CN111920875A - Patrinia extract with anti-atherosclerosis effect - Google Patents
Patrinia extract with anti-atherosclerosis effect Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/84—Valerianaceae (Valerian family), e.g. valerian
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/55—Liquid-liquid separation; Phase separation
Abstract
The invention discloses an extract, an effective part and a monomer component which are extracted and prepared from dried whole grass of a patrinia plant and have good Atherosclerosis (AS) resisting effect. Pharmacological experimental research shows that water, ethanol, petroleum ether, dichloromethane, ethyl acetate and n-butanol extracts remarkably reduce arterial plaque formation of AS model mice, reduce liver fat accumulation and improve heart function reduction and heart structure change caused by AS; the ethyl acetate extract has the best effect, and most of the contained components are not reported to have related drug effects; the total saponins, total flavonoids, total organic acids and active monomer components have good protection effect on HUVECs damaged by oxidative stress. The invention adopts the patrinia plant which has rich source and low price and is edible AS the wild vegetable AS the raw material, has simple and easy preparation method, provides wider AS treatment medicine source, has social value and economic value, and has important significance for developing high-efficiency and safe AS-resistant new medicines and health-care products.
Description
Technical Field
The invention relates to the technical field of separation and application of active parts of plants, in particular to an anti-atherosclerosis extract and an effective part of a patriniaceae plant and a preparation method thereof, and also relates to application of the patriniaceae plant in the aspect of anti-atherosclerosis and application of the extract, the effective part and an active monomer thereof.
Background
In recent years, with the increasing living standard of people, the eating habits and the living rhythm of people are also continuously changed, and patients with Atherosclerosis (AS) disease are increasing year by year and show a trend of being more and more younger. Atherosclerosis is a major cause of coronary heart disease, cerebral infarction, and peripheral vascular disease, and prevention and treatment of atherosclerotic disease has become an issue of global concern. Atherosclerosis is a cardiovascular disease which is mainly characterized by chronic inflammation and is participated in by multiple factors, lipid metabolism disorder is the basis of pathological changes of atherosclerosis, and the blood lipid level of Chinese people is gradually increased according to Chinese guidelines for preventing and treating dyslipidemia of adults (revised 2016). At present, chemical drugs for clinically treating atherosclerosis mainly focus on a certain link, such as regulation of lipid metabolism, prevention of lipid peroxidation, anti-inflammation and the like; secondly, the chemical drugs have large side effects on human bodies, and some drugs can not effectively control the occurrence of various complications, so the long-term use of the drugs in clinical treatment of atherosclerosis is limited to a certain extent, and the traditional Chinese medicine has the characteristics of multiple components, multiple targets, low side effect and the like, so the exploration, development and utilization of the traditional Chinese medicine for treating AS has great potential.
High-fat and high-cholesterol diet is known to increase the risk of obesity and hyperlipidemia, and finally, cause cardiovascular diseases such as atherosclerosis and coronary heart disease. At present, although the Chinese medicinal plants in multiple families have the effect of treating AS, no document reports that the patrinia plants have the effect of treating AS, and no extraction, purification and separation method aiming at the anti-atherosclerosis effective parts is reported for the patrinia plants.
The above background disclosure is only for the purpose of assisting understanding of the inventive concept and technical solutions of the present invention, and does not necessarily belong to the prior art of the present patent application, and should not be used for evaluating the novelty and inventive step of the present application in the case that there is no clear evidence that the above content is disclosed at the filing date of the present patent application.
Disclosure of Invention
Aiming at the defects in the prior art, the UPLC-QTOF-MS/MS technology and experimental research show that the patrinia (Patrinia villosa and Patrinia scabiosaefolia) has rich chemical components (figure 1 and table 1), the extract or the effective part and the monomer component of the extract can well reduce the formation of vascular plaques of a high-fat high-cholesterol diet mouse, have a good regulating effect on the blood lipid disorder condition of the mouse, improve the weight change and the heart function of a model mouse to a certain extent, well improve the liver lipid accumulation of an atherosclerosis model mouse, and have a good effect of resisting atherosclerotic diseases.
To achieve the purpose, the invention adopts the following technical scheme to provide the application of the patrinia plant extract or the effective part and the monomer component thereof in preparing the health care products or the medicines for preventing or treating the diseases related to atherosclerosis.
Preferably, the patrinia plant is selected from patrinia villosa and patrinia scabiosaefolia.
Preferably, the identification and confirmation method of the active substance of the patriniaceae plant is an UPLC-QTOF-MS/MS method.
Preferably, the disease is atherosclerotic disease caused by a high-fat high-cholesterol diet.
Preferably, the extract is an aqueous extract or an extract of an alcohol extract extracted by different polar solutions.
