Combination of Rhodiola and Astragalus for the treatment of neurodegenerative diseases

Abstract

The present invention relates to a product containing an extract of Rhodiola and an extract of Astragalus as products to be combined for simultaneous administration, separately or staggered over time, in the treatment of a neurodegenerative disease, and in particular for the treatment of Alzheimer's disease and Parkinson's disease.

Claims

1. A method of treating or providing a neuroprotective effect against a neurodegenerative disease comprising administering a therapeutically effective amount of each of a dried extract of Rhodiola rosea and a dried extract of Astragalus membranaceus simultaneously, to an individual in need thereof, wherein the dried extract of Rhodiola rosea contains at least 1.5% by dry weight of rosavin with respect to the total weight of dried extract of Rhodiola rosea, and wherein the dried extract of Astragalus membranaceus comprises at least 0.2% by dry weight of astragalosides with respect to the total weight of dry extract of Astragalus membranaceus and at least 10% by dry weight of polysaccharides with respect to the total weight of dried extract of Astragalus membranaceus.

2. The method of claim 1, wherein the neurodegenerative disease is Alzheimer's disease.

3. The method of claim 1, wherein the neurodegenerative disease is Parkinson's disease.

4. The method of claim 1, comprising administering the dried extract of Rhodiola rosea at a dose of at least 50 mg/day.

5. The method of claim 1, wherein the dried extract of Rhodiola rosea contains at least 0.25% by dry weight of salidroside with respect to the total weight of dried extract of Rhodiola rosea.

6. The method of claim 1, comprising administering the dried extract of Astragalus membranaceus at a dose of 50 mg/day.

7. The method of claim 1, wherein the ratio by dry weight of the dried extract of Astragalus membranaceus relative to the dried extract of Rhodiola rosea is between 0.5 and 1.5.

8. The method of claim 1, further comprising administering tocopherol, selenium, silicon, zinc, magnesium, polyphenols other than those derived from the Rhodiola rosea and Astragalus membranaceus extracts, or mixtures thereof.

9. A method for elongating neurites of neurons comprising administering a therapeutically effective amount of each of an extract of Rhodiola rosea and an extract of Astragalus membranaceus simultaneously to an individual in need thereof, wherein the dried extract of Rhodiola roseacontains at least 1.5% by dry weight of rosavin with respect to the total weight of dried extract of Rhodiola rosea, and wherein the dried extract of Astragalus membranaceus comprises at least 0.2% by dry weight of astragalosides with respect to the total weight of dry extract of Astragalus membranaceus and at least 10% by dry weight of polysaccharides with respect to the total weight of dried extract of Astragalus membranaceus.

10. The method of claim 9, comprising administering the extract of Rhodiola rosea at a dose of at least 50 mg/day.

11. The method of claim 9, wherein the extract of Rhodiola rosea contains at least 0.25% by weight of salidroside with respect to the total weight of dry extract of Rhodiola rosea.

12. The method of claim 9, comprising administering the extract of Astragalus membranaceus at a dose of 50 mg/day.

Description

(1) Other features and advantages of the invention will appear in the following examples, given by way of illustration, with reference to the Figures:

(2) FIG. 1 presents four diagrams illustrating the cytotoxicity results of the extract of Rhodiola, of Astragalus, of the combination of extracts (“Mix”) and the product “ADN-Téloméractives”, after 48 hours of exposure. The results are expressed in a mean standard deviation +/− of 3 replicates;

(3) FIG. 2 presents four diagrams illustrating the curative effect of extract of Rhodiola, of Astragalus, of the combination of extracts and of the product “ADN-Téloméractives” on the length of neurites after 48 hours in the Alzheimer model. The results are expressed in a mean standard deviation +/− of 3 replicates;

(4) FIG. 3 comprises three photographs illustrating the effect of 250 μg/mL of Rhodiola on differentiated SH-SY5Y (cells) exposed in advance to 10 μM of Aftin-5 for 48 hours. 20× magnification, nuclei in blue, tubulin B3 in green;

