METHOD OF PREPARING PENTACYCLIC TRITERPENOID SAPONINS AND DRUG COMPOSITION

Abstract

Disclosed are the preparing method of a pentacyclic triterpenoid saponin compound and a drug composition, and in particular the method of the pentacyclic triterpenoid saponin compounds as shown in formulae (I) to (XVI) in the preparation of a drug for preventing or treating a disease mediated by AMPK and/or ERRα, comprising the preparation of a drug for preventing or treating diseases such as a liver disease, respiratory system disease, metabolic disease, autoimmune disease, cardiovascular and cerebrovascular disease, kidney disease, central nervous system disease or muscular dystrophy. The definition of formulae (I) to (XVI) is the same as the definition in the specification.

##STR00001## ##STR00002## ##STR00003## ##STR00004##

Claims

1. A method of pentacyclic triterpenoid saponins represented by formulas (I)-(XVI) or pharmaceutically acceptable salts or solvates thereof in preparing a drug for preventing or treating AMPK and/or ERRα-mediated diseases: ##STR00019## ##STR00020## ##STR00021## ##STR00022##

2. The method according to claim 1, wherein the method of the pentacyclic triterpenoid saponins represented by the formulas (I)-(XIII) or the pharmaceutically acceptable salts or solvates thereof in preparing the drug for preventing or treating the AMPK-mediated diseases is: ##STR00023## ##STR00024## ##STR00025## ##STR00026##

3. The method according to claim 1, wherein the AMPK and/or ERRα-mediated diseases comprise hepatic diseases, respiratory system diseases, metabolic disorders, autoimmune diseases, cardiovascular and cerebrovascular diseases, kidney diseases, central nervous system diseases or muscular dystrophy.

4. The method according to claim 3, wherein the hepatic diseases comprise non-alcoholic fatty liver, non-alcoholic steatohepatitis, alcoholic fatty liver disease, hepatic fibrosis, hepatic cirrhosis, primary biliary cholangitis or primary sclerosing cholangitis.

5. The method according to claim 3, wherein the respiratory system diseases comprise cough, asthma, chronic obstructive pulmonary disease, bronchitis, pneumonia, respiratory distress syndrome, emphysema, idiopathic pulmonary fibrosis, cystic fibrosis pulmonary disease, allergic rhinitis or chronic rhinitis.

6. The method according to claim 3, wherein the metabolic disorders comprise insulin resistance, metabolic syndromes, diabetes and complications thereof, hyperlipidemia, obesity, hyperuricemia, gout or osteoporosis.

7. The method according to claim 3, wherein the autoimmune diseases comprise ulcerative colitis, Crohn's disease, systemic lupus erythematosus, rheumatoid arthritis, psoriasis, multiple sclerosis or Behcet's disease.

8. The method according to claim 3, wherein the cardiovascular and cerebrovascular diseases comprise hypertension, atherosclerosis, arrhythmia, coronary heart diseases, myocardial infarction, myocardial hypertrophy, carditis, heart failure, pulmonary arterial hypertension, cerebral apoplexy, stroke, cerebral infarction or cerebral edema.

9. The method according to claim 3, wherein the kidney diseases comprise autosomal dominant polycystic kidney disease or chronic nephritis.

10. The method according to claim 3, wherein the central nervous system diseases comprise Parkinson diseases, Alzheimer's diseases, α-synucleinopathy, depression, amyotrophic lateral sclerosis, fibromyalgia syndrome, neuralgia, Down's syndrome, Hallervorden-Spatz diseases, Huntington's diseases or Wilson's disease.

11. The method according to claim 3, wherein the muscular dystrophy is Duchenne muscular dystrophy.

12. A drug composition for preventing or treating the AMPK and/or ERRα-mediated diseases, comprising one or more compounds represented by formulas (I)-(XVI) or pharmaceutically acceptable salts or solvates thereof as active ingredients and pharmaceutically acceptable adjuvants ##STR00027## ##STR00028## ##STR00029## ##STR00030##

13. The drug composition according to claim 12, wherein a dosage form of the drug composition is a capsule, powder, a tablet, a granule, a pill, an injection, a syrup, oral liquid, an inhalant, a cream, an ointment, a suppository or a patch.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0062] FIG. 1 is an activation activity effect diagram of pentacyclic triterpenoid saponin on AMPK in Huh-7 cells (detected by Western Blot);

[0063] FIG. 2 is an activation activity effect diagram of pentacyclic triterpenoid saponin on AMPK in Huh-7 cells (detected by Western Blot);

[0064] FIG. 3 is an influence diagram of pentacyclic triterpenoid saponin on AMPK downstream signaling pathways (detected by Western Blot);

[0065] FIG. 4 is an influence diagram of ginsenoside Ro on serum lipid (TG, TC, LDL-c and HDL-c) level of high fat diet (HFD)-induced NASH model rats (n=8; compared with an HFD model group, *p<0.05, **p<0.01, ***p<0.001; and compared with a CHOW control group, ###p <0.001);

[0066] FIG. 5 is an influence diagram of ginsenoside Ro on liver lipid (TG and TC) level of high fat diet (HFD)-induced NASH model rats (n=8; compared with an HFD model group, *p<0.05, **p<0.01, ***p<0.001; and compared with a CHOW control group, ###p <0.001);

[0067] FIG. 6 is an influence diagram of ginsenoside Ro on mRNA expression levels of inflammatory factors (TNFa, IL-6 and IL-1) in high fat diet (HFD)-induced NASH model rats (n=8; compared with an HFD model group, **p<0.01, ***p<0.001; and compared with a CHOW control group, ###p<0.001);

[0068] FIG. 7 is an influence diagram of ginsenoside Ro on mRNA expression levels of cell factors (TGF and -SMA) related to fibrosis in livers of high fat diet (HFD)-induced NASH model rats (n=8; compared with an HFD model group, **p<0.01, ***p<0.001; and compared with a CHOW control group, ###p<0.001);

[0069] FIG. 8 is an influence diagram of ginsenoside Ro on levels of liver enzymes (ALT and AST) in serum of CCl.sub.4-induced hepatic fibrosis model mice (n=8; compared with a CCl.sub.4 model group, *p<0.05, **p<0.01; and compared with a control group, ###p<0.001);

[0070] FIG. 9 is an influence diagram of ginsenoside Ro on mRNA expression levels of cell factors (TGF , -SMA, MMP2 and collagen 1) related to fibrosis in livers of CCl.sub.4-induced hepatic fibrosis model mice (n=8; compared with a CCl.sub.4 model group, *p<0.05, **p<0.01, ***p<0.001; and compared with a control group, ###p<0.001);

[0071] FIG. 10 is an influence diagram of ziyuglycoside II on disease activity indexes (DAI) and colon lengths of ulcerative colitis model mice modeled with dextran sulphate sodium salt (DSS) (compared with the model group, ** P<0.01, *** P<0.001);

[0072] FIG. 11 is an influence diagram of ziyuglycoside II on colon histopathology of ulcerative colitis model mice modeled with dextran sulphate sodium salt (DSS);

[0073] FIG. 12 is an influence diagram of ziyuglycoside II on inflammatory factors (TNF- and IL-1), reactive oxygen species (ROS) and proteins related to tight junctions (ZO-1, Claudin-1 and Occludin) in colon tissues of the ulcerative colitis model mice modeled with dextran sulphate sodium salt (DSS) (compared with a model group, * P<0.05, ** P<0.01, *** P<0.001);

[0074] FIG. 13 is an influence diagram of ziyuglycoside II on weights and colon lengths of Crohn's disease model mice modeled with 2, 4, 6-trinitrobenzene sulfonic acid (TNBS) (compared with the model group, * P<0.05, *** P<0.001);

[0075] FIG. 14 is an influence diagram of ziyuglycoside II on inflammatory factors (TNF- and IL-1) and myeloperoxidase (MPO) in colon tissues of Crohn's disease model mice modeled with 2, 4, 6-trinitrobenzene sulfonic acid (TNBS) (compared with the model group, ** P<0.01, *** P<0.001);

[0076] FIG. 15 is an inhibit effect diagram of asperosaponin VI on polycystic kidney epithelial cell proliferation (detected with a CCK-8 method); and

[0077] FIG. 16 is an influence diagram of asperosaponin VI on signal molecules related to AMPK and proliferation of polycystic kidney epithelial cells (detected by Western Blot).