Preferably, the preparation of said aqueous extract comprises the steps of:
(1) taking dry whole herbal materials of the patrinia, crushing into coarse powder, adding 8-10 times of water into the herbal materials, and decocting for 0.5-1.5 h; filtering while hot to obtain residue and medicinal liquid;
(2) adding water into the dregs of the decoction, then soaking and decocting for 0.5-1.5 h for 3 times, combining water decoction, and concentrating to 1g crude drugs/ml liquid medicine.
Preferably, the ethanol extract is prepared by taking dry whole herbal medicines of the patriniaceae plants, crushing into coarse powder, heating and refluxing for 2 times by 8-10 times of 40-100% ethanol for 1-2.5 h each time, filtering to obtain primary filtrate, and recovering ethanol under reduced pressure to obtain ethanol extract.
Preferably, the preparation of the extract after the extraction with the solution of different polarities comprises the following steps:
(1) taking dry whole herbal materials of the patrinia, crushing into coarse powder, heating and refluxing for 2 times by using 8-10 times of 40-100% ethanol, extracting for 1-2.5 h each time, filtering to obtain primary filtrate, and recovering ethanol under reduced pressure to obtain ethanol extract;
(2) suspending the ethanol extract with appropriate amount of water, extracting with petroleum ether, dichloromethane, ethyl acetate, and n-butanol for 3 times respectively to obtain corresponding extractive solutions, and recovering solvent to obtain petroleum ether extract, dichloromethane extract, ethyl acetate extract, and n-butanol extract.
Preferably, the preparation of the effective part comprises the following steps:
taking dry whole herbal materials of the patrinia, crushing into coarse powder, heating and refluxing for 3 times by 10-20 times of 40-95% ethanol for 1-3 h each time, filtering to obtain primary filtrate, and recovering ethanol under reduced pressure to obtain ethanol extract;
(1) total saponins: taking 5 types of pretreated HPD-100, HPD-300, D101, X-5 and AB-8 macroporous resin, loading the macroporous resin on a column by a wet method, adding 0.20-1.00 g/mL of extracting solution according to the medicinal material quantity/resin volume ratio of 0.75g/mL and the volume flow of 1-4 BV/h, removing impurities by 1.5-3.5 BV of water, and eluting by 5BV 15-95% of ethanol to obtain the total saponin.
(2) Total flavonoids: after the macroporous adsorption resin is pretreated, the column is filled by a wet method, 1g of extract is taken, the extract is precisely weighed, a solution with the concentration of 3mg/ml is prepared by distilled water and is loaded on the column, deionized water is used for washing until effluent is colorless, elution is sequentially carried out at the flow rate of 1BV/h according to 10 to 75 percent ethanol, collection and drying are carried out, and the total flavone is obtained.
(3) Total organic acids: taking 5 types of pretreated HPD-100, HPD-600, D101, NKA-9 and AB-8 macroporous resin, loading the macroporous resin on a column by a wet method, wherein the volume of a column bed is 30mL, the volume ratio of 1g/mL medicinal material to the volume of the resin is 1BV/h, the pH value is 3.0, the flow rate is 2BV/h, loading the macroporous resin, then quickly leaching by 1BV purified water, and eluting by 2BV 10-95% ethanol in sequence to obtain the total organic acid.
Preferably, the active monomer component is saikosaponin d, betulinic acid, ecliptin A, oleanolic acid, saikosaponin a, a-hederin, buddlein IV, protocatechuic acid, chenodeoxycholic acid, protocatechuic aldehyde, 3, 4-dicaffeoylquinic acid, kaempferide, isorhamnetin, naringin dihydrochalcone, quercetin, raddeanin A, madecassic acid, aesculetin, 4, 5-dicaffeoylquinic acid, 2, 5-dicaffeoylquinic acid, isochlorogenic acid, neochlorogenic acid, liquidambaric acid, milkwort root-kaempferol VI, asiatic acid, sanguisorbin II, triptolide A, asperuloside, 8-epinuic acid, methylpriopsiloside A, belamcandoside, scopoletin, p-coumarin, cinnamic acid, quinic acid, and acetylization of resveratrol. .
The invention aims to provide an extract, an effective part and an active monomer which are extracted from patrinia plants and have the function of resisting atherosclerosis, a preparation method thereof and application thereof in preparing health-care products or medicaments for preventing or treating atherosclerosis and related diseases.
Advantageous effects
1. The raw materials of the invention have simple planting conditions and wide sources.
2. The invention preferably obtains the extract, the effective part and the active monomer with obvious anti-atherosclerosis effect by a modern extraction and separation method and combined with pharmacological experimental screening, and the extract, the effective part and the active monomer have good drug effect, strong activity and safe and reliable quality.
3. The extract, the effective part and the active monomer with the function of resisting atherosclerosis provided by the invention can be prepared into medicines or health-care products, and can be conveniently applied clinically. Meanwhile, the preparation method of the extract and the effective parts provided by the invention is simple and easy to operate, the yield of the extracted effective parts is high, the operability is strong, the production cost is low, and the preparation method is suitable for industrial large-scale production.