(5) FIG. 4 comprises three photographs illustrating the effect of 250 μg/mL of Astragalus on differentiated SH-SY5Y (cells) exposed in advance to 10 μM of Aftin-5 for 48 hours. 20× magnification, nuclei in blue, tubulin B3 in green;

(6) FIG. 5 comprises three photographs illustrating the effect of 250 μg/mL of the combination of extracts of Rhodiola and Astragalus on differentiated SH-SY5Y (cells) exposed in advance to 10 μM of Aftin-5 for 48 hours. 20× magnification, nuclei in blue, tubulin B3 in green;

(7) FIG. 6 comprises three photographs illustrating the effect of 25.00 μg/mL of “ADN-Téloméractives” product on differentiated SH-SY5Y (cells) exposed in advance to 10 μM of Aftin-5 for 48 hours. 20× magnification, nuclei in blue, tubulin B3 in green;

(8) FIG. 7 is a diagram illustrating the effect of the product “ADN-Téloméractives” on the repair of DNA lesions (8oxoG, Etheno, Glycols, AbaS, CPD-64) in HepG2 cells;

(9) FIG. 8 is a diagram illustrating the effect of the genotoxic EMS on the repair of DNA lesions (8oxoG, Etheno, Glycols, AbaS, CPD-64) in HepG2 cells;

(10) FIG. 9 is a diagram illustrating the effect of pre-treatment and post-treatment with the product “ADN-Téloméractives” on repair of EMS-induced DNA lesions (8oxoG, Etheno, Glycols, AbaS, CPD-64) in HepG2 cells;

(11) FIG. 10 presents three diagrams illustrating the effect of a combination of extracts of Rhodiola and Astragalus not including astragalosides (Combination 1), of a combination of extracts of Rhodiola and Astragalus not including polysaccharides (Combination 2) and of the combination of extracts of Rhodiola and Astragalus described in Example 1 (Combination 4) on the length of neurites after 48 hours in the Alzheimer's model; and

(12) FIG. 11 shows two diagrams illustrating the effect of a combination of Rhodiola and Astragalus extracts not including rosavin (Combination 3) and of the combination of extracts of Rhodiola and Astragalus described in Example 1 (Combination 4) on neurite length after 48 hours in the Alzheimer's model.

Example 1: Preparation of Products for Use According to the Invention

(13) 1. Materials and Methods

(14) 1.1 Products

(15) The following products were used: A Rhodiola extract obtained by hydroalcoholic extraction from ground roots of Rhodiola rosea, the extraction solvent consisting of 70% by volume of water and 30% by volume of ethanol, in a weight ratio of solvent to root powder of the order of 8:1. This extract of Rhodiola comprises at least 3% by weight of rosavin and at least 1% by weight of salidroside, with respect to the total weight of dry extract of Rhodiola; A first extract of Astragalus obtained by hydroalcoholic extraction from ground roots of Astragalus membranaceus, the extraction solvent consisting of 15% by volume of water and 85% by volume of ethanol, in a weight ratio of solvent to root powder of the order of 125:1; A second Astragalus extract obtained by extraction with pure water from ground roots of Astragalus membranaceus, in a weight ratio of water to root powder of the order of 5:1; The “ADN-Téloméractives” product marketed by HBN, comprising the combination of an extract of Rhodiola and of an extract of Astragalus. The product also contains tocopherol, selenium in the form of yeasts, silicon, particularly in the form of marine diatoms, zinc, magnesium, an extract of Vitis vinifera as well as an extract of Polygonum cuspidatum containing 25% resveratrol. The product also contains various vitamins.
Preparation of the Combined Extract of Astragalus:

(16) The two above-mentioned Astragalus extracts are mixed so as to obtain a combined Astragalus extract comprising at least 16% by weight of polysaccharides and at least 0.8% by weight of a mixture of astragalosides I, II, III, IV, V, VI and VII, based on the total weight of dry extract of Astragalus.

(17) Preparation of the Combination of Extracts of Rhodiola and of Astragalus:

(18) The combination of an extract of Rhodiola and of an extract of Astragalus for use according to the invention is prepared by mixing the above-mentioned extract of Rhodiola with the combined extract of Astragalus in a weight ratio of 1:1.

