Therapeutic use of a Fraximus augustifolia extract

Abstract

The present invention relates to extracts from Fraxinus angustifolia samara, processes for providing such extracts, and methods and uses of the extracts obtained. In particular, the present invention relates to the use of such extracts in reversing obesity-related and/or metabolic syndrome-related gut microbiota dysbiosis treatment, treating or preventing hepatic steatosis, non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH), and modulating and/or adjusting gut microbiota.

Claims

1. A method for: (a) reversing metabolic syndrome-related gut microbiota dysbiosis; (b) treating hepatic steatosis, non-alcoholic fatty liver disease (NAFLD) and/or non-alcoholic steatohepatitis (NASH); (c) treating gut microbiota dysbiosis-induced cardiovascular diseases and/or cardiometabolic diseases; and/or (d) delaying the progression of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH); comprising the administration of a therapeutically effective amount of a Fraxinus angustifolia samara extract to a subject in need thereof, wherein the extract comprises: (i) from about 1% to about 16% by weight of nuzhenide; (ii) from about 1% to about 18% by weight of GL3; (iii) oleoside methyl ester; (iv) excelside B; (v) GL5; and (vi) salidroside.

2. The method according to claim 1, wherein a disease or disorder to be reversed, treated, or delayed is selected from the group consisting of: metabolic syndrome-related gut microbiota dysbiosis; and/or hepatic steatosis, non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH).

3. A method of modulating or adjusting gut microbiota for treating non-alcoholic fatty liver disease (NAFLD) and/or delaying the progression of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) comprising the administration of an effective amount of a Fraxinus angustifolia samara extract to a subject in need thereof, wherein the extract comprises: (i) from about 1% to about 16% by weight of nuzhenide; (ii) from about 1% to about 18% by weight of GL3; (iii) oleoside methyl ester; (iv) excel side B; (v) GL5; and (vi) salidroside.

4. The method according to claim 3, wherein the modulating or adjusting increases bacterial groups selected from the genus consisting of Betaproteobacteria and Enterorhabdus.

5. The method according to claim 3, wherein the modulating or adjusting increases bacterial groups selected from the genus consisting of Prevotellaceae, Flavoifractor, Clostridium IV and Butyricicoccus.

6. The method according to claim 3, wherein the modulating or adjusting increases bacterial groups selected from the families comprising Coriobacteriaceae, Lactobacillaceae and Rikenellaceae.

7. The method according to claim 1, wherein the extract comprises: (i) from about 1% to about 15% by weight of nuzhenide; (ii) from about 1% to about 17% by weight of GL3; (iii) from about 0.5% to about 1% by weight of oleoside methyl ester; (iv) from about 0.03% to about 0.12% by weight of excelside B; (v) from about 0.1% to about 1.7% by weight of GL5; and (vi) from about 0.08% to about 0.7% by weight of salidroside.

8. The method according to claim 1, wherein the extract comprises about 10% by weight nuzhenide and about 10% by weight GL3.

9. The method according to claim 1, wherein the extract is a hydro-ethanolic extract.

10. The method according to claim 9, wherein the hydro-ethanolic extract is obtained using a solvent containing from about 30% to about 75% ethanol.

11. The method according to claim 1, wherein the extract is administered in the form of: (a) a pharmaceutical composition comprising the Fraxinus angustifolia extract and optionally a pharmaceutically acceptable excipient; or (b) a food composition comprising the Fraxinus angustifolia extract and optionally a food acceptable ingredient.

12. The method according to claim 11, wherein the composition is for oral administration.

13. The method according to claim 1, wherein the method is performed on a human subject.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 depicts: (A) body weight, (B) body weight gain of mice during the 12 weeks of consumption of the high fat diet (60%) with or without Fraxinus angustifolia extract at 200 mg/kg body weight, and (C) body composition of mice after the 12-week treatment.

(2) * indicates the result is statistically different from control, p<0.05.

