Method to prepare Hirsutella sinensis polysaccharides possessing protective activities on fatty liver disease

Abstract

The present invention provides a method to prepare polysaccharides from Hirsutella sinensis. The prepared polysaccharides can reduce liver size, weight and the number and size of liver lipid vacuoles, as well as serum triglycerides and serum aspartate aminotransferase levels in humans and animals. The prepared polysaccharides can therefore be used to prevent and treat fatty liver disease.

Claims

1. A method for treating fatty liver disease, comprising administering an effective amount of a polysaccharide extracted from Hirsutella sinensis to a subject having fatty liver disease, wherein the polysaccharide is isolated from a water extract of a H. sinensis mycelium and contains at least mannose, galactose, and glucose wherein a weight ratio of mannose, galactose, and glucose is 50:23:12 to 51:24:13.

2. The method of claim 1, wherein the polysaccharide further contains fucose, rhamnose, arabinose, glucosamine, and galactosamine.

3. The method of claim 2, wherein a weight ratio of the fucose, rhamnose, arabinose, glucosamine, galactose, glucose, mannose, and galactosamine in the polysaccharide is 3:3:1:4:23:12:50:0.2 to 4:4:2:5:24:13:51:0.6.

4. The method of claim 1, wherein the polysaccharide has a molecular weight ranging from 15,776 Da to 1,231,969 Da, and a polydispersity index (Mw/Mn) of 7.475.

5. The method of claim 1, wherein an average molecular weight of the polysaccharide is 312 kDa.

6. The method of claim 1, wherein the polysaccharide reduces liver size, liver weight, the size and number of liver lipid vacuoles, serum triglycerides and serum aspartate aminotransferase levels of the subject.

7. The method of claim 1, wherein the effective amount of the polysaccharide is from 0.001 mg/kg to 1 g/kg.

8. The method of claim 1, wherein the effective amount of the polysaccharide is 0.0646 g per kilogram of body weight.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The nature of the present invention will be apparent to those skilled in the art by reading the following detailed description of the preferred embodiments, with reference to the attached drawings:

(2) FIG. 1 shows a simplified flowchart for the isolation of the H. sinensis water extracts and polysaccharide sub-fractions described in the present invention.

(3) FIG. 2 shows the monosaccharide analysis of H. sinensis polysaccharide sub-fraction CS-1. The analysis was performed using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD).

(4) FIG. 3 shows the gel permeation chromatogram of H. sinensis polysaccharide sub-fraction CS-1.

(5) FIG. 4 shows the graph of weight fraction (WF)/dLog molecular weight (MW) vs. log molecular weight (MW) for H. sinensis polysaccharide sub-fraction CS-1.

(6) FIG. 5 shows the graph of cumulative weight fraction vs. log molecular weight for H. sinensis polysaccharide sub-fraction CS-1.

(7) FIGS. 6A-6E show the effects of H. sinensis polysaccharide sub-fractions on fatty liver disease in mice. Male C57BL/6 mice (n=10 per group) are fed with either standard chow (13.5% of energy from fat) or high-fat diet (HFD, 60% of energy from fat). The mice are supplemented with 10 μL of polysaccharide fraction (CS-1, CS-2, CS-3, or CS-4) or double-distilled water for 3 months by intragastric gavage. FIG. 6A shows the mice are sacrificed and livers are photographed; scale bars: 5 mm FIG. 6B shows the livers of mice are weighted. FIG. 6C shows the Liver tissue slides are prepared and stained with hematoxylin and eosin (H&E) to assess histological features; scale bars: 20 μm. FIG. 6D shows the determined serum TG level. FIG. 6E shows the determined serum AST levels. The results show that CS-1, CS-2 and CS-3 reduce signs of fatty liver disease in HFD mice compared to control HFD mice (***p<0.001, one-way ANOVA; ns, non-significant). CS-4 improves liver weight, vacuolation status, and serum TG levels compared to control HFD mice, but this sub-fraction produces non-significant results on serum AST levels. The results shown represent means±standard error of the mean (SEM).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

(8) In the following detailed description of the embodiments of the present invention, reference is made to the accompanying drawings, which are shown to illustrate the specific embodiments in which the present disclosure may be practiced. These embodiments are provided to enable those skilled in the art to practice the present disclosure. It is understood that other embodiments may be used and that changes can be made to the embodiments without departing from the scope of the present invention. The following description is therefore not to be considered as limiting the scope of the present invention.

