CN112546060B - Lipid-lowering traditional Chinese medicine monomer composition, preparation and use - Google Patents
Lipid-lowering traditional Chinese medicine monomer composition, preparation and use Download PDFInfo
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- CN112546060B CN112546060B CN202011479399.8A CN202011479399A CN112546060B CN 112546060 B CN112546060 B CN 112546060B CN 202011479399 A CN202011479399 A CN 202011479399A CN 112546060 B CN112546060 B CN 112546060B
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/472—Non-condensed isoquinolines, e.g. papaverine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/473—Quinolines; Isoquinolines ortho- or peri-condensed with carbocyclic ring systems, e.g. acridines, phenanthridines
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
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- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
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- General Health & Medical Sciences (AREA)
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- Engineering & Computer Science (AREA)
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- Obesity (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本发明公开了降脂的中药单体组合物、制剂及用途。该降脂的中药单体组合物由质量比为9.1～9.6：1：4.2～4.7：1.6～2.1：2.2～2.7：1.7～2.2的槲皮素‑3‑O‑β‑D‑葡萄糖醛酸苷、金丝桃苷、红镰霉素龙胆二糖苷、大黄素‑8‑O‑β‑D‑葡萄糖苷、O‑去甲荷叶碱和杏黄罂粟碱组成。本发明中药单体组合物可以降低血脂，特别对于高甘油三酯血症，降脂效果优于现有常用中成药降脂药降脂宁颗粒。The invention discloses lipid-lowering traditional Chinese medicine monomer compositions, preparations and uses. The lipid-lowering traditional Chinese medicine monomer composition is composed of quercetin-3-O-β-D-glucuronic acid whose mass ratio is 9.1-9.6:1:4.2-4.7:1.6-2.1:2.2-2.7:1.7-2.2 glycoside, hypericin, erythromycin gentiobioside, emodin-8-O-β-D-glucoside, O-norflavine and apricot papaverine. The traditional Chinese medicine monomer composition of the present invention can reduce blood lipids, especially for hypertriglyceridemia, the lipid-lowering effect is better than the existing commonly used traditional Chinese medicine lipid-lowering medicine Jiangzhining granules.
Description
Technical Field
The invention relates to a traditional Chinese medicine monomer composition for reducing blood fat, a preparation and application.
Background
Hyperlipidemia is a common disease in the current society, is manifested by overhigh cholesterol and triglyceride in serum, and can directly cause diseases seriously harming human health, such as atherosclerosis, coronary heart disease, pancreatitis and the like. The incidence of hyperlipidemia increases year by year, and in adults over 30 years of age in the country, the incidence of hyperlipidemia is 10% to 20%.
At present, many blood fat reducing medicines are available in the market, and western medicines are mainly used because of quick response and definite curative effect. Common medicines such as simvastatin can be used for reducing elevated total cholesterol, low density lipoprotein cholesterol, apolipoprotein B and triglyceride, but have obvious side effects and can cause abnormal blood sugar, general muscular soreness, weakness and the like of patients.
According to traditional Chinese medicine, the blood fat is generally considered to be the syndrome of qi stagnation and blood stasis caused by the fact that the blood-fat is increased seriously by phlegm-dampness in vivo and further influences the circulation of qi and blood, so traditional Chinese medicines with the functions of tonifying spleen and removing dampness, and promoting qi and activating blood circulation are selected for treatment. The commonly used lipid-lowering Chinese patent medicines comprise lipid-lowering Ning granules, ginkgo leaf capsules, blood lipid-lowering Kangjiao capsules and the like, and although the lipid-lowering side effect of the Chinese patent medicines is small, the dosage of the Chinese patent medicines is large, and the lipid-lowering effect is not as obvious as that of western medicines.
CN104173495A discloses Jiangzhining granules, which are prepared from the following raw materials in parts by weight: 25-27.5 parts of hawthorn, 1-1.25 parts of prepared fleece-flower root, 1-1.25 parts of cassia seed and 3.25-3.75 parts of lotus leaf. Although the Jiangzhining granules have the effects of expanding coronary vessels, increasing coronary flow, reducing cholesterol absorption, accelerating cholesterol excretion, inhibiting serum cholesterol increase and formation of atherosclerotic plaques, reducing blood fat and cholesterol and the like, the effect of reducing hyperlipidemia (TG) is not obvious, and the Jiangzhining granules need to be taken 3 times a day, 10g each time, the oral dosage is large, the administration is inconvenient, and the granules are easy to get damp.
CN104352594A discloses a Jiangzhining dispersible tablet, which is prepared by extracting hawthorn, prepared fleece flower root, cassia seed and lotus leaf, adding filler, disintegrating agent, flavoring agent, wetting agent, lubricant and glidant. The dispersible tablet is convenient to take, is suitable for the old and patients with swallowing difficulty, but still can not solve the problems of unobvious effect of reducing hyperlipidemia and large oral dosage.
