AU2021257523A1

AU2021257523A1 - Ginsenoside composition having alcoholic fatty liver-preventing and -treating function

Info

Publication number: AU2021257523A1
Application number: AU2021257523A
Authority: AU
Inventors: Zhiguang DUAN; Daidi Fan; Jing He; Jianya YAN
Original assignee: Shaanxi Giant Biotechnology Co Ltd
Current assignee: Shaanxi Giant Biotechnology Co Ltd
Priority date: 2020-04-17
Filing date: 2021-04-19
Publication date: 2022-12-22
Anticipated expiration: 2041-04-19
Also published as: CN111388490B; AU2021257523B2; WO2021209070A1; CN111388490A

Abstract

Disclosed in the present invention is a ginsenoside composition having an alcoholic fatty liver-preventing and -treating function. The pharmaceutical composition able to be used to prevent and/or treat alcoholic fatty liver of the present invention contains a prophylactically and/or therapeutically effective dose of ginsenoside Rk3 and ginsenoside Rh4 as active ingredients, and a pharmaceutically acceptable carrier. In the pharmaceutical composition, the mass ratio between the ginsenoside Rk3 and the ginsenoside Rh4 is 1:0.5-2, and is preferably 1:0.9-1.1. The ginsenoside composition of the present invention is able to markedly improve liver function index abnormalities caused by a large amount of alcohol consumption, reducing the generation of inflammatory infiltrates and fat vacuoles in liver tissue. The present invention provides a synergistically interacting pharmaceutical composition containing ginsenosides Rk3 and Rh4 and having alcoholic fatty liver-preventing and/or -treating effects.

Description

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GINSENOSIDE COMPOSITION HAVING ALCOHOLIC FATTY LIVER-PREVENTING AND -TREATING FUNCTION FIELD OF THE INVENTION

The application relates to the field of biomedicine, in particular to a ginsenoside Rk3/Rh4

composition for preventing and treating alcoholic fatty liver.

BACKGROUND OF THE INVENTION

Fatty liver disease is a common disease. According to the history of heavy drinking, it can be divided

into alcoholic fatty liver (AFL) disease and non-alcoholic fatty liver (NAFLD) disease. AFL is

alcoholic liver damage caused by long-term alcohol consumption. The main reason for the alcoholic

liver damage is the toxic metabolites, oxidative stress and metabolic disorders caused by alcohol

metabolism in liver cells. The pathogenesis of NAFLD is mainly related to IR, abnormal liver fat

metabolism, mitochondrial dysfunction and oxidative stress, genetic variation and metabolic changes,

and susceptibility to cell damage. Although the major cause of the liver diseases is viral hepatitis,

with the rising status of alcohol in the human diet, the incidence of alcoholic fatty liver disease is

getting higher and higher, and the chances of developing liver cirrhosis and liver cancer are much

higher than NAFLD. It has become the second leading cause of liver damage after viral hepatitis.

Polyene phosphatidylcholine is a commonly used drug for adjuvant treatment of alcoholic fatty liver

disease. It can effectively reduce oxygen free radicals, protect liver cell membrane, delay liver

fibrosis, and thus promote the recovery of patients. However, it is clinically believed that single-drug

therapy will cause slower recovery of patients, which is likely to cause intestinal disorders and lead

to diarrhea.

Ginsenoside, the active ingredient in ginseng, is a steroid compound that can affect multiple

metabolic pathways in the body. Studies have reported that ginsenosides Rk3 and Rh4 have obvious

anti-tumor, anti-oxidant, anti-inflammatory, and anti-apoptotic effects. There are also reports in the

literature that ginsenosides CK, Rhl and compositions thereof can be used to improve non-alcoholic

fatty liver fibrosis and insulin resistance drugs, but no research has been conducted on alcoholic fatty

liver; ginsenoside Rgl has a certain effect in the treatment of metabolic disorders caused by drinking,

but whether it has a protective effect on the liver has not been studied, while the combination of ginsenoside Rk3/Rh4 has never been used to improve alcohol-induced fatty liver, liver disease, and hepatic antioxidant reduction, etc.

SUMMARY OF THE INVENTION

The purpose of the present application is to provide a ginsenoside Rk3/Rh4 composition with the

function of preventing and treating alcoholic fatty liver disease.

The inventors found that the combination of ginsenoside Rk3/Rh4 can effectively improve the degree

of liver damage and steatosis caused by drinking, and thus the ginsenoside Rk3/Rh4 composition was

further applied to the treatment and remission of alcoholic fatty liver disease, and it is the first time

that the ginsenoside Rk3/Rh4 composition is used as the main component in the prevention and

treatment of alcoholic fatty liver disease, and the composition has good treatment and improvement

effects on alcoholic fatty liver disease. In a mouse model of alcoholic fatty liver disease, the

Rk3/Rh4 composition can significantly improve the survival rate of drinking mice, reduce the

content of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and reduce

the content of cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL) in serum and liver,

reduce lipid accumulation (oil droplets), inflammatory infiltration of liver tissue and fat vacuoles in

liver caused by alcohol consumption.