Definition

(9) The “effective dosage” or “effective amount” described in the present disclosure represents the dosage of H. sinensis polysaccharide sub-fraction that can reduce signs of fatty liver disease in animals and humans. The appropriate effective dosage may vary depending on the organism or individual treated but can be determined experimentally using various techniques, including a dose escalation study.

(10) The data shown in the present disclosure represent approximated experimental values that can vary within a range of ±20%, preferably ±10%, and most preferably ±5%.

(11) The present invention provides H. sinensis polysaccharide sub-fractions which are able to prevent or treat fatty liver disease, and the embodiments below show that the H. sinensis polysaccharides of the present invention reduce liver weight, vacuolation status, serum TG and serum AST levels of a subject. Generally, the polysaccharides of the present invention can be given to mammals and humans at a dose of 0.001-1,000 mg/kg of body weight per day. The details of the invention are given below.

(12) Characterization of the H. sinensis polysaccharide of the present invention is presented first, followed by experimentations showing that the isolated H. sinensis polysaccharide sub-fractions reduce signs of fatty liver disease.

Example 1

Preparation of H. sinensis Water Extracts and Polysaccharide Sub-Fractions

(13) In the present invention, the polysaccharide sub-fractions isolated from H. sinensis can effectively reduce liver weight, liver vacuolation, serum TG and AST levels. The polysaccharide sub-fractions of the present invention can be added to the diet as a drink, daily supplement, or food, without requiring significant lifestyle change, or without incurring in toxicity or other unfavorable health conditions.

(14) 1.1 Preparation of H. sinensis Water Extracts

(15) As shown in FIG. 1, a water extract is prepared by mixing 500 g of H. sinensis mycelium obtained from Chang Gung Biotechnology (Taipei, Taiwan) into 10 liters of distilled water using a 20 liter-stirred tank reactor. The 5% (w/v) mixture is agitated at a speed of 150 revolutions per minute (RPM) for 30 min at 121° C. The mixture is centrifuged to remove insoluble material and to obtain a supernatant (water extract of H. sinensis). The supernatant is concentrated to a final volume of 2.5 liters using a vacuum concentrator. The concentrated supernatant is sterilized at high temperature and pressure for 20 min in an autoclave to obtain a 20% (w/v) concentrated H. sinensis water extract labeled as W1 (FIG. 1).

(16) 1.2 Preparation of H. sinensis Crude Polysaccharide Extract

(17) Referring to FIG. 1, 120 mL of the W1 20% (w/v) concentrated H. sinensis water extract (which contains 2.09 g of total water-soluble carbohydrates; see Table 1) is mixed with 5 volumes (600 mL) of 95% ethanol and incubated at 4° C. for 16 hours to obtain a precipitate of crude polysaccharide extract. The mixture is centrifuged to isolate the precipitate (pellet). The supernatant is removed and 120 mL of 70% ice-cold ethanol is used to wash and resuspend the precipitate (pellet) and obtain a mixture. The mixture is centrifuged to obtain a supernatant and a pellet. The supernatants from three such washing-resuspension-centrifugation steps are combined to give a supernatant with a volume of 1,040 mL (labeled as sub-fraction CS-4, total water-soluble carbohydrates of 0.83 g; see Table 1). The crude polysaccharide extract is dissolved into 1,000 mL of distilled water and concentrated to a final volume of 700 mL using the vacuum concentrator in order to remove residual ethanol. Finally, distilled water is added to obtain a H. sinensis crude polysaccharide extract with a final volume of 2,400 mL (labeled as W2, total water-soluble polysaccharide of 1.26 g; see Tables 1 and 2).