CN108635426A discloses a Jiangzhining Chinese medicinal concentrated watered pill, which is prepared by extracting fructus crataegi, radix Polygoni Multiflori Preparata, semen Cassiae and folium Nelumbinis, and making into watered pill. The watered pill has small volume, is convenient to swallow, is not easy to absorb moisture, but still can not solve the problems of unobvious effect of reducing hyperlipemia and large oral dosage.
In conclusion, although simvastatin, a western lipid-lowering drug, has a good lipid-lowering effect, it has significant side effects and is not suitable for long-term consumption by patients. Although the Chinese patent medicine lipid-lowering Ning granules and the improved preparation thereof have small side effect on treating hyperlipidemia and are suitable for long-term eating, the Chinese patent medicine lipid-lowering Ning granules have the problems of large dosage, unobvious lipid-lowering effect and the like.
Disclosure of Invention
In view of the above, an object of the present invention is to provide a lipid-lowering Chinese medicinal monomer composition. The Chinese medicinal monomer composition can reduce blood fat, and has remarkable effect of reducing blood fat, especially for hypertriglyceridemia.
The invention also aims to provide a traditional Chinese medicine preparation for reducing blood fat. The Chinese medicinal preparation has good blood fat reducing effect, is safe to use and has small dosage.
The invention also aims to provide application of the traditional Chinese medicine monomer composition.
On one hand, the invention provides a traditional Chinese medicine monomer composition for reducing blood fat, which is prepared from the following components in parts by mass of 9.1-9.6: 1: 4.2-4.7: 1.6-2.1: 2.2-2.7: 1.7-2.2 of quercetin-3-O-beta-D-glucuronide, hyperoside, rubrofusarin gentiobioside, emodin-8-O-beta-D-glucoside, O-nornuciferine and papaverine.
According to the traditional Chinese medicine monomer composition, preferably, the traditional Chinese medicine monomer composition is prepared by mixing, by mass, 9.25-9.45: 1: 4.35-4.55: 1.75-1.95: 2.35-2.55: 1.85-2.05 of quercetin-3-O-beta-D-glucuronide, hyperoside, rubrofusarin gentiobioside, emodin-8-O-beta-D-glucoside, O-nornuciferine and papaverine.
According to the traditional Chinese medicine monomer composition, preferably, the traditional Chinese medicine monomer composition is prepared from the following components in a mass ratio of 9.32: 1: 4.45: 1.85: 2.41: 1.92 quercetin-3-O-beta-D-glucuronide, hyperoside, rubrofusarin gentiobioside, emodin-8-O-beta-D-glucoside, O-nornuciferine and apricot yellow papaverine.
According to the traditional Chinese medicine monomer composition, preferably, the traditional Chinese medicine monomer composition is prepared from the following components in a mass ratio of 9.28: 1: 4.51: 1.78: 2.52: 1.87 composed of quercetin-3-O-beta-D-glucuronide, hyperoside, rubrofusarin gentiobioside, emodin-8-O-beta-D-glucoside, O-nornuciferine and apricot yellow papaverine.
According to the traditional Chinese medicine monomer composition, preferably, the traditional Chinese medicine monomer composition is prepared from the following components in a mass ratio of 9.42: 1: 4.38: 1.93: 2.38: 2.04 quercetin-3-O-beta-D-glucuronide, hyperoside, rubrofusarin gentiobioside, emodin-8-O-beta-D-glucoside, O-nornuciferine and apricot yellow papaverine.
On the other hand, the invention provides a traditional Chinese medicine preparation for reducing blood fat, which comprises the traditional Chinese medicine monomer composition and pharmaceutically acceptable auxiliary materials.
According to the traditional Chinese medicine preparation of the present invention, preferably, the traditional Chinese medicine preparation comprises any one of capsules, tablets, granules, pills, powders, ointments, pellets, decoctions, medicated liquors, granules, oral liquids, injections, patches, sustained-release preparations and controlled-release preparations.
According to the traditional Chinese medicine preparation disclosed by the invention, preferably, a unit traditional Chinese medicine preparation contains 10-200 mg of traditional Chinese medicine monomer composition.
In another aspect, the invention also provides an application of the traditional Chinese medicine monomer composition in preparing a hypolipidemic drug.
According to the use of the present invention, preferably, the Chinese medicinal monomer combination is used for preparing a pharmaceutical preparation for reducing triglyceride.
