CN117379483A

CN117379483A - Traditional Chinese medicine composition for treating hyperlipidemia and preparation method and application thereof

Info

Publication number: CN117379483A
Application number: CN202311615174.4A
Authority: CN
Inventors: 张彤; 韩涵; 李玲; 陈泽璇; 唐洁
Original assignee: Shanghai University of Traditional Chinese Medicine
Current assignee: Shanghai University of Traditional Chinese Medicine
Priority date: 2023-11-29
Filing date: 2023-11-29
Publication date: 2024-01-12

Abstract

The invention relates to a traditional Chinese medicine composition for treating hyperlipidemia, a preparation method and application thereof, wherein the traditional Chinese medicine composition is an extract of three raw material mixtures of ginseng, hawthorn and pagodatree flower, and the proportion of the ginseng to the hawthorn is 2:1 to 1:2, the ratio of the hawthorn to the pagodatree flower is 2: 1-2: 2. the invention solves the problems of large toxic and side effects, complex taste, strong taste, difficult long-term taking and the like of the traditional lipid-lowering medicine, and the traditional Chinese medicine composition has simple components and ideal lipid-lowering effect.

Description

Traditional Chinese medicine composition for treating hyperlipidemia and preparation method and application thereof

Technical Field

The invention relates to a traditional Chinese medicine composition, in particular to a traditional Chinese medicine composition for treating hyperlipidemia, a preparation method and application thereof.

Background

Hyperlipidemia, medically considered dyslipidemia, symptoms including elevated serum Total Cholesterol (TC) levels, reduced serum Triglycerides (TG), reduced low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) levels, wherein elevated levels of non-high density lipoprotein cholesterol (non-HDL-C), i.e., serum LDL-C and residual cholesterol carried by triglyceride-rich lipoproteins, are a major risk factor for dyslipidemia in the global world, as well as for atherosclerotic cardiovascular disease (ASCVD). At present, the clinical treatment of the hyperlipidemia is mainly used for improving life style, taking lipid-lowering drugs and the like to control blood lipid.

Clinically accepted first-line medicines are statin lipid-lowering medicines which can play a lipid-lowering role by inhibiting cholesterol synthesis and accelerating low-density lipoprotein clearance, but according to the monitoring data of the adverse reaction of the national medicines, the statin medicines can be started to be taken for 1-3 months to cause abnormal blood sugar, and in addition, patients also have adverse reactions such as muscle discomfort, neurocognitive disorder, glutamic-pyruvic transaminase rise and the like. Fibrates are also common drugs for treating hyperlipidemia, which activate proteolytic enzymes and increase catabolism of very low density lipoproteins, thereby reducing TG levels, and thus are mainly used for treating hypertriglyceridemia or mixed hyperlipidemia mainly comprising elevated triglyceride levels, but have a cholesterol-lowering effect inferior to statin drugs, and simultaneously, have adverse effects such as myalgia, muscle weakness, and impaired liver function, which are easily caused within the first 3 months of administration of fibrates.

At present, the Chinese patent medicines for reducing blood fat are gradually increased, and the prescription thought of the Chinese patent medicines for reducing blood fat or modern prescription is as follows: based on the medicines for activating blood circulation and dissolving stasis and combining different indications, and the medicines for tonifying liver and kidney, reducing turbidity and resolving dampness are combined, for example, chinese patent CN116211974A discloses a traditional Chinese medicine composition for improving human microcirculation, which is characterized by complete formulation angles, including cooling blood and activating blood, resolving dampness and reducing turbidity, tonifying qi and nourishing yin and the like, can improve human microcirculation, and has the functions of reducing blood fat and reducing blood sugar.

Chinese patent CN110624077A discloses a traditional Chinese medicine composition for external application, chinese patent CN1879792A discloses a traditional Chinese medicine preparation capable of treating hypertension and cerebrovascular diseases, and the composition disclosed by the patent is prepared from more blood circulation promoting and stasis removing medicines, but contains more blood circulation promoting and stasis removing medicines such as polygonum multiflorum, safflower, rhizoma sparganii and the like, and can cause subcutaneous bleeding symptoms such as rash after long-term administration; also contains more Chinese medicines with strong smell, such as rhizoma Ligustici Chuanxiong, lignum Dalbergiae Odoriferae, radix aucklandiae, etc. which can cause gastrointestinal tract reaction.

The main problems of the existing lipid-lowering drugs include: (1) The chemical drugs have large toxic and side effects, can cause myalgia and myasthenia after being taken for a short time, can cause liver and kidney injury after being taken for a long time, and clinically needs to monitor liver function; (2) The Chinese patent medicine or the composition contains more traditional Chinese medicines with strong medicinal properties such as blood activating medicines, qi-flowing medicines and the like, needs to be applied to the patients for symptoms, and has adverse reactions after long-term administration.

Disclosure of Invention

The invention aims to solve the problems of toxic and side effects of lipid-lowering drugs, complex taste of traditional Chinese medicine prescription, strong nature and taste, difficult long-term administration and the like, and provides a traditional Chinese medicine composition for treating hyperlipidemia, which has simple components and ideal effects, and a preparation method and application thereof.

The aim of the invention is achieved by the following technical scheme:

the traditional Chinese medicine composition for treating hyperlipidemia is an extract of three raw material mixtures of ginseng, hawthorn and pagodatree flower, wherein the proportion of the ginseng to the hawthorn is 2:1 to 1:2, the ratio is 1:1, 1:1.5, 1:2, 1.5:1, 2:1, etc.; the ratio of the hawthorn to the pagodatree flower is 2: 1-2: the ratio of 2 to 1, 2 to 1.3, 2 to 2, etc. may be exemplified.

As a preferable technical scheme of the invention, the proportion of the ginseng, the hawthorn and the pagodatree flower is 1-4 according to parts by weight: 2:1 to 2. Preferably, 1:2: 1-2, the ratio of 1:2:2, 1:2:1.3, 1:2:1, etc., most preferably 1:2:2, may be exemplified.

According to the preferred technical scheme, the ginseng is a processed product of ginseng and red ginseng, and comprises ginseng slices and red ginseng slices, and the ginseng-based medicine according to Chinese pharmacopoeia (2020 edition) comprises ginseng and red ginseng, and the processed product comprises dried ginseng slices with the following content requirements: the total amount of ginsenoside Rg1 and ginsenoside Re is not less than 0.27%, and ginsenoside Rb1 is not less than 0.18%; the content of dry red ginseng tablet is required to be less than 0.22% of total ginsenoside Rg1 and ginsenoside Re, and less than 0.18% of ginsenoside Rb 1.

The fructus crataegi is processed product of fructus crataegi, the processed product of fructus crataegi comprises clean fructus crataegi, parched fructus crataegi and burnt fructus crataegi, the flos Sophorae Immaturus is processed product of flos Sophorae Immaturus, the processed product of flos Sophorae Immaturus comprises clean flos Sophorae Immaturus and parched flos Sophorae Immaturus, and the content measurement requirement of 2 processed products is that dry product contains rutin not less than 6.0%.

According to the preferred technical scheme, the traditional Chinese medicine composition is prepared by extracting ginseng, hawthorn and pagodatree flower with solvent (such as water or ethanol), extracting ginseng, hawthorn and pagodatree flower with solvent, concentrating, drying to obtain dry extract, crushing to obtain the traditional Chinese medicine composition, or adding proper auxiliary materials, and preparing into granules, pills, capsules and the like.

As a preferable technical scheme of the invention, the effective components contained in the traditional Chinese medicine composition comprise: the content of the active ingredients of the dry extract per unit mass is as follows: rg1+Re is more than or equal to 1.24mg/g; rb1 is more than or equal to 2.13mg/g; rutin is more than or equal to 37.13mg/g.

A preparation method of a traditional Chinese medicine composition for treating hyperlipidemia comprises the following steps: weighing Ginseng radix, fructus crataegi and flos Sophorae Immaturus at a certain proportion, adding extraction solvent, extracting, filtering to obtain medicinal liquid extract, concentrating, drying to obtain dry extract, and pulverizing to obtain the final product.

As a preferable technical scheme of the invention, the extracting solvent is water or ethanol, and the dosage of the extracting solvent is 5-10 times of the total weight of ginseng, hawthorn and pagodatree flower;

the extraction process comprises heating under reflux, maintaining the solvent in micro-boiling state, extracting for 1-5 times for 1-3 hr, and concentrating under reduced pressure at 50-90deg.C.

As a preferable technical scheme of the invention, the extraction solvent is ethanol with the concentration of 30% -80%. Ethanol having a concentration of 50% to 60% is preferred, and ethanol having a concentration of 50% is most preferred.

The application of a traditional Chinese medicine composition for treating hyperlipidemia is provided, wherein the traditional Chinese medicine composition is used for preparing a medicine for treating hyperlipidemia.