(18) 1.3 Fractionation of H. sinensis Crude Polysaccharide Extract

(19) 2,400 mL of H. sinensis crude polysaccharide extract is placed into a beaker and incubated at 50° C. in a water bath. The extract is fractionated by using TFF system (KrosFlo, Spectrum Laboratories) with a 0.2-μm hollow fiber membrane (1,500 cm.sup.2, PES). The trans-membrane pressure (TMP) is set at 15-16 psi. 600 mL of distilled water is added into the retentate during filtration when the volume of the retentate ranges from 800 to 1,000 mL. Addition of water is repeated two times (a total of 1,800 mL distilled water is added to the retentate). A 1,250 mL retentate (labeled as CS-1-1, total water-soluble polysaccharide of 0.24 g) and 3,600 mL of filtrate are obtained this way.

(20) The above-mentioned 3,600 mL of 0.2-μm filtrate is placed into a beaker and incubated at 50° C. in a water bath. The 3,600 mL of filtrate is fractionated by using TFF as above with a 300-kDa cassette membrane (50 cm.sup.2, PES). The TMP is set between 18-20 psi. 600 mL of distilled water is added into the retentate during filtration when the volume of the retentate ranges from 1,000 mL to 1,200 mL. 1,040 mL of retentate (labeled as CS-1-2, total water-soluble polysaccharide of 0.18 g) and 3,600-mL filtrate are obtained. Fractions CS-1-1 and CS-1-2 are combined to obtain a volume of 2,290 mL (labeled as sub-fraction CS-1, which contains 0.42 g of total water-soluble polysaccharides; see Table 2).

(21) The above-mentioned 3,600 mL of 300-kDa filtrate is placed into a beaker and the latter is incubated at 50° C. in a water bath. The 300-kDa filtrate is fractioned using TFF with a 10-kDa cassette membrane (50 cm.sup.2, PES). The TMP is set between 18-20 psi. 600 mL of distilled water is added into the retentate during filtration when the retentate ranges between 1,000 mL and 1,200 mL. The operation is repeated to obtain 990 mL of 10-kDa-to-300-kDa retentate (labeled as sub-fraction CS-2, containing 0.64 g of total water-soluble polysaccharides; see Table 2) and 3,600 mL of 10-kDa filtrate (labeled as sub-fraction CS-3, total water soluble polysaccharides of 0.16 g; see Table 2).

(22) The CS-1, CS-2, CS-3 and CS-4 fractions are concentrated separately using a vacuum concentrator to obtain a final volume of 120 mL. Concentrated fractions are sterilized at 121° C. for 20 min in an autoclave and stored at 4° C.

(23) 1.4 Determination of Total Water-Soluble Carbohydrates and Polysaccharides in H. Sinensis Water Extracts and Polysaccharide Sub-Fractions

(24) The phenol-sulfuric acid assay is used to determine the level of total water-soluble carbohydrates and polysaccharides in the water extracts and polysaccharide sub-fractions isolated from H. sinensis, including: the W1 20% (w/v) concentrated H. sinensis water extract (120 mL), the W2 H. sinensis crude polysaccharide extract (2400 mL), a combination of the retentate of the 0.2-μm filtration and 300-kDa-cutoff filtration (labeled as CS-1 sub-fraction; 2,290 mL), the retentate of the 10-kDa membrane filtration (labeled as CS-2; 990 mL), the filtrate of the 10-kDa-cutoff membrane filtration (labeled as CS-3; 3,600 mL), and the supernatants of the 95% ethanol precipitation and washing steps (labeled as sub-fraction CS-4; 1,040 mL). To establish a standard curve for the phenol-sulfuric acid assay, glucose standard solutions are prepared at concentrations of 0, 0.02, 0.04, 0.06, 0.08, 0.10, 0.12, 0.14, 0.16, 0.18, and 0.20 mg/mL. 200 μL of each solution is placed into 1.5-mL tubes. 200 μL of 5% phenol is added and the solution is mixed. 1 mL of sulfuric acid is added and the solution is mixed. After incubation for 20 min, absorbance is monitored at 490 nm using a spectrophotometer. The calibration curve of glucose standard solutions is prepared (calculated R.sup.2>0.99). The sample solutions are appropriately diluted. 200 μL of each diluted solution is placed into 1.5-mL tubes. Phenol and sulfuric acid are added and absorbance is monitored as above. The values obtained are plotted onto the calibration curve of glucose standard solutions to determine the concentration of total water-soluble carbohydrates or polysaccharide of the samples.

