CN118615340A

CN118615340A - Use of extract of apices Apii for preventing or treating liver diseases

Info

Publication number: CN118615340A
Application number: CN202310261198.8A
Authority: CN
Inventors: 高永良; 王伽伯; 杨红军; 马志涛
Original assignee: Individual
Current assignee: Individual
Priority date: 2023-03-10
Filing date: 2023-03-10
Publication date: 2024-09-10

Abstract

The present invention relates to the use of an extract of apices Apii for the prevention or treatment of liver diseases. The invention particularly relates to application of a celery-locust extract or a pharmaceutical composition containing the same in preparing medicines for preventing and/or treating liver diseases, wherein the celery-locust extract is an alcohol extract of celery seeds and pagodatree flower buds in a weight ratio of 1:1-4:1, and the liver diseases comprise hepatitis, alcoholic fatty liver, non-alcoholic fatty liver, liver fibrosis and liver cirrhosis, and particularly the alcoholic liver diseases or the non-alcoholic liver diseases. On the basis of evaluating the drug effect through a mouse model, clinical experiments of adult patients prove that the celery-locust extract can be effectively used for preventing or treating various liver diseases, is safe, effective and controllable in quality, and has clinical application value.

Description

Use of extract of apices Apii for preventing or treating liver diseases

Technical Field

The invention relates to application of an extract of apices sophorae in preventing or treating various liver diseases, in particular to application in preparing medicines for preventing or treating liver diseases of mammals including human beings.

Background

Common liver diseases include hepatitis, liver cirrhosis, liver abscess, primary liver cancer, etc., and the hepatitis is mainly chronic hepatitis and is classified into chronic viral hepatitis, autoimmune hepatitis, drug-induced toxic hepatitis, hereditary disease, and other chronic hepatitis with unknown causes. Viral hepatitis is most common with hepatitis b.

Liver disease is very dull and the most prominent symptoms are tiredness, weakness and poor appetite. Common symptoms include distending pain or discomfort, nausea, greasiness, fullness after eating or jaundice, dry mouth, dry or loose stool, yellow urine, low fever, dizziness, tinnitus, sallow complexion, etc. In the case of cirrhosis, in addition to the clinical manifestations of hepatitis, ascites, abdominal wall vascular herniation, edema around the body, oliguria, palms of the liver, spider nevi, and severe cases may also have major bleeding.

Liver disease types can be classified into viral liver disease and non-viral liver disease according to pathogenesis:

Viral liver disease: is a group of infectious diseases mainly caused by liver injury and is diagnosed according to etiology, and at least 5 hepatitis viruses, namely hepatitis A, B, C, D and E viruses, respectively cause viral hepatitis A, B, C, D and E.

Non-viral liver disease includes the following:

Alcoholic fatty liver disease: is a liver injury disease caused by long-term drinking (alcoholism).

Nonalcoholic fatty liver disease: refers to a clinical pathological syndrome which is mainly characterized by excessive deposition of fat in liver cells caused by alcohol removal and other clear liver damage factors.

Drug or poison liver disease: the toxic hepatitis is hepatitis or liver lesion caused by chemical poison (such as phosphorus, arsenic, carbon tetrachloride, etc.), medicine or biotoxin.

Metabolic abnormality liver disease: liver diseases caused by poor metabolism of certain substances in the body.

Fatty liver disease: refers to the pathological changes of excessive fat accumulation in liver cells caused by various reasons. The increase in fat content of hepatocytes may be caused by alcoholism, diabetes, hyperlipidemia, overweight, and the like. Clinically, the pathogenesis of fatty liver is represented by increased in vivo adipose tissue, increased in vivo fatty acid and free fatty acid release, and the fatty liver becomes a main energy supply substance of the organism, and the utilization of glucose is reduced. In general, the decrease in glucose utilization and the increase in glucose content in blood can stimulate insulin secretion to inhibit free fatty acid release, but when the body fat is greatly increased, the absolute amount of free fatty acid release is increased even if it is inhibited by insulin, so that excessive fatty acid is greatly entered into liver to be synthesized into triglyceride, thus fatty liver is formed.

Liver disease including fatty liver has become a killer for human health, but drugs for preventing and treating liver disease are not common in the prior art.

Chinese patent ZL201610313303.8 discloses an extract of apices for preventing or treating gout, and unexpectedly, the inventors continue to find that the extract of apices (chinese patent ZL 201610313303.8) has preventive and/or therapeutic uses for liver diseases in mammals including humans.

Disclosure of Invention

The invention aims to provide a use of a celery-sophora japonica extract in preparing a medicament for preventing and/or treating liver diseases of mammals including human beings, wherein the celery-sophora japonica extract is an alcohol extract of celery seeds (CELERY SEEDS) and sophora japonica (sophora flower bud) in a weight ratio of 1:1-4:1.

The invention also aims at providing the use of a pharmaceutical composition in the preparation of a medicament for preventing and/or treating liver diseases in mammals including humans, wherein the pharmaceutical composition comprises an extract of apices comprising celery seed and an alcohol extract of sophora flower bud in a weight ratio of 1:1-4:1 and one or more pharmaceutically acceptable carriers.

In one embodiment, the use according to the invention, wherein the extract of celery seed is an alcoholic extract of celery seed and sophora flower bud in a weight ratio of 2:1-4:1.

In another embodiment, the use according to the invention, wherein the alcohol extract is an extract in a C1-C4 alcohol solvent.

In a preferred embodiment, the use according to the invention, wherein the alcoholic extract is an extract in a C1-C4 alcoholic solvent selected from methanol, ethanol, isopropanol, n-butanol.

In another preferred embodiment, the use according to the invention, wherein the alcohol extract is an extract in ethanol.

In another embodiment, the use according to the invention, wherein the alcoholic extract is an extract in an aqueous alcoholic solution.

In a preferred embodiment, the use according to the invention, wherein the alcoholic extract is an extract in an aqueous alcoholic solution and the concentration of the aqueous alcoholic solution is 50% to 80%, preferably 50% to 70% by volume.

In another preferred embodiment, the use according to the invention, wherein the alcoholic extract is an extract in an aqueous ethanol solution and the concentration of the aqueous ethanol solution is 50% to 80%, preferably 50% to 70% by volume.

In a preferred embodiment, the use according to the invention, wherein the extract of apices graveolens is obtained by the following method:

1) Adding the celery seed and the pagodatree flower bud into an alcohol solvent, and carrying out ultrasonic extraction to obtain an extracting solution;

2) Concentrating the extractive solution under reduced pressure, precipitating with water, and vacuum drying the filtrate to obtain herba Apii Graveolentis extract.

In another preferred embodiment, the use according to the invention, wherein the extract of apices graveolens is obtained by the following method:

1) Adding the celery seed and the pagodatree flower bud into an alcohol solvent, and carrying out ultrasonic extraction for 2-4 times, wherein the ultrasonic time is preferably 30-60 minutes each time, so as to obtain an extracting solution;

1) Adding the celery seed and the pagodatree flower bud into the alcohol solvent according to the weight ratio of the alcohol solvent to the celery seed and the pagodatree flower bud of 10:1-8:1, and carrying out ultrasonic extraction for 2-4 times, wherein the ultrasonic time is preferably 30-60 minutes each time to obtain an extracting solution;

1) Adding celery seed and pagodatree flower bud into the ethanol water solution according to the weight ratio of the ethanol water solution to the celery seed and pagodatree flower bud of 10:1-8:1, and carrying out ultrasonic extraction for 2-4 times, wherein the ultrasonic time is preferably 30-60 minutes each time to obtain an extracting solution; the ethanol water solution is 50-80 percent by volume, preferably 50-70 percent by volume;

In one embodiment, the use according to the invention, wherein the liver disease comprises liver injury, hepatitis, fatty liver, liver fibrosis, cirrhosis.

In a preferred embodiment, the use according to the invention, wherein the liver disease is an alcoholic liver disease, e.g. alcoholic liver disease including liver injury, hepatitis, fatty liver, liver fibrosis, cirrhosis.

In another preferred embodiment, the use according to the invention, wherein the liver disease is a non-alcoholic liver disease, e.g. non-alcoholic liver disease including liver injury, hepatitis, fatty liver, liver fibrosis, cirrhosis.

In one embodiment, according to the use according to the invention, wherein the extract of Apium graveolens can improve liver function, liver injury, lipid accumulation, collagen deposition, liver index change, liver blood biochemical index change, weight loss or exercise capacity change associated with liver disease.

In another embodiment, the use according to the invention, wherein the extract of apices may improve the upregulation of lipid synthesis gene transcription associated with liver diseases, the upregulation of inflammation-associated gene transcription, the upregulation of pro-fibrotic gene transcription, etc.

In a preferred embodiment, the use according to the invention, wherein the pharmaceutical composition may be in the form of granules, tablets, pellets, drops or capsules.

The pharmaceutical composition according to the invention comprises the extract of apices of the invention as active ingredient and one or more pharmaceutically acceptable carriers.

The extract of the celery-sophora japonica can be used as the only active ingredient in a pharmaceutical composition, and can also be combined with other active agents for preventing and treating liver diseases.