(3) FIG. 2 depicts: (A) Blood glucose before and after 15, 30, 60, 90 and 120 minutes after oral glucose administration by oral gavage and (B) respective Area under the curve (AUC) values of mice at the end of the 12-week consumption of the high fat diet (60%) with or without Fraxinus angustifolia extract at 200 mg/kg body weight.

(4) * indicates the result is statistically different from control, p<0.05.

(5) FIG. 3 depicts: (A) liver section prepared from frozen liver and stained with oil red O of control and Fraxinus angustifolia treated mice (magnification ×20); and (B) fat percentage by area; Oil red O-stained slides were analyzed with ImageJ analysis software to obtain a quantitative histologic measurement of steatosis; a histogram of pixel intensity was generated from the image, the area was measured and the results were expressed as fat percentage by area. Data are represented by box plot showing median, first quartile, third quartile, minimum and maximum.

(6) ** indicates the result is statistically different from control, p=0.004 (Mann-Whitney test)

(7) FIG. 4 depicts relative proportion of taxonomic groups at the class level showing individual study samples for each sample type per group: High fat diet treated mice (HFD60) at 1 month (_T1) or 3 months (_T3) or High fat diet and Fraxinus angustifolia extract treated mice (HFD60+F. angust) at 1 month (_T1) or 3 months (_T3).

(8) FIG. 5 depicts relative proportion of taxonomic groups at the class level showing the average for each sample type per group: High fat diet treated mice (HFD60) at 1 month (_T1) or 3 months (_T3) or High fat diet and Fraxinus angustifolia extract treated mice (HFD60+F. angust) at 1 month (_T1) or 3 months (_T3).

(9) FIG. 6 depicts relative proportion of taxonomic groups at the family level showing individual study samples for each sample type per group: High fat diet treated mice (HFD60) at 1 month (_T1) or 3 months (_T3) or High fat diet and Fraxinus angustifolia extract treated mice (HFD60+F. angust) at 1 month (_T1) or 3 months (_T3).

(10) FIG. 7 depicts relative proportion of taxonomic groups at the family level showing the average for each sample type per group: High fat diet treated mice (HFD60) at 1 month (_T1) or 3 months (_T3) or High fat diet and Fraxinus angustifolia extract treated mice (HFD60+F. angust) at 1 month (_T1) or 3 months (_T3).

(11) FIGS. 8 and 9 depict Principal Coordinate Analysis (PCoA) to compare samples from the different groups of mice (high fat diet treated mice (HFD60) at 1 month (_T1) or 3 months (_T3) or High fat diet and Fraxinus angustifolia extract treated mice (HFD60+F. angust) at 1 month (_T1) or 3 months (_T3)) based on the Generalized UniFrac distance metrics. Both PCoA plots with alpha values of 0 and 1 are shown.

(12) FIG. 10 depicts comparison at 3 months between mice fed a high fat diet without supplementation and mice fed a high fat diet with supplementation with Fraxinus angustifolia extract. The linear discriminant analysis effect size was determined using default values (alpha value of 0.5 for both the factorial Kruskal-Wallis test among classes and the pairwise Wilcoxon test between subclasses, threshold of 2.0 for the logarithmic LDA score for discriminative features) and the strategy for multi-class analysis set to ‘all-against-all’. (B) LEfSe cladogram from the LDS effect size data were generated with Bacteria as the tree root with six genus maximum taxonomic levels. The highlights (green or red) represent enrichment of the indicated taxonomic groups in the corresponding group.

(13) FIG. 11 depicts comparison at 3 months between mice fed a high fat diet without supplementation and mice fed a high fat diet with supplementation with Fraxinus angustifolia extract. LEfSe cladogram from the LDS effect size data were generated with Bacteria as the tree root with six genus maximum taxonomic levels. The highlights (green or red) represent enrichment of the indicated taxonomic groups in the corresponding group.

(14) FIGS. 12, 13 and 14 depict regression analyses of Random Forest Identified Family taxonomic groups and the O red oil liver stain percentage as steatosis severity.