The traditional Chinese medicine monomer composition for reducing blood fat consists of quercetin-3-O-beta-D-glucuronide, hyperoside, rubrofusarin gentiobioside, emodin-8-O-beta-D-glucoside, O-nornuciferine and apricot yellow papaverine in a specific dosage ratio, is used for reducing blood fat and treating hyperlipidemia, is small in dosage and safe to take, can be taken for a long time, particularly has a remarkable blood fat reducing effect on hypertriglyceridemia, and is obviously superior to the conventional Chinese patent medicine lipid-reducing medicine lipid-lowering Ningke granules.
Detailed Description
The present invention will be further described with reference to the following specific examples, but the scope of the present invention is not limited thereto.
By analyzing the chemical components of the traditional Chinese medicine Jiangzhining granules and performing systematic research on pharmacology, the monomer composition disclosed by the invention is found to be the most key chemical component of the Jiangzhining granules for playing the role of reducing blood fat. The monomeric compounds are prepared into the composition according to the dosage proportion, and the lipid-lowering effect of the composition is obviously better than that of Jiangzhining granules, so that a new and more effective pharmaceutical composition for lowering blood lipid is provided, and a basis is provided for explaining the connotation of the blood lipid-lowering effect of the Jiangzhining granules of Chinese patent medicine and standardizing the preparation.
< Chinese medicinal monomer composition >
The traditional Chinese medicine monomer composition for reducing blood fat comprises the following components in parts by mass of 9.1-9.6: 1: 4.2-4.7: 1.6-2.1: 2.2-2.7: 1.7-2.2 of quercetin-3-O-beta-D-glucuronide, hyperoside, rubrofusarin gentiobioside, emodin-8-O-beta-D-glucoside, O-nornuciferine and papaverine.
In the invention, preferably, the traditional Chinese medicine monomer composition is prepared from the following components in a mass ratio of 9.25-9.45: 1: 4.35-4.55: 1.75-1.95: 2.35-2.55: 1.85-2.05 of quercetin-3-O-beta-D-glucuronide, hyperoside, rubrofusarin gentiobioside, emodin-8-O-beta-D-glucoside, O-nornuciferine and papaverine.
According to a preferred embodiment of the present invention, the traditional Chinese medicine monomer composition is prepared from 9.32: 1: 4.45: 1.85: 2.41: 1.92 quercetin-3-O-beta-D-glucuronide, hyperoside, rubrofusarin gentiobioside, emodin-8-O-beta-D-glucoside, O-nornuciferine and apricot yellow papaverine.
According to another preferred embodiment of the present invention, the Chinese medicinal monomer composition is prepared from 9.28 mass percent: 1: 4.51: 1.78: 2.52: 1.87 composed of quercetin-3-O-beta-D-glucuronide, hyperoside, rubrofusarin gentiobioside, emodin-8-O-beta-D-glucoside, O-nornuciferine and apricot yellow papaverine.
According to still another preferred embodiment of the present invention, the Chinese medicinal monomer composition is prepared from 9.42: 1: 4.38: 1.93: 2.38: 2.04 quercetin-3-O-beta-D-glucuronide, hyperoside, rubrofusarin gentiobioside, emodin-8-O-beta-D-glucoside, O-nornuciferine and apricot yellow papaverine.
In the lipid-lowering Chinese medicinal monomer composition, all the raw materials are known compounds, and can be obtained by commercially available methods or prepared by methods known in the art, such as extraction and separation from medicinal materials according to methods described in literatures. The composition may be prepared by a method of mixing conventionally used in the art, or may be prepared by other methods, and is not particularly limited.
In the invention, the English name of the used Quercetin-3-O-beta-D-Glucuronide is Quercetin 3-O-beta-D-Glucuronide with the chemical formula of C21H18O13And the molecular weight is 478.37.
The Hyperoside has English name of Hyperoside and chemical formula of C21H20O12And the molecular weight is 464.3763.
The Rubrofusarin gentiobioside has the English name of Rubrofusarin-6-O-beta-gentiobioside and the chemical formula of C27H32O15And the molecular weight is 596.53.
The Emodin-8-O-beta-D-glucoside has English name of Emodin-8-glucoside, and chemical formula of C21H20O10And the molecular weight is 432.41.
The O-Nornuciferine (2-hydroxy-1-methoxy-aporphine) is Ortho-Nornuciferine with chemical formula C18H19NO2And the molecular weight is 281.35.
The papaverine has the English name Armepavire and chemical formula C19H23NO3And the molecular weight is 313.39.
In the traditional Chinese medicine monomer composition, the six components are chemical components contained in the Chinese patent medicine Jiangzhining granules. The six monomers form the composition according to the dosage proportion of the invention, and the composition has remarkable lipid-lowering effect, is obviously superior to the lipid-lowering Ning granules, and is also superior to the compositions with other proportions.