The term "pharmaceutically acceptable" as used herein is useful in preparing a pharmaceutical composition that is generally safe, neither biologically nor otherwise undesirable, and is acceptable for veterinary and human pharmaceutical use.

As used herein, "carrier" refers to any pharmaceutically acceptable carrier, such as diluents, lubricants, or excipients, that is administered with the compound. The carrier may be selected from, for example: filler, disintegrating agent, binder and lubricant.

The filler includes, but is not limited to: starch or its derivatives such as corn starch, pregelatinized starch, modified starch, etc.; cellulose or its derivatives such as microcrystalline cellulose, ethylcellulose, methylcellulose, and the like; sugars such as glucose, sucrose, lactose, mannitol, sorbitol; neutral minerals such as calcium carbonate, calcium hydrogen phosphate, and the like, and combinations thereof. As used herein, the term "diluent" or "filler" is defined as an inert material used to increase the weight and/or size of a pharmaceutical composition, which may be present in the composition in the form of a substance or in the form of a mixture of compounds. Preferably, diluents or fillers are added when the amount of active ingredient and other excipients is too small to obtain a tablet of suitable size. The weight percentage of diluent or filler necessary for the pharmaceutical composition according to the present invention can be determined according to conventional methods well known to those skilled in the art, in particular, after the amount of other excipients such as disintegrants, binders, lubricants, etc. is determined, the diluent or filler is used in an appropriate amount according to the size requirements of the formulation.

Such disintegrants include, but are not limited to: crosslinked polyvinylpyrrolidone, sodium starch glycolate, crosslinked sodium carboxymethylcellulose, low-substituted hydroxypropyl cellulose, and combinations thereof. Further, the pharmaceutical composition comprises 0 to 10% of disintegrant, for example 0 to 8% of disintegrant, for example 0 to 5% of disintegrant, relative to the total weight of the pharmaceutical composition, which may also be used according to the experience of the person skilled in the art; pharmaceutical formulations that use no or little disintegrant are also common. Whereas the solid pharmaceutical compositions of the present invention in the form of a formulation may not have disintegrating properties in some cases, for example when they are in the form of capsules, no disintegrating agent may be added to the solid pharmaceutical compositions of the present invention in these cases.

The adhesive includes, but is not limited to: polyethylene glycol, starch, polyvinylpyrrolidone, hydroxypropyl methylcellulose, and combinations thereof. In view of the inherent adhesiveness of many solid pharmaceutical formulation materials and the ability to wet granulate with water as a wetting agent, it is also possible in the present invention to use water as a wetting agent as a potential binder, although the solid pharmaceutical composition of the present invention removes this water as a wetting agent in the final product. In addition, many solid pharmaceutical formulations are inherently cohesive and can be encapsulated by dry-pressing the powder to directly granulate. It can be seen that the binder may or may not be added to the solid pharmaceutical composition of the present invention. Even with wet granulation techniques, the binder may not be added. If added, the binder may be used in an amount of 0.1 to 10%, 0.2 to 5%, or 0.5 to 2.5% relative to the total weight of the pharmaceutical composition, which may also be used according to the experience of one skilled in the art.

The lubricant (including the glidant) has the function of enabling the powder material to be successfully molded into the preparation, for example, the powder material can be uniformly filled into a capsule shell when a capsule is prepared, for example, the powder material can be uniformly filled into a die of a tablet press when a tablet is prepared, and the sticking is avoided. Examples of lubricants include, but are not limited to: magnesium stearate, calcium stearate, talc, starch, stearic acid, colloidal silicon dioxide, polyethylene glycol. If added, the lubricant may be used in an amount of 0.1 to 10%, 0.2 to 5%, or 0.2 to 2% relative to the total weight of the pharmaceutical composition, which may also be used according to the experience of one skilled in the art.

In a preferred embodiment, the use according to the invention, wherein the daily dose of the extract of apices graveolens ranges from 225mg to 2250mg.

In another preferred embodiment, the use according to the invention, wherein the daily dose of the extract of apices graveolens ranges from 5.5mg/kg to 22mg/kg.

It is well known to those skilled in the art that the amount of drug administered depends on a variety of factors, including but not limited to the following: the activity of the particular compound used, the age of the patient, the weight of the patient, the health of the patient, the patient's integument, the patient's diet, the time of administration, the mode of administration, the rate of excretion, the combination of the drugs, etc. In addition, the optimal mode of treatment, such as the mode of treatment, the daily amount of the compound of formula (I) or the type of pharmaceutically acceptable salt, can be verified according to conventional treatment protocols.

The present invention is illustrated in detail below by way of the drawings and the specific examples, but it should be understood that they are merely illustrative of the invention and do not limit the scope of the invention in any way.

Drawings

FIG. 1 is a photograph of HE staining of liver tissue of a mouse in test example 1. ctrl: control group, etOH: alcoholic liver group, etOH+QH 50mg/kg: 50mg/kg of the extract of apiary and the intervention alcoholic liver group; etOH+QH 100mg/kg: 100mg/kg of the celery-locust extract intervenes in the alcoholic liver group; etOH+QH 200mg/kg: 200mg/kg of the extract of apiary and the intervention alcoholic liver group.

FIG. 2 is a photograph of oil red O staining of liver tissue of a mouse in test example 1. ctrl: control group, etOH: alcoholic liver group, etOH+QH 50mg/kg: 50mg/kg of the extract of apiary and the intervention alcoholic liver group; etOH+QH 100mg/kg: 100mg/kg of the celery-locust extract intervenes in the alcoholic liver group; etOH+QH 200mg/kg: 200mg/kg of the extract of apiary and the intervention alcoholic liver group.

FIG. 3 shows the effect of the extract of Apium graveolens on the weight change of mice caused by alcohol in test example 1. ctrl: control group, etOH: alcoholic liver group, etOH+QH 50mg/kg: 50mg/kg of the extract of apiary and the intervention alcoholic liver group; etOH+QH 100mg/kg: 100mg/kg of the celery-locust extract intervenes in the alcoholic liver group; etOH+QH 200mg/kg: 200mg/kg of the extract of apiary and the intervention alcoholic liver group.

FIG. 4 shows the effect of the extract of Apium graveolens on the liver index of mice induced by alcohol in test example 1. ctrl: control group, etOH: alcoholic liver group, etOH+QH 50mg/kg: 50mg/kg of the extract of apiary and the intervention alcoholic liver group; etOH+QH 100mg/kg: 100mg/kg of the celery-locust extract intervenes in the alcoholic liver group; etOH+QH 200mg/kg: 200mg/kg of the extract of apiary and the intervention alcoholic liver group. * P <0.05, etOH group vs ctrl group; the group EtOH+QH vs EtOH is < 0.05.

FIG. 5 shows the results of the test of the biochemical index of alcohol-induced liver injury by the extract of Apium graveolens of test example 1. FIG. 5A shows the serum glutamic pyruvic transaminase (ALT) index; FIG. 5B shows the serum glutamic-oxaloacetic transaminase (AST) index; in fig. 5C is a serum Triglyceride (TG) index. ctrl: control group, etOH: alcoholic liver group, etOH+QH 50mg/kg: 50mg/kg of the extract of apiary and the intervention alcoholic liver group; etOH+QH 100mg/kg: 100mg/kg of the celery-locust extract intervenes in the alcoholic liver group; etOH+QH 200mg/kg: 200mg/kg of the extract of apiary and the intervention alcoholic liver group. * P <0.05, < P <0.01, < P <0.001, etoh group vs ctrl group; #P <0.05, #P <0.01EtOH+QH group vs EtOH group.

FIG. 6 shows the effect of the extract of Apium graveolens on hair status of mice model of MCD in test example 2. ctrl: control group, MCD: MCD model group, mcd+qh 50mg/kg: 50mg of celery and locust bean extract kg intervention MCD group; MCD+QH 100mg/kg: intervention of 100mg/kg of the extract of apiary in the MCD group; MCD+QH 200mg/kg: 200mg/kg of the extract of Apium graveolens intervenes in MCD.

FIG. 7 shows the effect of the extract of Apium graveolens on the weight change of mice with MCD model in test example 2. ctrl: control group, MCD: MCD model group, mcd+qh 50mg/kg: 50mg of celery and locust bean extract kg intervention MCD group; MCD+QH 100mg/kg: intervention of 100mg/kg of the extract of apiary in the MCD group; MCD+QH 200mg/kg: 200mg/kg of the extract of Apium graveolens intervenes in the MCD group.

FIG. 8 shows the effect of the extract of Apium graveolens on liver index of MCD model mice in test example 2. ctrl: control group, MCD: MCD model group, mcd+qh 50mg/kg: 50mg of celery and locust bean extract kg intervention MCD group; MCD+QH 100mg/kg: intervention of 100mg/kg of the extract of apiary in the MCD group; MCD+QH 200mg/kg: 200mg/kg of the extract of Apium graveolens intervenes in the MCD group. nsP >0.05, mcd group vs ctrl group.