(15) The present invention will be further described by reference to the following, non-limiting examples.

EXAMPLES

Example 1—Extraction of Fraxinus angustifolia with Water

(16) A total of 2.5 kg of the samara of F. angustifolia were dried in air and then ground into coarse powder with a particle size approximately 1-2 mm. The coarse powder was soaked in water in a percolator at 80-90° C. for 5 hours and the water extract was drained from the percolator. The extraction process was repeated three times. All the water extracts were combined together and concentrated in a rotary vacuum evaporator. After water was evaporated, a total of 550 grams of dried powdered extract was obtained. The HPLC analysis indicates that this powdered extract contained two major secoiridoids, 11.4% (weight/weight) of nuzhenide and 6.2% of GB. The composition also contained 0.19% oleoside-1 1-methyl ester, 0.41% excelside B, 0.63% GI5, 0.2% salidroside, together with some minor secoiridoids including, ligstroside, oleoside dimethyl ester, and excelside A.

Example 2—Extraction of Fraxinus angustifolia with Water, Water-EtOH, and EtOH

(17) 5 samples were prepared and each sample contained 5 grams of F. angustifolia samara. Each sample was milled into powder and was subjected to solvent extraction with 200 niL of water, 25% EtOH/75% water, 50% EtOH/50% water, 75% EtOH/25% water, and EtOH, respectively. After extraction for 24 hours at room temperature (22-24° C.), the solvents were evaporated and the residual solids were analyzed by HPLC. The secoiridoid contents and salidroside are listed in Table 1.

(18) TABLE-US-00001 TABLE 1 Major secoiridoid contents and salidroside using different solvents (results expressed as percent by weight) Compounds EtOH 75% EtOH 50% EtOH 25% EtOH water Nuzhenide 9.05 15.04 15.43 14.10 1.50 GI 3 9.20 14.77 17.06 9.18 1.14 Oleoside dimethyl 0.57 0.91 0.78 0.74 0.96 ester Excelside B 0.06 0.09 0.10 0.12 0.03 GI 5 0.91 1.45 1.70 0.83 0.10 Salidroside 0.08 0.17 0.16 0.18 0.74

Example 3—Isolation of Secoiridoids from Fraxinus angustifolia

(19) 3.5 L of methanol were added and mixed with 500 grams of powdered extract obtained from the procedure shown in Example 1, for 3 hours at room temperature. The methanol solution was separated from the powder by a filtration process. The same process was repeated once and the two methanol extracts were combined and concentrated under reduced pressure to yield a total of 54 grams of dried methanol extract. The methanol extract was re-dissolved in water and filtered to remove non-water soluble substances. The filtrate was further subjected to reverse-phase column chromatographic separation over C-18 resin washed with water and gradient MeOH-water solvent system from 10% MeOH in water to 100% MeOH. A total of 7 fractions were collected. Each fraction eluted from column was evaporated under vacuum and combined by HPLC analysis. Fractions 2, 3 and 7 were loaded on a chromatographic column filled with silica gel resin and eluted with chloroform-methanol system started from CHCl.sub.3, 10% MeOH/CHCl.sub.3, 20% MeOH/CHCl.sub.3, to 100% MeOH. Fractions collected from silica gel column were compared by HPLC analysis and each separated eluate was repeatedly subjected to column chromatographies over MCI GEL CHP-20P and/or Sephadex LH-20 resins and eluted with water-methanol system until a single pure compound was obtained. The compounds excelside A, excelside B, nuzhenide, GI3, GI5, ligstroside, oleoside dimethyl ester, oleoside-1,1-methyl ester, and salidroside were identified. All the chemical structures were elucidated by spectroscopic methods.

Example 4—Testing the Effect of Fraxinus angustifolia Extract on Liver Steatosis in Mice

(20) 9-week-old adult male C57BL/6 mice were purchased from Charles River (Charles River Laboratories, L'Arbresle, Rhône, France) and housed at a constant room temperature and humidity and maintained in a 12/12h light/dark cycle in SPF conditions. They were fed with a high-fat diet (HFD) with 60% energy from fat obtained by SAFE (Scientific Animal Food & Engineering, Augy, France) for 12 weeks and water was given ad libitum. Tables 2 and 3 give the list of ingredients and the nutritional values of the HFD respectively.