(19) Aqueous solutions of extract of Rhodiola, of the combined extract of Astragalus, of the combination of extracts and of the product “ADN-Téloméractives” were freshly prepared with a range of 8 final concentrations (0.00125-0.0025-0.0050-0.0125-0.025-0.05-0.125-0.25 mg/mL).

(20) 1.2 Cell Culture and Differentiation

(21) SH-SY5Y (ATCC, CRL-2266) cells were cultured and transplanted according to the supplier's recommendations.

(22) All tests were performed in three technical replicates using 96-well microplates. After inoculation, SH-SY5Y cells were differentiated by exposure to retinoic acid (10 μM) for 4 days.

(23) 1.3 Cell Fixation and Staining

(24) After each test, the cells were fixed using a mixture of methanol and acetone at room temperature for 5 minutes, before staining the nuclei with Hoechst and immunostaining the B3 tubulin (secondary antibody conjugated to an Alexa 488 fluorochrome).

(25) 1.4 Cytotoxicity

(26) Cytotoxicity was evaluated using staining and counting of cell nuclei (cell counts were evaluated using MetaXpress (Molecular Devices)).

(27) 1.5 Statistical Analysis

(28) Statistical comparison of the results to the control conditions was performed using a t-test. The significance was expressed with the symbol “*” when p<0.05.

(29) 2. Results on the Cytotoxicity of Astragalus, Rhodiola, the Combination of Extracts and the Product “ADN-Téloméractives” (ADN-T)

(30) Cytotoxicity was evaluated on differentiated SH-SY5Y (cells) after 48 hours of exposure to Astragalus extract, to extract of Rhodiola, to the combination of extracts and to the product “ADN-Téloméractives”. None of the products showed a cytotoxic effect (see FIG. 1). Astragalus and Rhodiola induced an increase in viability compared to the control. The viability of SHSY-5Y (cells) exposed to the combination of extracts and the product “ADN-Téloméractives” is close to the viability of the control, regardless of the doses tested.

Example 2: Synergistic Effect of an Extract of Rhodiola and of an Extract of Astragalus for Use According to the Invention

(31) 1. Materials and Methods

(32) The products, cell culture and differentiation, cell fixation and staining, cytotoxicity and statistical analysis are as shown in Example 1.

(33) 1.1 Curative Test

(34) Models of neurodegenerative diseases can be established in the laboratory using differentiated neuronal cell lines. Using chemical inducers (Aftin-5, in this case), a model of Alzheimer's disease can be obtained with the human neuroblastoma cell line SH-SY5Y. The neuroprotective and curative properties of the combination of extracts are evaluated following neurotoxic aggression by counting neurons and measuring the length of neurites.

(35) The cells were exposed for 2 days with 10 μM of Aftin-5 alone. They were then treated for 48 hours with aqueous dilutions of Rhodiola, of Astragalus, of a combination of extracts, of “ADN-Téloméractives” product or simply water (control), in co-incubation with Aftin-5 (10 μM). After fixation and labeling, neurite outgrowth and cell counts were evaluated using MetaXpress (Molecular Devices).

(36) 1.2 Cellular Imaging and Image Analysis

(37) The images were obtained using an ImageXpress® Micro XLS (Molecular Devices) automated microscope with a 20× objective. Two dichroic filters were used simultaneously to specifically detect the different probes used. Four fields per well were recorded and analyzed individually.

(38) The images were analyzed using MetaXpress (Molecular Devices) software.

(39) 2. Results: Curative Effect

(40) The results are presented in FIG. 2.

(41) In this Figure, it may be observed that the Rhodiola and the Astragalus did not cure the neurodegenerative phenotype induced by 48 hours exposure to 10 μM d'Aftin-5. On the contrary, at all the tested doses, a length of neurites less than that presented by the control was observed.