FIG. 9 is a photograph of HE-stained liver tissue of a mouse in test example 2. ctrl: methionine-choline replete group, MCD: methionine-choline deficient group, mcd+qh 50mg/kg, apices extract 50mg/kg intervening MCD group; MCD+QH 100mg/kg Cress Sophora japonica extract 100mg/kg intervenes in the MCD group; MCD+QH 200mg/kg Cress Sophora japonica extract 200mg/kg interfere with the MCD group.

FIG. 10 is a photograph of oil red O staining of liver tissue of a mouse in test example 2. ctrl: control group, MCD: MCD model group, mcd+qh 50mg/kg: 50mg of celery and locust bean extract kg intervention MCD group; MCD+QH 100mg/kg: intervention of 100mg/kg of the extract of apiary in the MCD group; MCD+QH 200mg/kg: 200mg/kg of the extract of Apium graveolens intervenes in the MCD group.

FIG. 11 is a photograph of a Masson stain of liver tissue of a mouse in test example 2. ctrl: control group, MCD: MCD model group, mcd+qh 50mg/kg: 50mg of celery and locust bean extract kg intervention MCD group; MCD+QH 100mg/kg: intervention of 100mg/kg of the extract of apiary in the MCD group; MCD+QH 200mg/kg: 200mg/kg of the extract of Apium graveolens intervenes in the MCD group.

FIG. 12 shows the results of biochemical index detection of liver injury of mice with MCD model by using the extract of Apium graveolens of test example 2. FIG. 12A is an indicator of serum glutamic pyruvic transaminase (ALT); FIG. 12B is a serum glutamic-oxaloacetic transaminase (AST) index. ctrl: control group, MCD: MCD model group, mcd+qh 50mg/kg: 50mg of celery and locust bean extract kg intervention MCD group; MCD+QH 100mg/kg: intervention of 100mg/kg of the extract of apiary in the MCD group; MCD+QH 200mg/kg: 200mg/kg of the extract of Apium graveolens intervenes in the MCD group. * P <0.05, < P <0.01, < P <0.001, < P <0.0001, < mcd group vs ctrl group; #P <0.05, #P <0.01, #P <0.001, MCD+QH group vs MCD group.

FIG. 13 shows the effect of the extract of Apium graveolens on the motor ability of mice with MCD model in test example 2. FIG. 13A is total course data; b of fig. 13 is the rest time data; c of fig. 13 is edge crossing number data; d in fig. 13 is center pass count data. ctrl: control group, MCD: MCD model group, mcd+qh 50mg/kg: 50mg of celery and locust bean extract kg intervention MCD group; MCD+QH 100mg/kg: intervention of 100mg/kg of the extract of apiary in the MCD group; MCD+QH 200mg/kg: 200mg/kg of the extract of Apium graveolens intervenes in the MCD group.

FIG. 14 shows the effect of RT-qPCR on the transcriptional upregulation of lipid synthesis genes in mice with MCD model in test example 2. FIG. 14A is data for Acly (ATP-citrate lyase); FIG. 14B is data for ACC1 (acetyl-CoA carboxylase 1); FIG. 14C is FASN (fatty acid synthetase) data; FIG. 14D is data for SCD1 (stearoyl-CoA 1); FIG. 14E is Srebp a (cholesterol regulatory element binding protein 1 a) data; FIG. 14F is Srebp c (cholesterol regulatory element binding protein 1 c) data; g of fig. 14 is Srebp (cholesterol regulatory element binding protein 2) data. ctrl: control group, MCD: MCD model group, mcd+qh 50mg/kg: 50mg of celery and locust bean extract kg intervention MCD group; MCD+QH 100mg/kg: intervention of 100mg/kg of the extract of apiary in the MCD group; MCD+QH 200mg/kg: 200mg/kg of the extract of Apium graveolens intervenes in the MCD group.

FIG. 15 shows the effect of RT-PCR on the transcriptional upregulation of inflammation-associated genes in mice with MCD model in test example 2. FIG. 15A is data for TNF- α (tumor necrosis factor- α); FIG. 15B is data for IL-6 (interleukin-6); FIG. 15C is IFN- β (interferon- β) data; FIG. 15D is Cxcl (C-X-C motif chemokine 10) data; FIG. 15E is data for IL-1β (interleukin-1β); f in FIG. 15 is data for IL-18 (interleukin-18). ctrl: control group, MCD: MCD model group, mcd+qh 50mg/kg: 50mg of celery and locust bean extract kg intervention MCD group; MCD+QH 100mg/kg: intervention of 100mg/kg of the extract of apiary in the MCD group; MCD+QH 200mg/kg: 200mg/kg of the extract of Apium graveolens intervenes in the MCD group.

FIG. 16 shows the effect of RT-PCR on the transcription of upregulated pro-fibrotic genes in mice in the MCD model tested in test example 2. FIG. 16A is data for α -SMA (α -smooth muscle actin); FIG. 16B is the data of COL1A1 (collagen 1A 1); FIG. 16C is data for TGF-. Beta.1 (transforming growth factor-. Beta.). ctrl: control group, MCD: MCD model group, mcd+qh 50mg/kg: 50mg of celery and locust bean extract kg intervention MCD group; MCD+QH 100mg/kg: intervention of 100mg/kg of the extract of apiary in the MCD group; MCD+QH 200mg/kg: 200mg/kg of the extract of Apium graveolens intervenes in the MCD group.

FIG. 17 shows the effect of the extract of Apium graveolens on hair status of HFD model mice in test example 2. ctrl: a control group; HFD: a set of HFD models; HFD+QH2 50mg/kg: 50mg/kg of the extract of apices Apii have been used to intervene in HFD group; HFD+QH 100mg/kg: intervention HFD group of 100mg/kg of celery-locust extract; HFD+QH 200mg/kg: 200mg/kg of the extract of Apium graveolens intervenes in the HFD group.

FIG. 18 shows the effect of the extract of Apium graveolens on body weight of HFD model mice in test example 2. ctrl: a control group; HFD: a set of HFD models; HFD+QH2 50mg/kg: 50mg/kg of the extract of apices Apii have been used to intervene in HFD group; HFD+QH 100mg/kg: intervention HFD group of 100mg/kg of celery-locust extract; HFD+QH 200mg/kg: 200mg/kg of the extract of Apium graveolens intervenes in the HFD group. (nsP >0.05, P <0.05, HFD group vs ctrl group; nsP >0.05hfd+qh group vs HFD group).

FIG. 19 shows the effect of the extract of Apium graveolens on the feeding of mice with HFD model in test example 2. ctrl: a control group; HFD: a set of HFD models; HFD+QH2 50mg/kg: 50mg/kg of the extract of apices Apii have been used to intervene in HFD group; HFD+QH 100mg/kg: intervention HFD group of 100mg/kg of celery-locust extract; HFD+QH 200mg/kg: 200mg/kg of the extract of Apium graveolens intervenes in the HFD group. nsP >0.05, P <0.05, hfd group vs ctrl group.

FIG. 20 is a photograph of HE-stained liver tissue of a mouse in test example 2. ctrl: a control group; HFD: a set of HFD models; HFD+QH2 50mg/kg: 50mg/kg of the extract of apices Apii have been used to intervene in HFD group; HFD+QH 100mg/kg: intervention HFD group of 100mg/kg of celery-locust extract; HFD+QH 200mg/kg: 200mg/kg of the extract of Apium graveolens intervenes in HFD.

FIG. 21 is a photograph of a mouse liver tissue stained with oil red O in test example 2. ctrl: a control group; HFD: a set of HFD models; HFD+QH2 50mg/kg: 50mg/kg of the extract of apices Apii have been used to intervene in HFD group; HFD+QH 100mg/kg: intervention HFD group of 100mg/kg of celery-locust extract; HFD+QH 200mg/kg: 200mg/kg of the extract of Apium graveolens intervenes in the HFD group.

FIG. 22 shows the effect of the extract of Apium graveolens on the biochemical index of liver injury in HFD model mice in test example 2. FIG. 22A is an indicator of serum glutamic pyruvic transaminase (ALT); FIG. 22B is a serum glutamic-oxaloacetic transaminase (AST) index; FIG. 22C is a serum Triglyceride (TG) index; FIG. 22D is a serum total cholesterol (T-CHO) index; FIG. 22E is a serum high density lipoprotein cholesterol (HDL-C) index; FIG. 22F is an index of serum low density lipoprotein cholesterol (LDL-C). Ctrl: a control group; HFD: a set of HFD models; HFD+QH2 50mg/kg: 50mg/kg of the extract of apices Apii have been used to intervene in HFD group; HFD+QH 100mg/kg: intervention HFD group of 100mg/kg of celery-locust extract; HFD+QH 200mg/kg: 200mg/kg of the extract of Apium graveolens intervenes in the HFD group. nsP >0.05, < P <0.01, < P <0.001, < P <0.0001, < hfd group vs ctrl group; nsP >0.05, #p <0.01, #p <0.001, #p <0.0001, #hfd+qh group vs HFD group.

Detailed Description

The following examples are for illustrative purposes only and are not intended to, nor should they be construed to, limit the invention in any way. Those skilled in the art will appreciate that conventional variations and modifications may be made to the following embodiments without departing from the spirit or scope of the invention.