< preparation and use >
The invention provides a traditional Chinese medicine preparation for reducing blood fat, which comprises a traditional Chinese medicine monomer composition and pharmaceutically acceptable auxiliary materials. The dosage form of the Chinese medicinal preparation of the present invention is not particularly limited, and may be any pharmaceutical dosage form. Preferably any one of capsule, tablet, granule, pill, powder, unguent, pellet, decoction, medicated liquor, granule, oral liquid, injection, patch, sustained release preparation and controlled release preparation. More preferably any one of tablets, pills, capsules, granules and oral liquids.
In the invention, the lipid-lowering Chinese medicinal preparation can contain pharmaceutically acceptable auxiliary materials. The kind of the pharmaceutically acceptable auxiliary material is not limited. When the lipid-lowering Chinese medicinal preparation is an oral preparation, the auxiliary material may be one or more selected from diluents, binders, disintegrants and lubricants. The diluent includes, but is not limited to, one or more of mannitol, microcrystalline cellulose, lactose, sucrose, pregelatinized starch, or dextrin, preferably one or more of microcrystalline cellulose, lactose, or dextrin. The binder is selected from one or more of methylcellulose, sodium carboxymethylcellulose, ethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, gelatin, povidone or polyethylene glycol, preferably one or more of sodium carboxymethylcellulose, gelatin or povidone. The disintegrant includes but is not limited to one or more of sodium carboxymethyl starch, croscarmellose sodium and crospovidone. The lubricant is selected from one or more of polyethylene glycol, sodium lauryl sulfate, talcum powder or magnesium stearate, and is preferably crospovidone or croscarmellose sodium.
In the invention, the dosage of the traditional Chinese medicine monomer composition in a unit traditional Chinese medicine preparation is 10-200 mg, preferably 50-100 mg. The unit traditional Chinese medicine preparation refers to a preparation and application unit preparation, such as a tablet, a bag of granules, a capsule, a bottle of oral liquid and the like. When the dosage of the traditional Chinese medicine monomer composition in the prepared unit traditional Chinese medicine preparation is within the range, the dosage is small, the medication is safe, the traditional Chinese medicine preparation can be eaten for a long time, and the lipid-lowering effect is obvious.
The traditional Chinese medicine monomer composition has a remarkable effect of treating hyperlipidemia, and particularly has an exact curative effect on hypertriglyceridemia.
The following examples and comparative examples use the starting materials.
Jiangzhining granules: purchased from general Giantong pharmaceuticals Ltd under the batch number 20200702 and having a format of 10g by 18 bags.
Simvastatin: available from Adamas Reagent CO., Ltd., LOT: P03869.
quercetin-3-O- β -D-glucuronide: from Dowman Biotechnology, Inc., LOT: MUST-20070805.
Hyperin: purchased from Shanghai leaf Biotechnology, Inc., LOT: P14A11F 121347.
Rubrofusarin gentiobioside: purchased from Shanghai Hotan Biotechnology, Inc., LOT: 16120621.
emodin-8-O- β -D-glucoside: purchased from Shanghai leaf Biotechnology, Inc., LOT: P17A10F 86138.
O-nornuciferine: purchased from Shanghai-derived leaf Biotechnology, Inc., LOT: M16N10S 103163.
Apricot yellow papaverine: purchased from Shanghai leaf Biotechnology, Inc., LOT: P22M10F 88910.
Example 1
The traditional Chinese medicine monomer composition for reducing blood fat of the embodiment is prepared from the following components in a mass ratio of 9.32: 1: 4.45: 1.85: 2.41: 1.92 quercetin-3-O-beta-D-glucuronide, hyperoside, rubrofusarin gentiobioside, emodin-8-O-beta-D-glucoside, O-nornuciferine and apricot yellow papaverine.
Example 2
The traditional Chinese medicine monomer composition for reducing blood fat of the embodiment is prepared from the following components in a mass ratio of 9.28: 1: 4.51: 1.78: 2.52: 1.87 composed of quercetin-3-O-beta-D-glucuronide, hyperoside, rubrofusarin gentiobioside, emodin-8-O-beta-D-glucoside, O-nornuciferine and apricot yellow papaverine.
Example 3
The lipid-lowering traditional Chinese medicine monomer composition comprises the following components in a mass ratio of 9.42: 1: 4.38: 1.93: 2.38: 2.04 quercetin-3-O-beta-D-glucuronide, hyperoside, rubrofusarin gentiobioside, emodin-8-O-beta-D-glucoside, O-nornuciferine and apricot yellow papaverine.
Comparative example 1
The traditional Chinese medicine monomer composition for reducing blood fat of the comparative example comprises the following components in percentage by mass of 2.22: 1: 0.73:
1.44: 0.24: 1.23 quercetin-3-O-beta-D-glucuronide, hyperoside, rubrofusarin gentiobioside, emodin-8-O-beta-D-glucoside, O-nornuciferine and apricot yellow papaverine.