DETAILED DESCRIPTION OF THE INVENTION

[0078] The present invention will be further described below in detail in combination with the accompanying drawings and the embodiments. The present invention is not limited to these embodiments. One of skill in the art may properly improve process parameters in view of the disclosure herein. Of particular note, all similar substitutes and modifications are apparent to one of skill in the art and are considered to be included within the present invention.

[0079] Compounds represented by formulas (I)-(XVI) in the embodiments of the present invention may be prepared with extraction, separation and purification from Chinese herbal medicines such as ginseng, radix dipsaci, Calendula officinalis, fructus kochiae, asiatic pennywort herb, radix bupleuri, Polygala fallax hemsl, Caulis et Folium Hederae Sinensis and radix sanguisorbae with reference to methods in literatures (Chinese Patent No. CN102125570B; Natural Product Reports 2011, 28, 543); may also be subjected to semi-synthetic preparation with reference to methods in literatures (Synlett 2004, 2, 259; Natural Product Reports 2011, 28, 543; Arch. Pharm. Chem. Life Sci. 2015, 348, 615); and may also be commercially purchased. The purity of each compound commercially purchased or prepared with semi-synthesis is 95% or above.

Embodiment 1

Synthesis of Asperosaponin C (XIV, CAS: 60252-11-1)

[0080] ##STR00013## ##STR00014##

[0081] L-arabinose (1 g, 6.66 mmol) was dissolved into pyridine (20 mL), and benzoyl chloride (4.6 mL, 39.97 mmol) was slowly dripped into an ice-water bath for overnight reaction at room temperature. The reaction was monitored with TLC; ethyl acetate (15 mL) was added to dilute a reaction liquid after the reaction was completed; the reaction liquid was washed with water (20 mL), a 1N aqueous hydrochloric acid solution (20 mL), an aqueous saturated NaHCO.sub.3 solution (20 mL) and saturated NaCl (20 mL) in sequence; an organic layer was dried with anhydrous sodium sulfate, concentrated and purified by silica gel chromatography (petroleum ether: ethyl acetate=5: 1) to obtain a compound XIV-1 (white solids, 3.2 g, yield: 84%).

[0082] The compound XIV-1 (3.2 g, 5.65 mmol) was dissolved into dichloromethane (20 mL), a solution of 35% HBr in acetic acid (3.5 mL) was added under the ice-water bath, stirring was conducted at the room temperature for reaction, and the reaction was monitored with with TLC.

[0083] After the reaction was completed, a reactant was washed with the water (20 mL), the aqueous saturated NaHCO3 solution (20 mL) and the saturated NaCl (20 mL) in sequence; an organic layer was dried with the anhydrous sodium sulfate and concentrated to obtain a compound XIV-2. The compound XIV-2 was dissolved into acetone/water (25:1, 26 mL), silver carbonate (1.85 g, 6.71 mmol) was added, and stirring was conducted at the room temperature for reaction overnight. After the reaction was completed, suction filtration was conducted with diatomite, and a filtered product was subjected to concentration and purified by silica gel chromatography (petroleum ether: ethyl acetate=3: 1) to obtain a compound XIV-3 (white solids, 950 mg, two-step yield: 37%).

[0084] The compound XIV-3 (950 mg, 2.05 mmol) was dissolved into anhydrous dichloromethane (15 mL), and trichloroacetonitrile (2.2 mL, 21.94 mmol) and DBU (166 μL, 1.11 mmol) were added for reaction for 3h at the room temperature. After the reaction was completed, a reaction liquid was directly concentrated and purified by silica gel chromatography (petroleum ether: ethyl acetate=3: 1) to obtain a compound XIV-4 (white solids, 1.05 g, yield: 84%).

[0085] Benzyl oleanolate (1 g, 1.829 mmol) and the compound XIV-4 (1.33 g, 2.192 mmol) were dissolved into the anhydrous dichloromethane (15 mL), a 4A zeolite (1.9 g) was added, trimethylsilyl trifluoromethanesulfonate (2.5 μL, 0.013 mmol) was added in the ice-water bath, and stirring was conducted for reaction overnight at the room temperature in an argon protection environment. After the reaction was completed, suction filtration was conducted, the zeolite was removed, and concentration was conducted. A residue was purified by silica gel chromatography (petroleum ether: ethyl acetate=10: 1) to obtain a compound XIV-5.

[0086] The compound XIV-5 was dissolved into a mixed solution of methanol and the dichloromethane (methanol: dichloromethane=2:1, 24 mL); sodium methoxide (119 mg, 2.20 mmol) was added; reaction was conducted for 5 h at the room temperature; DOWEX5OWX2-100 cation exchange resin was added to adjust a pH value of a reaction liquid to be 7; suction filtration was conducted; the resin was removed; concentration was conducted; and a residue was purified by silica gel chromatography (dichloromethane: methanol=10:1) to obtain a compound XIV-6 (white solids, 424 mg, two-step yield: 34%).

[0087] The compound XIV-6 (289 mg, 0.426 mmol) was dissolved into tetrahydrofuran (15 mL), 10% Pd/C (30 mg) was added, a hydrogen balloon was added, and stirring was conducted at the room temperature for reaction overnight. After the reaction was completed, a solution was spun dried, diethyl ether (15 mL) was added for stirring overnight, suction filtration was conducted, and a filter cake was washed with the diethyl ether to obtain a compound XIV (white solids, 158 mg, yield: 63%): .sup.1H NMR (300 MHz, DMSO-d.sub.6) .sup.1H-NMR (pyridine-d.sub.5) δ:5.47 (br s, 1H), 4.77 (d, 1H, J=7.0), 4.43 (t, 1H, J=8.7), 4.31 (m, 2H), 4.16 (dd, 1H, J=8.7, 3.1), 3.83 (m, 1H), 3.37-3.27 (m, 2H), 1.29, 1.27, 1.00, 0.99, 0.94, 0.93, 0.83 (s, 7×3H, CH3); ESI-MS: 611.5 [(M+Na).sup.+]. HRMS: m/z 587.3962 [M−H].sup.− ([C.sub.35H.sub.55O.sub.7]=587.3953).

Embodiment 2

Synthesis of -Hederin (XV, CAS: 35790-95-5)

[0088] ##STR00015## ##STR00016## ##STR00017##

[0089] The compound XIV-6 (424 mg, 0.624 mmol) prepared with reference to Embodiment 1 was dissolved into anhydrous N, N-dimethylformamide (15 mL); toluenesulfonic acid (18 mg, 0.095 mmol) and 2, 2-dimethoxypropane (230 1.872 mmol) were added in an ice-water bath; and reaction was conducted for 5 h at room temperature. After the reaction was completed, 0.3 mL of triethylamine was dripped for quenching reaction, 20 mL of ethyl acetate was added to dilute a reaction liquid, the reaction liquid was washed with water (30 mL) and a saturated salt solution (30 mL) separately, and an organic layer was dried with anhydrous sodium sulfate and was concentrated. A residue was purified by silica gel chromatography (petroleum ether: ethyl acetate=5: 1) to obtain a compound XV-1 (white solids, 426 mg, yield: 95%).