The minimum dosage of the traditional Chinese medicine composition to be administered by mice is 1.0g/kg; the clinical minimum dose is 0.08g/kg based on 60kg for a standard adult.

Compared with the existing lipid-lowering drugs, such as statins and other chemical drugs, the lipid-lowering cassia seed tablet has the advantages of complex formula, strong sex flavor, easy gastrointestinal tract irritation and possibility of causing various problems of burden on liver and kidney functions after long-term administration.

According to the invention, an L02 cell steatosis model with a higher reference value is selected, the modeling period of the L02 cell steatosis model is shorter, the induction success rate is higher, the model is more stable, the model can be more fit with the actual human in-vivo metabolic process, the oleic acid-palmitic acid induced L02 cell steatosis model is adopted for optimizing the preparation method and screening the formula of the traditional Chinese medicine composition, and then the C57BL/6J mice are fed with long-term high-fat feed for inducing the edible hyperlipidaemia model for in-vivo efficacy verification and dosage comparison of the traditional Chinese medicine composition, so that the traditional Chinese medicine composition provided by the invention has excellent lipid-lowering effect and low toxic and side effect.

Drawings

FIG. 1 shows cytotoxicity screening of different Chinese medicinal compositions;

FIG. 2 shows the effect of different Chinese medicinal compositions on TG levels contained in steatosis L02 cells;

FIG. 3 shows the oil red O staining of L02 cells after administration of different traditional Chinese medicine compositions;

FIG. 4 shows cytotoxicity of different ginseng-haw compositions;

FIG. 5 is a graph showing the effect on TG levels in L02 cells after administration of different ginseng-haw compositions;

FIG. 6 shows the O-staining of oil red after the human being participates in the administration of L02 cells of the model in different proportions of hawthorns;

FIG. 7 shows cytotoxicity of different ginseng-haw-pagodatree flower compositions;

FIG. 8 shows the effect of different ratios of ginseng, haw and pagodatree flower on the TG levels contained in the L02 cells of the model;

FIG. 9 shows the O-staining of oil after administration of L02 cells of the model to ginseng-haw-pagodatree flower of different compositions;

FIG. 10 is a different component oil red O-stained lipid assay;

FIG. 11 shows cytotoxicity of Ginseng radix tablet-fructus crataegi-flos Sophorae Immaturus composition, or Ginseng radix tablet-parched fructus crataegi-flos Sophorae Immaturus composition, or Ginseng radix tablet-charred fructus crataegi-flos Sophorae Immaturus composition, or Ginseng radix tablet-fructus crataegi-parched flos Sophorae Immaturus composition;

FIG. 12 is the effect of ginseng slice-Jiedu haw-Jiedu pagodatree flower composition, or red ginseng slice-Jiedu haw-Jiedu pagodatree flower composition, or ginseng slice-fried haw-Jiedu pagodatree flower composition, or ginseng slice-burnt haw-Jiedu pagodatree flower composition, or ginseng slice-Jiedu haw-fried pagodatree flower composition on the level of TG contained in L02 cells;

FIG. 13 is an oil red O stain of 200ug crude drug/mL of the composition of ginseng slice-Jiedu haw-Jiedu pagodatree flower, or of the composition of red ginseng slice-Jiedu haw-Jiedu pagodatree flower, or of the composition of ginseng slice-fried haw-Jiedu pagodatree flower, or of the composition of ginseng slice-burnt haw-Jiedu pagodatree flower after administration of fat-denatured L02 cells;

FIG. 14 shows the cytotoxicity of aqueous, alcoholic and aqueous extracts;

FIG. 15 is a graph showing the effect of aqueous, alcoholic, and aqueous-first extracts on the level of TG in L02 cells;

FIG. 16 shows oil red O staining of a water extract, an alcohol extract and a water extract of a Chinese medicinal composition after 200ug crude drug/mL administration of steatosis L02 cells;

FIG. 17 shows the trend of the extraction efficiency of different ethanol concentrations for the extraction of the active ingredients;

FIG. 18 is a graph showing the comprehensive scores of the active ingredients of the extracts obtained from different extraction solvents;

FIG. 19 is a comparison of blood lipid levels for each group;

FIG. 20 is a comparison of liver HE staining morphology;

FIG. 21 is a comparison of liver oil red O staining patterns.

Detailed Description

[ medicinal materials, reagents and instruments ]

The raw materials adopted by the invention are specifically sourced as follows:

ginseng (Sichuan lotus Chinese herbal pieces stock, lot 2207131, origin Jilin);

hawthorns (Sichuan new lotus Chinese medicinal decoction pieces stock, inc., lot number 2211006, hebei, origin);

pagodatree flower (Sichuan lotus Chinese herbal pieces stock, inc., lot number 2206033, hebei, origin);

red ginseng (Sichuan lotus Chinese herbal pieces stock, lot number 2310103, origin Jilin);

fried hawthorn (Sichuan new lotus Chinese medicinal decoction pieces stock, inc., lot number 2309004, hebei, producing area);

Fructus crataegi (Sichuan lotus Chinese medicinal decoction pieces stock, inc., lot number 2309049, hebei, origin);

fried pagodatree flower (Sichuan new lotus Chinese herbal pieces stock, inc., lot number 2203103, hebei, origin);

95% ethanol (national pharmaceutical group chemical reagent limited, analytical grade), RPMI-1640 medium, fetal bovine serum, diabody, PBS buffer, L02 cells, triglyceride kit, oleic acid, palmitic acid, bovine Serum Albumin (BSA), sodium hydroxide, modified oil red O staining kit (bi yun tian biotechnology, C0158M);

vertical laminar flow clean bench, bench type high-capacity low-speed centrifuge, 6-hole cell culture plate, 96-hole culture plate and enzyme-labeling instrument.

[ preparation of cell administration mother liquor, medium, free fatty acid Molding solution ]

Cell administration mother liquor of the traditional Chinese medicine composition is prepared: taking a proper amount of traditional Chinese medicine composition, performing ultrasonic dissolution by using a sterilized RBS buffer solution, filtering by using a 0.22um filter membrane to obtain mother liquor with the concentration of about 10mg crude drug/mL, subpackaging, and storing at-20 ℃.

Preparing a culture medium: complete medium containing 10% fetal bovine serum: under aseptic conditions, 40mL of RPMI-1640 medium was transferred to a sterilized 50mL centrifuge tube, 4.5mL of fetal bovine serum and 0.45mL of diabody (penicillin and streptomycin) were added, and after the preparation date was marked, the cells were stored at 4 ℃.

Preparing Free Fatty Acid (FFA) molding liquid:

(1) Preparation of 10% Bovine Serum Albumin (BSA): 5g BSA was weighed separately and dissolved in 50mL RPMI-1640 medium (without fetal bovine serum and diabody);

(2) Preparation of 0.1M sodium hydroxide: weighing 0.2g of sodium hydroxide to 50mL of sterilized deionized water, slowly adding the solution in portions, and dissolving the solution;

(3) Preparation of 10mM sodium Palmitate (PA) mother liquor: weighing a proper amount of PA, and dissolving the PA by using 0.1M sodium hydroxide to prepare 100mM PA; 2mL of 100mM PA was taken, 18mL of 10% BSA was added to make 20mL of 10mM PA, and stored at 4 ℃.

(4) Preparation of 20mM sodium Oleate (OA) mother liquor: weighing a proper amount of OA, and dissolving the OA by using 0.1M sodium hydroxide to prepare 100mM OA; 4mL of 100mM OA was taken, 16mL of 10% BSA was added thereto to prepare 20mL of 20mM OA, and the mixture was stored at 4 ℃.

(5) Preparation of control group medium: 1mL of 0.1M sodium hydroxide was taken and 10% BSA was added to 10mL and stored at 4 ℃.

Preparation of complete Medium containing 10% CCK-8: 700uL of CCK-8 is taken and dissolved in 7mL of complete medium, and after uniform blowing, the complete medium containing 10% of CCK-8 is obtained, and 110uL of complete medium is obtained per hole.

Experimental methods

Cell culture: normal human liver cell strain L02 was routinely cultured in RPMI 1640 complete medium containing 10% fetal bovine serum at 37℃under conditions of 5% volume fraction of CO2 and saturated humidity. All experiments were performed in the logarithmic cell growth phase. When the cells grow to the fusion degree of 80% -90%, the cells are digested and passaged by pancreatin for subsequent experiments.

Modeling a hepatic cell steatosis model: after L02 cells are paved in a 96-well plate for culture, after adherence, when the cells grow to 60% of fusion degree, FFA (OA: PA=2:1) with 1mM respectively is induced for 24 hours, a large amount of lipid droplets are accumulated in the cells through oil red staining, the intracellular TG level is obviously increased, and the cell activity is not influenced, namely the molding is successful.