(21) TABLE-US-00002 TABLE 2 List of ingredients of the Purified Diet 260HF diet from SAFE (Augy, France) Purified Diet 260HF Quantity (g) Casein 22.800 DL-methionine 0.200 Maldodextrin 17.015 Sucrose 16.633 Anhydrous butter 33.350 Soybean oil 2.500 Minerals premix AIN93G-mx 4.550 Sodium bicarbonate 1.050 Potassium citrate 0.400 Vitamins premix AIN93G-vx 1.300 Choline bitartrate 0.200 Antioxidant 0.002 Total 100

(22) TABLE-US-00003 TABLE 3 Nutritional values of the Purified Diet 260HF diet from SAFE (Augy, France) Total energy (kcal/kg) 5283 Energy from protein in  776 (14.7%) kcal/kg (%) Energy from fat in kcal/kg 3222 (61%) (%) Energy from carbohydrates 1285 (24.3%)

(23) In the treatment group, the Fraxinus angustifolia (Vahl) extract was directly mixed in the diet and thus administered through oral route at 200 mg/kg/day, which represents a human equivalent dose of 1 g/day according to the formula from FDA (2005): Human equivalent dose HED (mg/kg)=animal dose in mg/kg×(animal weight in kg/human weight in kg). The dried extract of Fraxinus angustifolia samara was obtained by extraction with 30% (v/v) ethanol in water as described herein. The extract can preferably contain approximately 10% (% w/w) of nuzhenide and GL3 based on the total dry weight of the herbal extract. The effect of Fraxinus angustifolia (Vahl) extract consumption was analysed by comparing the different parameters in rats consuming both the HFD and the extract (F. angustifolia group) in comparison to rats consuming the HFD alone (control group).

(24) Body weight and body weight gain was followed during the 12 weeks and body composition (percentage of fat mass, lean mass and water) was evaluated by NMR at the end of treatment. An oral glucose tolerance test (OGTT) was done after the 12 weeks of treatment by administrating 2 g per kg body weight of glucose in fasted mice and by following glycaemia during the 2 hours following glucose administration.

(25) At sacrifice, liver was carefully removed, weighted and conditioned for both histological analyses. For the detection of lipid deposition in liver, liver section were prepared from frozen liver and stained with oil red O as previously reported (Fowler, S. D., Greenspan, P., O. J. Histochem Cytochem, 33, 833-836 (1985)). Oil red O-stained slides were analyzed with ImageJ analysis software (National Institute of Mental Health, Bethesda, Md., USA) to obtain a quantitative histologic measurement of steatosis. Five random images at ×20 magnification for each liver biopsy were taken to ensure a representative sample for each specimen. A histogram of pixel intensity was generated from the image, the area was measured and the results were expressed as fat percentage by area.

(26) As shown in FIG. 1, the 12-week consumption of a high fat diet induced a strong body weight gain that was counteracted by the simultaneous consumption of the Fraxinus angustifolia extract. Particularly, the Fraxinus angustifolia extract was able to reduce fat mass in mice fed a HFD during 12 weeks.

(27) As shown in FIG. 2, consumption of the Fraxinus angustifolia extract was also able to significantly reduce glucose intolerance in mice fed a HFD as shown by the significantly reduced blood glucose concentration 30 and 60 minutes after the glycemic load (p<0.05) and by the significant reduction of the area under the (glycaemia versus time) curve (AUC) (p=0.07).