[0090] A compound XV-2 was prepared with reference to methods in literatures (Genetics & Molecular Research, 2016, 15, doi: 10.4238/gmr.15038998). The compound XV-1 (150 mg, 0.209 mmol), the compound XV-2 (169 mg, 0.272 mmol) and a 4A zeolite (225 mg) were added in anhydrous dichloromethane (15 mL), a mixed solution was stirred for 40 min at the room temperature and then was cooled to −78° C., a boron trifluoride-diethyl ether complex (18 μL, 0.146 mmol) was added at this temperature, and reaction was conducted for 3 h at −78° C. . After the reaction was completed, 0.2 mL of triethylamine was dripped for quenching reaction, and suction filtration and concentration were conducted. A residue was purified by silica gel chromatography (petroleum ether: ethyl acetate=8:1) to obtain a compound XV-3 (colorless oily liquid, 154 mg, yield: 63%).

[0091] The compound XV-3 (150 mg, 0.128 mmol) was dissolved into a mixed solvent of the dichloromethane and methanol (15 mL, dichloromethane: methanol=1:2), toluenesulfonic acid was added (24 mg, 0.128 mmol), stirring was conducted for reaction at the room temperature, and the reaction was monitored with TLC. After the reaction was completed, a few drops of the triethylamine were dripped to terminate the reaction, a solvent was spun dried, and a residue was purified by silica gel chromatography (petroleum ether: ethyl acetate=2:1) to obtain a compound XV-4 (white solids, 120 mg, yield: 83%).

[0092] The compound XV-4 was dissolved into a mixed solvent of the methanol and the dichloromethane (methanol: dichloromethane=2:1, 15 mL), and sodium methoxide (23 mg, 0.426 mmol) was added for reaction for 5 h at the room temperature. After the reaction was completed, DOWEX5OWX2-100 cation exchange resin was added to adjust a pH value of reaction liquid to be 7 and was removed by suction filtration, and concentration was conducted. A residue was purified by silica gel chromatography (dichloromethane: methanol=20: 1) to obtain a compound XV-5 (white solids, 50 mg, yield: 57%).

[0093] The compound XV-5 (50 mg) was dissolved into a mixed solvent of the methanol and tetrahydrofuran (methanol: tetrahydrofuran=1: 1, 15 mL), 10% Pd/C (5 mg) was added, a hydrogen balloon was added, and stirring was conducted at the room temperature for reaction overnight. After the reaction was completed, a reactant was subjected to suction filtration with diatomite, a filtrate was spun dried, and a residue was purified by silica gel chromatography (dichloromethane: methanol=10:1) to obtain a compound XV (white solids, 34 mg, yield: 76%): .sup.1H NMR (300 MHz, Pyridine-d.sub.5) δ 6.14 (s, 1H), 5.49 (s, 1H), 4.93 (d, J=4.9 Hz, 1H), 4.77-4.72 (m, 1H), 4.67-4.54 (m, 3H), 4.41-4.23 (m, 4H), 3.89-3.77 (m, 1H), 3.43-3.22 (m, 2H), 1.65 (d, J=6.0 Hz, 3H), 1.32 (s, 3H), 1.20 (s, 3H), 1.08 (s, 3H), 1.03 (s, 3H), 1.01 (s, 3H), 0.97 (s, 3H), 0.86 (s, 3H).

Embodiment 3

Synthesis of Hederin A2 (XVI, CAS: 3391-80-8)

[0094] ##STR00018##

[0095] A compound XVI-1 was prepared with reference to methods in literatures (J. Am. Chem. Soc., 1999, 121(51): 12196).

[0096] With reference to the methods in Embodiment 1, with benzyl oleanolate as a raw material, XIV-4 was replaced by the compound XVI-1, compounds XVI-2 and XVI-3 were prepared in sequence, and finally, the compound XVI-1 was prepared with catalytic hydrogenation and debenzylation on the compound XVI-3: .sup.1H-NMR (pyridine-d.sub.5) δ 5.44 (br s, 1H), 4.93 (d, 1H, J=7.7), 4.57 (br d, 1H, J=11.3), 4.43 (m, 1H), 4.25 (m, 2H), 4.01 (m, 2H), 3.46 (dd, 1H, J=12.5, 2.2), 3.37 (dd, 1H, J=8.0, 2.4), 1.31, 1.30, 1.01, 0.98, 0.93, 0.91, 0.80 (s, 7×3H, CH3); ESI-MS: 641.4 [(M+Na).sup.+]. HRMS: m/z 617.4071 [M−H].sup.− ([C.sub.36H.sub.57O.sub.8]=617.4059).

Embodiment 4

Activation Activity of Pentacyclic Triterpenoid Saponin on AMPK in Huh-7 Cells

[0097] The activation activity of a compound on AMPK in Huh-7 cells was detected by using Western Blot.

[0098] Cell culture conditions: Huh-7 cells were cultured in an incubator with 5% CO.sub.2 at 37° C. in DMEM complete medium (containing 10% fetal calf serum and 1% streptomycin/penicillin).

[0099] Antibody: anti-AMPK (CST, 2532S); anti-pAMPK (CST, 2535S).

[0100] Western Blot experiment: the influence of the compound on phosphorylation level of the AMPK in the Huh-7 cells was detected: cells with more than 90% cells alive were taken for the experiment. In a 12-well plate, the Huh-7 cells were plated in the plate at 250000 cells per well, the plate was placed in the incubator with 5% CO.sub.2 at 37° C. for culture, an original culture medium was discarded after 12 h, a complete medium containing the pentacyclic triterpenoid saponin was added, a final concentration of subject pentacyclic triterpenoid saponin was set to be 10 μM, and an administration time was set to be 12 h. A positive control compound employs AICAR (100 μM), metformin (70 μM) or A-769662 (100 μM). Then proteins were extracted for Western Blot detection.

[0101] Experimental result: a Western Blot experimental result was subjected to gray scale scanning, a p-AMPK/AMPK ratio of negative control DMSO was defined as 1, and a p-AMPK/AMPK ratio of the subject compound was a relative ratio of a negative control group. The larger the numerical value was, the stronger the activation activity of the AMPK of the compound was. An activity data result is shown in Table 1.

TABLE-US-00001 TABLE 1 Activation activity of pentacyclic triterpenoid saponins on AMPK (The final concentration of the pentacyclic triterpenoid saponin is set to be 10 μM) Compound pAMPK/AMPK ratio DMSO 1.0 Metformin (70 μM) 1.22 AICAR (100 μM) 1.47 A-769662 (100 μM) 2.59 (I) 2.39 (II) 2.90 (III) 1.54 (IV) 1.75 (V) 1.81 (VI) 2.27 (VII) 1.76 (VIII) 2.0 (IX) 1.88 (X) 3.33 (XI) 1.95 (XII) 2.45 (XIII) 2.51 (XIV) 2.23 (XV) 2.87 (XVI) 2.05

[0102] The experimental result (Table 1) shows that all the compounds represented by the formulas (I)-(XVI) have very strong AMPK activation activity which are significantly superior to recognized AMPK activators such as the metformin, the AICAR and the A-769662.

Embodiment 5

Influence of Pentacyclic Triterpenoid Saponins on AMPK Downstream Signaling Pathways of Huh-7 Cells

[0103] The influence of a compound on AMPK downstream signaling pathways of Huh-7 cells was detected by using Western Blot.

[0104] Cell culture conditions: Huh-7 cells: a DMEM complete medium (containing 10% fetal calf serum and 1% streptomycin/penicillin) were cultured in an incubator with 5% CO.sub.2 at 37° C. . Antibody: anti-ACC (CST, 3676S); anti-pACC (CST, 11818S); anti-mTOR (CST, 2983S); anti-pmTOR (CST, 5536S); anti-PGC1a (Abcam, ab54481); anti-ERRα (Abcam, ab76228).

[0105] Western Blot experiment: the influence of the compound on the AMPK downstream signaling pathways of the Huh-7 cells was detected.

[0106] Cells with more than 90% cells alive were taken for the experiment. In a 12-well plate, the Huh-7 cells were plated in the plate at 250000 cells per well, the plate was placed in the incubator with 5% CO.sub.2 at 37° C. for culture, an original culture medium was discarded after 12 h, a complete medium containing the pentacyclic triterpenoid saponin was added, a final concentration of subject pentacyclic triterpenoid saponin was set to be 10 μM, and an administration time was set to be 12 h. A positive control compound employed AICAR (100 μM). Then proteins were extracted for Western Blot detection, and the protein change conditions of pACC/ACC, pmTOR/mTOR, PGC-1a and ERRα were detected.