That is, the present application comprises:

1. A pharmaceutical composition for the preventing and/or treating alcoholic fatty liver disease,

comprising prophylactically and/or therapeutically effective amount of ginsenoside Rk3 and

ginsenoside Rh4 as active ingredients, and a pharmaceutically acceptable carrier,

wherein in the pharmaceutical composition, the weight ratio of the ginsenoside Rk3 to the

ginsenoside Rh4 is 1:0.5-2, preferably 1:0.9~1.1.

2. The pharmaceutical composition according to item 1, wherein in the pharmaceutical composition,

the weight ratio of the ginsenoside Rk3 to the ginsenoside Rh4 is 1:1.

3. The pharmaceutical composition according to item 1, wherein, based on 100 parts by weight of the

total ginsenosides comprised in the pharmaceutical composition, the total amount of the ginsenoside

Rk3 and the ginsenoside Rh4 comprised in the pharmaceutical composition is 10 parts by weight or

more.

4. The pharmaceutical composition according to item 1, wherein, based on 100 parts by weight of

Rk3 and the ginsenoside Rh4 comprised in the pharmaceutical composition is 100 parts by weight.

5. The pharmaceutical composition according to item 1, wherein the pharmaceutical composition

does not comprise other active ingredients other than the ginsenoside Rk3 and the ginsenoside Rh4

for preventing and/or treating alcoholic fatty liver disease.

6. Use of ginsenoside Rk3 and ginsenoside Rh4 in the preparation of a pharmaceutical composition

for preventing and/or treating alcoholic fatty liver disease, wherein, in the pharmaceutical

composition, the weight ratio of the ginsenoside Rk3 to the ginsenoside Rh4 is 1:0.5-2, preferably

1:0.9~1.1.

7. The use according to item 6, wherein, in the pharmaceutical composition, the weight ratio of the

ginsenoside Rk3 to the ginsenoside Rh4 is 1:1.

8. The use according to item 6, wherein, based on 100 parts by weight of total ginsenosides

comprised in the pharmaceutical composition, the total amount of the ginsenoside Rk3 and the

ginsenoside Rh4 comprised in the pharmaceutical composition is 10 parts by weight or more.

9. The use according to item 6, wherein, based on 100 parts by weight of total ginsenosides

ginsenoside Rh4 comprised in the pharmaceutical composition is 100 parts by weight.

10. The use according to item 6, wherein the pharmaceutical composition does not comprise other

active ingredients other than the ginsenoside Rk3 and the ginsenoside Rh4 for preventing and/or

treating alcoholic fatty liver disease.

The ginsenoside Rk3/Rh4 composition for preventing and treating alcoholic fatty liver disease

according to the present application can be either a medicine or any other preparation form; it can be

used for the purpose of treating related diseases, or it can be used to improve or prevent fatty liver

caused by alcohol consumption. The preparation can be ginsenoside Rk3/Rh4, or a compound

preparation composed of ginsenoside Rk3/Rh4 and other components, wherein ginsenoside Rk3/Rh4

is used as the main component.

The ginsenoside Rk3/Rh4 of the present application can be obtained by extraction, transformation

and separation of ginseng or American ginseng, or by chemical synthesis. Specifically, the ginsenoside Rk3/Rh4 can be obtained by extracting, transforming and separating from the roots, stems and/or leaves of ginseng or American ginseng by utilizing the process methods of the prior art.

In addition, the inventors also found that in the mouse model of alcoholic fatty liver disease, Rk3 or

Rh4 can improve the survival rate of drinking mice, reduce the content of alanine aminotransferase

(ALT) and aspartate aminotransferase (AST) in serum, and reduce the content of cholesterol (TC),

triglyceride (TG), and low-density lipoprotein (LDL) in serum and liver, reduce alcohol-induced

lipid accumulation (oil droplets), inflammatory infiltration of liver tissue, and fat vacuoles in the

liver.

Therefore, the present application also includes:

11.Use of ginsenoside Rk3 in the preparation of a pharmaceutical composition for preventing and/or

treating alcoholic fatty liver disease.

12.The use according to item 11, wherein based on 100 parts by weight of total ginsenosides

comprised in the pharmaceutical composition, the amount of the ginsenoside Rk3 comprised in the

pharmaceutical composition is 5 parts by weight or more, preferably 10 parts by weight or more

, more preferably 20 parts by weight or more, more preferably 50 parts by weight or more, more

preferably 70 parts by weight or more, more preferably 80 parts by weight or more, more preferably

parts by weight or more, more preferably 95 parts by weight or more, more preferably 99 parts by

weight or more, more preferably, 100 parts by weight.

13.The use according to item 11, wherein the pharmaceutical composition does not comprise other

active ingredients other than the ginsenoside Rk3 for preventing and/or treating alcoholic fatty liver

disease.

14.Use of ginsenoside Rh4 in the preparation of a pharmaceutical composition for preventing and/or

treating alcoholic fatty liver disease.

15.The use according to item 14, wherein, based on 100 parts by weight of total ginsenosides

comprised in the pharmaceutical composition, the amount of the ginsenoside Rh4 comprised in the

pharmaceutical composition is 5 parts by weight or more, preferably 10 parts by weight or more,

more preferably 20 parts by weight or more, more preferably 50 parts by weight or more, more

weight or more, more preferably, 100 parts by weight.