Comparative example 2
The traditional Chinese medicine monomer composition for reducing blood fat of the comparative example comprises the following components in percentage by mass of 0.178: 1: 0.94: 0.38: 0.16: 0.38 percent of quercetin-3-O-beta-D-glucuronide, hyperoside, rubrofusarin gentiobioside, emodin-8-O-beta-D-glucoside, O-nornuciferine and apricot yellow papaverine.
Comparative example 3
The traditional Chinese medicine monomer composition for reducing blood fat of the comparative example comprises the following components in percentage by mass of 1.03: 1: 2.08: 0.45: 0.44: 0.04 parts of quercetin-3-O-beta-D-glucuronide, hyperoside, rubrofusarin gentiobioside, emodin-8-O-beta-D-glucoside, O-nornuciferine and apricot yellow papaverine.
Experimental example 1-hypolipidemic Activity test
In order to compare the difference of the blood fat reducing activity between the traditional Chinese medicine monomer composition in the embodiment 1 and the traditional Chinese medicine monomer compositions, the Chinese patent medicine lipid-lowering Ning particles and the western medicine lipid-lowering medicine simvastatin in the comparative examples 1-3, the invention adopts a high fat model established by sodium oleate induced HepG2 cells to carry out the research of the blood fat reducing activity.
1. Preparation of the principal solution
RMPI-1640 complete medium: the RMPI-1640 medium was added with 1 wt% of a cyan-streptavidin antibody and 10 wt% of fetal bovine serum, and stored at 4 ℃.
Phosphate Buffered Saline (PBS): taking a fixed package of phosphate powder, adding 2L of double distilled water, continuously stirring to uniformly dissolve PBS, subpackaging into a serum bottle, autoclaving, and storing at 4 ℃.
Cell cryopreservation solution: contains 20 wt% of fetal calf serum, 10 wt% of DMSO solution and 70 wt% of RMPI-1640 culture medium.
MTT solution: 150mg of thiazole blue (MTT) powder is dissolved in 30mL of PBS buffer solution, and is protected from light to be uniformly dissolved, and after being filtered by a 0.22 mu m microporous filter, the mixture is subpackaged and is placed at-20 ℃ for being stored in the absence of light.
Sodium oleate solution: weighing a certain amount of sodium oleate reference substance, adding a small amount of PBS to completely dissolve, adding RMPI-1640 complete culture medium to prepare a solution with a certain concentration, filtering by a 0.22 mu m microporous filter, and storing at 4 ℃.
Jiangzhining granule solution: weighing 3.12mg of JIANGZHINING granule, adding 15.600mL of RMPI-1640 complete culture medium, dissolving completely, and making into 200 μ g/mL solution. Filtering with 0.22 μm sterile filter membrane.
The traditional Chinese medicine monomer composition solution of the invention: according to the weight percentage of quercetin-3-O-beta-D-glucuronide: hyperin: rubrofusarin gentiobioside: emodin-8-O- β -D glucoside: o-nornuciferine: apricot yellow papaverine 9.32: 1: 4.45: 1.85: 2.41: 1.92, finally obtaining a traditional Chinese medicine monomer composition solution with the concentration of 0.2mg/mL, filtering the solution by a 0.22 mu m microporous filter, and storing the solution at 4 ℃.
Comparative example 1 traditional Chinese medicine monomer composition solution: according to the weight percentage of quercetin-3-O-beta-D-glucuronide: hyperin: rubrofusarin gentiobioside: emodin-8-O- β -D glucoside: o-nornuciferine: apricot yellow papaverine 2.22: 1: 0.73: 1.44: 0.24: 1.23 to obtain a Chinese medicinal monomer composition solution with a concentration of 0.2mg/mL, filtering with a 0.22 μm microporous filter, and storing at 4 deg.C.
Comparative example 2 traditional Chinese medicine monomer composition solution: according to the weight percentage of quercetin-3-O-beta-D-glucuronide: hyperin: rubrofusarin gentiobioside: emodin-8-O- β -D glucoside: o-nornuciferine: apricot yellow papaverine is 0.178: 1: 0.94: 0.38: 0.16: 0.38, to obtain a Chinese medicinal monomer composition solution with a concentration of 0.2mg/mL, filtering with a 0.22 μm microporous filter, and storing at 4 deg.C.
Comparative example 3 traditional Chinese medicine monomer composition solution: according to the weight percentage of quercetin-3-O-beta-D-glucuronide: hyperin: rubrofusarin gentiobioside: emodin-8-O- β -D glucoside: o-nornuciferine: apricot yellow papaverine 1.03: 1: 2.08: 0.45: 0.44: 0.04 to obtain a Chinese medicinal monomer composition solution with a concentration of 0.2mg/mL, filtering with 0.22 μm microporous filter, and storing at 4 deg.C.