(28) As shown in FIG. 3, the 12-week consumption of a high fat diet induced a high level of fat deposit into the liver, i.e steatosis in the control group (35.2%), which is classical with this type of diet as a model of diet induced obesity, diabetes and liver steatosis (see: Takahashi Y, Soejima Y, Fukusato T, World J Gastroenterol, 18(19), 2300-2308 (2012); and Zhou, Y. and Xie, L., Am J Digest Dis, 2(1), 60-67 (2015)). The 12-week treatment with the Fraxinus angustifolia extract was able to significantly reduce the severity of steatosis (p=0.004), as only 22.8 percent of fat was found into the liver of mice treated with the extract, which corresponds to a reduction of 35% of steatosis. According to the World Gastroenterology Organisation histological scoring system (2012) and the classification from Kleiner and Brunt (Kleiner, D. E. and Brunt, E. M., Semin Liver Dis, 32, 3-13 (2012)), which classify the severity of steatosis according to fat content (Grade 0: <5%, Grade 1: 5-33%, Grade 2: 34-66%, Grade 3: >67%), the treatment with the Fraxinus angustifolia warranted the reduction of steatosis severity from grade 2 to grade 1.

Example 5—Testing the Effect of Fraxinus angustifolia Extract on Gut Microbiota Dysbosis in Mice

(29) In order to evaluate gut microbiota modification induced by Fraxinus angustifolia extract consumption in mice fed the high fat diet, a 16S rDNA metagenomics study was performed on murine fecal samples at the beginning (4 weeks) and after 12 weeks of HFD consumption with or without the Fraxinus angustifolia extract (10 mice par groups, total number of 40 mice). Bacterial populations contained in the samples were determined using next generation high throughput sequencing of variable regions (V3-V4) of the 16S rDNA bacterial gene.

(30) The metagenomics workflow is used to classify organisms from a metagenomic sample by amplifying specific regions in the 16S ribosomal RNA gene. This metagenomics workflow is exclusive to bacteria. The main output is a classification of reads at several taxonomic levels: phylum, class, order, family and genus. The microbial population present in the samples has been determined using next generation high throughput sequencing of variable regions of the 16S rRNA bacterial gene. The workflow included the following steps:

(31) (1) Library Construction and Sequencing

(32) PCR amplification was performed using 16S universal primers targeting the V3-V4 region of the bacterial 16S ribosomal gene. The joint pair length was set to encompass 476 base pairs amplicon thanks to 2×300 paired-end MiSeq kit V3. For each sample, a sequencing library was generated by addition of sequencing adapters. The detection of the sequencing fragments was performed using MiSeq Illumina® technology.

(33) (2) Bioinformatics Pipeline

(34) The targeted metagenomic sequences from microbiota were analysed using the following bioinformatics pipeline; briefly, after demultiplexing of the bar coded Illumina paired reads, single read sequences were cleaned and paired for each sample independently into longer fragments. After quality-filtering and alignment against a 16S reference database, a clustering into operational taxonomic units (OTU) with a 97% identity threshold, and a taxonomic assignment were performed in order to determine community profiles.

(35) Based on these results, graphical representations were made of the relative proportion of taxonomic groups (phylum, class, order, family, and genus) present in 1) individual study samples and 2) the average for each sample type/group.

(36) As shown in FIGS. 4, 5, 6 and 7, the gut microbiota profiles were similar at the beginning of high fat diet consumption (T1) although some minor differences could be seen at the class and more intensively at the family levels in mice that have consumed the Fraxinus angustifolia extract in comparison to mice that have not consumed the extract concomitantly with the HFD.

(37) Principal Coordinate Analysis (PCoA) was performed to compare samples based on the Generalized UniFrac distance metrics (Lozupone C, Lladser M E, Knights D, Stombaugh J, Knight R (2011) UniFrac: an effective distance metric for microbial community comparison. ISME J. 5(2): 169-172) in order to illustrate the differences into groups of mice.

(38) As shown in FIGS. 8 and 9, although the gut microbiota composition of mice were similar at the beginning of high fat diet consumption (T1) as demonstrated by the stackable profiles of the individuals' distribution, it could be seen that after 3 months of treatment with the Fraxinus angustifolia extract, treated individuals could be differentiated from untreated individuals according to their gut microbiota composition.