The celery seed and the pagodatree flower bud are purchased from the special market of Anguo Chinese medicinal materials in Hebei province.

Example 1 preparation of an extract of Apium graveolens 1

500G of celery seeds and 500g of pagodatree flower bud are taken, 60% (V/V) ethanol with the weight being 10 times that of the celery seeds is added for ultrasonic extraction for 3 times, and 45 minutes each time. The extract was concentrated under reduced pressure and dried in vacuo to give extract 1 (30 g) of the present invention.

Mixing the above obtained extract 1 with dextrin with equal weight, and encapsulating, wherein each capsule contains extract equivalent to 8g of total medicinal materials (total weight of semen Apii Graveolentis and flos Sophorae Immaturus).

Example 2 preparation of Apium graveolens extract example two

Taking 1000g of celery seeds and 500g of pagodatree flower bud, adding 50% (V/V) ethanol with the weight being 8 times of that of the celery seeds, and carrying out ultrasonic extraction for 2 times each time for 30 minutes. The extract was concentrated under reduced pressure and dried in vacuo to give extract 2 (26 g) of the present invention.

The extract 2 obtained above was mixed with starch of equal weight, and filled into capsules, each capsule containing a composition equivalent to 6g of total medicinal material weight (sum of weight of celery seed and pagodatree flower bud).

Example 3 preparation of an extract of Cress Sophora japonica III

2000G of celery seeds and 500g of pagodatree flower bud are taken, and 70% (V/V) ethanol with the weight being 10 times of that of the celery seeds is added for ultrasonic extraction for 4 times, and 60 minutes each time. The extract was concentrated under reduced pressure and dried in vacuo to give extract 3 (44 g) of the present invention.

Mixing the above obtained extract 3 with dextrin with equal weight, and encapsulating, wherein the weight of the composition contained in each capsule is equivalent to 5g of the total medicinal materials (the sum of the weight of the celery seed and the pagodatree flower bud).

Example 4 preparation of tablets

Using extract 1 prepared in example 1, tablets were prepared according to the following formulation:

mixing extract 1 with microcrystalline cellulose, granulating with HPMC solution (5% water solution), drying, adding crospovidone and micropowder silica gel, mixing, and tabletting.

EXAMPLE 5 preparation of pellets/capsules

Using extract 2 prepared in example 2, pellets (capsules) were prepared according to the following formulation:

The preparation is carried out according to the following method:

(1) Weighing extract 2 of prescription amount, adding microcrystalline cellulose and crospovidone, pulverizing, and mixing;

(2) Adding HPMC solution (5% aqueous solution);

(3) The pellet is prepared by adopting an extrusion-spheronization pellet preparation method: extruding the mixed materials into strips at an extrusion speed of 10-40 r/min; transferring into a rounding device, cutting off extrudate at 40-70Hz, and rounding at 30-50Hz for 10-20 min to obtain micropill;

(4) And (3) transferring the prepared pellets into a drying room for drying under the drying conditions of 30+/-2 ℃ for 12 hours, and encapsulating after drying.

Test example 1 evaluation and study of the effectiveness of the Aphis Sophora extract on the alcoholic fatty liver model of mice

1. Experimental method

1. Evaluation of apiary extract on alcoholic fatty liver animal model

Experimental C57BL/6 male mice, 8-10 weeks old, weight 20-22g, randomly divided into: control group (ctrl group), alcoholic liver group (EtOH group), alcohol+celery tree extract intervention group (EtOH+QH group), 6-10 mice per group, wherein the administration dose of the celery tree extract is 50, 100, 200mg/kg. The extract of the apices and the sophora japonica are prepared into suspension solution by adopting 0.9% sterile physiological saline. In the experiment, NIAAA alcoholic liver disease models are adopted.

Referring to the conversion formula of the dosing amount of human and animals, the dosing amount of the celery-locust extract of the experimental mice is equivalent to 330mg, 659mg and 1319mg oral doses of 60kg adult individuals clinically, and the comparison is shown in table 1.

Table 1 administration of the mouse apices extract with an adult (60 kg) dose control

The specific embodiment is as follows: first, all mice were acclimatized with control fluid feed for 5 days. Thereafter, ctrl groups were fed with control fluid feed for 10 days and given an equal volume of physiological saline; etOH groups were fed with 5% (v/v) alcoholic fluid feed for 10 days and given an equal volume of physiological saline; the EtOH+QH (50, 100, 200 mg/kg) groups were fed with 5% (v/v) alcohol fluid feed for 10 days and administered by gavage once a day at 50, 100, 200mg/kg doses, respectively. On day 16 of molding 6:00, etOH+QH (50, 100, 200 mg/kg) groups were filled with an extract solution of Oenanthe Javanica, and two other groups were filled with an equal volume of physiological saline. After 1 hour, ctrl group was perfused with 9g/kg 45% (wt/v) maltodextrin solution, etOH group and etoh+qh (50, 100, 200 mg/kg) group were perfused with 31.5% (v/v) alcohol solution, and after 9 hours, materials were drawn and mouse serum and liver were collected.

2. Serum collection

The orbit was collected, the blood was collected in a 1.5mL EP tube, allowed to stand at room temperature for 1 hour, centrifuged at 3000rpm for 15 minutes, and serum was collected and stored at-80℃for further use.

3. Pathology detection

Mouse liver tissue was removed and fixed in 4% paraformaldehyde for 24 hours for HE and oil red O staining.

4. Blood biochemical index detection

The "alanine aminotransferase (glutamic pyruvic transaminase/alt/GPT) kit (Lishi method)" kit (microplate method) "," aspartic aminotransferase (glutamic oxaloacetic transaminase/ast/GOT) kit (microplate method) ", and" Triglyceride (TG) assay kit (single reagent GPO-PAP method) (microplate reader and biochemical analyzer) "were used for the measurement of ALT, AST, TG, T-CHO, respectively.

And (3) according to the instruction of the kit, preparing standard curves of all detection indexes and simultaneously measuring a sample to be detected. OD values at detection wavelengths corresponding to the respective indexes were measured using a microplate Reader (bio tek), and ALT/AST/TG values of the respective samples were calculated from the measured standard curves.

(^ns P >0.05, <0.01, <0.001, < P <0.001, < EtOH group vs ctrl group; ^nsP>0.05,^#P<0.05,^## P <0.01, < EtOH+QH group vs EtOH group).

2. Experimental results

The celery-locust extract can effectively interfere with the related liver pathology detection condition of the alcoholic fatty liver of mice

FIG. 1 is a photograph of HE staining of liver tissue of mice. As shown in fig. 1, the ctrl group liver cells are orderly arranged, the liver rope structure is clear, the structure is complete, and no obvious pathological change exists; the EtOH group shows the steatosis of the mixture of severe vesicular property and microvesicle property, the focal balloon-like deformation, the shrinkage of a large number of cell nuclei and the unobvious hepatic cable structure; compared with the EtOH group, each dosage group of EtOH+QH can effectively improve the fatty degeneration of the liver of mice caused by alcohol, obviously reduce fatty cavitation, ensure clear and complete liver cable arrangement, ensure no obvious water degeneration of liver cells, ensure that the prevention and treatment effects of each dosage group of EtOH+QH on alcoholic hepatitis have no obvious difference from pathological results, and ensure good treatment effects at low dosage (50 mg/kg).

(II) the extract of Oenanthe Javanica is effective in improving lipid accumulation in liver of mice

FIG. 2 is a photograph of oil red O staining of liver tissue of mice. As shown in fig. 2, the ctrl group liver cells are orderly arranged, the liver rope structure is clear, no obvious oil red O staining trace exists, and no obvious lipid accumulation exists; the EtOH group has large-area darker oil red O dyeing; each dosage group of EtOH+QH can effectively reduce the dyeing area and intensity of oil red O. And no significant differences were seen between etoh+qh dose groups, good therapeutic effect was seen at low doses. This result suggests that the extract of apices Apii has greatly reduced liver lipid accumulation levels caused by alcoholic liver injury.

(III) the extract of Oenanthe Javanica is effective in improving weight change and liver index change of mice caused by drinking

Figure 3 is the effect of the extract of apices on alcohol-induced weight change in mice, FIG. 4 is the effect of Sophora japonica extract on alcohol-induced liver index changes in mice. As shown in fig. 3, the EtOH group body weight continuously decreased and the etoh+qh groups body weight each appeared to decrease and then increase compared to ctrl group. As shown in fig. 4, the liver index of EtOH group significantly increased compared to ctrl group and the liver coefficients of etoh+qh groups were significantly decreased compared to EtOH group. The result suggests that the extract of apices Apii has the effect of improving weight change and liver coefficient change caused by drinking.

(IV) the extract of the celery-locust is effective in improving changes of liver blood biochemical indexes ALT, AST, TG caused by drinking

FIG. 5 shows the results of the detection of the effect of the extract of Apium graveolens on the biochemical index of alcohol-induced liver injury. Table 2 shows biochemical index data of the extract of apices Apii and Sophora japonica for liver injury caused by alcohol. As shown in fig. 5 and table 2, etOH group ALT, AST, TG was significantly elevated compared to ctrl group; serum ALT, AST, TG was significantly reduced for each dose group of etoh+qh compared to EtOH group. The result shows that the celery-locust extract can effectively improve blood biochemical index ALT, AST, TG change caused by drinking.