2. Experimental methods
2.1 cell recovery, culture and cryopreservation
2.1.1 cell recovery
The RMPI-1640 complete culture medium is placed to the room temperature, the temperature of the constant-temperature water bath is adjusted to 37 ℃, and the HepG2 cells are taken out from the ultra-low temperature refrigerator and quickly placed in the constant-temperature water bath for melting. The cell suspension in the tube was gently pipetted evenly and added to a 15mL sterile centrifuge tube containing 4mL of RMPI-1640 complete medium for centrifugation (1000r, 2 min). Discarding supernatant, collecting cells, adding 2mL of RMPI-1640 complete culture medium, blowing into cell suspension, transferring into cell culture bottle, placing at 37 deg.C and 5% CO2Culturing in a constant temperature incubator.
2.1.2 cell culture and passage
HepG2 cells were cultured in sterile RMPI-1640 complete medium prepared, and cultured in a cell incubator set at 37 ℃ and 5% CO2Observing the growth state of the cells and carrying out passage; when the cell growth density reaches 80%, passage is carried out, the cells are washed for 2 times by sterile PBS (phosphate buffer solution) placed to room temperature, and sterile pancreatin is added for digestionObserving the cell morphology under a microscope, removing pancreatin after digestion, adding 3mL of sterile RMPI-1640 complete culture medium, lightly blowing and beating the bottom cells of the bottle by using a sterile straw, transferring the bottom cells to a 15mL centrifuge tube, centrifuging, removing supernatant, adding the prepared sterile RMPI-1640 complete culture medium, blowing and beating the cells until the cells are completely suspended, and carrying out passage according to the number of the cells in proportion.
2.1.3 cell cryopreservation
Taking HepG2 cells in good growth state, collecting the cells according to the steps in passage, adding a frozen stock solution precooled at 4 ℃, blowing and beating the cells until the cells are completely suspended, subpackaging the cells into labeled frozen stock tubes, wherein the labeled contents comprise labeled cell names, generation numbers and frozen stock dates, and placing the cells into liquid nitrogen for long-term storage after gradient cooling.
2.2 cell viability assay
The cell viability value is detected by an MTT colorimetric method, MTT can be reduced into water-insoluble blue-purple crystalline formazan by succinate dehydrogenase in mitochondria of living cells and is deposited in the cells, but dead cells do not have the phenomenon. Blue-violet formazan crystals in cells can be dissolved by DMSO, and the absorbance value of the blue-violet formazan crystals can be detected under an enzyme labeling instrument, so that the activity of the cells can be indirectly reflected.
Collecting HepG2 cells in logarithmic growth phase, making into cell suspension, inoculating into 96-well plate at 3000/well density, adding 100 μ L per well, and placing in 5% CO2Incubation in a cell incubator at 37 ℃ for 24 h. Dissolving rubrofusarin gentiobioside, hyperin, quercetin-3-O-beta-D-glucuronide, emodin-8-O-beta-D-glucoside, amygdaline, O-demethylnuciferine, sodium oleate and simvastatin by using an RMPI-1640 complete culture medium. Filtering with 0.22 μm microporous filter, and adjusting sodium oleate concentration to 20, 40, 60, 80, 100 μ g/mL; simvastatin concentrations of 30, 35, 40, 50, 60, 70, 80, 90 μ M; the rubrofusarin gentiobioside, hyperin, quercetin-3-O-beta-D-glucuronide, emodin-8-O-beta-D-glucoside, amygdaline and O-demethylnuciferine are respectively provided with six concentrations of 10, 20, 40, 80, 160 and 320 mu M. Discarding the culture medium, adding 150 μ L of each concentration of drug-containing culture medium into each well, and setting blank control group6 multiple wells at 5% CO were placed at each concentration2Incubate at 37 ℃ for 24 h. The medium was discarded, 100. mu.L of MTT solution was added, and the mixture was placed in 5% CO2Incubate at 37 ℃ for 4 h. MTT solution is discarded, 150. mu.L DMSO solution is added to each well, the mixture is placed on a microplate oscillator and shaken for 10min, blue-violet formazan crystals are fully dissolved, and the absorbance (OD) value is measured at 490nm wavelength. And drawing a cell viability diagram by taking different administration concentrations as an abscissa and an absorbance value as an ordinate.