16.The use according to item 14, wherein the pharmaceutical composition does not comprise other

active ingredients of preventing and/or treating alcoholic fatty liver disease other than the

ginsenoside Rh4.

The above-mentioned pharmaceutical composition may be, for example, an oral preparation or an

injection. The oral preparation can be, for example, hard capsules, soft capsules, slow-release

capsules, sugar-coated tablets, powders, granules, tablets, medicinal granules, dropping pills,

honeydew pills, syrups or oral liquids; the injections are solution type, mixed suspension type,

emulsion type or lyophilized powder. The above-mentioned pharmaceutical compositions may also

be in external dosage forms, such as ointments, gels, sprays or patches. The above-mentioned

pharmaceutical compositions may comprise adjuvants or other pharmaceutically acceptable carriers.

The ratio of the adjuvant to the active ingredient of the drug can be, for example, the active

ingredient accounts for 30% to 50% by weight, and the adjuvant accounts for 50% to 70% by weight.

The adjuvant can be, for example, one or more of sodium hyaluronate, magnesium stearate, sodium

alginate, starch, microcrystalline cellulose, chitosan, stachyose, adhesive or collagen.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the chemical structural formula of the ginsenoside Rk3

Figure 2 is the chemical structural formula of the ginsenoside Rh4

Figure 3 is a graph showing the results of HE staining of mouse liver tissue in Example 4. Wherein,

1: a graph showing HE staining of liver tissue of mice in control group;

2: a graph showing HE staining of liver tissue of mice in model group;

3: a graph showing HE staining of liver tissue of mice in positive drug control group;

4: a graph showing HE staining of liver tissue of mice in ginsenoside Rk3 group;

: a graph showing HE staining of liver tissue of mice in ginsenoside Rh4 group;

6: a graph showing HE staining of liver tissue of mice in ginsenoside Rk3/Rh4 composition group 1;

7: a graph showing HE staining of liver tissue of mice in ginsenoside Rk3/Rh4 composition group 2;

8: a graph showing HE staining of liver tissue of mice in ginsenoside Rk3/Rh4 composition 3

groups;

9: a graph showing HE staining of liver tissue of mice in ginsenoside Rgl group;

: a graph showing HE staining of liver tissue of mice in ginsenoside CK/Rhl group.

DETAIL DESCRIPTION OF THE INVENTION

First, in one aspect, the present application provides a pharmaceutical composition that can be used

for the prevention and/or treatment of alcoholic fatty liver disease, comprising prophylactically

and/or therapeutically effective amount of ginsenoside Rk3 and ginsenoside Rh4 as active

ingredients, and a pharmaceutically acceptable carrier,

In the pharmaceutical composition, the weight ratio of the ginsenoside Rk3 to the ginsenoside Rh4 is

1:0.5-2.

In this specification, the ginsenoside Rk3 refers to the compound shown in Figure 1, and its

molecular formula is C 3 6H 6 0 0 8 .

The above-mentioned ginsenoside Rk3 is a known compound, and can be prepared by methods

known in the art, for example, by adding an organic acid solution to to triol group ginsenosides in a

directional conversion reaction under high temperature and high pressure conditions to obtain a

higher-purity ginsenoside Rk3 crude product, and then purified by high-performance liquid phase

separation and other methods to obtain pure ginsenoside Rk3 with a purity of >98%.

In this specification, the ginsenoside Rh4 refers to the compound shown in Figure 2, and its

molecular formula is C 3 6H 6 0 0 8 .

The above-mentioned ginsenoside Rh4 is a known compound and can be prepared by methods

known in the art. For example, triol group ginsenosides are added with organic acid solutions, and

after a directional conversion reaction under high temperature and high pressure conditions, the pure

ginsenoside Rh4 with a purity of >98% is obtained by separation and purification.

The inventor found that the combination of the ginsenoside Rk3 and the ginsenoside Rh4 according

to the ratio range defined in the present application showed synergistic effect in the prevention and/or

treatment of alcoholic fatty liver disease (CIvalue is less than 1, preferably CI value is less than 0.8,

more preferably less than 0.7). The inventors also found that within the above ratio range, the

ginsenoside Rk3 and the ginsenoside Rh4 are also low in toxicity. Moreover, the inventors found that

if the contents of the ginsenoside Rk3 and the ginsenoside Rh4 in the pharmaceutical composition

are not within the ratio range defined in the present application, there is no synergistic effect.

Considering the significant synergistic effect, in the composition, the weight ratio of the ginsenoside

Rk3 and the ginsenoside Rh4 is preferably 1:0.9~1.1, more preferably 1:1.

In the pharmaceutical composition of the present application, other ginsenosides may or may not be

comprised. When the pharmaceutical composition of the present application comprises other

ginsenosides, from the perspective of better playing the role of synergy, the content of the

ginsenoside Rk3 and the ginsenoside Rh4 is preferably 50 parts by weight or more, more preferably

parts by weight or more, more preferably 70 parts by weight or more, more preferably 80 parts by

weight or more, more preferably 90 parts by weight or more, more preferably 95 parts by weight or

more, more preferably 99 parts by weight or more, more preferably 100 parts by weight (that is, the

pharmaceutical composition comprises only the above two ginsenosides), based on 100 parts by

weight of total ginsenosides comprised in the pharmaceutical composition. The purity of ginsenoside

Rk3 used in the pharmaceutical composition of the present application can be more than 98%, and

the purity of ginsenoside Rh4 used in the pharmaceutical composition of the present application can

be more than 98%.