Drug cytotoxicity concentration screening: filtering mother liquor of each screening product with 0.22um filter membrane, and gradually diluting with complete culture medium to obtain complete culture medium containing medicinal liquid with concentration of 400ug crude drug/mL, 200ug crude drug/mL, 100ug crude drug/mL, 50ug crude drug/mL, 20ug crude drug/mL, 10ug crude drug/mL, 5ug crude drug/mL, 2.5ug crude drug/mL, and 100uL per well. After 24 hours, the drug-containing medium was discarded, and after 1 hour incubation at 37℃with complete medium containing 10% CCK-8, the absorbance was measured at 450 nm. The drug concentration with the cell viability rate of more than 80% is screened.

Effects of drug on hepatocyte steatosis model: spreading L02 cells in a 96-well plate for culture, after adherence, taking mother liquor of each screening product when the cells grow to 60% of fusion degree, filtering with a 0.22um filter membrane, gradually diluting with a complete culture medium containing 1mM FFA, and preparing into a certain concentration of 100uL per well; the model group is cultured by a control group culture medium, and the blank group is cultured by a normal complete culture medium; after 24 hours of incubation, the cultured cells were collected, washed 2 times with PBS, and sub-packaged, with a portion for TG determination and a portion for oil red O staining observation.

Intracellular TG content assay: the well culture plate is taken out and placed on an ice box, the ice box is washed for 2 times by using PBS buffer solution, cell lysate is added into each well, cells are scraped off after short incubation, cell fragments and lysate are moved into a tube, and incubation is carried out on ice, and the shaking time is carried out during the period of incubation. Centrifuging to collect supernatant, and detecting TG content according to the instruction of the kit; the intracellular total protein content was measured by BCA protein concentration assay and the results were calculated and the final results were expressed as TG content per unit mass of protein (mmol/g). The experiment was repeated three times, the measurement result was expressed as the standard deviation of the mean soil.+ -. And the data were statistically analyzed by SPSS26.0 software, p <0.05 indicated that the difference was statistically significant.

Oil red staining was used to observe intracellular lipid droplet formation: discarding the original culture medium of each hole in the 6-hole culture plate, and washing 2 times by using PBS buffer solution; fixing with 4% paraformaldehyde for 10min, adding 1mL modified oil red O staining solution, staining at room temperature for 30min under dark condition, and sealing; washing with a staining wash (or 60% isopropyl alcohol) for 30s until the background is transparent; the stained cells were kept moist by the addition of 1mL of sterilized PBS and observed microscopically for oil red O staining.

[ preparation of Chinese medicinal composition ]

Respectively weighing fructus crataegi-flos Sophorae Immaturus, ginseng radix-flos Sophorae Immaturus and Ginseng radix-fructus crataegi, ginseng radix-fructus crataegi-flos Sophorae Immaturus, respectively adding 8 times of 50% ethanol, heating and reflux extracting for 1.5 hr (micro boiling), and filtering; adding corresponding extraction solvent each time, and extracting for 2 times; mixing the obtained 2 times of medicinal liquids, concentrating at 60deg.C under reduced pressure to a certain volume, drying under reduced pressure to obtain dry extract, and pulverizing to obtain Ginseng radix-fructus crataegi composition, ginseng radix-flos Sophorae Immaturus composition, fructus crataegi-flos Sophorae Immaturus and Ginseng radix-fructus crataegi-flos Sophorae Immaturus composition.

Taking proper amounts of Ginseng radix-fructus crataegi composition, ginseng radix-flos Sophorae Immaturus composition, fructus crataegi-flos Sophorae Immaturus composition and Ginseng radix-fructus crataegi-flos Sophorae Immaturus composition, dissolving with sterilized RBS buffer solution by ultrasonic, filtering with 0.22um filter membrane to obtain mother liquor with concentration of about 10mg crude drug/mL, packaging, and storing at-20deg.C.

Experimental results

1. Cytotoxicity screening of different Chinese medicinal compositions

As can be seen from FIG. 1, the concentration of the haw-pagodatree flower composition, ginseng-haw composition and ginseng-haw-pagodatree flower composition administered is 0-200 ug crude drug/mL, and thus, the concentration of the Chinese medicinal composition administered in the in vitro pharmacodynamic experiment is 200ug crude drug/mL.

2. Effect of different Chinese medicinal compositions on TG level contained in steatosis L02 cells

FIG. 2 shows the effect of different Chinese medicinal compositions on TG levels in steatosis L02 cells (p <0.01 compared to the blank group, #p0.05 compared to the model group, #p0.05 compared to the group, #p0.05)

As can be seen from fig. 2, the triglyceride level in the L02 cells after molding was significantly increased compared to the blank group; compared with the model group, when 200ug crude drug/mL haw-pagodatree flower composition, ginseng-haw composition and ginseng-haw-pagodatree flower composition are administered, the ginseng-haw composition and the ginseng-haw-pagodatree flower composition exhibit the effect of remarkably reducing the level of fatty degeneration L02 cell TG; compared with the ginseng-hawthorn composition, the ginseng-hawthorn-pagodatree flower composition has better lipid-lowering effect.

3. Oil red O staining of different traditional Chinese medicine composition 200ug crude drug/mL administration fatty degeneration L02 cells

FIG. 3 shows oil red O staining (x 400) of L02 cells of different Chinese medicinal composition after 200ug crude drug/mL injection molding, wherein A blank group, B model group, C fructus crataegi-flos Sophorae Immaturus composition, D Ginseng radix-flos Sophorae Immaturus composition, E Ginseng radix-fructus crataegi composition, and F Ginseng radix-fructus crataegi-flos Sophorae Immaturus composition

As can be seen from fig. 3, compared with the blank group, the molded L02 cells can be seen to have orange-yellow oil droplets densely distributed and accumulated in the cells; after 200ug crude drug/mL haw-pagodatree flower composition is administrated, a large amount of lipid drops still accumulate and distribute; and after 200ug crude drug/mL of the ginseng-pagodatree flower composition, the ginseng-hawthorn composition and the ginseng-hawthorn-pagodatree flower composition are administrated, the distribution density degree of intracellular lipid droplets is obviously reduced, and the aggregation phenomenon of lipid droplets is obviously reduced.

The experiment can confirm that the ginseng, the hawthorn and the pagodatree flower can synergistically play the role of reducing lipid, and the in vitro efficacy experiment is utilized to compare the influence of the combination of the three traditional Chinese medicines or the combination of the three traditional Chinese medicines on lipid accumulation of the fat-modified L02 cells. According to the TG level and the oil red O dyeing experimental result, after the ginseng-hawthorn-pagodatree flower is combined, the intracellular TG level of the steatosis L02 can be obviously reduced, the intracellular lipid accumulation is reduced, and the better lipid-lowering effect is embodied.

[ determination of the Components of Ginseng-Crataegus pinnatifida-Sophora flower composition ]

1. Preparation of different ginseng-haw composition and ginseng-haw-pagodatree flower composition

Weighing ginseng and hawthorn decoction pieces, respectively 1:1, 1:1.5, 1:2, 1.5:1 and 2:1, adding 8 times of 50% ethanol, heating and refluxing for 1.5 hours (micro boiling), filtering, and extracting for 2 times; mixing the obtained 2 times of medicinal liquids, concentrating at 60deg.C under reduced pressure to a certain volume, drying under reduced pressure to obtain dry extract, and pulverizing to obtain Ginseng radix-fructus crataegi composition.

Weighing ginseng and haw decoction pieces with optimal proportion, adding a certain amount of pagodatree flower decoction pieces, respectively enabling the components of the haw and the pagodatree flower to be 2:1, 2:1.3 and 1:1, adding 8 times of 50% ethanol, heating, refluxing and extracting for 1.5 hours (micro boiling), filtering, and extracting for 2 times; mixing the obtained 2 times of medicinal liquids, concentrating at 60deg.C under reduced pressure to a certain volume, drying under reduced pressure to obtain dry extract, and pulverizing to obtain Ginseng radix-fructus crataegi-flos Sophorae Immaturus composition.

2. Cell administration mother liquor preparation of traditional Chinese medicine composition

Taking a proper amount of ginseng-haw composition or ginseng-haw-pagodatree flower composition, using sterilized RBS buffer solution to make ultrasonic dissolution, using 0.22um filter membrane to make filtration so as to obtain mother liquor whose concentration is about 10mg crude drug/mL, packaging and storing at-20 deg.C.