(42) By contrast, it can be observed that the combination of extracts of Rhodiola and of Astragalus showed a dose-dependent increased curative effect against the induced Alzheimer's phenotype. The effect of the dose increased from 12.50 μg/mL to 250 μg/mL. At 250 μg/mL, the curative effect was significantly different from the control with a 119.1% increase in neurite length. A U-shaped dose effect was also observed with the product “ADN-Téloméractives” with a peak at 25.00 μg/mL for a neurite length at 125.9. This value was not significantly different from the control, but compared to the whole cell population, a statistical difference is shown by the t-test.

(43) It follows that Rhodiola alone and Astragalus alone do not enable the length of the neurites to be improved: on the contrary, it is deteriorated relative to the control.

(44) By contrast the combination of the extracts of Rhodiola and of Astragalus extracts enables, surprisingly, a maintenance and even an extension of the length of the neurites.

(45) It follows that the combination of extracts has a synergistic curative effect in a model of neurodegenerative diseases and more specifically, a model of Alzheimer's disease.

(46) FIG. 3 shows three photographs illustrating the effect of 250 μg/mL of Rhodiola on differentiated SH-SY5Y (cells) exposed in advance to 10 μM of Aftin-5 for 48 hours. It can be observed from this Figure that, compared to the control, cells exposed to Aftin-5 show a significant reduction in neurite length. The cells co-exposed to Aftin-5 and Rhodiola show a shorter neurite length than those exposed to Aftin-5 alone.

(47) FIG. 4 shows three photographs illustrating the effect of 250 μg/mL of Astragalus on differentiated SH-SY5Y (cells) exposed in advance to 10 μM of Aftin-5 for 48 hours. As in the previous Figure, it can be observed in this Figure that, compared to the control, the cells exposed to Aftin-5 show a significant reduction in neurite length. The cells co-exposed to Aftin-5 and Astragalus show a shorter neurite length than those exposed to Aftin-5 alone.

(48) FIG. 5 comprises three photographs illustrating the effect of 250 μg/mL of the combination of extracts of Rhodiola and of Astragalus on differentiated SH-SY5Y (cells) exposed in advance to 10 μM of Aftin-5 for 48 hours. As in the previous Figures, it is observed in this Figure that, compared to the control, the cells exposed to Aftin-5 show a significant reduction in length of the neurites. The cells co-exposed to Aftin-5 and to 250 μg/mL of the combination of extracts of Rhodiola and of Astragalus show better viability and greater neurite length than those exposed to Aftin-5 alone.

(49) FIG. 6 comprises three photographs illustrating the effect of 25.00 μg/mL of “ADN-Téloméractives” product on differentiated SH-SY5Y (cells) exposed in advance to 10 μM of Aftin-5 for 48 hours. As in the previous Figures, it is observed in this Figure that, compared to the control, the cells exposed to Aftin-5 show a significant reduction in length of the neurites. The cells co-exposed to Aftin-5 and to 25.00 μg/mL of “ADN-Téloméractives” product show better viability and greater length of neurites than those exposed to Aftin-5 alone.

Example 3: Neuroprotective Effect of an Extract of Rhodiola and of an Extract of Astragalus for Use According to the Invention

(50) 1. Materials and Methods

(51) The products, cell culture and differentiation, cell fixation and staining, cytotoxicity and statistical analysis are as shown in Example 1.

(52) 1.1 Neuroprotection Test

(53) The cells were exposed for 48 hours to aqueous dilutions of Rhodiola, Astragalus, a combination of extracts and the product “ADN-Téloméractives”, with 30 μM of Aftin-5 (a phenotypic inducer of Alzheimer's disease). After fixation and labeling, neurite outgrowth and cell counts were evaluated using MetaXpress (Molecular Devices).

(54) 2. Results: Neuroprotective Effect

(55) Rhodiola, Astragalus, the extract combination and the product “ADN-Téloméractives” showed a significant protective effect against the phenotype of Alzheimer's disease induced by Aftin-5 (30 μM). Both the Astragalus and the Rhodiola showed a neuroprotective effect at 1.25 and 2.50 μg/mL, increasing the length of neurites to 129.9 and 127.9 respectively. The mixture of extracts of Astragalus and of Rhodiola induced a neuroprotective effect at 1.25 μg/mL, with an increase in neurite length of 125.6% compared to the control. The product “ADN-Téloméractives” induced a neuroprotective effect at 2.50 μg/mL, the length of the neurites being 126.8% compared to the control.