TABLE 2 Biochemical index data (mean+ -SEM) of liver injury caused by alcohol with Apium graveolens extract

The pathological and biochemical experimental results indicate that the celery-sophora japonica extract can effectively prevent/interfere with the occurrence of alcoholic fatty liver. In the experiment, the lowest dosage of 50mg/kg is adopted once a day (equivalent to 5.5mg/kg of human oral dosage), which can obviously inhibit liver injury, lipid accumulation, liver index change and liver blood biochemical index (ALT, AST, TG) change caused by drinking.

Test example 2 evaluation and study of the effectiveness of the Cress Sophora extract on non-alcoholic fatty liver disease in mice

1. Experimental method

1. Animal experiments of intervention of celery-locust extract (QH) on non-alcoholic fatty liver disease

The gene expression profile of the mouse model of nonalcoholic hepatitis of methionine/choline deficiency model (MCD model) is relatively close to that of human nonalcoholic steatohepatitis, and can induce visible liver steatosis and rapidly progress to hepatitis and liver fibrosis. However, the MCD model lacks features of clinical nonalcoholic hepatitis metabolic disorders and significant weight loss with test cycle. The non-alcoholic hepatitis model of the high fat diet model (HFD model) is more similar to human non-alcoholic steatohepatitis in pathogenesis. Therefore, the experiment adopts the MCD model and the HFD model to detect the intervention effect of the celery-sophora japonica extract on the non-alcoholic hepatitis.

The herba Apii Graveolentis extract is prepared into suspension with 0.9% sterile physiological saline. The specific experimental scheme is as follows:

Intervention of apiary extract in MCD non-alcoholic steatohepatitis mice experiments: the 6-7 week old C57BL/6 male mice were randomly grouped, with 6-10 mice per group. The method is divided into: control group (ctrl group), MCD non-alcoholic steatohepatitis model group (MCD model group), MCD model and administration of apices extract intervention group (mcd+qh group). QH administration doses were 50, 100, 200mg/kg. Each group treatment was specifically as follows: ctrl groups were fed with methionine/choline rich feed and MCD groups and mcd+qh (50, 100, 200 mg/kg) groups were fed with methionine/choline deficient feed. On the basis, 50 mg/kg of the solution of the apices sophorae extract, 100 mg/kg of the solution of the apices sophorae extract and 200mg/kg of the solution of the apices sophorae extract are respectively administrated to the stomach once a day in week 1-2; the ctrl group and the MCD group were given equal volumes of saline for gastric lavage. The MCD+QH (50, 100, 200 mg/kg) groups at weeks 3-6 were respectively administered with 50, 100, 200mg/kg of the solution of the apices, and the solution of the extracts of the apices were irrigated once every two days, and the ctrl group and the MCD group were administered with an equal volume of physiological saline for the same period of time. After 6 weeks, the serum and liver of the mice were collected. During the treatment, MCD model experiments were photographed 4 weeks after modeling to record the hair status of each group of mice. The influence of QH on the mobility of MCD (micro-control device) caused by nonalcoholic steatohepatitis is examined by using an open field experiment the day before drawing materials, each mouse is independently placed in the open field, the mouse is allowed to freely move for 5 minutes, and data such as the movement track and total movement distance, the resting time, the edge passing times, the central passing times and the like of the mouse are recorded through software.

Intervention of apiary extract in HFD non-alcoholic steatohepatitis mice experiments: c57BL/6 wild male mice at 8 weeks of age were randomly grouped, with 6-10 mice per group. The method is divided into: control group (ctrl group), HFD non-alcoholic steatohepatitis model group (HFD model group), HFD model and administration of apices extract intervention group (hfd+qh group). QH administration doses were 50, 100, 200mg/kg. Each group treatment was specifically as follows: ctrl groups were fed with normal feed, HFD groups and HFD+QH (50, 100, 200 mg/kg) groups were fed with 60kcal% high fat feed. On this basis, 50, 100, 200mg/kg of the solution of the extract of Apium graveolens are respectively administered to the stomach once a day with HFD+QH (50, 100, 200 mg/kg). ctrl group and HFD group were given equal volumes of saline for the same period of time to lavage. After molding for 4 weeks, the status of the hair of the mice was recorded by photographing. Mice feed was recorded 5 consecutive days after 8 weeks. After 12 weeks, mouse serum and liver were collected.

Referring to the formula for converting the dosage of human and animal, the dosage of the apices extract of the mice in the experiment is equivalent to 330mg, 659mg and 1319mg oral dosage of 60kg adult in clinic, and the dosage is shown in the table 1.

2. Serum collection

3. Pathology detection

Mouse liver tissue was fixed with 4% paraformaldehyde for 24 hours and HE stained, oil red O stained, masson stained.

4. Blood biochemical index detection

The "alanine aminotransferase (glutamic pyruvic transaminase/alt/GPT) kit (Lishi method)" kit (microplate method) "," aspartic aminotransferase (glutamic oxaloacetic transaminase/ast/GOT) kit (microplate method) ", and" Triglyceride (TG) assay kit (single reagent GPO-PAP method) (microplate reader and biochemical analyzer) "were used for the measurement of ALT, AST, TG, T-CHO, respectively. HDL-C, LDL-C was measured using Elabscience "Low Density lipoprotein cholesterol (LDL-C) colorimetric kit (two reagent direct method)" and "high Density lipoprotein cholesterol (HDL-C) colorimetric kit (two reagent direct method)", respectively.

And (3) according to the instruction of the kit, preparing standard curves of all detection indexes and simultaneously measuring a sample to be detected. OD values at detection wavelengths corresponding to the respective indexes are measured by using a enzyme-labeled instrument (Synergy H1 Hybrid Reader, bioTek), and ALT/AST/TG/T-CHO/HDL-C/LDL-C values of the respective samples are calculated according to the measured standard curve.

(^ns P >0.05, # P <0.01, # P <0.001, # P <0.0001, MCD/HFD group vs ctrl group; ^ns P >0.05, # P <0.01, # P <0.001, # P <0.0001, MCD+QH/HFD+QH group vs MCD/HFD group).

5. RT-qPCR detection

Total liver RNA was extracted by FastPure Cell/Tissue Total RNA Isolation Kit (vazyme). UsingQuantStudio ^TM 6Flex Real-TIME PCR SYSTEM,384 wells (Thermo Scientific) were subjected to RT-qPCR detection and amplified in a two-step method using a SYBR GREEN QPCR MASTER Mix formulated as a 10ul system. The CT values of the respective primers were analyzed by the DeltaCT method.

2. Experimental results

Effective intervention of the extract of apices Apii and Sophora japonica in MCD non-alcoholic fatty liver

1. Cress Sophora japonica extract for improving hair state of MCD non-alcoholic steatohepatitis model mice

FIG. 6 is the effect of the extract of Apium graveolens on hair status of MCD model mice. As shown in fig. 6, ctrl group had smooth and clean hair and MCD model group had a messy hair. After the gastric lavage treatment of the celery-sophora japonica extract, the MCD+QH group improves the hair state in a QH dose-dependent manner, and the MCD+QH 200mg/kg group hair state is similar to that of the ctrl group.

2. Effect of celery-locust extract on MCD non-alcoholic steatohepatitis mice weight change and liver index

Fig. 7 shows the effect of the extract of apices in case of weight change in MCD model mice, and fig. 8 shows the effect of the extract of apices in case of liver index in MCD model mice. As shown in fig. 7, ctrl group mice continued to gain in body weight. The MCD model group had similar weights to the mcd+qh groups and all continued to decrease. As shown in fig. 8, there was no significant difference in liver index between ctrl group, MCD model group and mcd+qh groups. The extract of the celery-locust has no obvious influence on the body weight change and liver index of the MCD non-alcoholic steatohepatitis mice.

3. The extract of the celery-locust effectively intervenes in liver pathology related to a mouse model of MCD non-alcoholic steatohepatitis

FIG. 9 is a photograph of HE staining of liver tissue of mice. As shown in fig. 9, the ctrl group liver cells are orderly arranged, the liver rope structure is clear, the structure is complete, and no obvious pathological change exists; the MCD model group has severe mixed vesicular and microvesicle fat transformation and focal balloon-like transformation, the cytoplasms of the liver cells are different in depth, a large number of cell nuclei are condensed, the hepatic cable structure is not obvious, and the severe inflammatory cell infiltration is accompanied; the MCD+QH groups are dose-dependently reduced in liver injury caused by MCD nonalcoholic steatohepatitis, and the MCD+QH 200mg/kg groups only have visible fat vacuoles locally, the whole liver tissue structure is complete, the hepatic chordae are arranged clearly, and balloon-like changes are not seen.