(1) Intracellular TG assay

The well culture plate is taken out and placed on an ice box, the ice box is washed for 2 times by using PBS buffer solution, cell lysate is added into each well, cells are scraped off after short incubation, cell fragments and lysate are moved into a tube, and incubation is carried out on ice, and the shaking time is carried out during the period of incubation. Centrifuging to collect supernatant, and detecting TG content according to the instruction of the kit; the intracellular total protein content was measured by BCA protein concentration assay and the results were calculated and the final results were expressed as TG content per unit mass of protein (mmol/g). The experiment was repeated three times, the measurement result was expressed as the standard deviation of the mean soil.+ -. And the data were statistically analyzed by SPSS26.0 software, p <0.05 indicated that the difference was statistically significant.

(2) Oil red staining to observe formation of intracellular lipid droplets

Discarding the original culture medium of each hole in the 6-hole culture plate, and washing 2 times by using PBS buffer solution; fixing with 4% paraformaldehyde for 10min, adding 1-2 mL of oil red O working solution, dyeing at room temperature for 30min in a dark place, and sealing; wash with 60% isopropanol for 30s to background transparency; sucking out the dye liquor, adding hematoxylin dye liquor for counterstaining for 5min (dying cell nuclei); washing with water to turn blue, and sealing with glycerol gelatin; and observing the formation of orange lipid drops in the cells under a light microscope.

(3) Intracellular lipid quantification

Discarding the original culture medium of each hole in the 6-hole culture plate, and washing 2 times by using PBS buffer solution; fixing with 4% paraformaldehyde for 10min, adding 1-2 mL of oil red O working solution, dyeing at room temperature for 30min in a dark place, and sealing; wash with 60% isopropanol for 30s to background transparency; the plates were placed in a 37℃incubator to bake out the water, then 1mL of isopropanol was added to each well, and after shaking on a shaker for 10min, 100uL was aspirated and added to a 96-well plate, and the OD of each well was measured at a wavelength of 510nm with an microplate reader. The experiment was repeated three times, the measurement results were expressed as mean ± standard deviation ± and the data were statistically analyzed by SPSS26.0 software, p <0.05 indicated that the differences were statistically significant.

Experimental results

Determination of Ginseng radix-fructus crataegi composition components

(1) Cytotoxicity screening of different ginseng-haw compositions

Fig. 4 shows cytotoxicity of different ginseng-haw combinations, p <0.05 compared to the blank. From FIGS. 7-11, it can be seen that the concentrations of ginseng and haw (1:1), ginseng and haw (1:1.5), ginseng and haw (1:2), ginseng and haw (1.5:1), ginseng and haw (2:1) are 0-200 ug crude drug/mL, and have no significant effect on L02 cell activity, and the concentrations of ginseng and haw (1:2) are 400ug crude drug/mL.

(2) Effect of Ginseng-Crataegus pinnatifida composition in different proportions on the level of TG contained in steatosis L02 cells

FIG. 5 is a graph showing the effect of different ginseng-haw composition on TG levels contained in steatosis L02 cells after administration of 200 ug/mL, p <0.05 compared to the blank; compared to the model group, #p <0.05. As can be seen from fig. 12, the triglyceride level in the L02 cells after molding was significantly increased compared to the blank group; compared with a model group, when 200ug crude drugs/mL of ginseng and hawthorn (1:1), ginseng and hawthorn (1:1.5), ginseng and hawthorn (1:2), ginseng and hawthorn (1.5:1) and ginseng and hawthorn (2:1) are dosed, the lipid accumulation of the molding L02 cells is reduced to a certain extent by the 5 kinds of liquid medicine in proportion to the ginseng and the hawthorn, wherein the lipid accumulation of the molding L02 cells can be remarkably relieved by the ginseng and the hawthorn (1:1), the ginseng and the hawthorn (1:2) and the ginseng and the hawthorn (2:1).

(3) Different proportions of people participate in oil red O staining of 200ug crude drug/mL drug administration modeling L02 cells of hawthorn liquid medicine

FIG. 6 shows oil red O staining (x 800) of L02 cells after administration of crude drug/mL of molded form at a ratio of 200ug crude drug/mL of 1:1, 1:1.5, 1:2, 1:1, and 2:1, wherein the blank group A, the model group B, the person participating in haw (1:1), the person participating in haw (1:1.5), the person participating in haw (1:2), the person participating in haw (1.5:1), and the person participating in haw (2:1) are shown as the blank group A, the person participating in haw (1:1) are shown as the person participating in haw (D). Compared with a blank group, the molded L02 cells can be seen to have orange-yellow oil drops densely distributed and aggregated in the cells; compared with the model group, when 200ug crude drugs/mL of ginseng and hawthorn (1:1), ginseng and hawthorn (1:1.5), ginseng and hawthorn (1:2), ginseng and hawthorn (1.5:1) and ginseng and hawthorn (2:1) are dosed, the liquid medicine of the ratio of 5 ginseng and hawthorn all causes a certain reduction trend of lipid drop accumulation of the model L02 cells, wherein the reduction of lipid drop accumulation of the ginseng and the hawthorn (1:2) is most remarkable.

From the above experiments, the conventional ratio of ginseng to haw is 2:1-1:2, so that the ratio of fat degeneration L02 cell model is utilized to carry out drug effect comparison, and the intracellular TG level and oil red O staining condition are used as in vitro drug effect evaluation indexes of different ginseng to haw compositions, and it is known that when 200ug crude drug/mL is administered, the ginseng to haw (1:2) composition shows better drug effect than other ginseng to haw compositions for improving the fat degeneration of L02 cells, and in the subsequent experiments, the components of ginseng to haw to pagodatree flower are further determined under the optimal ratio of ginseng to haw (1:2).

The invention further determines the components of ginseng-hawthorn-pagodatree flower, and the specific experimental results are as follows.

(1) Cytotoxicity screening of different component ginseng-haw-pagodatree flower compositions

FIG. 7 shows cytotoxicity of various ginseng-haw-pagodatree flower compositions, and it can be seen that the ginseng-haw-pagodatree flower (1:2:2) composition, the ginseng-haw-pagodatree flower (1:2:1.3) composition, and the ginseng-haw-pagodatree flower (1:2:1) composition have no significant effect on L02 cell activity when the administration concentration is 0-400 ug crude drug/mL.

(2) Effect of Ginseng radix-fructus crataegi-flos Sophorae Immaturus composition of different components on TG level contained in L02 cells of molding

Fig. 8 shows the effect of ginseng, haw, and pagodatree flower at 1:2:2, 1:2:1.3, 1:2:1 ratios on TG levels contained in model L02 cells, compared to the blank group, < p <0.01; compared to the model group, #p <0.05. As can be seen from fig. 8, the triglyceride level in the L02 cells after molding was significantly increased compared to the blank group; compared with a model group, when 200ug crude drugs/mL of ginseng, hawthorn and pagodatree flower are administered in the proportions of 1:2:2, 1:2:1.3 and 1:2:1, the lipid accumulation of the molding L02 cells is reduced to a certain extent by 3 kinds of liquid medicine in the proportion of ginseng and hawthorn, wherein the lipid accumulation of the molding L02 cells can be remarkably relieved by the ginseng, the hawthorn and the pagodatree flower (1:2:2), and the ginseng, the hawthorn and the pagodatree flower (1:2:1).

(3) Oil red O staining of L02 cells of model for drug administration of 200ug crude drug/mL of liquid medicine of ginseng, hawthorn and pagodatree flower in different proportions

FIG. 9 shows oil red O staining (x 400) of L02 cells after drug administration of different components of ginseng-haw-pagodatree flower composition 200ug crude drug/mL, A blank group, B model group, C ginseng-haw-pagodatree flower (1:2:2) composition, D ginseng-haw-pagodatree flower (1:2:1.3) composition, E ginseng-haw-pagodatree flower (1:2:1) composition. Fig. 10 shows oil red O-stained lipid assay of different composition ginseng-haw-pagodatree flower composition 200ug crude drug/mL after administration of model L02 cells, compared to blank group, <0.01; compared to the model group, #p <0.05, #p <0.01; inter-group comparison, with p <0.05.

As can be seen from fig. 9 and 10, compared with the blank group, the molded L02 cells were densely distributed and aggregated with orange-yellow oil droplets; compared with the model group, when 200ug crude drugs/mL of ginseng, hawthorn and pagodatree flower are administered in the proportions of 1:2:2, 1:2:1.3 and 1:2:1, the lipid drop accumulation of the model L02 cells is reduced to a certain extent by 3 kinds of liquid medicine of the ginseng and the hawthorn, wherein the lipid drop accumulation of the ginseng-hawthorn-pagodatree flower (1:2:2) composition is most obviously reduced.

According to the experiment, on the basis that the ginseng-haw component is 1:2, the pagodatree flower is added for component determination, and according to the TG level and the change condition of oil red O dyeing after the administration of molding cells, the ginseng-haw-pagodatree flower combination effect is better, and particularly, the effect of improving the lipid accumulation in the fat degeneration L02 cells of the ginseng-haw-pagodatree flower (1:2:2) combination is most remarkable.

