:7.5:1;
(iii) when the composition or kit of parts contains D-pinitol, ursolic acid, DHA and ginkgo flavonoid, the weight ratio of the components is about 63.3:7.5:40:1;
composition or kit of parts. 15. The composition or kit of parts according to any one of claims 1 to 14, further comprising a carrier. 16. The composition of claim 15, wherein the concentration of carrier is between 20% and 90% (w/w). 17. A pharmaceutical product comprising a composition according to any one of claims 1 to 16 and a pharmaceutically active carrier. 17. A food or dietary supplement comprising a composition according to any one of claims 1 to 16 and a nutritionally acceptable carrier. A composition or kit of parts according to any one of claims 1 to 16, a pharmaceutical product according to claim 17 or a food or dietary supplement according to claim 18 for use in medicine. A composition or kit of parts according to any one of claims 1 to 16, a pharmaceutical product according to claim 17 or a food or dietary supplement according to claim 18 for use in at least one of the prevention and treatment of cognitive impairment. The composition, kit of parts, pharmaceutical product or food or dietary supplement of claim 20 , wherein the cognitive impairment is selected from the list consisting of mild cognitive impairment, Alzheimer's disease and Parkinson's disease. 22. The method of claim 20 or 21, wherein at least one component of the composition, kit of parts, pharmaceutical product or food or dietary supplement is administered separately from the remaining components of the composition, kit of parts, pharmaceutical product or food or dietary supplement. composition, kit of parts, pharmaceutical product or food or dietary supplement. 23. The method of any one of claims 20-22, wherein administration of the composition, component included in the kit of parts, pharmaceutical product, or food or dietary supplement comprises administering at least one of (i) to (iv) A composition, part part, pharmaceutical product or food or dietary supplement that:
(i) about 150 mg/day of a vegetable 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
(iv) about 200 mg/day of D-pinitol. 23. The method of any one of claims 20-22, wherein administration of the composition, component included in the kit of parts, pharmaceutical product, or food or dietary supplement comprises administering at least one of (i) to (iv) A composition, kit of parts, pharmaceutical product or food or dietary supplement that:
(i) about 2.5 mg/kg of a vegetable extract rich in ursolic acid;
(ii) about 8 mg/kg of a fatty acid composition rich in DHA;
(iii) about 8.33 mg/kg of Ginkgo biloba; and
(iv) about 3.33 mg/kg of D-pinitol. 25. The composition, kit of parts, pharmaceutical according to any one of claims 20 to 24, wherein the treatment comprises administering multiple doses of the composition, component included in the kit of parts, pharmaceutical product, food or dietary supplement for at least 1 month. product or food or dietary supplement. 26. A kit of parts for use according to any one of claims 20 to 25, wherein the parts of the kit of parts are combined prior to said administration.

Images