(25) Total water-soluble carbohydrates and polysaccharides measured in the extracts and sub-fractions isolated from H. sinensis are shown in Tables 1 and 2. The analysis in Table 2 shows that the W2 crude polysaccharide extract contains 0.42 g of total water-soluble polysaccharides with a molecular weight above 300 kDa (CS-1), which accounts for 33.3% of the total polysaccharides found in the H. sinensis crude polysaccharide extract (W2). The W2 extract also contains 0.64 g of polysaccharides between 10 kDa to 300 kDa (CS-2), which accounts for 50.8% of the total polysaccharides found in the W2 extract. The W2 extract also contains 0.16 g of polysaccharides with a molecular weight below 10 kDa (CS-3), which accounts for 12.7% of the total polysaccharides found in the W2 extract.

(26) TABLE-US-00001 TABLE 1 Determination of water-soluble carbohydrates and polysaccharides in water extracts and CS-4 polysaccharide sub-fraction isolated from H. sinensis Content Percentage Fraction (g) (%) W1 Total water-soluble carbohydrates 2.09 100 W2 Total water-soluble polysaccharides 1.26 60.3 CS-4 Mono-, di-, oligo-saccharides 0.83 39.7

(27) TABLE-US-00002 TABLE 2 Polysaccharide distribution of W2 concentrated water extract and polysaccharide sub-fractions isolated from H. sinensis Content Percentage Fraction (g) (%) W2 Total water-soluble carbohydrates 1.26 100 CS-1 MWCO > 300 kDa 0.42 33.3 CS-2 300 kDa > MWCO > 10 kDa 0.64 50.8 CS-3 10 kDa > MWCO 0.16 12.7
MWCO: Molecular Weight Cut-Off
1.5 Monosaccharide Composition of the CS-1 Polysaccharide Sub-Fraction

(28) High pH anion exchange chromatography-pulsed amperometric detection (HPAEC-PAD) is used to analyze the monosaccharide composition of the CS-1 sub-fraction, which is selected here as a representative polysaccharide sub-fraction possessing insulin-sensitizing effects. Monosaccharide standard solutions of L-fucose, L-rhamnose, D-galactosamine, D-arabinose, D-glucosamine, D-galactose, D-glucose and D-mannose are prepared at 0.1, 0.5, 1, 2, and 5 mg/L. 25 μL of each solution is submitted to ionic chromatography analysis with the HPAEC-PAD Dionex ICS-5000 System (CarboPacPA1 column with an internal diameter of 4×250 mm; Thermo Scientific). Elution is performed with 16 mM NaOH (which corresponds to a mixture of water and 200 mM NaOH at the volume ratio of 92:8). The flow rate is set at 1 mL/min. Temperature of column is set at 30° C. After 30 min of analysis, the peak area of each monosaccharide standard is determined and the standard curve of monosaccharide standards is prepared (R.sup.2>0.99).