[0107] The experimental result (FIG. 3) shows that the pentacyclic triterpenoid saponin compounds of the present invention can effectively cause changes of AMPK downstream proteins, and a change trend is related to AMPK activation. It should be particularly noted that, as being capable of inhibiting mTOR signaling pathways and increasing protein levels of the PGC1α and the ERRα at the same time, the pentacyclic triterpenoid saponin may promote mitochondrial synthesis and oxidative metabolism, induce protective autophagy and then exert the efficacy of reducing blood fat, resisting inflammation, apoptosis and fibrosis and the like.

Embodiment 6

Protection Effect of Ginsenoside Ro (Compound Represented by the Formula (I)) on High Fat Diet (HFD) Induced NASH Rat Model

[0108] Animal: 32 SD male rats of SPF level with 6 weeks old and 220 g were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All the animals maintained 12 h alternative circadian rhythm with free access to food.

[0109] Instrument: animal weight scale; slicing machine; full-automatic biochemical analyzer; inverted microscope

[0110] Reagent: the ginsenoside Ro (I) was purchased from Shanghai Boka Chemistry Technology Co., Ltd; obeticholic acid (OCA) was purchased from Jiangsu Vcare PharmaTech Co., Ltd; high fat diets were purchased from Research Diets (D12492, 60 kcal %); and standard feeds were purchased from Research Diets (D12450B, 10 kcal %).

Experimental Process:

1. Animal Grouping and Modeling

[0111] The rats were randomly divided into 4 groups based on the weight: a control group (CHOW), a high fat diet model group (HFD), a positive drug obeticholic acid (OCA, 5 mg/Kg) group (HFD+OCA) and a ginsenoside Ro (Ro for short, 5 mg/kg) group (HFD+Ro). The control group was fed with the standard feeds and drank water normally; and the rest groups were fed with the high fat diets, drank 10% aqueous sucrose solution for replacing the water and were modeled for 20 weeks.

2. Administration

[0112] After 20 weeks, the control group and the high fat diet model group were given with a CMC-Na solution with the volume fraction of 0.5% intragastrically every day, the HFD+OCA group was given with the obeticholic acid (5 mg/Kg) intragastrically every day, and the HFD+Ro was given with the ginsenoside Ro (5 mg/Kg) intragastrically every day. Administration was conducted for 8 weeks, during which, the control group was given with the standard feeds and drank the water normally, and the rest three groups were given with the high fat diets and drank 10% aqueous sucrose solution. The rats in each group were weighed every day, weights, hairs, feces and activity conditions were carefully observed and recorded.

3. Material Obtaining

[0113] The rats were deprived of food but not water for 12 h in advance; and the next morning, blood was taken from eye sockets, the rats were sacrificed, and livers were harvested. Right liver lobule tissues were fixed with 4% paraformaldehyde for HE stained slicing. A part of liver tissues were divided into 3 parts, which were snap-frozen in liquid nitrogen for subsequent detection of other indexes.

4. Determination of Biochemical Indexes

[0114] Whole blood was left to still standing for 2 h at room temperature, centrifugation was conducted for 15 min at 3000 rpm, and a serum was collected. 100 Mg of liver tissues were taken and homogenized with 0.9 mL of precooled normal saline, a homogenate was centrifuged for 15 min at 4° C. and 3000 rpm, and a supernatant was taken. Both of the serum and the supernatant were sent to Pathologic Department of Hospital of Integrated Traditional Chinese and Western Medicine in Jiangsu Province for determination of levels of triglyceride (TG), total cholesterol (TC), low density lipoprotein cholesterin (LDL-c) and high density lipoprotein cholesterol (HDL-c) in the serum and levels of the TG and the TC in the supernatant of the homogenate of the liver tissues with the full-automatic biochemical analyzer.

5. Liver Tissue Slicing

[0115] Pretreated tissues were sent to Google Biotechnology Co., Ltd for preparing HE stained slices and oil red stained slices.

6. Extraction of Proteins from Liver Tissues and Western Blot (WB) Detection

[0116] Liver tissues preserved at −80° C. were put in the liquid nitrogen, about 10 mg of the liver tissues were quickly dissected out, 500 μL of precooled tissue lysate was added, and the tissues were homogenized with a homogenizer and then left to lyse on ice for 30 min; and centrifugation was conducted for 15min at 4° C. and 12000 rpm, 170 mL of a supernatant was sucked to a clean EP tube, 10 mL of the supernatant was taken for BCA protein quantification, 40 mL of 5*loading buffer was added in the rest protein supernatant for denaturing by boiling, SDS-PAGE electrophoresis was conducted subsequently, the proteins were transferred onto a PVDF membrane, then incubation and elution of a relevant antibody were conducted, and finally, a chemiluminescence apparatus was employed for imaging analysis.

7. Extraction of Liver Tissue RNA and q-PCR Detection

[0117] Liver tissues preserved at −80° C. were put in the liquid nitrogen, about 10 mg of the liver tissues were quickly dissected out, about 500 μL of a precooled Trizol reagent was added, the tissues were homogenized with the homogenizer and then left to lyse on the ice for 15 min, 100 μL of chloroform was added for vigorous shaking for 15 s, and a product was put on the ice for 10 min and then was subjected to centrifugation for 15 min at 4° C. and 12000 rpm. An upper aqueous phase is transferred into a clean 1.5 mL EP tube, 200 mL of isopropanol was added for RNA precipitation, the EP tube was put on the ice for 10 min and then was subjected to centrifugation for 10 min at 4° C. and 12000 rpm. The supernate was discarded, precipitates were washed one time with 75% ethyl alcohol, a supernate was discarded after centrifugation, and precipitates of RNA dissolved with the water were treated with 40 mL of DEPC. The concentration of RNA was quantified with Nano, a reverse transcription reagent from Takara company was added according to the specification, and mRNA was reverse transcribed into cDNA with a common PCR instrument. Finally, forward primers and reverse primers of target genes, a q-PCR reagent (SYBR Green) and the cDNA were added in a special 96-well plate for q-PCR, and a q-PCR instrument was used for amplification and quantification. A difference between gene expressions was characterized by selecting a ΔΔCt value, and a GraphPad Prism 7 software was employed for data processing and statistical test.

8. Experimental Results

[0118] Results in FIG. 4 and FIG. 5 show that OCA and the ginsenoside Ro can both significantly lower the levels of the TG in the serum and the liver, and the curative effect of the ginsenoside Ro is equivalent to that of the OCA. It is noted that the ginsenoside Ro may effectively lower the levels of the TC, the LDL-c and the HDL-c in the serums of the rats fed with the high fat diets, whereas the OCA cannot lower but slightly up-regulates the levels of the TC and the LDL-c in the serums of the rats fed with the high fat diets. In the HFD group, the contents of the TC in the livers were increased to about 3 times of that of the control group and were significantly lowered after the rats were treated with the ginsenoside Ro.

[0119] In order to further detect the effect of the ginsenoside RO on lowering inflammations and fibrosis of the livers of NASH animals, the mRNA expression conditions of related inflammatory factors and fibrosis-related cell factors in the liver tissues were determined. A result in FIG. 6 shows that the ginsenoside Ro may significantly inhibit increase, caused by high fat diets, in mRNA expression levels of TNF-α, IL-6 and IL-10, and therefore, it is considered that the ginsenoside Ro can effectively resist liver inflammatory responses induced by the high fat diets. A result in FIG. 7 shows that the ginsenoside Ro may significantly inhibit increase, caused by the high fat diets, in mRNA expression levels of TGF-β and α-SMA, and therefore, it is considered that the ginsenoside Ro can effectively resist liver fibrosis induced by the high fat diets.