The content of the total ginsenosides can be determined by the vanillin method, and the contents of

the ginsenosides Rk3 and Rh4 can be determined by the HPLC method.

The pharmaceutical composition of the present application may comprise other active ingredients for

preventing and/or treating alcoholic fatty liver disease, or may not comprise active ingredients for

preventing and/or treating alcoholic fatty liver disease (that is, using the ginsenoside Rk3 and the

ginsenoside Rh4 as the only active ingredient for preventing and/or treating alcoholic fatty liver

disease).

On the other hand, the inventors found that the pharmaceutical composition of the present application

has a significant preventive and/or therapeutic effect on alcoholic fatty liver disease. Therefore, the

present application also provides the use of the pharmaceutical composition of the present

application in the preparation of a medicament for preventing and/or treating alcoholic fatty liver

disease.

Pharmaceutically acceptable carriers, such as adjuvants, may be comprised in the pharmaceutical

compositions of the present application. There are no special restrictions on the adjuvants in the

pharmaceutical composition of the present application, for example, the adjuvants commonly used in

medicines or health products in the technical field can be used. Specifically, the adjuvants are starch,

dextrin, lactose, mannitol, sodium hypromellose, xanthan gum, protein sugar, etc.

The dosage form of the pharmaceutical composition of the present application is not particularly

limited, for example, it may be an oral dosage form or an injection dosage form. The oral dosage

form can be a liquid dosage form or a solid dosage form. The oral dosage forms can be, for example,

hard capsules, soft capsules, slow-release capsules, compressed tablets, sugar-coated tablets, powders,

granules, dropping pills, honeydew pills, syrups or oral liquids; the injection dosage forms can be, for

example, solution forms, suspension type, emulsion type or lyophilized powder. The administration

mode of the pharmaceutical composition for preventing and/or treating alcoholic fatty liver disease

can be, for example, oral administration, drip or injection.

When preparing a solid preparation for oral use, after adding an excipient and optionally a binder, a

disintegrating agent, a lubricant, a coloring agent, a flavoring agent, etc. to the main drug, tablets,

coated tablets, granules, fine granules, powders, capsules, etc can be prepared according to a

conventional method.

As an excipient, for example, lactose, corn starch, white sugar, glucose, sorbitol, crystalline cellulose,

silicon dioxide, etc. can be used; as a binder, for example, polyvinyl alcohol, ethyl cellulose, methyl

cellulose, Arabic gum, hydroxypropyl cellulose, hydroxypropyl methylcellulose, etc. can be used; as

a lubricant, for example, magnesium stearate, talc, silicon dioxide, etc. can be used; as a coloring

agent, coloring agents allowed to be added in drugs can be used; as a flavoring agent, cocoa powder,

menthol, aromatic acid, peppermint oil, borneol, cinnamon powder can be used. Of course, the

above-mentioned tablets and granules can also be coated with sugar coating, gelatin coating, and

other necessary outer coatings.

When preparing injections, pH adjusters, buffers, suspending aids, solubilizers, stabilizers, isotonic

agents, preservatives, etc. can be added to the main drug as required, and then intravenous,

subcutaneous, and intramuscular injections can be prepared according to conventional methods. At

this time, if necessary, a freeze-dried product can be prepared by a conventional method.

Examples of the suspending aid include methyl cellulose, Tween 80, hydroxyethyl cellulose, Arabic

gum, tragacanth gum powder, sodium carboxymethyl cellulose, polyoxyethylene sorbitan

monolaurate, and the like.

Examples of the solubilizer include polyoxyethylene hydrogenated castor oil, Tween 80, niacinamide,

polyoxyethylene sorbitan monolaurate, polyethylene glycol, castor oil fatty acid ethyl ester, and the

like.

Moreover, examples of the stabilizer include sodium sulfite, sodium metasulfite, etc., examples of the preservative include methylparaben, ethylparaben, sorbic acid, phenol, cresol, chlorocresol, etc.

Tumors can be treated by administering the pharmaceutical composition of the present application to

a subject. The subject may be a mammal, such as a human, a rat, a rabbit, a sheep, a pig, a cow, a cat,

a dog, a monkey, etc., preferably a human.

The pharmaceutical composition of the present application can be administered orally or

parenterally.

The dosage varies depending on the degree of symptoms, patient age, sex, body weight, difference in

sensitivity, administration method, administration period, administration interval, the nature of the

pharmaceutical preparation, the type of active ingredient, etc. There is no particular limitation, but

usually the dosage for an adult (with body weight 60Kg) is g-30000mg daily, preferably 10tg~

3000mg daily, more preferably 100g~2000mg daily, more preferably 1mg~1000mg daily, more

preferably 5~900mg daily, more preferably 10mg~500mg daily, more preferably 100mg~300mg

daily (based on the total amount of the ginsenoside Rk3 and the ginsenoside Rh4), and the

above-mentioned dosage can usually be administered 1 to 3 times a day.