(29) 1 mL of CS-1 sub-fraction (containing 3 mg of total water-soluble polysaccharides) is hydrolyzed with 1.79 mL of distilled water and 1.33 mL of trifluoroacetic acid at 112° C. for 12 hours. Acid is removed by co-distillation with water after the hydrolysis is complete. Each hydrolysate (1 mg) is dissolved in pure water (1 mg/mL). After a 4-fold dilution of the hydrolysate with pure water (0.25 mg/mL), 25 μL of the hydrolysate solution is used for ionic chromatography analysis using the HPAEC-PAD system. Elution is performed as above. After 30 min of analysis, the analytic HPAEC-PAD profile of hydrolysate solution is acquired. The monosaccharide composition and molar ratio of the CS-1 sub-fraction is determined by comparison with the standard curve. The CS-1 sub-fraction is found to contain 3.2% fucose, 3.4% rhamnose, 1.7% arabinose, 4.6% glucosamine, 23.8% galactose, 12.5% glucose, 50.4% mannose, and 0.4% galactosamine (Tables 3 and 4 and FIG. 2).

(30) TABLE-US-00003 TABLE 3 Monosaccharide composition of CS-1 polysaccharide sub-fraction isolated from H. sinensis and analyzed using HPAEC-PAD Monosaccharide Percentage (%) Fucose 3.2 Rhamnose 3.4 Arabinose 1.7 Glucosamine 4.6 Galactose 23.8 Glucose 12.5 Mannose 50.4 Galactosamine 0.4

(31) TABLE-US-00004 TABLE 4 Monosaccharide molar ratio of CS-1 polysaccharide sub-fraction isolated from H. sinensis Monosaccharide Molar ratio Fucose 0.07 Rhamnose 0.07 Arabinose 0.04 Glucosamine 0.09 Galactose 0.47 Glucose 0.25 Mannose 1 Galactosamine 0.01
1.6 Molecular Weight Distribution of CS-1 and CS-2 Polysaccharide Sub-Fractions Isolated from H. sinensis

(32) The molecular weights of the CS-1 and CS-2 sub-fractions are analyzed by size-exclusion chromatography (SEC) and high performance liquid chromatography with a refractive index detector (Waters, model 2410) and a dual detector (Viscotek, model 270). Dextran 670 (1.5 mg/mL) is used as a standard marker to calibrate the system. 100 μL of sample is analyzed on two connected GPC columns (TSKgel G5000PWxL and TSKgel G6000PWxL; 7.8×300 mm) Elution is performed with 0.02% NaNO.sub.3 in pure water. The flow rate is set at 0.5 mL/min (column temperature of 45° C.).

(33) Molecular weight parameters of the CS-1 and CS-2 sub-fractions are calculated using the OmniSEC software (Viscotek) and the following equations: Mn: number average molecular weight

(34) $\mathrm{Mn}=\frac{.Math.\mathrm{NiMi}}{.Math.\mathrm{Ni}}$ Mw: weight average molecular weight

(35) $\mathrm{Mw}=\frac{.Math.{\mathrm{NiMi}}^2}{.Math.\mathrm{NiMi}}$ Mz: higher average molecular weight

(36) $\mathrm{Mz}=\frac{.Math.{\mathrm{NiMi}}^3}{.Math.{\mathrm{NiMi}}^2}$ Mp: molecular weight at peak maximum, which is measured at the point of the molecular weight distribution maximum Mi: molecular weight of a chain Ni: number of chains of that molecular weight

(37) Molecular weight analysis of the CS-1 sub-fraction (total water-soluble polysaccharide of 4 mg/mL) is performed using the GPC/SEC system; refractive index (RI) and light scattering (LS) data are obtained (FIG. 3). The polysaccharide molecular weight distribution is calculated using Viscotek OmniSEC software (FIG. 4) and the cumulative weight fraction is determined (FIG. 5).

(38) The cumulative weight fraction values of CS-1 at 0.95 (5%) and 0.05 (95%) correspond to molecular weights of 15,776 Da and 1,231,969 Da, respectively. Polysaccharides between 15,776 Da and 1,231,969 Da thus represent approximately 90% of total polysaccharide weight of the sub-fraction. The polydispersity index (Mw/Mn) is measured as 7.475. Table 5 shows a comparison of the molecular weights of the CS-1 and CS-2 polysaccharide sub-fractions.