In conclusion, the invention uses the fatty degeneration L02 cell model, takes the intracellular TG level and the oil red O dyeing condition as evaluation indexes, and is divided into different traditional Chinese medicine compositions, namely, the improvement effect of ginseng-hawthorn, hawthorn-pagodatree flower, ginseng-pagodatree flower and ginseng-hawthorn-pagodatree flower on the fatty degeneration L02 cell model, and determines that the lipid-lowering effect of the ginseng-hawthorn-pagodatree flower composition is most prominent. The optimum proportion of the ginseng-hawthorn composition is determined, and the optimum components of the ginseng-hawthorn-pagodatree flower are further determined to be 1:2:2.

[ determination of processed Chinese medicinal composition ]

1. Preparation of Chinese medicinal composition comprising different processed materials

Weighing Ginseng radix slice, fructus crataegi and flos Sophorae Immaturus, respectively, mixing the processed materials at a ratio of 1:2:2, adding 8 times of 50% ethanol, heating and reflux extracting for 1.5 hr, filtering, and extracting for 2 times; mixing the obtained 2 times of medicinal liquids, concentrating under reduced pressure at 60-80deg.C to a certain volume, drying under reduced pressure at 60-80deg.C to obtain dry extract, and pulverizing to obtain the composition of radix Ginseng slice, fructus crataegi and flos Sophorae Immaturus.

Weighing Ginseng radix Rubri tablet, fructus crataegi and flos Sophorae Immaturus, respectively, mixing the processed materials at a ratio of 1:2:2, adding 8 times of 50% ethanol, heating and reflux extracting for 1.5 hr, filtering, and extracting for 2 times; mixing the obtained 2 times of medicinal liquids, concentrating under reduced pressure at 60-80deg.C to a certain volume, drying under reduced pressure at 60-80deg.C to obtain dry extract, and pulverizing to obtain Ginseng radix Rubri tablet-fructus crataegi-flos Sophorae Immaturus composition.

Weighing ginseng slices, fried hawthorns and clean pagodatree flowers, wherein the ratio of each processed product is 1:2:2, adding 8 times of 50% ethanol, heating and refluxing for extraction for 1.5 hours, filtering, and extracting for 2 times; mixing the obtained 2 times of medicinal liquids, concentrating under reduced pressure at 60-80deg.C to a certain volume, drying under reduced pressure at 60-80deg.C to obtain dry extract, and pulverizing to obtain Ginseng radix tablet-parched fructus crataegi-purified flos Sophorae Immaturus composition.

Weighing Ginseng radix slice, fructus crataegi preparata and flos Sophorae Immaturus, respectively, mixing the processed materials at a ratio of 1:2:2, adding 8 times of 50% ethanol, heating and reflux extracting for 1.5 hr, filtering, and extracting for 2 times; mixing the obtained 2 times of medicinal liquids, concentrating under reduced pressure at 60-80deg.C to a certain volume, drying under reduced pressure at 60-80deg.C to obtain dry extract, and pulverizing to obtain Ginseng radix tablet-fructus crataegi preparata-flos Sophorae Immaturus composition.

Weighing Ginseng radix slice, fructus crataegi and flos Sophorae Immaturus preparata, respectively, mixing the processed materials at a ratio of 1:2:2, adding 8 times of 50% ethanol, heating and reflux extracting for 1.5 hr, filtering, and extracting for 2 times; mixing the obtained 2 times of medicinal liquids, concentrating under reduced pressure at 60-80deg.C to a certain volume, drying under reduced pressure at 60-80deg.C to obtain dry extract, and pulverizing to obtain Ginseng radix tablet-fructus crataegi-parched flos Sophorae Immaturus composition.

Taking a proper amount of ginseng slice-clean haw-clean pagodatree flower composition, or red ginseng slice-clean haw-clean pagodatree flower composition, or ginseng slice-fried haw-clean pagodatree flower composition, or ginseng slice-burnt haw-clean pagodatree flower composition, or ginseng slice-clean haw-fried pagodatree flower composition, carrying out ultrasonic dissolution by using a sterilizing RBS buffer solution, filtering by using a 0.22um filter membrane, obtaining mother liquor with the concentration of about 10mg crude drug/mL, sub-packaging, and storing at-20 ℃.

(1) Intracellular TG assay

(2) Oil red staining to observe formation of intracellular lipid droplets

Experimental results

Determination of processed Chinese medicinal composition

1. Cytotoxicity screening of different traditional Chinese medicine composition preparations

FIG. 11 shows cytotoxicity of various ginseng-haw composition, it can be seen that the ginseng slice-haw-sophora japonica composition, or the red ginseng slice-haw-sophora japonica composition, or the ginseng slice-fried haw-sophora japonica composition, or the ginseng slice-haw-fried sophora japonica composition has no significant effect on L02 cell activity when the administration concentration is 0-200 ug crude drug/mL.

2. Effect of Chinese medicinal composition of different component preparations on TG level contained in L02 cells for molding

Fig. 12 is the effect of ginseng slice-net hawkthorn-net pagodatree flower composition, or red ginseng slice-net hawkthorn-net pagodatree flower composition, or ginseng slice-fried hawkthorn-net pagodatree flower composition, or ginseng slice-burnt hawkthorn-net pagodatree flower composition, or ginseng slice-net hawkthorn-fried pagodatree flower composition on TG level contained in modeling L02 cells, compared to blank group, p <0.01; compared to the model group, #p <0.05. As can be seen from fig. 12, the triglyceride level in the L02 cells after molding was significantly increased compared to the blank group; compared with a model group, when 200ug crude drug/mL of the ginseng slice-clean hawthorn-clean pagodatree flower composition, or the red ginseng slice-clean hawthorn-clean pagodatree flower composition, or the ginseng slice-fried hawthorn-clean pagodatree flower composition, or the ginseng slice-burnt hawthorn-clean pagodatree flower composition, or the ginseng slice-clean hawthorn-fried pagodatree flower composition, the lipid accumulation of the L02 cells of the model is reduced to a certain extent by the liquid medicine with the ratio of 5 ginseng to hawthorn, wherein the lipid accumulation of the L02 cells of the model can be remarkably relieved by the ginseng slice-clean hawthorn-clean pagodatree flower composition; compared with the ginseng slice-clean haw-clean pagodatree flower composition, the red ginseng slice-clean haw-clean pagodatree flower composition, or the ginseng slice-fried haw-clean pagodatree flower composition, or the ginseng slice-burnt haw-clean pagodatree flower composition, or the ginseng slice-clean haw-fried pagodatree flower composition, has no inter-group difference in lipid accumulation effect of the molding L02 cells.

3. Oil red O staining of drug-delivery fatness L02 cells of 200ug crude drug/mL of traditional Chinese medicine composition with different preparations

FIG. 13 shows the oil red O staining (x 400) of L02 cells after drug administration of 200 ug/mL of different Chinese medicinal composition, A blank group, B model group, C ginseng slice-clean haw-clean pagodatree flower composition, D red ginseng slice-clean haw-clean pagodatree flower composition, E ginseng slice-fried haw-clean pagodatree flower composition, F ginseng slice-fried haw-clean pagodatree flower composition, G ginseng slice-clean haw-fried pagodatree flower composition.

As can be seen from fig. 13, compared with the blank group, the molded L02 cells were seen to have orange-yellow oil droplets densely distributed and aggregated in the cells; 200ug crude drug/mL of ginseng slice-clean haw-clean pagodatree flower composition, or red ginseng slice-clean haw-clean pagodatree flower composition, or ginseng slice-fried haw-clean pagodatree flower composition, or ginseng slice-clean haw-fried pagodatree flower composition, the distribution density of intracellular lipid drops has a certain tendency to be reduced, wherein, the alleviating effect of ginseng slice-clean haw-clean pagodatree flower is most remarkable.

The experiment proves that the combination of the ginseng, the hawthorn and the pagodatree flower in different processing methods has a certain improvement effect on the L02 cell steatosis model, wherein the lipid-lowering effect of the combination of the ginseng slice, the clean hawthorn and the clean pagodatree flower has statistical significance, so that compared with the combination of other processed products, the lipid-lowering effect is better.

[ determination of preparation Process of Chinese medicinal composition ]

1. Preparation of Chinese medicinal composition by different extraction methods

(1) Aqueous extract of Chinese medicinal composition

Weighing decoction pieces of Ginseng radix, fructus crataegi and flos Sophorae Immaturus at a certain proportion, adding 8 times of distilled water, heating and reflux extracting for 1.5 hr (micro boiling), filtering, extracting for 2 times, mixing the medicinal liquids, concentrating at 60deg.C under reduced pressure to a certain volume, drying under reduced pressure to obtain dry extract, and pulverizing to obtain the Chinese medicinal composition water extract.