Examples The present application will be further described below through specific examples. The following embodiments describe the present application by way of example only, but the protection scope of the present application is not limited thereto.

Example 1 Preparation of the ginsenoside Rk3/Rh4 composition tablets for preventing and treating alcoholic fatty liver disease 100 g of ginsenoside Rk3 with a purity of > 98%, 200 g of ginsenoside Rh4 with a purity of > 98%, and 500 g of medicinal adjuvants commonly used in tablet preparation were taken, mixed evenly, pressed into tablets, dried and packed to obtain 2000 tablets of the present application, each tablet comprising 150mg of active ingredients.

Example 2 Preparation of the ginsenoside Rk3/Rh4 composition capsules for preventing and treating alcoholic fatty liver disease

100 g of ginsenoside Rk3 with a purity of >98%, 100 g of ginsenoside Rh4 with a purity of >98%,

and 600 g of commonly used medicinal adjuvants were taken and mixed evenly, and then put into

capsules currently on the market to obtain 2000 capsules of the present application, each capsule

comprising 100mg of active ingredients.

Example 3 Preparation of the ginsenoside Rk3/Rh4 composition oral liquid for preventing and

treating alcoholic fatty liver disease

g of ginsenoside Rk3 with a purity of > 98% and 80 g of ginsenoside Rh4 with a purity of > 98%

were taken, mixed evenly, and then an appropriate amount of flavorings, such as crystalline fructose,

stachyose, citric acid, and pectin, and diluent water were added by conventional methods. The

resulting mixture was heated to dissolve, homogenized, filled, and sterilized to prepare 1,000 bottles

of the oral liquid of the present application, each bottle of the oral liquid comprising 120 mg of active

ingredients.

Example 4 Efficacy verification test of ginsenoside Rk3/Rh4 composition in preventing and

treating alcoholic fatty liver disease

100 adult male mice (18-22 grams) were randomly divided into normal control group; model group;

drug administration group: the composition group 1 of the present application in Example 2 (Rk3

mg/kg, Rh4 15mg); the composition group 2 of the present application (Rk3 7.5mg/kg, Rh4

22.5mg), the composition group 3 of the present application (Rk3 22.5mg/kg, Rh4 7.5mg),

ginsenoside Rk3 (30mg/kg) group, ginsenoside Rh4 (30mg/kg) kg) group, polyene

phosphatidylcholine (30mg/kg) positive drug control group, ginsenoside Rgl (30mg/kg) group,

ginsenoside CK/Rhl (CK 15mg/kg, Rhl 15mg/kg) group, a total of 10 groups, except for the normal

control group, the mice in the other groups were given 50% ethanol by gavage once a day, and the

ethanol dose was 12 mL/kg BW for 4 consecutive weeks. Mice in the normal control group were

given an equal volume of purified water by gavage. The test drug was prepared into a suspension

with purified water, and it was administered by gavage once a day. The normal control group and the

model control group were given the same amount of purified water by gavage, and the gavage

volume was 10ml/kg/d. During the experiment, the mice had free access to food and water, and were

weighed twice a week, and the tested sample amount was adjusted according to body weight. The model control group and each sample group were given 50% ethanol by gavage at the end of the experiment, and the mice were given 12ml/kg BW by gavage. The negative control group was given purified water and fasted for 16 hours. After the animals were anesthetized by intraperitoneal injection of 60 mg/kg BW pentobarbital sodium solution, blood was collected from the abdominal aorta, and liver tissue was collected to detect various indicators: serum ALT, AST and LDL, serum

TC, serum TG and LDL content, liver tissue TG content and histopathological examination. The test

results were expressed as mean plus or minus standard deviation( xs), and the statistical processing

method adopts one-way analysis of variance; the two-drug interaction index CI=AB/(AxB) was

calculated, wherein T was the measured index value of the drug administration group, and C was the

measured index value of the model group, AB was the T/C value of the two-drug combination group,

and A and B were the T/C values of the single-drug action group. When CI < 1, the two drugs had

synergistic effect, and when CI < 0.7, the synergistic effect was very significant (refer to David

H.Kem, Carol R.Morgan, and Susanne U.Hildebrand-Zanki. In vitro and in vivo interaction between

cisplatin and topotecan in ovarian carcinoma systems[J]. Cancer Research, 1988, 48.; and Li Xingqi.

High potency of lidamycin on glioma suppression and its synergism with temozolomide:

involvement of enhanced antiangiogenesis and apoptosis induction[D]. China Union Medical

College, 2009.).