(39) TABLE-US-00005 TABLE 5 Molecular weight comparison of the CS-1 and CS-2 sub-fractions Parameter CS-1 CS-2 Mn (Daltons) 41,731 38,842 Mw (Daltons) 311,921 49,215 Mz (Daltons) 7,589,000 79,949 Mw/Mn (Polydispersity index) 7.475 1.267 MW of 5% of cumulative WF (Daltons) 15,776 22,563 MW of 95% of cumulative WF (Daltons) 1,231,969 109,219 MW: molecular weight

Example 2

Effects of H. sinensis Polysaccharide Sub-Fractions on Signs of Fatty Liver Disease in HFD-Fed Mice

(40) FIG. 6 shows the effects of H. sinensis polysaccharide sub-fractions on experimental fatty liver disease induced in mice by feeding with HFD. Male C57BL/6 mice (n=10 per group) are fed with either chow (13.5% of energy from fat) or HFD (60% of energy from fat) and supplemented with 100 μL of polysaccharide sub-fraction (CS-1, CS-2, CS-3, or CS-4) or double-distilled water for 3 months by intragastric gavage. HFD feeding induces signs of fatty liver disease, including increase of liver size (FIG. 6A), liver weight (FIG. 6B), and the size and number of liver lipid vacuoles (FIG. 6C), as well as increased levels of serum TG (FIG. 6D) and AST (FIG. 6E), compared to chow-fed mice. Notably, treatment of HFD mice with CS-1, CS-2 and CS-3 for 3 months reduces liver size (FIG. 6A), liver weight (FIG. 6B), and the size and number of lipid vacuoles (FIG. 6C), as well as serum TG (FIG. 6D) and AST levels (FIG. 6E) compared to control HH) mice. In comparison, H. sinensis polysaccharide sub-fraction CS-4 reduces liver size (FIG. 6A), liver weight (FIG. 6B), lipid vacuolization (FIG. 6C), and serum TG levels (FIG. 6D) compared to control HFD mice, but this sub-fraction produces non-significant results on AST levels (FIG. 6E). In addition, the effects of CS-4 on TG levels are less pronounced than those produced by CS-1, CS-2 and CS-3 sub-fractions (FIG. 6D).

(41) Based on the concentration of polysaccharides found in each sub-fraction (CS-1, 0.35 g/100 mL; CS-2, 0.53 g/100 mL; CS-3, 0.13 g/100 mL), we calculate the effective amount of polysaccharide sub-fraction that reduces signs of fatty liver disease in the treated mice (which have an average body weight of 30 g): 0.00035 g of CS-1/mouse; 0.00053 g of CS-2/mouse; and 0.00013 g of CS-3/mouse. By extension, the effective dosage of H. sinensis polysaccharide sub-fraction reducing fatty liver disease in a human subject (with a body weight of 70 kg) is estimated as follows: 0.82 g of CS-1/subject; 1.24 g of CS-2/subject; and 0.30 g of CS-3/subject. In other words, the effective dosage of H. sinensis polysaccharide sub-fraction in a human subject is: 0.012 g/kg (CS-1), 0.018 g/kg (CS-2), and 0.0043 g/kg (CS-3).

(42) The present invention provides H. sinensis polysaccharide sub-fractions, which can reduce signs of fatty liver disease in mammals. The H. sinensis polysaccharide sub-fractions of the present invention are therefore valuable for the development of new preventive strategies and treatments for fatty liver disease. The embodiments presented in the present disclosure are given as representative results that can be obtained with the polysaccharide sub-fractions, but they do not, however, limit the scope of the invention. It will be apparent to those skilled with the art that modifications can be made to the embodiments, without departing from the scope of the present invention and the appended claims.