Cell viability value (%). OD addition/OD control X100%
2.3 modeling of hyperlipidemic cells and cellular administration
Culturing HepG2 cells in RMPI-1640 complete medium, standing at 37 deg.C and 5% CO2In an incubator. Preparing cell suspension from logarithmic growth phase cells, adjusting density to 1 × 105one/mL was seeded in 6-well plates, each set of 5 duplicate wells per concentration, in 5% CO2Incubate at 37 ℃ for 24 h. Discarding the culture medium, adding 2mL of the culture medium into blank group wells, adding 2mL of the culture medium containing sodium oleate into the rest wells, respectively, inducing to establish a high lipid model, and adding 5% CO2Incubate at 37 ℃ for 24 h. Subsequently, the medium was discarded, and a blank group, an administration group, a JIANGZHINING granule group, a model group, a comparative example 1 group, a comparative example 2 group, a comparative example 3 group, and a positive drug group (simvastatin 35. mu.M) were set. Adding 2mL of culture medium into blank group wells, adding 2mL of culture medium containing sodium oleate into model group wells, adding 2mL of culture medium containing the Chinese medicinal monomer composition of the invention into administration group wells, adding 2mL of culture medium containing the Chinese medicinal monomer composition of comparative example 1 into comparative example 1 group wells, adding 2mL of culture medium containing the Chinese medicinal monomer composition of comparative example 2 into comparative example 2 group wells, adding 2mL of culture medium containing the Chinese medicinal monomer composition of comparative example 3 into comparative example 3 group wells, adding 2mL of culture medium containing Jiangzhining granules into Jiangzhining granules, adding 2mL of culture medium containing simvastatin into positive group wells, placing in 5% CO2Incubate at 37 ℃ for 24 h.
2.4TG content determination
Establishing a HepG2 cell high fat model, and determining the content of TG in cells after 24h of administration. The medium was discarded, and 3mL of PBS solution was added for washing, and the washing was repeated twice. Adding 1mL of pancreatin for digestion, placing under an inverted microscope for observation, and discarding the pancreatin solution after digestion is finished. Cells in the wells were collected by adding 1mL of RMPI-1640 complete medium and the cell suspension was transferred to a sterile centrifuge tube for centrifugation (1000r, 2 min). After centrifugation, the supernatant was discarded, and 1mL of PBS buffer was added to the tube and centrifuged (1000r, 10min) twice. Adding 200 mu L of 2% Triton X-100 lysate, blowing and beating for several times, then placing on ice for cracking for 40min, carrying out low-temperature centrifugation (1000r, 10min) after the cells are completely cracked, taking supernatant, and detecting according to an experimental operation method of a TG kit: detecting the OD value of TG at the wavelength of 510nm, and calculating the content of TG according to the following formula:
the TG content measurement results were converted into the inhibition rates (i.e., potency values) of each drug against TG according to the following formula:
statistical analysis is carried out on experimental data by adopting SPSS 22.0 software, and results are expressed as mean value +/-standard deviation
Indicated, ANOVA analysis was used. And (4) carrying out normal and variance homogeneity tests, then carrying out comparison among groups, and if the variances are not uniform, carrying out comparison among groups by adopting a nonparametric test. Statistical analysis and plotting of experimental data was performed using GraphPad Prism 7.0 software.
3. Results of the experiment
The invention adopts an MTT method to analyze the influence of 6 monomer compounds in the traditional Chinese medicine monomer composition on the activity of HepG2 cells. The results show that the safe concentration range of each monomeric compound is shown in the following table 1, which indicates that the medicament is safe and nontoxic under the administration concentration of each monomeric compound, can be eaten for a long time, and solves the problems that the existing western medicament, namely the lipid-lowering medicament simvastatin, has large side effect and can not be eaten for a long time.
TABLE 1 safe concentration ranges for each of the monomeric compounds in the inventive monomeric compositions
| Compound (I) | Safe concentration range |
| Rubrofusarin gentiobioside | 10～320μM |
| Hyperoside A | 10～160μM |
| Quercetin-3-O-beta-D-glucuronide | 10～320μM |
| emodin-8-O-beta-D-glucoside | 10～320μM |
| Almond and papaverine | 10～80μM |
| O-nornuciferine | 10～80μM |
The results of lipid-lowering experiments showed that the inventive monomer composition inhibited the sodium oleate-induced intracellular TG content in HepG2 at 74.03%, the JIANGZHINING granule at 36.21%, the comparative example 1 at 34.92%, the comparative example 2 at 33.33%, and the comparative example 3 at 41.77%. The traditional Chinese medicine monomer composition has the highest inhibition rate on TG content, and the inhibition rate is remarkably different from that of Jiangzhining granules (P is less than 0.01). Therefore, the traditional Chinese medicine monomer composition has better effect on reducing the lipid of triglyceride than the traditional Chinese medicine lipid-reducing Ning granules.
Moreover, the lipid-lowering effect of the traditional Chinese medicine monomer composition (the mass ratio of the quercetin-3-O-beta-D-glucuronide, the hyperoside, the rubrofusarin gentiobioside, the emodin-8-O-beta-D glucoside, the O-nornuciferine and the amygdaline is 9.32: 1: 4.45: 1.85: 2.41: 1.92) which is prepared according to the specific proportion is far better than that of the traditional Chinese medicine monomer composition prepared according to other proportions.