(2) Alcohol extract of Chinese medicinal composition

Weighing decoction pieces of Ginseng radix, fructus crataegi and flos Sophorae Immaturus at a certain proportion, adding 8 times of 75% ethanol, reflux-extracting for 1.5 hr (micro boiling), filtering, extracting for 2 times, mixing the decoctions, concentrating under reduced pressure at 60deg.C to a certain volume, drying under reduced pressure to obtain dry extract, and pulverizing to obtain the Chinese medicinal composition ethanol extract.

(3) Alcohol extraction and then water extraction of traditional Chinese medicine composition

Weighing decoction pieces of Ginseng radix, fructus crataegi and flos Sophorae Immaturus at a certain proportion, adding 8 times of 75% ethanol, heating and reflux extracting for 1.5 hr (micro boiling), and filtering; adding 8 times of distilled water into the residue, heating and reflux extracting for 1.5 hr (slightly boiling), and filtering; mixing the ethanol extract and the water extract, concentrating at 60deg.C under reduced pressure to a certain volume, drying under reduced pressure to obtain dry extract, and pulverizing to obtain the final product.

Experimental results

1. Cytotoxicity screening of aqueous extracts, alcoholic extracts and aqueous extracts of Chinese medicinal compositions

FIG. 14 shows cytotoxicity of aqueous extracts, alcoholic extracts and aqueous extracts after alcoholic extracts of the Chinese medicinal composition, and shows that the complete culture medium containing the aqueous extracts, alcoholic extracts and aqueous extracts after alcoholic extracts of the Chinese medicinal composition is used for L02 cells, and the administration concentration is 0-200 ug crude drug/mL, and has no obvious influence on the activity of the L02 cells. Thus, subsequent cell administration will select 200ug crude drug/mL for in vitro efficacy studies.

2. Effect of aqueous, alcoholic and aqueous extracts of Chinese medicinal composition on TG levels contained in fat-modified L02 cells

Fig. 15 is a graph showing the effect of aqueous, alcoholic and aqueous extracts of a Chinese medicinal composition on TG levels contained in steatosis L02 cells, compared to the blank, <0.01; compared to the model group, #p <0.01. It can be seen that the triglyceride level contained in the L02 cells is significantly increased after molding compared with the blank group; compared with a model group, when 200ug crude drug/mL of the traditional Chinese medicine composition aqueous extract, alcohol extraction without and after the alcohol extraction are added, the lipid accumulation of L02 cells after molding tends to be reduced, wherein the traditional Chinese medicine composition alcohol extract has remarkable relieving effect on the lipid accumulation of L02 cells after molding; in comparison between groups, the influence of the water extract, the alcohol extract and the water extract on the TG level of the fatty degeneration L02 cells is not significantly different.

3. Oil red O staining of traditional Chinese medicine composition water extract, alcohol extract and water extract 200ug crude drug/mL drug administration fatty degeneration L02 cells

FIG. 16 shows oil red O staining (x 400) of a crude drug/mL-administered steatosis L02 cells, wherein the crude drug/mL-administered steatosis L02 cells are a water extract of a Chinese medicinal composition, an alcohol extract of a Chinese medicinal composition, a model group, a water extract of a Chinese medicinal composition, an alcohol extract of a Chinese medicinal composition, and an alcohol extract of a Chinese medicinal composition. Compared with a blank group, the L02 cells after molding can be seen to have orange-yellow oil drops densely distributed and the phenomenon of aggregation of the oil drops is obvious; compared with the model group, when 200ug crude drug/mL of the traditional Chinese medicine composition aqueous extract, the alcohol extract and the aqueous extract are added, lipid drop accumulation of the model L02 cells has a certain reduction trend, and the phenomenon of drop aggregation is reflected to be reduced, and the distribution density of the drops is reduced.

The above-mentioned results show that the extraction solvent of the Chinese medicinal composition has a certain influence on the in vitro efficacy, and compared with the model group, the aqueous extract, the alcohol extract and the aqueous extract after alcohol extraction all have a certain tendency to relieve the intracellular lipid accumulation of the steatosis L02 cells, wherein the lipid-lowering effect of the alcohol extract has statistical significance. Therefore, the alcohol extraction method is superior to the water extraction method and the alcohol extraction method and the water extraction method.

2. Comparison of extraction efficiency of Chinese medicinal composition extracted with different ethanol concentrations

Reagents and instrumentation used: high performance liquid chromatography apparatus (Agilent technologies (China) Co., LC-1200series, USA), chromatography column (Agilent technologies (China) Co., ZORBAX Eclipse XDB-C18, analytical 4.6X105 nm 5-Micron, USA); acetonitrile (sigma aldrich (Shanghai) trade company, chromatographic purity, china), methanol (sigma aldrich (Shanghai) trade company, chromatographic purity, china), phosphoric acid (national drug group chemical company, analytical purity, lot number 220220823, china), ginsenoside Rg1 control, ginsenoside Re control, ginsenoside Rb1 control, rutin control, methanol (national drug group chemical company, analytical purity, china).

Preparation of a traditional Chinese medicine composition test article: adding 8 times of ethanol (80%, 70%, 60%, 50% or 0% ethanol) with different concentrations into decoction pieces of Ginseng radix, fructus crataegi and flos Sophorae Immaturus, reflux extracting under heating for 1.5 hr, filtering, extracting for 2 times, and mixing filtrates; concentrating under reduced pressure at 60deg.C to give extractive solution. Precisely removing 0.20mL of the extract, adding 30mL of methanol, ultrasonically extracting for 40 min, and filtering for later use.

Preparing a ginsenoside mixed reference substance: precisely weighing ginsenoside Rg1 reference substance, ginsenoside Re reference substance and ginsenoside Rb1 reference substance, adding methanol to obtain mixed solution containing 0.2mg of each 1mL, and shaking.

Preparation of rutin reference substance: and (3) taking a proper amount of rutin reference substance, precisely weighing, and adding methanol to prepare a solution containing 0.1mg per 1 mL.

The method for detecting the ginsenoside comprises the following steps: octadecylsilane chemically bonded silica is used as a filler; acetonitrile (A) -water (B) is taken as a mobile phase, and gradient elution is carried out: 0-12 min,19% A; 12-60 min,19% -36% A; the detection wavelength was 203nm.

The detection method of rutin comprises the following steps: octadecylsilane chemically bonded silica is used as a filler; gradient elution was performed with acetonitrile (a) -0.3% phosphoric acid water (B) as mobile phase: 0-26 min,20% → 39.5% A; 26-26.1 min,39.5% -20% A; 26.1-30 min,20% A; the detection wavelength was 360nm.

Record ginsenoside Re, rg ₁ The peak area of corresponding retention time of Rb1 and rutin is calculated, the content obtained by extracting effective components of crude drug per unit weight is calculated, and comprehensive evaluation is performed, wherein the calculation formula is shown as formula 1-1.

Formula 1-1: composite score = ginsenoside Re content/max (ginsenoside Re content obtained by different extraction solvents) ×0.2+ginsenoside Rg ₁ Content/max (ginsenoside Rg obtained by different extraction solvents) ₁ Content) 0.2+ ginsenoside Rb ₁ Content/max (ginsenoside Rb obtained by different extraction solvents) ₁ Content) 0.2+rutin content/max (rutin content obtained from different extraction solvents) 0.4

Experimental results

TABLE 1 content of ginsenoside Re, rg1, rb1 and rutin and comprehensive score

FIG. 17 shows the trend of the extraction efficiency of different ethanol concentrations for the extraction of the active ingredients; fig. 18 shows the comparison of the active ingredient composite scores of the extracts obtained with different extraction solvents, the comparison between groups, p <0.05, p <0.01.

From the above test results, as the ethanol concentration increases, the extraction efficiency of ginsenoside Rg1 and Re increases, while the extraction efficiency of ginsenoside Rb1 decreases, and the extraction efficiency of rutin increases and decreases, and the extraction efficiency at 50% ethanol concentration is optimal. The effective components in the traditional Chinese medicine composition extracted by ethanol with different concentrations are comprehensively evaluated, and compared with 80 percent ethanol, 70 ethanol, 60 percent ethanol and 0 percent ethanol, 50 to 60 percent ethanol can better extract the effective components in the traditional Chinese medicine composition, and the traditional Chinese medicine composition extracted by 50 percent ethanol can furthest extract index components in pharmacopoeias such as ginsenoside Rg1, re, rb1, rutin and the like.