Example 4: Effect of the Product “ADN-Téloméractives” on DNA Repair Systems

(56) 1. Materials and Methods

(57) 1.1 Solubilization of the Product “ADN-Téloméractives” (ADN-T)

(58) After milling “ADN-Téloméractives” product, an aqueous stock solution is prepared at 12.5 mg/mL. It is stirred at 37° C. for 1 hour. The residues are then removed by centrifugation at 1500 rpm for 5 minutes.

(59) 1.2 Model

(60) The model consists in studying the involvement of the DNA repair systems in the protective effect of the product “ADN-Téloméractives” against genotoxins, using an in vitro approach on HepG2 cells, brought into contact with the genotoxic agent EMS (ethyl methane sulfonate).

(61) 1.3 Experimental Design

(62) 1. Pretreatment of cells with the solution of “ADN-Téloméractives” product.

(63) Day 0: at 9:00 a.m., inoculation of the wells with the cells, then at 3:00 p.m., addition of “ADN-Téloméractives” product at 40 μg/mL.

(64) 2. Introduction of the Genotoxic Compound EMS

(65) Day 2: at 3:00 p.m. introduction of the EMS at two concentrations: 0.2 mM and 1 mM.

(66) 3. Putting the cells back in the presence of the “ADN-Téloméractives” product.

(67) Day 3: at 8:00 a.m., addition of “ADN-Téloméractives” at 40 μg/mL.

(68) 4. Harvesting and analysis

(69) Day 4: at 8:00 a.m., harvesting

(70) A control without “ADN-Téloméractives” product is conducted in parallel.

(71) TABLE-US-00001 TABLE 1 Lists of the conditions tested ADN-T pre-treatment Genotoxic treatment ADN-T post-treatment (μg/mL) (mM) (μg/mL) 0 0 0 0 0.2 0 0 1 0 40 0 40 40 0.2 40 40 1 40

(72) 1.4 Analyses

(73) The extracts are deposited on a chip functionalized by plasmids comprising series of specific DNA lesions: 8oxoG (8oxoG) ethenobases (Etheno) Glycols of thymine and cytosine (Glycols) abasic sites (AbaS) photoproducts (pyrimidine dimers and 6-4 photoproducts) (CPD-64)

(74) The DNA repair enzymes, contained in the extracts, excise the lesions (or the DNA fragment surrounding the lesions) and incorporate a fluorescent marker (dCTP-Cy3) during DNA resynthesis.

(75) The fluorescent signal is quantified using a scanner. It is proportional to the Excision/Resynthesis capacities of each extract with respect to each lesion.

(76) This test is used to characterize the Basic Excision Repair (BER) and Nucleotide Excision Repair (NER) systems.

(77) The extract concentration is 0.2 mg/mL.

(78) Each sample is tested on 2 chips. Each chip has 4 spots per lesion. The results are then normalized (NormalizeIt).

(79) The value of the Control plasmid (no lesion) is subtracted from the total intensity value obtained for each lesion.

(80) The results are given in Total Fluorescence Intensity for each lesion+/−Standard Deviation.

(81) 2. Results

(82) The results are presented in FIGS. 7, 8 and 9.

(83) In FIG. 7, it is observed that at the non-cytotoxic dose tested, the product “ADN-Téloméractives” had no effect on the DNA repair systems, except for a clear repair simulating effect on glycols.

(84) In FIG. 8, it is observed that at least at the highest concentration tested, EMS exclusively stimulates Basic Excision Repair (BER). The repair of photoproducts (CPD-64) managed by Nucleotide Excision Repair (NER) is not affected. This result is in favor of the formation by EMS of small alkylation or oxidation-type lesions, which would induce the activation of the enzymes managing them.

(85) This result conforms to what is expected.

(86) Lastly, in FIG. 9, it is observed that the pre-treatment of the cells with the product “ADN-Téloméractives” completely cancels the impact of EMS used at the lowest concentration, on the DNA repair systems.