[0120] The above results show that the ginsenoside Ro may significantly lower lipid accumulation, caused by the high fat diets, of the rat livers, inhibits the inflammatory responses and the liver fibrosis and has the curative effect equivalent to that of the obeticholic acid (OCA) as a positive control drug. It is noted that, clinical researches and animal experiments discover that the OCA may increase the level of the LDL-c in the serum although lowering lipid accumulation of the livers, whereas the ginsenoside Ro does not have such side effects in the animal experiments. This hints that the ginsenoside Ro may avoid the side effects of the rising the LDL-c in the serum of the OCA in clinical application and thus may be used for preventing and treating chronic hepatic diseases such as fatty liver diseases (particularly, non-alcohol steatohepatitis), chronic hepatitis and hepatic fibrosis.

[0121] Other types of the pentacyclic triterpenoid saponin in the present invention also show the fatty liver disease resistant activity similar to the ginsenoside Ro. For example, all of asiaticoside (V), madecassoside (VI), saikosaponin A (VII), saikosaponin D (VIII), ziyuglycoside I (XII) and ziyuglycoside II (XIII) can effectively lower lipid accumulation, caused by the high fat diets, of the rat livers (result shown in Table 2) and inhibit the inflammatory responses of the livers (result shown in Table 3). This hints that the pentacyclic triterpenoid saponin provided by the present invention may be used for preventing and treating the chronic hepatic diseases such as the fatty liver diseases (particularly, non-alcohol steatohepatitis) and chronic hepatitis.

TABLE-US-00002 TABLE 2 Influence of pentacyclic triterpenoid saponins on lipid levels of serums and livers of high fat diet induced NASH model rats Serum Liver TG Liver TC Serum TG LDL-c Group n Dosage (mmoL/gprot) (mmoL/gprot) (mg/dL) (mg/dL) Control group 8 — 0.932 ± 0.171  0.301 ± 0.081  12.52 ± 1.37  35.54 ± 3.24  Model group 8 — .sup. 1.951 ± 0.319.sup.### .sup. 0.881 ± 0.204.sup.### .sup. 25.67 ± 3.78.sup.### .sup. 57.32 ± 6.22.sup.### Ziyuglycoside 8 5 mg/Kg 1.302 ± 0.443** 0.547 ± 0.272*  18.32 ± 2.51** 48.52 ± 6.98*  I Ziyuglycoside 8 5 mg/Kg 1.299 ± 0.387** 0.503 ± 0.256** 19.41 ± 4.37*  45.36 ± 5.37** II Saikosaponin 8 5 mg/Kg 1.268 ± 0.294** 0.518 ± 0.133** 20.43 ± 4.04*  47.06 ± 7.83*  A Saikosaponin 8 5 mg/Kg  1.173 ± 0.606*** 0.509 ± 0.248** 19.66 ± 5.12*  46.75 ± 7.35** D Madecassoside 8 5 mg/Kg 1.240 ± 0.213** 0.472 ± 0.201** 18.83 ± 4.11** 47.25 ± 7.45*  Asiaticoside 8 5 mg/Kg 1.235 ± 0.316** 0.513 ± 0.279** 18.00 ± 4.25** 45.15 ± 4.53** (n = 8, compared with the control group, .sup.###p < 0.001; and compared with the model group, *p < 0.05, **p < 0.01, ***p < 0.001)

TABLE-US-00003 TABLE 3 Influence of pentacyclic triterpenoid saponins on levels of ALT (glutamic- pyruvic transaminase), AST (glutamic oxalacetic transaminase) and MDA (malonaldehyde) in serums of high fat diet induced NASH model rats ALT AST MDA Group n Dosage (U/L) (U/L) (U/L) Control group 8 — 43.94 ± 5.38  95.41 ± 8.12  7.68 ± 2.47 Model group 8 — 90.36 ± 17.34.sup.### 150.32 ± 24.36.sup.### 16.26 ± 5.18.sup.### Ziyuglycoside I 8 5 mg/Kg 74.03 ± 12.38* 120.91 ± 19.32** 11.74 ± 3.26* Ziyuglycoside II 8 5 mg/Kg 69.28 ± 12.33** 122.16 ± 17.38** 10.41 ± 2.02** Saikosaponin A 8 5 mg/Kg 71.36 ± 9.26* 115.24 ± 19.23*** 10.03 ± 3.03** Saikosaponin D 8 5 mg/Kg 67.74 ± 8.29** 121.43 ± 9.28**  9.58 ± 3.15** Madecassoside 8 5 mg/Kg 65.86 ± 10.43*** 127.21 ± 9.34* 11.59 ± 2.88* Asiaticoside 8 5 mg/Kg 67.38 ± 13.46** 116.40 ± 11.35*** 10.14 ± 3.52** (n = 8, compared with the control group, *p < 0.05, **p < 0.01; and compared with the model group, #p < 0.05, ##p < 0.01)

Embodiment 7

Protection Effect of Ginsenoside Ro (the Compound Represented by the Formula (I)) on CCl.SUB.4.-Induced Hepatic Fibrosis Mice

[0122] Animal: 32 C57BL/6J male mice of SPF level with 25 g were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All the animals maintained 12 h alternative circadian rhythm with free access to food.

[0123] Instrument: animal weight scale; slicing machine; full-automatic biochemical analyzer; inverted microscope.

[0124] Reagent: the ginsenoside Ro was purchased from Shanghai Boka Chemistry Technology Co., Ltd; CCl.sub.4 was purchased from sigma Company; and sunflower seed oil was purchased from Nanjing Yuanye Biotechnology Co., Ltd.

Experimental Process:

1. Animal Grouping

[0125] The mice were randomly divided into 4 groups based on weights: a blank control group (CTL), a model group (CCl.sub.4), a ginsenoside Ro low-dosage group (Low) (5 mg/kg) and a ginsenoside Ro high-dosage group (High) (10 mg/kg), totally 4 groups (with 8 mice each).

2. Modeling and Administration

[0126] A modeling mode was that the mice were given with a CCl.sub.4-contained sunflower seed oil solution (with a CCl.sub.4 content of 25%, v/v) in a dosage of 2mL/kg by intraperitoneal injection, and the mice in the control group were injected with a corresponding dosage of the sunflower seed oil solution two times per week. From experiment day 1 onward, the mice in the control group were given with a CMC-Na solution with the mass fraction of 0.5% intragastrically every day and with sunflower seed oil two times per week by intraperitoneal injection; the mice in the model group were given with the CMC-Na solution with the mass fraction of 0.5% intragastrically every day and with the CCl.sub.4-contained sunflower seed oil solution two times per week by intraperitoneal injection; the mice in the ginsenoside Ro low-dosage group (Low) were given with 5 mg/kg of the ginsenoside Ro intragastrically every day and with a CCl.sub.4 solution two times per week by intraperitoneal injection; and the mice in the ginsenoside Ro high-dosage group (High) were given with 10 mg/kg of the ginsenoside Ro intragastrically every day and with the CCl.sub.4-contained sunflower seed oil solution two times per week by intraperitoneal injection. Administration was conducted for a total of 4 weeks.

3. Material Obtaining

[0127] The mice were deprived of food but not water for 12 h in advance; and the next morning, blood was taken from eyeballs, the mice were sacrificed, and livers were harvested. Right liver lobule tissues were fixed with 4% paraformaldehyde for HE stained slicing. The rest liver tissues were divided into 3 parts, which were snap-frozen in liquid nitrogen for subsequent detection of other indexes.

4. Determination of Biochemical Indexes

[0128] Whole blood was left to still standing for 2 h at room temperature, centrifugation was conducted for 15 min at 3000 rpm, and a serum was collected and sent to Pathologic Department of Hospital of Integrated Traditional Chinese and Western Medicine in Jiangsu Province for determination of levels of glutamic oxalacetic transaminase (AST) and glutamic-pyruvic transaminase (ALT) in the serum with a full-automatic biochemical analyzer.