According to index components of ginseng and pagodatree flower in Chinese pharmacopoeia and content determination method thereof, ginsenoside Rg is adopted ₁ 、Re、Rb ₁ And comprehensively grading the extraction efficiency of the traditional Chinese medicine composition extraction method by taking rutin as an index component. According to the high performance liquid phase measurement result and the comprehensive score calculation, the extraction efficiency of 50% -60% ethanol for the 4 index components is better than that of 70% ethanol and 80% ethanol, wherein the extraction efficiency and economic benefit are better when the traditional Chinese medicine composition is extracted by 50% ethanol.

Therefore, the preferred preparation method of the traditional Chinese medicine composition is as follows: weighing decoction pieces of Ginseng radix, fructus crataegi and flos Sophorae Immaturus, adding 8 times of 50% ethanol, heating and reflux extracting for 1.5 hr (micro boiling), filtering, and extracting for 2 times; mixing the obtained 2 times of medicinal liquids, concentrating at 60deg.C under reduced pressure to a certain volume, drying under reduced pressure to obtain dry extract, and pulverizing to obtain the Chinese medicinal composition.

3. Verification and research of preferred preparation process of traditional Chinese medicine composition

The preparation method of the traditional Chinese medicine composition determined by the invention comprises the following steps: weighing the ginseng slice-clean haw-clean pagodatree flower composition, or the red ginseng slice-clean haw-clean pagodatree flower composition, or the ginseng slice-fried haw-clean pagodatree flower composition, or the ginseng slice-burnt haw-clean pagodatree flower composition, or the ginseng slice-clean haw-fried pagodatree flower composition decoction pieces, wherein the ratio of each processing product is 1:2:2, adding 8 times of 50% ethanol, heating and refluxing for extraction for 1.5 hours (micro boiling), filtering, and extracting for 2 times; mixing the obtained 2 times of medicinal liquids, concentrating at 60deg.C under reduced pressure to a certain volume, drying under reduced pressure to obtain dry extract, and pulverizing to obtain the Chinese medicinal composition. Precisely weighing 0.1g of the traditional Chinese medicine composition, adding 5mL of methanol, ultrasonically extracting for 40 min, centrifuging at 4000r/min for 10 min, transferring the supernatant to a 5mL volumetric flask, and fixing the volume with methanol for later use.

Detecting according to liquid chromatography conditions in comparison of extraction efficiency of the Chinese medicinal composition extracted by two different ethanol concentrations,

record ginsenoside Re, rg ₁ And calculating the transfer rate of the effective components by the peak areas of corresponding retention time of Rb1 and rutin.

Effective component transfer rate (%) = (Chinese medicinal composition mass×content)/(decoction piece amount×content in decoction pieces) =dry extract yield×dry extract content/content in decoction pieces;

the theoretical minimum content of active ingredient= (pagodatree flower weight pharmacopoeia minimum content) ×ingredient transfer rate/(pagodatree flower weight dry extract yield) =pharmacopoeia minimum content×ingredient transfer rate/dry extract yield.

TABLE 2

As can be seen from the data in table 2 above, the theoretical minimum content of the active ingredients, i.e. the active ingredient content per unit mass of the dry extract, is: rg1+Re is more than or equal to 1.24mg/g; rb1 is more than or equal to 2.13mg/g; rutin is more than or equal to 37.13mg/g.

[ in vivo efficacy experiment of Chinese medicinal composition for treating hyperlipidemia ]

Instrument and reagent information

UV6200H electronic balance (SHIMADZU Shimadzu, d=0.01 g, japan), AUW220D electronic balance (Shimadzu corporation, d=0.1 mg/0.01mg, japan), constant temperature shaker (Taiku Kui laboratory Equipment Co., pegyin 2-2 constant temperature shaker, china), high speed refrigerated centrifuge (Simer Feishmania technology (China), disposable vacuum blood collection tube (Hebei Kang Weishi medical technology Co., containing separation gel and coagulant, china), ultrasonic multichannel pulverizer (Ningbo Xinzhi biotechnology Co., scientz-24TD, china), 96-well cell culture plate (Corning Incorporated, ref_3599, china), enzyme-labeled instrument (BioTek Instruments, model Eon, USA)

60% lipid-powered high fat feed (Research diabetes, research diabetes_D 12492, lot 22110202, 22120607, USA), carboxymethylcellulose sodium 300-800 mPa.s (national pharmaceutical sciences chemical Co., ltd., chemical purity, china), total cholesterol test box (Nanj institute of bioengineering, A111-2-1, lot 20221220, china), triglyceride test box (Nanj institute of bioengineering, A110-2-1, lot 20221217, china), high density lipoprotein cholesterol test box (Nanj institute of bioengineering, A112-2-1, lot 20221205, china), low density lipoprotein cholesterol test box (Nanj institute of bioengineering, A113-2-1, lot 20221119, na), 4% tissue cell fixative (Shanghai, inc., 11 IA, china)

The preparation of the traditional Chinese medicine composition comprises the following steps: mixing Ginseng radix, fructus crataegi, and flos Sophorae Immaturus at a ratio of 1:2:2, adding 8 times of 50% ethanol, reflux extracting under heating for 1.5 hr, filtering, extracting for 2 times, and mixing filtrates; rotary steaming at 60deg.C to recover ethanol, concentrating under reduced pressure at 60deg.C to obtain 1.1g crude drug/mL thick extract, packaging, and storing at-20deg.C.

The preparation method of the oral liquid medicine of the traditional Chinese medicine composition comprises the following steps: taking a proper amount of Chinese medicinal composition thick extract, diluting with 0.5% sodium carboxymethylcellulose, and pulverizing by ultrasonic waves, and using the Chinese medicinal composition thick extract for gastric lavage administration of mice.

Experimental method

1. Feeding and modeling experimental animals: SPF-class male C57BL/6J mice, 5 weeks old, 18-20 g, supplied by Experimental animal technology Co., ltd., beijing, are raised in Shanghai university of Chinese medicine laboratory animal center, raised by special persons in a raising environment with temperature of 22-24 ℃ and relative humidity of 60-70% RH, and the bright and dark conditions are alternately carried out for 12 hours each. Mice were kept normally for one week to adapt to the feeding environment before the beginning of the formal experiments. The animal use license was SYXK 2020-0009, and all experimental animal care and protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai university of Chinese medicine.

2. Grouping and molding: mice were randomly divided into 7 groups: blank, model, high, medium and low dose composition groups of 7 each, for a total of 35. The blank group was given normal diet, and the model group and each dosing group were fed with 60% fat-powered high fat diet.

3. Dosing regimen: the mice in each group synchronously start to be dosed, and the model group and the normal group are dosed with sodium carboxymethyl cellulose with the same volume of 0.5 percent; the administration of mice in the traditional Chinese medicine composition group is converted from the body surface area method and the clinical dosage, the administration dosage of the dry extract powder of the mice in the high-dosage composition is 4.0g/kg, the administration dosage of the mice in the medium-dosage composition is 2.0g/kg, and the administration dosage of the mice in the low-dosage composition is 1.0g/kg. Converted into standard adult (60 kg), the clinical recommended dosage of the dry extract is as follows: the high dose is 0.32g/kg, the medium dose is 0.16g/kg, and the low dose is 0.08g/kg.

4. Evaluation of efficacy of Chinese medicinal composition for treating hyperlipidemia

(1) Blood lipid level: the main indexes of clinical evaluation of hyperlipidemia are serum Total Cholesterol (TC), serum Triglyceride (TG), serum high density lipoprotein cholesterol (HDL-C) and serum low density lipoprotein cholesterol (LDL-C), so the drug effect experiment related to the invention also uses the above 4 blood lipid indexes as key evaluation indexes.

The mice were taken every three days, and their body weights were measured weekly. After molding for 4 weeks, mice in the blank group and the model group were randomly drawn every 2 weeks, blood was examined, and blood lipid level was observed to change, when the blood lipid level of the molded mice was significantly changed as compared with that of the blank group (p)<0.05 And then the molding is successful. After 10 weeks of synchronous administration of the actual molding, taking materials of mice, taking blood from eyeballs, centrifuging for 15min at 3000r/min, taking upper serum, and measuring the 4 blood lipid indexes. Statistical processing results are adoptedAnd (3) representing. Statistical analysis with SPSS26.0 software, p<0.05 indicates that the difference is statistically significant.

(2) Liver tissue HE staining pathological section: the related literature shows that hyperlipidemia may cause accumulation of lipid droplets in the liver, so that pathological sections of the liver are also used as reference indexes for pharmacodynamic experiments in the experiment. Liver tissues obtained from mice molded for 10 weeks are separated into tissues with the size of 1cm multiplied by 1cm at a fixed position, fixed by 4% paraformaldehyde fixing solution, and subjected to steps such as fixing, dehydration, embedding, slicing, H & E staining and the like for histopathological analysis and observation.