(87) Still, induction of the DNA repair systems at the highest concentration of EMS persists. This induction is however lower than with EMS alone, except for glycol lesion. Stimulation of repair of the Glycols lesion evokes the effect of the product “ADN-Téloméractives”.

(88) Thus: The “ADN-Téloméractives” product alone, at a non-cytotoxic dose, has a significant action of stimulating glycol repair. Pretreatment with the product “ADN-Téloméractives” cancels the effects of the lowest dose of EMS and reduces the effects of the highest dose. At the lowest dose of EMS, the “ADN-Téloméractives” product, in sufficient quantity, inactivates EMS with respect to its genotoxic effects on DNA. At the highest dose of EMS, the “ADN-Téloméractives” product, probably in a limiting quantity, would not be sufficient to inactivate all of the EMS present, but would reduce its impact.

Example 5: Effect of Certain Active Ingredients of an Extract of Rhodiola and of an Extract of Astragalus for Use According to the Invention

(89) 1. Materials and methods

(90) 1.1 Products

(91) The following products were used: The first extract of Astragalus as described in Example 1 (obtained by hydroalcoholic extraction from ground roots of Astragalus membranaceus). This extract comprises at least 4% by weight of a mixture of astragalosides I, II, III, IV, V, VI and VII, with respect to the total weight of dry extract of Astragalus, but does not comprise polysaccharides; The second extract of Astragalus as described in Example 1 (obtained by extraction with pure water from ground roots of Astragalus membranaceus). This extract comprises at least 20% by weight of polysaccharides, with respect to the total weight of dry extract of Astragalus, but does not contain astragalosides; The Rhodiola extract as described in Example 1 (Rhodiola Extract 1), comprising at least 3% by weight of rosavin and at least 1% by weight of salidroside, with respect to the total weight of dry extract of Rhodiola; A Rhodiola extract comprising at least 3% by weight of salidroside (Rhodiola Extract 2), with respect to the total weight of dry extract of Rhodiola, but not comprising rosavin (supplied by Gonmisol); The combination of extracts of Rhodiola and of Astragalus described in Example 1, hereinafter referred to as combination 4, the extract of Rhodiola comprising at least 3% by weight of rosavin and at least 1% by weight of salidroside, with respect to the total weight of dry extract of Rhodiola, and the extract of Astragalus comprising at least 16% by weight of polysaccharides and at least 0.8% by weight of a mixture of astragalosides I, II, III, IV, V, VI and VII, with respect to the total weight of dry extract of Astragalus.
Preparation of Combinations of Extracts

(92) In addition to combination 4, the preparation of which is described in Example 1, three other combinations of extracts are prepared: Combination 1: This combination is prepared by mixing the second extract of Astragalus (not comprising astragalosides) and Rhodiola extract 1 in a weight ratio of 4:5; Combination 2: This combination is prepared by mixing the first extract of Astragalus (comprising no polysaccharides) and Rhodiola extract 1 in a weight ratio of 1:5; Combination 3: This combination is prepared by mixing the first Astragalus extract, the second Astragalus extract and the above-mentioned Rhodiola extract 2 not comprising rosavin in a weight ratio of 3:12:5.
Preparation of Aqueous Solutions from Combinations of Extracts

(93) For each combination, two aqueous solutions were freshly prepared to compare the effects of astragalosides, polysaccharides and rosavin, the concentrations being given in Tables 2 and 3 below.