4. The extract of the celery-locust is effective in improving liver lipid accumulation of MCD non-alcoholic steatohepatitis mice

FIG. 10 is a photograph of oil red O staining of liver tissue of mice. As shown in fig. 10, the ctrl group hepatocytes are orderly arranged, the liver rope structure is clear, the structure is complete, and no obvious oil red O staining trace exists; the MCD model group had a large area of darker oil red O staining and a large number of dark red lipid accumulation points; the MCD and QH groups are used for reducing liver lipid accumulation caused by an MCD model in a dose-dependent manner, and the oil red O staining area and lipid accumulation point of liver tissues are reduced along with the increase of the administration dose of the apices extract. This result suggests that the extract of apices Apii has greatly improved liver lipid accumulation level caused by MCD non-alcoholic steatohepatitis.

5. The extract of the celery-locust is effective in improving liver fibrosis of MCD non-alcoholic steatohepatitis mice

FIG. 11 is a photograph of a Masson stain of mouse liver tissue. As shown in fig. 11, ctrl group hepatocytes were aligned neatly, liver rope structure was clear, structure was complete, and no obvious collagen deposition was observed; MCD model groups had more collagen deposition. After MCD non-alcoholic steatohepatitis mice were treated with the apices, collagen deposition in liver tissue decreased with increasing doses of apices extract. Among them, the MCD+QH 200mg/kg group did not see significant collagen deposition. This result suggests that the extract of apices Apii has greatly reduced liver fibrosis progression caused by MCD non-alcoholic steatohepatitis.

6. The extract of the celery-locust effectively reduces the change of liver blood biochemical indexes caused by MCD non-alcoholic steatohepatitis

FIG. 12 shows the results of biochemical index detection of liver injury of mice with MCD model by using the extract of Apium graveolens. Table 3 shows biochemical index data of liver injury of the mice with MCD model by using the extract of Apium graveolens. As shown in fig. 12 and table 3, both ALT and AST were significantly increased in the MCD model group compared to ctrl group. Serum ALT and AST were significantly reduced in each of the mcd+qh dose groups compared to the MCD group. The result shows that the celery-sophora japonica extract can effectively improve blood biochemical index change caused by MCD non-alcoholic steatohepatitis.

TABLE 3 Biochemical index data (mean+ -SEM) of liver injury of Cress Sophora extract on MCD model mice

7. Open field experiments indicate that the celery-locust extract can improve the exercise capacity of MCD non-alcoholic steatohepatitis model mice

FIG. 13 is the effect of the extract of Apium graveolens on motor ability of MCD model mice. As shown in fig. 13, the MCD model mice showed a significant decrease in total distance traveled, number of edge passes, and number of center passes, and a significant increase in resting time, compared to ctrl mice, indicating that MCD modeling made the mice weak and the motor ability relatively decreased. Compared with the MCD group, the total movement distance, the edge passing times and the center passing times of each group of MCD and QH are obviously increased, and the rest time is obviously reduced. This result shows that the extract of apices Apii can improve the decrease of exercise ability caused by MCD non-alcoholic steatohepatitis and recover the physical state of mice.

8. The extract of Apium graveolens can effectively improve lipid synthesis gene transcription up-regulation caused by MCD non-alcoholic steatohepatitis

FIG. 14 shows the effect of RT-qPCR on the transcriptional upregulation of lipid synthesis genes in mice with MCD models. As shown in fig. 14, the expression of lipid synthesis pathway genes Acly, ACC1, FASN, SCD1, srebp a, srebp1c, srebp2 was all significantly increased in the MCD model group compared to ctrl group. After treatment with the extract of apices, the above lipid synthesis pathway genes were significantly decreased in the mcd+qh group. This result suggests that the extract of apices Apii has reduced liver lipid accumulation by regulating the expression of lipid synthesis genes in mice of MCD model.

9. The extract of apices Apii and Sophora japonica can effectively improve the up-regulation of inflammation-related genes of MCD model mice

FIG. 15 shows the effect of RT-PCR on the transcriptional upregulation of the inflammation-associated genes in mice with the MCD model. As shown in FIG. 15, the transcription levels of the inflammatory-related genes TNF-. Alpha., IL-6, IFN-. Beta., cxcl-10, IL-1. Beta., IL-18 were significantly increased in the MCD model group compared to the ctrl group. After treatment with the extract of apices, the above-mentioned inflammation-related genes were significantly reduced in the mcd+qh group. Suggesting that the celery-locust extract may reduce liver inflammation level of MCD non-alcoholic steatohepatitis mice by regulating related inflammatory gene transcription.

10. The extract of the celery-locust effectively improves the up-regulation of the fibrosis related genes of the MCD model mice

FIG. 16 shows the effect of RT-PCR on the transcription of the upregulated pro-fibrotic genes in mice with the MCD model. As shown in FIG. 16, the fibrosis-related genes α -SMA, COL1A1, and TGF-. Beta.1 were all significantly increased in the MCD model group compared to the ctrl group. After treatment with the extract of apices, the above-mentioned fibrosis-related genes were significantly reduced in the mcd+qh group. The celery-locust extract was suggested to reduce liver fibrosis levels in MCD model mice by modulating transcription of the associated fibrosis genes.

(II) effective intervention of the extract of Apium graveolens for evaluating HFD non-alcoholic fatty liver disease

1. Herba Apii Graveolentis extract for improving hair state of HFD non-alcoholic steatohepatitis mice

Fig. 17 is the effect of the extract of apices on hair status in HFD model mice. As shown in fig. 17, ctrl group hair was smooth and clean, and HFD model group hair was messy. After the treatment with the extract of apices, the HFD+QH group improved the hair status in a dose-dependent manner. The HFD+QH 200mg/kg group hair status was similar to the ctrl group.

2. The extract of Apium graveolens slows the weight gain of mice with HFD non-alcoholic steatohepatitis, and is independent of food intake

Figure 18 is the effect of the extract of apices on body weight of HFD model mice. FIG. 19 shows the effect of the extract of Apium graveolens on the feeding of HFD model mice. As shown in fig. 18, the HFD model group mice increased greatly in body weight gain compared to ctrl group. The mice in the HFD+QH groups after treatment with the apices extract had significantly lower body weight gain rate and significantly showed dose dependence compared to the HFD model group. The body weight increasing speed of the HFD+QH50mg/kg group is slightly higher than that of the ctrl group, the body weight increasing speed of the HFD+QH100deg.C/kg group is similar to that of the ctrl group, and the body weight increasing speed of the HFD+QH200mg/kg group is slightly lower than that of the ctrl group.

During the study period, as shown in fig. 19, mice in the HFD model group and hfd+qh groups did not see significant differences in feed intake, but were all significantly lower than ctrl group.

This result demonstrates that the extract of apices can effectively relieve weight gain due to high fat diet without affecting the feeding amount of mice.

3. The extract of herba Apii Graveolentis can effectively interfere with liver pathological conditions related to HFD non-alcoholic steatohepatitis mice

FIG. 20 is a photograph of HE staining of liver tissue of mice. As shown in fig. 20, the ctrl group liver cells are orderly arranged, the liver rope structure is clear, the structure is complete, and no obvious pathological change exists; the HFD model group has severe foaming and micro foaming mixed fat transformation and extensive balloon transformation, liver cells are obviously enlarged, liver cell cytoplasm is obviously shallowed, a large number of cell nuclei are condensed, and the hepatic cable structure is not obvious; the HFD+QH groups are dose-dependent, so that liver injury caused by HFD nonalcoholic steatohepatitis is reduced, no obvious lipid accumulation is seen in the HFD+QH 100mg/kg group, the whole liver tissue structure is complete, the hepatic cable arrangement is clear, and balloon-like changes are not seen.

4. The extract of Apium graveolens is effective in improving liver lipid accumulation of HFD non-alcoholic steatohepatitis mice

FIG. 21 is a photograph of oil red O staining of liver tissue of mice. As shown in fig. 21, the ctrl group liver cells are orderly arranged, the liver rope structure is clear, the structure is complete, and no obvious oil red O staining trace exists; the HFD model group had a large area of darker oil red O staining and a large number of deep red lipid accumulation points; the HFD and QH groups are used for reducing liver lipid accumulation caused by HFD nonalcoholic steatohepatitis in a dose-dependent manner, and the liver tissue oil red O staining area and lipid accumulation point are reduced along with the increase of the administration dose of the celery fruit extract; the HFD+QH 100mg/kg group had a significantly reduced oil red O staining area, similar to the ctrl group. This result suggests that the extract of apices Apii has greatly relieved liver lipid accumulation levels in mice with HFD model.

5. The herba Apii Graveolentis extract can effectively improve liver injury biochemical index ALT, AST, TG, T-CHO, HDL-C, LDL-C change of HFD non-alcoholic steatohepatitis mice

FIG. 22 shows the effect of the extract of Apium graveolens on the biochemical index of liver injury in HFD model mice. Table 4 shows the effect data of the extract of Apium graveolens on the biochemical index of liver injury in HFD model mice. As shown in FIG. 22 and Table 4, HFD model group ALT, AST, TG, T-CHO, HDL-C, LDL-C were significantly elevated compared to ctrl group. Serum ALT, AST, TG, T-CHO and HDL-C, LDL-C were significantly reduced in each of the HFD+QH dose groups compared to the HFD model group. The result shows that the celery-locust extract can effectively improve blood biochemical indexes ALT, AST, TG, T-CHO and HDL-C, LDL-C of HFD model mice.