(39) The Linear Discriminant Analysis (LDA) Effect Size (LEfSe) (Segata, N. et al., Genome Biol, 12(6), R60 (2011)) method was then used to analyze the high-dimensional class comparisons of the metagenomics data. LefSe is an algorithm for high-dimensional biomarker discovery and explanation that can identify taxonomic groups characterizing the differences between two or more biological conditions. It emphasizes both statistical significance and biological relevance, allowing researchers to identify differentially abundant features that are also consistent with biologically meaningful categories (subclasses). LEfSe first robustly identifies features that are statistically different among biological classes. It then performs additional tests to assess whether these differences are consistent with respect to expected biological behavior. The linear discriminant analysis effect size was determined using default values (alpha value of 0.5 for both the factorial Kruskal-Wallis test among classes and the pairwise Wilcoxon test between subclasses, threshold of 2.0 for the logarithmic LDA score for discriminative features) and the strategy for multi-class analysis set to ‘all-against-all’.

(40) As shown in FIGS. 10 and 11, LefSe analysis revealed that, at the genus or OTU levels, there was enrichment of different taxonomic groups (Burkholderiales, Sutterellacae, Parasutterella, Betaproteobacteria, Enterorhabdus and other OTUs) in mice treated with the Fraxinus angustifolia extract in comparison to untreated mice fed a HFD. Conversely, Prevotellaceae, Flavonifractor, Clostridium IV, Butyricicoccus and other taxonomic groups were less represented in mice treated with the Fraxinus angustifolia extract in comparison to untreated mice fed a HFD. These results highlight the modification of gut microbiota induced by the Fraxinus angustifolia extract supplementation.

(41) Correlation between microbiome analysis and steatosis severity was analysed by using the Random Forest Analysis methodology (Touw, W. G. et al., Brief Bioinform, 14(3), 315-26 (2013)). As shown in FIGS. 12, 13 and 14, the relative abundance of several taxonomic groups at the family or the genus level respectively are correlated with the steatosis severity in mice fed a high fat diet, clearly showing that Fraxinus angustifolia extract was able to modify the gut microbiota and particularly the relative abundance of some families or genus (Coriobacteriaceae, Lactobacillaceae, Rikenellaceae) and that these modifications could reduce the development of steatosis. The results of the analysis of the correlation between abundance of taxonomic groups (by family and genus) and steatosis severity is shown in Tables 4 and 5 below.

(42) TABLE-US-00004 TABLE 4 Statistical analysis for the correlation between abundance of taxonomic groups by Family and steatosis severity. Random Forest (increased mean P Taxonomic Classification square error) Spearman r (two-tailed) Family Actinobacteria|Actinobacteria| 7.98 −0.47 0.05 Coriobacteriales|Coriobacteriaceae Family Fimicutes|Bacilli|Lactobacillales| 2.98 −0.53 0.03 Lactobacillaceae Family Bacteroidetes|Bacteroidia| 4.02 −0.40 0.11 Bacteroidales|Rikenellaceae Family Firmicutes|Clostridia|unclassified 1.04 0.49 0.05 |unclassified

(43) TABLE-US-00005 TABLE 5 Statistical analysis for the correlation between abundance of taxonomic groups by Genus and steatosis severity. Random Forest (increased mean P Taxonomic Classification square error) Spearman r (two-tailed) Genus Actinobacteria|Actinobacteria| 9.33 −0.51 0.04 Coriobacteriales|Coriobacteriaceae Genus Fimicutes|Bacilli|Lactobacillales| 0.88 −0.53 0.03 Lactobacillaceae Genus Bacteroidetes|Bacteroidia| 2.73 −0.40 0.12 Bacteroidales|Rikenellaceae Genus Firmicutes|Clostridia|unclassified| 0.14 0.49 0.04 unclassified|unclassified Genus Firmicutes|Clostridia|Clostridiales| 1.84 0.51 0.04 Ruminococcaceae|Butyricicoccus