The experimental results were as follows:

Table 1. The influence of the composition of the present application on mouse body weight, liver

weight, liver coefficient (xs)

group dose (mg/kg) weight (g) liver weight (g) liver coefficient(%)

blank control group 36.35±1.20 1.42±0.21AA 3.91±0.20AA

model group 35.62±2.18 1.81±0.37** 5.08±0.33**

polyene 30 36.02±2.13 1.67±0.36A 4.63±0.36*A phosphatidylcholine

Ginsenoside Rk3 30 36.21±1.43 1.75±0.31*A 4.82±0.55*A

Ginsenoside Rh4 30 35.83±1.35 1.74±0.28*A 4.85±0.44*A

Composition 1 30 36.12±1.09 1.46±0.23AA 4.04±0.36AA

Composition 2 30 36.08±1.23 1.71±0.21*A 4.76±0.52*A

Composition 3 30 35.96±1.31 1.77±0.26*A 4.71±0.34*A

Ginsenoside Rgl 30 36.18±1.25 1.72±0.19*A 4.75±0.36A

Ginsenoside CK/Rh1 30 35.92±1.92 1.75±0.23A 4.88±0.29A

CI index of Composition - - - - 0.88 0.88 1

CI index of Composition - - - - 1.02 1.03 2

CI index of Composition - - - - 1.05 1.02 3

Note: **P<0.01, *P<0.05 vs. blank control; AAP<0.01,AP<0.05 vs. model group

There was no significant difference in body weight among the groups (P>0.05); compared with the

blank control group, the liver weight and liver coefficient of the model group were significantly

increased (P<0.01). Compared with the model group, the liver coefficient of each administration

group decreased significantly (P < 0.05), among which the liver coefficient of the Rk3/Rh4 group

decreased extremely significantly (P<0.01). The results of this experiment showed that each drug

administration group had obvious inhibitory effect on the hepatomegaly of mice caused by alcohol,

and under the same dose, the composition 1 of the present application was more effective than each

single component, and had a synergistic effect (CI value < 1); compared with polyene

phosphatidylcholine, ginsenoside Rgl and ginsenoside CK/Rhl, the effect of reducing the liver

coefficient of the composition 1 of the present application was more significant.

Table 2. The influence of the composition of the present application on serum ALT, and AST

activity of mice with alcoholic fatty liver disease( xs)

dose ALT activity AST activity group (mg/kg) (U/L) (U/L)

blank control group 32.43±3.07AA 52.17±8.29AA model group 61.82±8.62** 126.93±19.45** polyene 30 43.93±4.16AA 82.12±11.33AA phosphatidylcholine

Ginsenoside Rk3 30 53.29±7.3 1A 91.23±11.85A*

Ginsenoside Rh4 30 51.87±6.92A 93.58±19.96A*

Composition 1 30 40.28±3.59AA 62.98±9.73AA

Composition 2 30 49.87±4.96A 83.59±19.01A*

Composition 3 30 50.06±6.38A 77.42±11.98A*

Ginsenoside Rg1 30 54.84±6.11A 94.73±9.87A

Ginsenoside CK/Rhl 30 49.72±4.93AA 93.09±11.36A

CI index of Composition 1 0.89 0.94

CI index of Composition 2 1.24

CI index of Composition 3 1.15

Note: **P<0.01, *P<0.05 vs. blank control; AAP<0.01,AP<0.05 vs. model group

ALT and AST are the landmark indicators of liver damage. The content of ALT and AST in normal

serum is very low. Only after liver damage, ALT and AST in hepatocytes are released into the blood

by cell rupture, and the reactive oxygen species generated during the metabolism of ethanol by the

liver can directly cause oxidative damage to liver cells. As could be seen from Table 2, the animal

serum ALT and AST activities of each administration group and the model group had significant

differences (P<0.05), and the animal serum ALT and AST activities of the composition Rk3/Rh4

group of the present application were extremely significant different from those of the model group

(P<0.01), significant lower than those of the model group, but not significantly different from those

of the blank control group (P>0.05). The test results showed that the composition of the present

application could significantly reduce the activities of abnormally elevated ALT and AST in the

serum of mice with alcoholic fatty liver disease, under the same dose, the composition 1 of the

present application had a stronger effect on reducing the activity of ALT and AST than each single

component, as well as polyene phosphatidylcholine, ginsenoside Rgl, and ginsenoside CK/Rhl.

After calculation, the combination index CI of composition 1 of the ginsenoside Rk3 and the ginsenoside Rh4 was less than 1, while the combination index CI of composition 2 and composition

3 was greater than 1, indicating that the composition 1 of the present application had a good

synergistic effect and had a good protective effect on liver cell damage caused by drinking.

Table 3. The influence of composition of the present application on serum TC, TG, LDL content and

liver tissue TG content of mice with alcoholic fatty liver disease (xs) Serum TC Serum TG Serum LDL liver tissue TG group (mmol/L) (mmol/L) (mmol/L) (mmol/L)