(3) Liver tissue oil red O staining pathological section: liver tissues obtained from mice molded for 10 weeks are separated into tissues with the size of 1cm multiplied by 1cm at a fixed position, fixed by 4% paraformaldehyde fixing solution, and subjected to steps such as fixing, dehydration, embedding, slicing, oil red O staining and the like for histopathological analysis and observation.

Experimental results

1. Blood lipid level

Table 3 each index level (LDL-C) in serum of mice of each group (n=7,)/>

note that: p <0.01 compared to the blank; compared to the model group, #p <0.05, #p <0.01; compared with the model group, ∈r shows a decreasing trend, but no significant difference.

As can be seen from table 3, the model group 4 blood lipid level indexes showed that the mice blood lipid level was abnormal after 10 weeks of feeding the high fat feed, i.e. the TC, TG and LDL-C levels were significantly increased and the HDL-C level was significantly decreased, compared with the blank group, so that the long-term feeding of the high fat feed was considered to induce the model of hyperlipidemia in C57 mice to be successful.

Fig. 19 shows comparison of blood lipid levels for each group, with p <0.01 compared to the blank group; compared with the model group, the composition with the dosage of 3 doses has certain lipid-lowering efficacy, compared with the model group, the efficacy of the composition with the dosage of 3 doses is mainly reflected in remarkably improving HDL-C and LDL-C, the efficacy of the medium-dosage group can remarkably improve TG, HDL-C and LDL-C, the efficacy of the high-dosage group is best, 4 blood lipid indexes of the composition with the dosage of 4 doses are improved, namely, the level of TC, TG, LDL-C is remarkably reduced, and the level of HDL-C is remarkably increased.

2. Liver HE staining section

FIG. 20 shows a comparison of HE staining patterns (x 200) for liver, A for the normal group, B for the model group, C for the high dose group, D for the medium dose group, and E for the low dose group; the blank small circles indicated by the red scissors are fat drops. After 10 weeks of molding by feeding the high-fat feed, compared with the blank group, the liver of the mice in the model group has a large quantity of fat drops spread over, and the fat drops are large and dense; compared with a model group, the composition has obviously reduced lipid drops in the livers of the high-dose group, the medium-dose group and the low-dose group, wherein the liver morphology of the high-dose group and the medium-dose group of the composition is close to that of a blank group of livers, which indicates that the traditional Chinese medicine composition can obviously improve liver lipid accumulation and has the best lipid lowering effect of the high dose.

3. Liver viscera oil red O staining slice

FIG. 21 shows comparison of liver oil red O staining patterns (x 200), wherein A is a normal group, B is a model group, C is a high-dose group of the composition, D is a medium-dose group of the composition, and E is a low-dose group of the composition, and after molding for 10 weeks by feeding a high-fat feed, the liver of mice in the model group stained with oil red O has a large number of lipid droplets spread over, and the lipid droplets are dense and aggregated, compared with a blank group; compared with the model group, the composition high dose group, the composition medium dose group and the composition low dose group have obviously reduced liver lipid drops, which suggests that the composition has a certain alleviation effect on liver lipid accumulation of the hyperlipidemic mice.

Based on the in vitro cell experimental result, the lipid-lowering effect of the ginseng-hawthorn-pagodatree flower (1:2:2) alcohol extract is optimal, and the traditional Chinese medicine composition is used for in vivo experiments of animals to verify the efficacy.

The method adopts the high-fat feed to feed the C57BL/6J mice to form a hyperlipidemia model, sets three doses of the traditional Chinese medicine composition, synchronously feeds the traditional Chinese medicine composition, takes the blood lipid level and liver staining slices of the mice as evaluation indexes after 10 weeks of molding feeding, and can improve the abnormal blood lipid level and liver lipid accumulation to a certain extent, wherein the high-dose drug effect of the traditional Chinese medicine composition is optimal, namely the TC, TG, LDL-C level can be obviously reduced, the HDL-C level can be obviously improved, and the lipid accumulation in the liver can be obviously reduced.

In conclusion, based on the series of in vitro experimental results, the ginseng-hawthorn-pagodatree flower (1:2:2) composition with lipid-lowering potential is used for feeding a lipid-lowering feed to induce a C57BL/6J mouse hyperlipidemia model to verify lipid-lowering efficacy. After 10 weeks of mould making and synchronous administration, TC, TG, HDL-C, LDL-C level and liver pathology staining slices are taken as evaluation indexes, the lipid-lowering effect of the ginseng-hawthorn-pagodatree flower (1:2:2) composition is verified, and the drug effect of the composition under different administration doses is compared, so that the following is known: the low-dose (the dosage specified by pharmacopoeia), the medium-dose (1.2 times of the dosage specified by pharmacopoeia) and the high-dose (2.4 times of the dosage specified by pharmacopoeia) of the traditional Chinese medicine composition can play a role in improving abnormal blood lipid level and liver lipid accumulation to a certain extent, wherein the lipid-lowering efficacy of the traditional Chinese medicine composition with the medium-low dose is mainly reflected in improving the levels of TG, HDL-C and LDL-C, and the traditional Chinese medicine composition has a certain role in relieving liver lipid accumulation, and the high-dose efficacy of the traditional Chinese medicine composition is optimal, namely the level of TC, TG, LDL-C can be obviously reduced, the level of HDL-C can be obviously improved, and the lipid accumulation in the liver can be obviously reduced. By combining the lipid-lowering effect of the traditional Chinese medicine composition with medium and low doses in vivo experiments, low doses can be recommended to be taken for a long time.

The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and those skilled in the art, based on the present disclosure, should make improvements and modifications without departing from the scope of the present invention.

Claims

1. The traditional Chinese medicine composition for treating the hyperlipidemia is characterized by being prepared from traditional Chinese medicine raw materials, wherein the traditional Chinese medicine raw materials comprise ginseng, hawthorn and pagodatree flower;

the traditional Chinese medicine is prepared from the following raw materials in parts by weight: 1 to 1:2, the ratio of the hawthorn to the pagodatree flower is 2: 1-2: 2.

2. the traditional Chinese medicine composition for treating hyperlipidemia according to claim 1, wherein the proportion of ginseng, hawthorn and pagodatree flower is 1-4 in parts by weight: 2:1 to 2.

3. The traditional Chinese medicine composition for treating hyperlipidemia according to claim 1 or 2, wherein the ginseng is a processed product of ginseng and red ginseng, and comprises ginseng slices and red ginseng slices;

The fructus crataegi is processed product of fructus crataegi, including fructus crataegi, parched fructus crataegi, and baked fructus crataegi;

the flos Sophorae Immaturus is processed product of flos Sophorae Immaturus, including clean flos Sophorae Immaturus and parched flos Sophorae Immaturus.

4. The traditional Chinese medicine composition for treating hyperlipidemia according to claim 1 or 2, wherein the traditional Chinese medicine composition is a solvent extract of ginseng, hawthorn and pagodatree flower, and is obtained by extracting ginseng, hawthorn and pagodatree flower with solvent, concentrating and drying to obtain dry extract, and pulverizing to obtain the traditional Chinese medicine composition; or adding appropriate adjuvant, and making into granule, pill, or capsule.

5. The Chinese medicinal composition for treating hyperlipidemia according to claim 4, wherein the effective components contained in the Chinese medicinal composition comprise: the content of the active ingredients of the dry extract per unit mass is as follows: rg1+Re is more than or equal to 1.24mg/g; rb1 is more than or equal to 2.13mg/g; rutin is more than or equal to 37.13mg/g.

6. The method for preparing the traditional Chinese medicine composition for treating hyperlipidemia according to claim 1 or 2, which is characterized by comprising the following steps: weighing Ginseng radix, fructus crataegi and flos Sophorae Immaturus at a certain proportion, adding extraction solvent, extracting, filtering to obtain medicinal liquid extract, concentrating, drying to obtain dry extract, and pulverizing to obtain the final product.

7. The method for preparing a Chinese medicinal composition for treating hyperlipidemia according to claim 6, wherein the extraction solvent is water and/or ethanol, and the amount of the extraction solvent is 5-10 times of the total weight of ginseng, haw and pagodatree flower;

the extraction process comprises heating under reflux, keeping the extraction solvent in micro-boiling state, extracting for 1-3 hr each time, and concentrating under reduced pressure at 50-90deg.C.

8. The method for preparing a Chinese medicinal composition for treating hyperlipidemia according to claim 7, wherein the extraction solvent is ethanol with a concentration of 30% -80%.

9. The use of a Chinese medicinal composition for treating hyperlipidemia according to claim 1, wherein the Chinese medicinal composition is used for preparing a medicament for treating hyperlipidemia.

10. The use of a Chinese medicinal composition for treating hyperlipidemia according to claim 9, wherein the clinically minimum dose is 0.08g/kg based on 60kg of standard adult.