5. Liver Tissue Slicing

[0129] Pretreated tissues were sent to Google Biotechnology Co., Ltd for preparing HE stained slices.

6. Extraction of Liver Tissue RNA and q-PCR Detection Liver tissues preserved at −80° C. were put in the liquid nitrogen, about 10 mg of the liver tissues were quickly dissected out, about 500 μL of a precooled Trizol reagent was added, the tissues were homogenized with the homogenizer and then left to lyse on the ice for 15 min, 100 μL of chloroform was added for vigorous shaking for 15 s, and a product was put on the ice for 10 min and then was subjected to centrifugation for 15 min at 4° C. and 12000 rpm. An upper aqueous phase was transferred to a clean 1.5 mL EP tube, 200 mL of isopropanol was added for RNA precipitation, the EP tube was put on the ice for 10 min and then was subjected to centrifugation for 10 min at 4° C. and 12000 rpm. The supernate was discarded, precipitates were washed one time with 75% ethyl alcohol, a supernate was discarded after centrifugation, and precipitates of

[0130] RNA dissolved with the water were treated with 40 mL of DEPC. The concentration of RNA was quantified with Nano, a reverse transcription reagent from Takara Company was added according to the specification, and mRNA was reverse transcribed into cDNA with a common PCR instrument. Finally, forward primers and reverse primers of target genes, a q-PCR reagent

[0131] (SYBR Green) and the cDNA were added in a special 96-well plate for q-PCR, and a q-PCR instrument was used for amplification and quantification. A difference between gene expressions was characterized by selecting ΔΔa Ct value, and a GraphPad Prism 7 software was employed for data processing and statistical test.

7. Experimental Results

[0132] As shown in FIG. 8, compared with the blank control group (CTL), the levels of the ALT and the AST in the serums of the model group (CCl.sub.4) were significantly increased; and after the mice were treated with the ginsenoside Ro low-dosage group (Low) (5 mg/kg) and high-dosage group (High) (10 mg/kg), the levels of the ALT and the AST in the serums were both significantly lowered. The above results hint that the ginsenoside Ro may exert certain beneficial effects to the liver inflammations and the hepatic fibrosis by lowering the levels of transaminase ALT and AST in the serums.

[0133] As shown in FIG. 9, compared with the blank control group (CTL), the mRNA expression levels of the hepatic fibrosis related cell factors (TGF, -SMA, MMP2 and collagen 1) of the model group (CCl.sub.4) were significantly increased; and after the mice were treated with the ginsenoside Ro low-dosage group (Low) (5 mg/kg) and high-dosage group (High) (10 mg/kg), the mRNA expression levels of the hepatic fibrosis related cell factors (TGF, -SMA, MMP2 and collagen 1) were significantly lowered. The above results hint that the ginsenoside Ro may inhibit the hepatic fibrosis by lowering the expression levels of the hepatic fibrosis related cell factors (TGF, -SMA, MMP2 and collagen 1).

[0134] The above results show that the ginsenoside Ro may significantly inhibit the inflammatory responses and the hepatic fibrosis of hepatic fibrosis animal models and improve liver functions. Therefore, considered that the ginsenoside Ro may be used for preventing and treating the hepatic fibrosis and other diseases related to fibrosis.

[0135] Other types of pentacyclic triterpenoid saponin in the present invention also show the activity similar to the ginsenoside Ro in the aspect of inhibiting the inflammatory responses and the hepatic fibrosis of the hepatic fibrosis animal models. For example, a-hederin (X), hederacoside C (XI), ziyuglycoside I (XII) and ziyuglycoside II (XIII) can all effectively inhibit mouse hepatic fibrosis induced by CCl.sub.4 (see Table 4). This hints that the pentacyclic triterpenoid saponin provided by the present invention may be used for preventing and treating the hepatic fibrosis and other diseases related to fibrosis.

TABLE-US-00004 TABLE 4 Influence of pentacyclic triterpenoid saponin on hepatic fibrosis related gene expression of CCl.sub.4-induced hepatic fibrosis mice Group n Dosage TGF-β collagen 1 MMP2 Control group 8 — 1.00 ± 0.45  1.00 ± 0.37  1.00 ± 0.27 Model group 8 — 4.42 ± 1.53.sup.### 12.35 ± 3.30.sup.### 14.36 ± 4.39.sup.### Ziyuglycoside I 8 5 mg/Kg 2.91 ± 1.10*  7.65 ± 2.43**  8.38 ± 3.86* Ziyuglycoside II 8 5 mg/Kg 3.03 ± 0.76*  8.16 ± 3.37*  7.38 ± 3.48** α-hederin 8 5 mg/Kg 2.75 ± 0.99**  7.93 ± 2.28**  6.37 ± 5.73** Hederacoside C 8 5 mg/Kg 2.96 ± 1.01*  8.07 ± 3.27*  7.65 ± 4.03** (n = 8, data in the table is fold induction compared with the control group. Compared with the control group, ###p < 0.01; and compared with the model group, *p < 0.05, **p < 0.01)

Embodiment 8

Treatment Effect of Ziyuglycoside II (Compound Represented by the Formula (XIII)) on Ulcerative Colitis Model Mice Induced by Dextran Sulphate Sodium Salt (DSS)

[0136] C57BL/6 male mice with 6-8 weeks old were selected; a control group was fed with purified water; and a model group, a treatment group and a positive control group were fed with purified water containing 2% dextran sulphate sodium salt (DSS) (36000-50000 M.Wt) for 7 days and then with the purified water for 3 days, and drug intervention was conducted at the same time. The mice in the control group and the model group were orally administered with carboxymethyl cellulose sodium as a placebo, the mice in the treatment group were orally administered with the ziyuglycoside II (purchased from Shanghai Boka Chemistry Technology Co., Ltd) (with a dosage of 5 mg/kg), and the mice in the positive control group were orally administered with 5-aminosalicylic acid as a clinical drug (5-ASA, purchased from Shanghai Sangon Biological Engineering Co., Ltd) (with a dosage of 100 mg/kg). In the administration process, weight changes, feces characters and hematochezia conditions of the mice were observed and recorded every day and were scored, and disease activity indexes were calculated (Disease activity index, DAI).

[0137] A scoring rule and a calculation method were as follows: DAT=(weight score+diarrhea score+occult blood score)/3, wherein weight score: 0 (weight gained or unchanged), 1 (weight lost by 1-5%), 2 (weight lost by 6-10%), 3 (weight lost by 11-15%), 4 (weight lost by 15% or above); diarrhea score: 0 (normal feces), 2 (soft feces), 4 (feces sticking anuses or diarrhea); and occult blood score: 0 (negative occult blood), 2 (positive occult blood), and 4 (visible bleeding). Comparing differences in DAI of various groups, it can be known that the ziyuglycoside II has significant prevention and treatment effects on ulcerative colitis of the mice modeled by the dextran sulphate sodium salt (DSS). As shown in (A) of FIG. 10, after the mice were treated with the ziyuglycoside II (5 mg/kg), DAI scores of the mice were obviously lower than that of the model group , and the curative effect of the ziyuglycoside II treatment group was significantly superior to a positive drug group.

[0138] The above mice were sacrificed by cervical dislocation at day 11, colon tissues were harvested, lengths of the colon tissues were measured for statistical summary, and differences in colon length of various groups were compared, from which, the ziyuglycoside II has significant prevention and treatment effect on the ulcerative colitis model mice. As shown in (B) of FIG. 10, the colon lengths of the ziyuglycoside II treatment group were significantly larger than those of the model group and superior to the positive drug group. Surprisingly, there is nearly no difference in colon length of the mice in the ziyuglycoside II treatment group from the control group.

[0139] Middle segments, with lengths of about 1 cm, of the colon tissues of the above mice were selected, soaked in a formalin solution for fixing overnight, embedded with paraffin and sliced, and the slices were subjected to HE staining. As shown in FIG. 11, patterns of intestinal mucosal layers of the model group are disturbed, and a great amount of inflammatory cells infiltrate into the intestinal mucosal layers; whereas the patterns of the intestinal mucosal layers of the treatment groups are integral, and infiltration of the inflammatory cells is reduced, showing obvious treatment effect. Particularly, compared with the positive drug group, the ziyuglycoside II has the advantage of maintaining a thickness of a muscular layer.