blank control group 8.84±0.28AA 1.97±0.12AA 7.23±0.56AA 1.82±0.12AA

model group 12.59±0.27** 3.02±0.17** 13.28±0.67** 2.98±0.21**

polyene 10.29±0.21A 2.47±0.20AA 9.72±0.35A 2.61±0.18A phosphatidylcholine

Ginsenoside Rk3 11.39±0.25 2.83±0.18A 10.98±0.62*A 2.72±0.17*

Ginsenoside Rh4 11.25±0.17 2.76±0.21A 10.82±0.59*A 2.65±0.16*A

Composition 1 9.08±0.15AA 2.09±0.15AA 8.12±0.41AA 1.97±0.12AA

Composition 2 10.95±0.19 2.66±0.24A 9.87±0.53*A 2.61±0.13*A

Composition 3 11.03±0.18 2.59±0.19A 9.38±0.51*A 2.47±0.12*A

Ginsenoside Rgl 10.21±0.23A 2.44±0.19AA 9.62±0.16A 2.69±0.21A

Ginsenoside CK/Rh1 9.91±0.31A 2.69±0.21A 9.81±0.29A 2.33±0.18AA

CI index of Composition 1 0.89 0.81 0.91 0.81

CI index of Composition 2 1.08 1.03 1.10 1.08

CI index of Composition 3 1.08 1.00 1.05 1.02

Note: **P<0.01, *P<0.05 vs. blank control; AAPS0.01,AP<0.05 vs. model group

Long-term ingestion of alcohol will abnormally increase the serum TC and TG content of mice, and

the liver accumulates cholesterol and triglycerides. As shown in Table 3, the serum TC, TG, LDL

contents and liver tissue TG content of animals in each drug administration group were significantly

different from those in the model group (P<0.05), wherein the serum TC, TG, LDL contents and

liver tissue TG content of animals in the composition group of the present application were extremely significantly different from those of the model group (P<0.01). After calculation, the combination index (CI) of composition 1 was less than 1, while the combination index (CI) of composition 2 and composition 3 was greater than 1, indicating that the composition 1 of the present application had a good synergistic effect, while outside the ratio range of the composition of the present application, the combination of the ginsenoside Rk3 and the ginsenoside Rh4 had no synergistic effect, and the damage of alcohol to liver cells was achieved through lipid peroxidation mediated by free radicals. Composition 1 of the present application could effectively reduce the contents of TC and TG in the serum of mice, improved LDL levels, inhibited the synthesis of endogenous TC and TG, and improved lipid distribution in the body and deposition in internal organs.

From the pathological sections of the mouse livers, it could be clearly seen that the livers of the mice

in the control group were normal in size, red, soft and sharp; the livers of the mice in the model

group were significantly enlarged, gray-white, grainy, and greasy on the cut surface; the liver

morphology of each treatment group was improved compared with that of the model group, but the

liver morphology of the Rk3/Rh4 composition group was closer to that of the control group

compared to the other treatment groups. The results of HE staining of the liver (Figures 3, 1-10)

showed that the hepatocytes in the blank control group were normal in size and shape, neatly

arranged, with large and round nuclei in the center, and the cytoplasm was uniformly colored. There

was no lipid droplet deposition in the hepatocytes, the morphology and structure of the hepatocytes

were normal, and there was no steatosis and inflammatory changes; the hepatocytes in the model

group were swollen and the boundaries were unclear. A large number of vacuolar-like lipid droplets

could be seen in the cells with different sizes. Steatotic hepatocytes were distributed in foci. Liver

tissue was almost replaced by fat vacuoles. Ballooning change of hepatocyte took place, and there

was inflammatory cell infiltration in the portal area; in ginsenoside Rk3 group, ginsenoside Rh4

group, composition 2 group, composition 3 group, positive drug blank control group, ginsenoside

Rgl group and ginsenoside CK/Rhl group, the fat vacuoles in the liver tissue showed scattered

distribution, lipid accumulation (oil droplets) became smaller, and no obvious inflammatory

inflammatory cells were found. The mice fed with the Rk3/Rh4 composition 1 of the present

application had almost no lipid droplet deposition in the liver tissue, the morphological structure of

the hepatocytes were close to normal, andthere was no steatosis. The above effects were better at medium doses. It showed that the disclosed Rk3/Rh4 composition of the present application could better improve the degree of alcohol-induced liver steatosis in mice.

Example 5 Exploration on the mechanism of ginsenoside Rk3/Rh4 composition in preventing

and treating alcoholic fatty liver disease

In Example 4, the normal control group; model group; Rk3 group, Rh4 group, and Rk3/Rh4 group

were measured for liver superoxide dismutase (SOD), liver reduced glutathione (GSH), and liver

propylene glycol aldehyde (MDA) levels; and the expression levels of related genes in hepatocytes

were determination by molecular chip screening. The test results were expressed as mean plus or

minus standard deviation( xs), and the statistical processing method adopted one-way analysis of

variance;

The experimental results were as follows:

Table 4. The influence of the composition of the present application on liver SOD, GSH and MDA of

mice with alcoholic fatty liver disease( xs) SOD GSH MDA group (U/mg) (mmol/mg) (jtmol/mg)

blank control group 322.4±22.01AA 62.11±0.62AA 1.82±0.12AA

model group 154.34±17.3** 27.73±0.55** 2.98±0.21**

Ginsenoside Rk3 234.13±25.97AA 47.83±0.18AA 2.12±0.17AA

Ginsenoside Rh4 208.23±23.22AA 42.76±0.21*AA 2.25±0.16*AA

Ginsenoside Rk3/Rh4 189.02±20.25AA 52.02±0.72AA 2.03±0.18AA

Note: **P<0.01, *P<0.05vs blank control; AAP<0.01, AP<0.05vs model group

The liver cells of alcoholic fatty liver were very susceptible to oxidative stress, and the antioxidant

capacity of the mice in the model group was weakened, which was manifested as increased levels of

MDA, a harmful product of alcohol metabolism, decreased SOD activity in liver tissue, and

decreased GSH content of reduced glutathione. Compared with the model group, the activities of

SOD and GSH content in liver tissues of the Rk3 group, Rh4 group and Rk3/Rh4 group were significantly increased (P<0.01), and the MDA content was extremely significantly decreased

(P<0.01), indicating that Rk3 and Rh4 had antioxidant activities, which could effectively improve

liver oxidative damage caused by drinking.