TABLE 4 Biochemical index Effect of Cress Sophora extract on liver injury in HFD model mice (mean+ -SEM)

The above results show that the extract of the celery-locust has good treatment effect on the non-alcoholic steatohepatitis induced by the MCD and HFD models. The extract of herba Apii Graveolentis can remarkably improve liver injury and lipid accumulation caused by MCD and HFD models, and simultaneously relieve collagen deposition caused by the two models in a dose-dependent manner to delay the non-alcoholic steatohepatitis process. And effectively down-regulates ALT and AST rising in MCD and HFD models. The celery-locust extract can effectively relieve the disorder condition of the hair of mice with MCD and HFD models, and improve the change of the motor ability of the mice with MCD models. The extract of apices Apii could not alleviate the weight loss of MCD mice. The extract of the celery-locust can effectively regulate lipid synthesis, inflammation and fibrosis related gene transcription which are upregulated by MCD.

The results of the pathological and biochemical detection and the like indicate that the celery-sophora japonica extract can effectively prevent/intervene in the non-alcoholic liver disease. The celery-locust extract has great development value for preventing and treating the non-alcoholic fatty liver.

Test example 3 evaluation of clinical efficacy of Cress Sophora extract on patients with alcoholic fatty liver disease

Alcoholic liver disease is generally a liver injury caused by long-term high-volume drinking of patients, and mainly comprises alcoholic cirrhosis, hepatitis, fatty liver and the like. In recent years, the incidence of alcoholic liver disease has been increasing with the regulation of dietary structure of people, excessive drinking. The initial stage of alcoholic liver disease is alcoholic fatty liver, mainly because triglyceride is excessively accumulated in liver cells, liver fibrosis and hepatitis occur, and severe liver cirrhosis and liver cancer can be caused, so that physical and mental health of people is greatly affected. Therefore, the traditional Chinese medicine composition has positive significance for timely and effective treatment, improvement of liver functions and delay of liver fibrosis progress of patients with alcoholic fatty liver.

The research aims at using the celery-locust extract of the invention to treat alcoholic fatty liver so as to evaluate the clinical effect of the celery-locust extract on improving liver fibrosis and liver function, and provides the basis for evaluating the clinical effect of the celery-locust extract of the invention on the clinical treatment effect of the celery-locust extract on the alcoholic fatty liver of human body.

1. General data

68 Cases of alcoholic fatty liver disease patients who are collected and treated in 2021, 10 months and 2022, 10 months of certain public hospitals in Beijing city are selected as study subjects.

Inclusion criteria:

(1) Meets the diagnosis standard of alcoholic fatty liver patients;

(2) A history of drinking for more than 5 years, a drinking level of more than 20g/d for females and more than 40g/d for males, or a history of substantial drinking for nearly two weeks for patients, a drinking level of more than 80g/d;

(3) Clinical symptoms can be jaundice, hypodynamia, inappetence or upper abdominal pain, and can also have no typical symptoms;

(4) Abnormal liver function, elevated aspartate aminotransferase (ASPARTATE AMINOTRANSFERASE, AST) and alanine aminotransferase (alanine aminotransferase, ALT);

(5) The ultrasonic or CT and other influence and diagnosis of the liver accord with the expression of alcoholic fatty liver.

Exclusion criteria:

(1) Liver treatment drugs have recently been used;

(2) There are severe liver and kidney functional insufficiency:

(3) Treating allergic patients with drug.

The observation group and the control group were each divided into 34 cases according to the random number method. In the observation group, 23 men and 11 women; age 38-62 years, average (47.18.+ -. 5.23) years; the disease course is 3-10 years, and the average (6.17+/-2.76) years. In the control group, 24 men and 10 women; age 38-61 years, average (47.08+ -5.23) years; the disease course is 3-10 years, and the average (6.27+/-2.66) years. The difference between the general data of the two groups of patients is not statistically significant (P > 0.05), and the two groups of patients are comparable.

The study was reviewed and passed by the ethics committee of the hospital, and both the patient and family members had knowledge of the notes present during the treatment of alcoholic fatty liver disease and had voluntarily signed informed consent.

2. Therapeutic method

Hospitalized patients were given general precautions, including positive diet control, prohibition of drinking, weight control, and appropriate exercise.

Control group: polyene phosphatidyl choline capsules (national standard: H21058010, 228mg, produced by Beijing Sainophenanthrene Co.) were administered 3 times daily, 2 granules each time.

Observation group: 225mg of the extract of apices Apii of the present invention was administered 2 times daily, 1 granule each time.

Both groups were treated for 2 months.

3. Observation index and detection method

After the treatment, the treatment effect of the patient, the liver function state of the patient before and after the treatment, and the improvement degree of liver fibrosis of the patient before and after the treatment are compared.

Liver function: the patients were examined for aspartate Aminotransferase (AST) and alanine Aminotransferase (ALT), total cholesterol (total cholesterol, TC) and triglycerides (TRIACYLGLYCERIDE, TG) using a fully automated biochemical analyzer.

Liver fibrosis: the ELASA method was used to determine pre-type III collagen (PC III), hyaluronic Acid (HA), laminin (LN) and type IV collagen before and after patient treatment.

4. Evaluation of therapeutic Effect

The effect is shown: the liver function is recovered to be normal, and ALT, AST, TC, TG values are recovered to be normal ranges; symptoms such as jaundice, debilitation, inappetence and the like disappear, and no recurrence occurs within 2 months after stopping taking the medicine.

The method is effective: the liver function is recovered, ALT, AST, TC, TG values are reduced, symptoms such as jaundice, hypodynamia, inappetence and the like are relieved, and the medicine is stopped and then is repeated, so that the treatment is effective.

Invalidation: liver function was not significantly altered or worsened.

Total effective rate of treatment = (significant + effective) number of cases = total number of cases x 100%.

5. Statistical method

Carrying out data analysis by using SPSS22.0 statistical software, and comparing counting data by using X2 test; the metering data are in accordance with the normal too distribution, expressed as Mean ± standard deviation (Mean ± SEM), and the comparison between the two groups is tested, and the difference is statistically significant, expressed as P < 0.05.

6. Results

6.1 Comparison of the therapeutic Effect of the two groups of patients

The observed group had a total therapeutic efficiency of 89.66% significantly higher than 51.72% for the control group, the differences being statistically significant (x2=6.34.p < 0.05), see table 5 below.

Table 5 comparison of the effects of two groups of patients before and after treatment (mean±sem)

6.2 Liver function comparison before and after treatment of two groups of patients

Prior to treatment, the comparative differences in liver function indexes such as AST, ALT, TC and TG were not statistically significant (P > 0.05) in both groups. After treatment, the liver function index of AST, ALT, TC and TG etc. of the patients in the observation group was significantly lower than that of the control group, and the differences were statistically significant (P < 0.05), as shown in table 6 below.

Table 6 liver function comparison before and after treatment (mean+ -SEM) for two groups of patients

Note that: p <0.05 compared to pre-treatment for this group.

6.3 Comparison of liver fibrosis index before and after treatment of two groups of patients

The comparative differences of liver fibrosis indexes such as LN, HA, PCIII and IV type collagen of the two groups of patients before treatment have no statistical significance (P is more than 0.05); after treatment, liver fibrosis indexes such as LN, HA, PC III and IV type collagen of patients in the observation group are obviously lower than those of the control group, and differences have statistical significance (P < 0.05), and the following table 7.

Table 7 comparison of liver fibrosis index before and after treatment (mean+ -SEM) for two groups of patients

Note that: p <0.05 compared to pre-treatment for this group.

7. Conclusion(s)

Alcoholic fatty liver disease is a common disease among liver diseases, and is caused by long-term heavy drinking of patients. Alcohol metabolism in vivo causes liver cells to produce a large amount of harmful substances such as acetaldehyde and the like, secretes a large amount of hydroxyl and free radicals, causes metabolic disorder of patients, and further develops into important life-threatening serious diseases such as liver cirrhosis, liver cancer and the like. At present, alcohol withdrawal is the most critical measure for treating alcoholic fatty liver, but most people have generated dependence on alcohol due to insufficient understanding of alcohol withdrawal significance and harm of alcohol consumption of a plurality of patients, lack of understanding of fatty liver, and lack of typical clinical symptoms and signs in early stage of alcoholic fatty liver, so that early treatment time is often missed, and finally serious illness is caused to threaten life. Therefore, the treatment of alcoholic fatty liver patients is first to take positive alcohol-stopping measures, and at the same time, treatment with effective drugs must be combined.

In clinical researches, the total effective rate of oral apices extract treatment of patients in an observation group is 88.24%, the effective rate of oral polyene phosphatidylcholine in a control group on alcoholic fatty liver is 55.94%, and the total effective rate of oral apices extract treatment in the observation group is obviously higher than that in the control group. After a great deal of wine is drunk by patients, harmful substances such as acetaldehyde and the like are generated, so that liver cells are damaged, and liver fibrosis of the patients is gradually generated. The control group has reported the polyene phosphatidyl cholinergic to delay the progress of liver fibrosis and the action mechanism thereof. While observing that the treatment of alcoholic fatty liver by the celery-locust extract not only accelerates the metabolism of esters, but also finds the effect of resisting hepatic fibrosis or delaying hepatic fibrosis to be better than that of polyene phosphatidylcholine in a control group, which is clearly another important finding of the invention.