[0140] 10 mg of proximal rectum segments of the colon tissues of the above mice were selected, PBS was added for homogenization, and centrifugation was conducted for taking a supernate. The supernate was subjected to ELISA detection, and protein quantification was conducted for inflammatory factors TNF-α and IL-lb. As shown in (A) of FIG. 12, the expression levels of the inflammatory factors TNF- and IL-1 of the ziyuglycoside II treatment group were significantly lower than those of the model group, and the effect is superior to the positive drug group.

[0141] Under the pathological conditions of the ulcerative colitis, the content of reactive oxygen species (ROS) in colons may be continuously increased, and excessive ROS may strengthen destruction of the colon tissues. 10 mg of colon tissues of the above mice were selected for detection of ROS therein. As shown in (B) of FIG. 12, the content of the ROS in the tissues may be significantly lowered with administration with the ziyuglycoside II (5 mg/kg), and the effect is superior to the positive drug group.

[0142] When the ulcerative colitis occurs, the inflammatory factors may destruct tight junction between intestinal tracts, and destruction of the tight junction enables functions of mucosal barriers to to be lost and accelerates development of the diseases. 5 mg of colon tissues of the above mice were selected for qPCR analysis. As shown in (C) of FIG. 12, after the mice were given with the ziyuglycoside II (5 mg/kg), the expression levels of three main proteins, including ZO-1, Claudin-1 and Occludin, related to tight junction are significantly increased compared with the model group. This hints that the ziyuglycoside II further has the efficacy of increasing the expression level of the tight junction and then protecting a structure of each colon. The above results show that the ziyuglycoside II has significant protection and treatment effect on the ulcerative colitis of the DSS-induced mice, and the total curative effect is significantly superior to the 5-aminosalicylic acid as a clinical first-line drug. Therefore, the ziyuglycoside II is expected to be useful for preparing the drug for preventing or treating the inflammatory bowel diseases such as the ulcerative colitis.

[0143] Other types of pentacyclic triterpenoid saponin in the present invention also show the protection effect similar to the ziyuglycoside II on ulcerative colitis models of the DSS-induced mice. For example, after the mice were treated with the saikosaponin A (VII), the saikosaponin D (VIII), ziyuglycoside I (XII), ziyuglycoside II (XIII), ginsenoside Ro (I), asperosaponin VI (II), reinioside C (IX), α-hederin (X) or hederacoside C (XI), DAI scores of the treatment group were all obviously lower than those of the model group and significantly superior to the positive drug group.

Embodiment 9

Treatment Effect of Ziyuglycoside II on 2, 4, 6-Trinitrobenzene Sulfonic Acid (TNBS) Modeled Crohn's Disease Model Mice

[0144] C57BL/6 male mice with 6-8 weeks old were selected and randomly divided into a control group, a mode group, a treatment group and a positive control group. Expect for the control group, after the mice in the rest groups anesthetized with isoflurane, hoses were carefully inserted into colons of the mice from anuses, and 100 mL of 1.5% 2,4,6-trinitrobenzene sulfonic acid (TNBS) was slowly injected. Before the hoses were pulled out, the mice were placed upside down for 1 min. First intragastric administration was conducted at the day of enema treatment, the mice in the control group and the model group were orally administered with carboxymethyl cellulose sodium as a placebo, the mice in the treatment group were orally administered with the ziyuglycoside II (purchased from Shanghai Boka Chemistry Technology Co., Ltd) (with a dosage of 5 mg/kg), and the mice in the positive drug group were orally administered with 5-aminosalicylic acid as a clinical drug (5-ASA, purchased from Shanghai Sangon Biological Engineering Co., Ltd) (with a dosage of 100 mg/kg). Administration was continuously conducted for 7 days. In the administration process, the states of the mice were observed every day, and weight changes of the mice were recorded.

[0145] Differences in weight change of the mice in various groups were compared, from which, ziyuglycoside II has the treatment effect on the 2, 4, 6-trinitrobenzene sulfonic acid (TNBS) modeled Crohn's disease model mice. As shown in (A) of FIG. 13, after the mice were given with the ziyuglycoside II (5 mg/kg), the drop scope of weight loss of the mice was obviously lower than that of the model group, and the curative effect of the ziyuglycoside II treatment group was significantly superior to the positive drug group.

[0146] The above mice were sacrificed by cervical dislocation at day 8, colon tissues were harvested, lengths of the colon tissues were measured for statistical summary, and differences in colon length of various groups were compared, from which, the ziyuglycoside II has significant treatment effect on the Crohn's disease model mice. As shown in (B) of FIG. 13, the colon lengths of the ziyuglycoside II treatment group were significantly larger than those of the model group and superior to the positive drug group. Surprisingly, there is nearly no difference in colon length of the mice in the ziyuglycoside II treatment group from the control group. 10 mg of proximal rectum segments of the colon tissues of the above mice were selected, PBS was added for homogenization, and centrifugation was conducted for taking a supernate. The supernate was subjected to ELISA detection, and protein quantification was conducted for inflammatory factors TNF- and IL-1. As shown in (A) of FIG. 14, the expression levels of the inflammatory factors TNF- and IL-1 of the ziyuglycoside II treatment group were significantly lower than those of the model group and superior to the positive drug group. Myeloperoxidase (MPO) is an enzyme specific to neutrophils, and the amount of the neutrophils in the intestinal tracts may be reflected by detecting the MPO level. 5 mg of proximal rectum segments of the colon tissues of the above mice were selected, PBS was added for homogenization, and the content of the MPO in a homogenate was detected with a MPO kit (purchased from Nanjing Jiancheng Bioengineering Institute). As shown in (B) of FIG. 14, the content of the MPO in the tissues may be significantly lowered with administration with the ziyuglycoside II (5 mg/kg), and the effect is superior to the positive drug group. The above results show that the ziyuglycoside II has significant protection and treatment effect on the TNBS-induced Crohn's disease model mice, and the total curative effect is significantly superior to the 5-aminosalicylic acid as the clinical first-line drug. Therefore, the ziyuglycoside II is expected to be useful for preparing the drug for preventing or treating the inflammatory bowel diseases such as the Crohn's disease.

[0147] Other types of pentacyclic triterpenoid saponin in the present invention also show the protection and treatment effect similar to the ziyuglycoside II on TNBS-induced Crohn's disease model mice. For example, after the mice were treated with the saikosaponin A (VII), the saikosaponin D (VIII), ziyuglycoside I (XII), ziyuglycoside II (XIII), ginsenoside Ro (I), asperosaponin VI (II), reinioside C (IX), α-hederin (X) or hederacoside C (XI), there is nearly no difference in colon length of the mice in the treatment group from the control group. Furthermore, the expression levels of the inflammatory factors TNF-α and IL-1β of the treatment group were significantly lower than those of the model group and superior to the positive drug group.

Embodiment 10

Influence of Asperosaponin VI (Compound Represented by the Formula (II)) on Proliferation of Human Polycystic Kidney Cyst Lining Epithelial Cells

[0148] Cell and reagent: complete media for human ADPKD cyst lining epithelial cell lines WT9-12 and DMEM were purchased from ATCC Company; fetal calf serums were purchased from Gibco Company; Cell Counting Kit-8 was purchased from DOJINDO Company; Click-iT® EdU Imaging Kits were purchased from Invitrogen™ Thermo Fisher Scientific Company; Caspase -3/CPP32 Colorimetric Assay Kit was purchased from BioVision Company; Annexin V/PI double-staining flow type cell apoptosis detection kits were purchased from Bender Company; and anti-Phospho-(AMPKa, mTOR, p70S6K, 4E-BP1) antibodies were purchased from Cell Signaling Technology Company.