(94) TABLE-US-00002 TABLE 2 Concentrations of the aqueous solutions tested for Test A Concentra- tion combi- Salidro- Polysac- Astra- Combi- nation Rosavin side charides galosides nation (mg/mL) (mg/mL) (mg/mL) (mg/mL) (mg/mL) 1 0.0225 0.0003750 0.0001250 0.0020000 — 2 0.0150 0.0003750 0.0001250 — 0.0001000 3 0.0167 — 0.0001250 0.0020000 0.0001000 4 0.0250 0.0003750 0.0001250 0.0020000 0.0001000

(95) TABLE-US-00003 TABLE 3 Concentrations of the aqueous solutions tested for Test B Concentra- tion combi- Salidro- Polysac- Astra- Combi- nation Rosavin side charides galosides nation (mg/mL) (mg/mL) (mg/mL) (mg/mL) (mg/mL) 1 0.0450 0.0007500 0.0002500 0.0040000 — 2 0.0300 0.0007500 0.0002500 — 0.0002000 3 0.0333 — 0.0002500 0.0040000 0.0002000 4 0.0500 0.0007500 0.0002500 0.0040000 0.0002000

(96) 1.2 Cell Culture and Differentiation

(97) SH-SY5Y cells were cultured and transplanted according to the supplier's recommendations.

(98) All tests were performed in three technical replicates using 96-well microplates. After inoculation, the SH-SY5Y cells were differentiated by exposure to staurosporin (25 nM in 1% fetal calf serum complete culture medium) for 3 days.

(99) 1.3 Cell Fixation and Staining

(100) After each test, the cells were fixed using a mixture of formaldehyde and methanol (marketed by Sigma-Aldrich under the name “Formalin 10%”) at room temperature for 10 minutes, followed by permeabilization in 0.5% Tween 20 (P2287 marketed by Sigma-Aldrich) for 30 minutes and finally staining the nuclei with Hoechst and indirect immunocytochemical labeling of ß3 tubulin to distinguish neurites (rabbit anti ß3 tubulin antibody (ab18207 marketed by Abcam) and rabbit anti-IgG secondary antibody conjugated to Alexa 488 fluorochrome (marketed by Ozyme)) for 1 hour.

(101) 1.4 Curative Test

(102) The cells were exposed for 2 days with 10 μM Aftin-5 alone. They were then treated for 48 hours with the aqueous solutions given in Table 2 or simply with water (control), co-incubating with Aftin-5 (10 μM). After fixation and labeling, neurite outgrowth was evaluated using MetaXpress (Molecular Devices).

(103) 1.5 Cellular Imaging and Image Analysis

(104) The images were obtained using an ImageXpress® Micro Confocal System (Molecular Devices) automated microscope with a 20× objective. Two dichroic filters were used simultaneously to specifically detect the different probes used. Four images per well were recorded and analyzed individually.

(105) The images were analyzed using MetaXpress (Molecular Devices) software.

(106) 2. Results on Neurite Lengthening

(107) The results for neurite lengthening are shown in FIG. 10 for combinations 1, 2 and 4, and in FIG. 11 for combinations 3 and 4.

(108) 2.1 Influence of the Combination of Astragalosides and Polysaccharides

(109) The results of Tests A and B show that combinations 1 and 2 enable lengthening of the neurites compared to the control. As a matter of fact, combination 1 enables neurite lengthening of about 16% in Test A and of about 26% in Test B, and combination 2 enables neurite lengthening of about 24% in Test A and of about 21% in Test B.

(110) However, lengthening of the neurites obtained in the case of combination 1 (not comprising astragalosides) or combination 2 (not comprising polysaccharides) is less compared with the lengthening of the neurites obtained in the case of combination 4. As a matter of fact, combination 4 enables lengthening of the neurites by approximately 32% in Test A and even by approximately 40% in Test B.

(111) Therefore, a combination of extracts comprising both astragalosides and polysaccharides makes it possible to obtain greater lengthening of the neurites than a combination of extracts comprising only one of these active ingredients.

(112) 2.2 Influence of Rosavin

(113) The results of Tests A and B show that combination 3, which does not contain rosavin, enables lengthening of the neurites by about 5% in Test A and by approximately 8% in Test B, compared to the control.

(114) However, the lengthening of the neurites obtained in the case of that combination 3 is appreciably less compared with the lengthening of the neurites obtained in the case of combination 4. As a matter of fact, combination 4 enables lengthening of the neurites by approximately 32% in Test A and even by approximately 40% in Test B.

(115) Therefore, a combination of extracts comprising rosavin provides much greater lengthening of the neurites than a combination of extracts without rosavin.