TABLE 2 Effect of the present invention on the intracellular TG content in HepG2 cells
Note: VS model group, # P <0.05, # P < 0.01; VS positive group, # P <0.05, # P < 0.01; VS blank, P <0.05, P < 0.01.
TABLE 3 Effect of JIANGZHINING granule on the intracellular TG content in HepG2
Note: VS model group, # P <0.05, # P < 0.01; VS positive group, # P <0.05, # P < 0.01; VS blank, P <0.05, P < 0.01.
TABLE 4 Effect of the comparative example 1 Chinese medicinal monomer composition on the intracellular TG content in HepG2 cells
Note: VS model group, # P <0.05, # P < 0.01; VS positive group, # P <0.05, # P < 0.01; VS blank, P <0.05, P < 0.01.
TABLE 5 Effect of the comparative example 2 Chinese medicinal monomer composition on the intracellular TG content in HepG2 cells
Note: VS model group, # P <0.05, # P < 0.01; VS positive group, # P <0.05, # P < 0.01; VS blank, P <0.05, P < 0.01.
TABLE 6 Effect of the comparative example 3 Chinese medicinal monomer composition on the intracellular TG content in HepG2 cells
Note: VS model group, # P <0.05, # P < 0.01; VS positive group, # P <0.05, # P < 0.01; VS blank, P <0.05, P < 0.01.
The present invention is not limited to the above-described embodiments, and any variations, modifications, and substitutions which may occur to those skilled in the art may be made without departing from the spirit of the invention.
Claims (10)
1. The lipid-lowering traditional Chinese medicine monomer composition is characterized by comprising the following components in parts by mass of 9.1-9.6: 1: 4.2-4.7: 1.6-2.1: 2.2-2.7: 1.7-2.2 of quercetin-3-O-beta-D-glucuronide, hyperoside, rubrofusarin gentiobioside, emodin-8-O-beta-D-glucoside, O-nornuciferine and papaverine.
2. The traditional Chinese medicine monomer composition of claim 1, wherein the traditional Chinese medicine monomer composition is prepared from the following components in a mass ratio of 9.25-9.45: 1: 4.35-4.55: 1.75-1.95: 2.35-2.55: 1.85-2.05 of quercetin-3-O-beta-D-glucuronide, hyperoside, rubrofusarin gentiobioside, emodin-8-O-beta-D-glucoside, O-nornuciferine and papaverine.
3. The traditional Chinese medicine monomer composition of claim 1, wherein the traditional Chinese medicine monomer composition is prepared from the following raw materials in a mass ratio of 9.32: 1: 4.45: 1.85: 2.41: 1.92 quercetin-3-O-beta-D-glucuronide, hyperoside, rubrofusarin gentiobioside, emodin-8-O-beta-D-glucoside, O-nornuciferine and apricot yellow papaverine.
4. The traditional Chinese medicine monomer composition of claim 1, wherein the traditional Chinese medicine monomer composition is prepared from the following raw materials in a mass ratio of 9.28: 1: 4.51: 1.78: 2.52: 1.87 composed of quercetin-3-O-beta-D-glucuronide, hyperoside, rubrofusarin gentiobioside, emodin-8-O-beta-D-glucoside, O-nornuciferine and apricot yellow papaverine.
5. The traditional Chinese medicine monomer composition of claim 1, wherein the traditional Chinese medicine monomer composition is prepared from the following raw materials in a mass ratio of 9.42: 1: 4.38: 1.93: 2.38: 2.04 quercetin-3-O-beta-D-glucuronide, hyperoside, rubrofusarin gentiobioside, emodin-8-O-beta-D-glucoside, O-nornuciferine and apricot yellow papaverine.
6. A lipid-lowering Chinese medicinal preparation, which is characterized by comprising the Chinese medicinal monomer composition as claimed in any one of claims 1 to 5 and pharmaceutically acceptable auxiliary materials.
7. The Chinese medicinal preparation according to claim 6, wherein the Chinese medicinal preparation comprises any one of capsules, tablets, granules, pills, powders, ointments, pellets, decoctions, liquors, electuary, oral liquids, injections, patches, sustained-release preparations, and controlled-release preparations.
8. The Chinese medicinal preparation according to claim 7, wherein a unit Chinese medicinal preparation comprises 10-200 mg of the Chinese medicinal monomer composition.
9. Use of the monomeric composition of any one of claims 1-5 in the preparation of a lipid-lowering drug.
10. The use of claim 9, wherein the combination of herbal monomers is used to prepare a pharmaceutical formulation for lowering triglycerides.
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