[0149] The influence of 2-DG on proliferation of the polycystic kidney epithelial cells was detected with a CCK-8 method: a DMEM complete medium containing 10% fetal calf serum (FBS) was prepared. The polycystic kidney epithelial cells were cultured in an incubator with 5% CO.sub.2 at 37° C. after being recovered, a solution was replaced every other day, the polycystic kidney epithelial cells were passaged every 2-3 days, and the cells were in a growing state of adhering to the wall. After trypsin enzyme digestion, cells were resuspended by using a complete medium, the concentration of a polycystic kidney epithelial cell suspension was regulated to be 3×10.sup.7/L, the polycystic kidney epithelial cell suspension was inoculated to 96-well plate by 100 mL per well for culture for 24 h, and the suspension was replaced by a culture medium containing 0.1% FBS for synchronization for 24 h. The asperosaponin VI was dissolved into DMSO to prepare 20 μM of mother liquor, a drug liquid was diluted by the culture medium, administration concentrations were 0.234 μM, 0.469 μM, 0.938 μM, 1.875 μM, 3.75 μM, 7.5 μM, 15 μM, 30 μM and 60 μM respectively, 6 sub-wells were arranged for each concentration, and then different concentrations of the asperosaponin VI were given to various groups (only the culture medium was added for the blank group without cells and drugs; and the control group was a solvent control, and the DMSO contained culture medium consistent to the administration group was added) for culture for 48 h, 10 mL of Cell Counting Kit-8 reagent was added into each well for incubation for 4 h in the incubator at 37° C., and an absorbance value of each well at a wavelength of 450 nm was read by a microplate reader. Proliferation inhibition rate (%)=(A control-A exmpriment)/(A control-A blank)×100%. Experimental results are shown in FIG. 15.

[0150] Detection on protein expression with Western blot: cells in the control group and the treatment group (intervening the polycystic kidney epithelial cell with 10 μM of asperosaponin VI for 48 h) were collected, cells were lysed with a RIPA lysate for extraction of total proteins, the concentration of proteins was detected with a BCA method, and 50 mg of sample proteins were taken for sample addition in sequence; after SDS-PAGE electrophoresis, the sample proteins were transferred onto PVDF membranes at a constant current (I=250 mA), were blocked for 1 h with 5% BSA and were incubated for 24 h with different primary antibodies; after the membranes were washed four times with TBST, incubation was conducted for 1.5 h at a normal temperature by using HRP-labeled second antibodies (1:10000); and after the membranes were washed four times with TBST, the proteins were developed with an ECL, and protein bands of various groups were subjected to gray scale analysis with an Image-J gel image analysis software. Experimental results are shown in FIG. 16.

Experimental Results

[0151] CCK-8 experimental results (FIG. 15) show that with increase in concentration of the asperosaponin VI, the number of viable cells is gradually reduced, which hints that the asperosaponin VI is dose-dependent in the inhibition effect on proliferation of the polycystic kidney epithelial cells, and a median inhibitory concentration (IC.sub.50) is calculated to be 3.2 μM.

[0152] Western blot detection results show that after the mice are treated with the asperosaponin VI, the phosphorylation level of AMPKa in the polycystic kidney epithelial cells is increased (FIG. 16), which shows that the asperosaponin VI activates the AMPK in the polycystic kidney epithelial cells; and meanwhile, the phosphorylation levels of activation molecules mTOR, p70S6K and 4E-BP1 in proliferation-related signaling pathways are lowered (FIG. 16), which shows that the asperosaponin VI inhibits mTOR signaling pathways by activating the AMPK and then inhibits proliferation of the polycystic kidney epithelial cells.

[0153] The above results show that the asperosaponin VI may inhibit proliferation of the polycystic kidney epithelial cells and then may prevent and treat the polycystic kidney diseases (particularly, autosomal dominant polycystic kidney disease).

[0154] Other types of pentacyclic triterpenoid saponin in the present invention also show the proliferation inhibition effect similar to the asperosaponin VI on the polycystic kidney epithelial cells (Table 5).

TABLE-US-00005 TABLE 5 Proliferation inhibition effect of pentacyclic triterpenoid saponin on polycystic kidney epithelial cells Group n Inhibition rate (%) Control group 6  6.8 ± 2.3 Ginsenoside Ro 6 74.4 ± 2.3*** Ziyuglycoside I 6 69.2 ± 1.7*** Ziyuglycoside II 6 72.1 ± 2.0*** Asiaticoside 6 68.5 ± 1.8*** Madecassoside 6 76.3 ± 2.3*** α-hederin 6 80.5 ± 3.2*** Hederacoside C 6 64.4 ± 2.6*** (After the polycystic kidney epithelial cells were treated for 48 h with 10 μM pentacyclic triterpenoid saponin, the proliferation inhibition rate of the polycystic kidney epithelial cells was detected with a CCK-8 method; and compared with the control group, ***P < 0.001)

Embodiment 11

[0155] Any one of the compounds (50 g) represented by the formula (I)-(XVI), hydroxypropyl methylcellulose E (150 g), starch (200 g), a proper amount of povidone K30 and magnesium stearate (1 g) were mixed, pelletized and tableted to prepare tablets.

[0156] Meanwhile, the mixture could be prepared into oral liquids and capsules based on the oral solution conventional preparation method, the capsule and soft capsule conventional preparation method and other preparation methods in Chinese Pharmacopoeia (2015, 4th edition); or prepared into different dosage forms such as powder, granules, a pill, an injection, a syrup, an inhalant, an ointment, a suppository, a patch and other pharmacally conventional preparation forms by being combined with different carriers including an excipient, an adhesive, a disintegrating agent, a lubricant, a corrigent, a flavoring agent, a coloring agent and a sweetener. Various dosage forms of any one of the compounds represented by the formula (I)-(XVI) may be used for preventing and treating fatty liver diseases, including non-alcoholic fatty liver diseases, particularly, non-alcoholic fatty liver, non-alcoholic steatohepatitis and non-alcoholic steatohepatitis induced hepatic cirrhosis.

[0157] Pharmaceutically acceptable salts of any one of the compounds represented by the formula (I)-(XVI), metal ions (such as sodium, potassium and calcium) or salts formed by pharmaceutically acceptable amine (such as ethylendiamine and tromethamine) or ammonium ions may also be used for preventing and treating the fatty liver diseases, including the non-alcoholic fatty liver diseases, particularly, the non-alcoholic fatty liver, the non-alcoholic steatohepatitis and the non-alcoholic steatohepatitis induced hepatic cirrhosis.

[0158] Any one of the compounds represented by the formula (I)-(XVI) or pharmaceutically acceptable salts or solvates thereof may be used in combination with one or more drugs for resisting the non-alcoholic fatty liver diseases to be used for preventing and treating the fatty liver diseases, including the non-alcoholic fatty liver diseases, particularly, the non-alcoholic fatty liver, the non-alcoholic steatohepatitis and the non-alcoholic steatohepatitis induced hepatic cirrhosis with significant effect. These drugs include AMPK activators, farnesoid X receptor (FXR) activators (such as obeticholic acid, GS-9674, EDP-305 and LJN452), acetyl-CoA carboxylase (ACC) inhibitors (such as GS-0976), apoptosis signal regulating kinase-1 (ASK1) inhibitors (such as Selonsertib), PPAR activators (such as Elafibranor, Saroglitazar, IVA337 and MSDC-0602K), caspase inhibitors (such as Emricasan), stearoyl-CoA desaturase-1 (SCD1) inhibitors (such as Aramchol), long-acting glucagon-like peptide-1 (GLP-1) receptor activators (such as Semaglutide), apical sodium-dependent bile salt transporter (ASBT) inhibitors (such as Volixibat), vascular adhesion protein-1 (VAP-1) inhibitors (such as BI 1467335), CCR5R blocking agents (such as Cenicriviroc) and thyroid hormone receptor b (THR-b) activators (such as MGL-3196).