Table 5. Statistical table of the effect of the composition of the present application on the relative

expression levels of genes related to important metabolic pathways in mice with alcoholic fatty liver

disease

group Fasn LpL Scdl PPARu

model group vs. blank t4.17 13.18 t6.43 12.96 control group

Rk3 group vs. model 12.92 t 1.13 15.92 t1.84 group

Rh4 group vs. model 12.18 t2.68 13.57 t 1.97 group

Rk3/Rh4 group vs. 13.38 t2.97 17.18 t2.63 model group

Note: In the table, I indicates the down-regulated fold of the corresponding gene in hepatocyte mRNA expression,

and t indicates the up-regulated fold of the corresponding gene in the hepatocyte mRNA expression.

Fatty acid synthase (Fasn), which plays a crucial role in fatty acid synthesis and is responsible for all

catalytic steps in the de novo synthesis of a long-chain palmitate (palmitate) from acetyl-CoA and

malonyl-CoA. The gene expression directly affects the amount of fatty acid synthase and plays an

important role in controlling animal fat deposition. Lipoprotein lipase (LpL) is one of the key

enzymes in fat metabolism. It acts on lipoproteins and mainly decomposes triacylglycerols in

lipoproteins. It is the rate-limiting enzyme for removing triacylglycerols in plasma and promotes the

transfer of cholesterol from lipoproteins, phospholipids and apolipoproteins; LpL also has the ability

to increase the binding of chylomicrons to LP receptors, promoting chylomicron uptake. Sterol

coenzyme A desaturase 1 (Scdl) is the rate-limiting enzyme in the biosynthesis of monounsaturated

fatty acids, plays a central regulatory role in fatty acid metabolism, and is also one of the target genes for leptin; leptin is closely related to diabetes, obesity, and fatty liver, and is one of the hot areas of metabolic diseases in recent years.

Under the experimental conditions, the model group can increase the synthesis of Fasn and decrease

the synthesis of LpL, which is the main culprit leading to excessive deposition of fat in liver cells and

the primary factor causing fatty liver. Rh4 and the Rk3/Rh4 composition can significantly increase

the synthesis of LpL, reduce the synthesis of fatty acid in the liver tissue, strengthen the

decomposition of fat, and have positive significance for relieving liver steatosis. In addition, the

up-regulation of Scdl gene expression in the model group may be one of the most important

molecular mechanisms of alcoholic steatosis, but each drug administration group can significantly

down-regulate the expression of Scdl gene.

It can be known from the above pharmacodynamic experiments that the composition of the present

application has the effect of preventing and treating alcoholic fatty liver disease, and long-term

administration has no side effects.

Finally, it should be noted that the above description is only a preferred embodiment of the present

application, and under the enlightenment of the application, those ordinary skilled in the art can make

a variety of similar expressions without departing from the purpose and claims of the application,

and such transformations fall within the scope of protection of the application..

Industrial Applicability

The present application provides a pharmaceutical composition comprising ginsenosides Rk3 and

Rh4, which is synergistic and has the effect of preventing and/or treating alcoholic fatty liver disease.

Claims

ginsenoside Rh4 is 1:0.5-2, preferably 1:0.9~1.1.

2. The pharmaceutical composition according to claim 1, wherein in the pharmaceutical composition,

the weight ratio of the ginsenoside Rk3 to the ginsenoside Rh4 is 1:1.

3. The pharmaceutical composition according to claim 1, wherein based on 100 parts by weight of

the total ginsenosides comprised in the pharmaceutical composition, the total amount of the

ginsenoside Rk3 and the ginsenoside Rh4 comprised in the pharmaceutical composition is 10 parts

by weight or more.

4. The pharmaceutical composition according to claim 1, wherein based on 100 parts by weight of

5. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition

for preventing and/or treating alcoholic fatty liver disease.

for preventing and/or treating alcoholic fatty liver disease, wherein in the pharmaceutical

1:0.9~1.1.

7. The use according to claim 6, wherein in the pharmaceutical composition, the weight ratio of the

ginsenoside Rk3 to the ginsenoside Rh4 is 1:1.

8. The use according to claim 6, wherein based on 100 parts by weight of total ginsenosides

9. The use according to claim 6, wherein based on 100 parts by weight of total ginsenosides

10. The use according to claim 6, wherein the pharmaceutical composition does not comprise other

treating alcoholic fatty liver disease.

TFE00387AU 1/2

OH H

H

OH H O O OH

OH O OH HO O

OH

Figure 1 5

OH H

H

OH H O O OH

OH O OH HO O

OH

OH Figure 2 10

15

1005 96.7