The clinical efficacy and research of the human body show that after treatment, indexes of the observed group patients AST, ALT, TC, TG and the like which can reflect liver functions are obviously higher than those of the control group, and further the celery-locust extract is a great finding for improving liver functions and reversing liver fibrosis of alcoholic fatty liver patients.

In conclusion, the celery-sophora japonica extract has remarkable treatment effect on patients with alcoholic fatty liver, and can effectively improve liver function of the patients and inhibit or reverse liver fibrosis.

Test example 4 evaluation of clinical efficacy of Cress Sophora extract on patients with non-alcoholic fatty liver disease

Clinically, there is no history of excessive alcohol consumption, but the clinical pathological features of fat accumulation and hepatic parenchymal steatosis are called non-alcoholic fatty liver. It is counted that more than 40% of non-alcoholic fatty liver patients develop liver fibrosis gradually, and that more than 15% of patients develop cirrhosis, liver cancer, etc. with exacerbation. In recent years, the non-alcoholic fatty liver in China shows a severe trend of high onset and low age. Clinical studies have found that, in addition to drinking, unhealthy diet and lifestyle of patients, obesity, diabetes and the like are important factors leading to non-alcoholic fatty liver. As a chronic progressive disease, non-alcoholic fatty liver disease has become the second largest liver disease after secondary viral hepatitis, and complete cure is not achieved at present. Early discovery, early treatment, intervention through effective treatment measures and consolidation become the focus of clinical attention. The traditional Chinese medicine is classified into the research categories of phlegm syndrome and liver-kidney yin deficiency, and the principle of purging pathogenic fire, clearing damp, removing toxin and protecting liver is considered to be the primary principle in treatment, and the ideal effect is achieved through the treatment of the discovered prescription.

The research is to design the clinical drug effect evaluation and research of human body on the basis that the celery-locust extract has special drug effect evaluation on a mouse non-alcoholic fatty liver model.

1. General data

100 Non-alcoholic fatty liver disease patients participating in the study were all screened by a public hospital in Beijing city, 1 st 2021 st to 8 nd 2022 and then included in a control group (50 cases, blood lipid health treatment) and an observation group (50 cases, treatment with the apices of the present invention). Grouping was accomplished using a random number table method and different treatment regimens were applied. Control group: the ratio of the number of men to women is 28:22; the age range is 30-65 years old, and the median value (47.58 +/-3.31) is the year old. Observation group: the ratio of the number of men to women is 30:20; the age range is 31-66 years old, and the median value (48.12+/-3.36) is the year old. The verification shows that the difference of the 2 groups of baseline treatments has no statistical significance and has equilibrium comparability (P is more than 0.05).

2. Method of

The control group oral medicine Xuezhikang (national medicine standard Z10950029, produced by North Daweixin Biotech Co.) is treated. The oral dosage standard is 0.6 g/time, and the oral administration is carried out with warm water 2 times a day.

The observation group orally takes the extract of the apices sophorae (225 mg of the extract of the apices sophorae plus 225mg of auxiliary materials per capsule), and the treatment is carried out by taking 1 capsule each time, 2 times a day in the morning and evening, and warm water can be taken.

All patients had a course of treatment of 4 weeks for a total of 2 courses. During the treatment period, the wine is strictly forbidden, the scientific and reasonable healthy light diet is maintained, and a certain exercise amount is maintained.

3. Observation index and detection method

① Comparison of liver function indicators

Liver function indexes such as glutamic pyruvic transaminase (ALT), glutamyl transferase (GGT), alkaline phosphatase (ALT), total Bilirubin (TBIL) and the like before and after treatment of two groups of patients are effectively detected and counted.

② Comparison of fatty liver scoring conditions

Checking liver conditions of a patient before and after treatment by using color Doppler ultrasound, and referring and executing the following fatty liver grade scoring standard; severe fatty liver (score >12 points), moderate fatty liver (score 7-12 points), mild fatty liver (score <6 points).

4. Statistical method

All raw data analysis was performed by statistical software (version SPSS 21.0), and the metrology data was expressed in (mean±sem) and independent samples were tested for t-test, with P <0.05 being the difference statistically significant.

5. Results

5.1 Liver function comparison before and after treatment of two groups of patients

Before treatment, the differences of the liver function indexes of the two groups of patients have no statistical significance (P > 0.05). After treatment, the liver function index of the observed group was significantly lower than the value of the control group (P < 0.05), and the differences were statistically significant, as shown in table 8 below.

Table 8 liver function comparison before and after treatment (mean+ -SEM) for two groups of patients

5.2 Comparison of fatty liver score before and after treatment of two groups of patients

The differences in fatty liver scores for each grade of the two groups of patients before treatment were not statistically significant (P > 0.05). After treatment, the fatty liver scores of each grade of the observed group were significantly lower than the control group (P < 0.05), the differences were statistically significant, see table 9 below.

Table 9 comparison of fatty liver score for two groups of patients (mean+ -SEM)

5.3 Conclusion

Currently, western medicines are mostly adopted for treating the nonalcoholic fatty liver clinically, but as the pathogenesis of the nonalcoholic fatty liver is complex, no medicine can thoroughly cure the nonalcoholic fatty liver, and auxiliary treatment is carried out by maintaining nutrition for protecting the liver, the treatment is limited by the treatment mechanism and adverse reactions of the medicine, so that the clinical effect is not ideal. The traditional Chinese medicine is classified into the research categories of 'phlegm syndrome', 'liver and kidney yin deficiency', and symptoms such as blood and qi disorder, viscera disorder and finally phlegm syndrome, blood stasis and the like caused by improper diet and unsmooth emotion are taken as root causes, and the traditional Chinese medicine strictly follows and implements the treatment principles of eliminating phlegm and removing blood stasis, clearing heat and detoxicating and invigorating spleen and clearing liver from the whole through dialectical theory. Pharmacological studies of the extract of apices Apii have demonstrated compliance with the above therapeutic principles. In the clinical study of the invention, the liver function index and fatty liver score of the observed group of patients are significantly lower than the control group (P < 0.05), due to the discovery of effective doses of the celery-locust extract and treatment regimen.

The invention aims at the treatment of the non-alcoholic fatty liver disease patients by applying the celery-sophora extract, compared with western medicines, the invention can improve clinical symptoms and liver function indexes more rapidly, has better medication safety, and highlights the discovery and clinical application value of the invention.

Claims

1. Use of an extract of apices in the manufacture of a medicament for the prevention and/or treatment of liver diseases in a mammal, including a human, wherein the extract of apices is an alcoholic extract of apices and flos Sophorae Immaturus in a weight ratio of 1:1-4:1, preferably 2:1-4:1, wherein the liver diseases include liver injury, hepatitis, fatty liver, liver fibrosis, liver cirrhosis.

2. Use of a pharmaceutical composition in the manufacture of a medicament for the prevention and/or treatment of liver disease in a mammal, including a human, wherein the pharmaceutical composition comprises an extract of apices comprising celery seed and pagodatree flower bud in a weight ratio of 1:1-4:1, preferably 2:1-4:1, and one or more pharmaceutically acceptable carriers, wherein the liver disease comprises liver injury, hepatitis, fatty liver, liver fibrosis, cirrhosis.

3. The use according to claim 1 or 2, wherein the liver disease is an alcoholic liver disease or a non-alcoholic liver disease.

4. The use according to claim 1 to 3, wherein the extract of Apium graveolens can improve liver function, liver injury, lipid accumulation, collagen deposition, liver index change, liver blood biochemical index change, weight loss or exercise capacity change associated with liver disease.

5. Use according to any one of claims 1 to 4, wherein the alcoholic extract is an extract in a C1-C4 alcoholic solvent, preferably methanol, ethanol, isopropanol, n-butanol, more preferably ethanol.

6. Use according to any one of claims 1 to 5, wherein the alcoholic solvent is an aqueous alcoholic solution and the concentration of the aqueous alcoholic solution is 50-80%, preferably 50-70% by volume.

7. The use according to any one of claims 1 to 6, wherein the extract of apices comprising the following is obtained by:

1) Adding the celery seed and the pagodatree flower bud into an alcohol solvent, and carrying out ultrasonic extraction to obtain an extracting solution; the ultrasonic extraction is preferably carried out for 2-4 times, and the ultrasonic time is preferably 30-60 minutes each time; preferably, the weight ratio of the alcohol solvent to the celery seed and the pagodatree flower bud is 10:1-8:1;

8. The use according to claim 2, wherein the pharmaceutical composition is in the form of granules, tablets, pellets, drops or capsules.

9. The use according to any one of claims 1 to 8, wherein the daily dose of the extract of apices Apii is in the range of 225mg to 2250mg.

10. The use according to any one of claims 1 to 9, wherein the daily dose of the extract of apices graveolens is in the range of 5.5mg/kg to 22mg/kg.