Drawings
FIG. 1 is a total ion flow diagram of Patrinia villosa and Patrinia scabiosaefolia under positive and negative ion modes
FIG. 2 is a graph showing the effect of white flower extract on liver tissue of AS mice
FIG. 3 is a graph showing the effect of Hemerocallis citrina extract on liver tissue of AS mice
Detailed Description
The invention is further described with reference to specific examples, without limiting the scope of protection and the scope of application of the invention.
Example 1: preparation of medicinal materials
The experimental methods of the present invention, unless otherwise indicated, involve starting materials and reagents that are commercially available and commercially available.
The patrinia villosa medicinal material used in the invention is purchased from Jiangxi camphor tree Tianqitang Chinese herbal piece limited company with the batch number of 1808005, and the patrinia villosa medicinal material is purchased from Sichuan new lotus flower Chinese herbal piece limited company with the batch number of 18009044. Pulverizing dried whole herbs of herba Patriniae and herba Patriniae, sieving with No. 3 sieve, and making into medicinal powder.
The invention selects patrinia villosa and patrinia scabiosaefolia to prepare water extracts respectively according to the following methods:
(1) taking the medicinal material powder, adding 8-10 times of water into the medicinal material, and decocting for 0.5-1.5 h; filtering while hot to obtain residue and medicinal liquid;
(2) adding water into the dregs of the decoction, then soaking and decocting for 0.5-1.5 h for 3 times, combining water decoction, and concentrating to 1g crude drugs/ml liquid medicine.
The invention selects patrinia villosa and patrinia scabiosaefolia to prepare ethanol extracts according to the following methods respectively:
the medicinal powder is taken, heated and refluxed for 2 times with 8-10 times of 40-100% ethanol, the reflux is carried out for 2 hours each time, the filtration is carried out to obtain primary filtrate, and ethanol is recovered under reduced pressure to obtain ethanol extract.
The invention selects patrinia villosa and patrinia villosa juss to prepare extracts extracted by different solvents according to the following methods respectively:
(1) taking the medicinal powder, heating and refluxing the medicinal powder for 2 times by 8-10 times of 40-100% ethanol, filtering the medicinal powder for 2 hours each time to obtain primary filtrate, and recovering the ethanol under reduced pressure to obtain ethanol extract;
(2) suspending the ethanol extract with appropriate amount of water, extracting with petroleum ether, dichloromethane, ethyl acetate, and n-butanol for 3 times respectively to obtain corresponding extractive solutions, and recovering solvent to obtain petroleum ether extract, dichloromethane extract, ethyl acetate extract, and n-butanol extract.
The invention selects patrinia villosa and patrinia scabiosaefolia to prepare effective parts according to the following methods respectively:
(1) total saponins: taking dry whole herbal materials of the patrinia, crushing into coarse powder, heating and refluxing for 3 times by 10-20 times of 40-95% ethanol for 1 hour each time, filtering to obtain primary filtrate, and recovering ethanol under reduced pressure to obtain ethanol extract; and (3) taking the pretreated macroporous resin, loading the macroporous resin into a column by a wet method, adding 0.20-1.00 g/mL of extracting solution, sampling according to the proportion of 0.75g/mL (medicinal material amount/resin volume) and the volume flow of 1-4 BV/h, removing impurities by 1.5-3.5 BV of water, and eluting by 5BV and 15-95% of ethanol to obtain the total saponins.
(2) Total flavonoids: taking dry whole herbal materials of the patrinia, crushing into coarse powder, heating and refluxing for 3 times by 10-20 times of 40-95% ethanol for 1 hour each time, filtering to obtain primary filtrate, and recovering ethanol under reduced pressure to obtain ethanol extract; after the macroporous adsorption resin is pretreated, the column is filled by a wet method, 1g of extract is taken, the extract is precisely weighed, a solution with the concentration of 3mg/ml is prepared by distilled water and is loaded on the column, deionized water is used for washing until effluent is colorless, elution is sequentially carried out at the flow rate of 1BV/h according to 10 to 75 percent ethanol, collection and drying are carried out, and the total flavone is obtained.
(3) Total organic acids: taking dry whole herbal materials of the patrinia, crushing into coarse powder, heating and refluxing for 3 times by 10-20 times of 40-95% ethanol for 2 hours each time, filtering to obtain primary filtrate, and recovering ethanol under reduced pressure to obtain ethanol extract; and (2) loading the pretreated macroporous resin into a column by a wet method, wherein the volume of a column bed is 30mL, the ratio of 1g/mL (medicinal material amount/resin volume), the pH value is 3.0, and the flow rate is 2BV/h, loading the macroporous resin into the column by volume flow, then quickly leaching by using 1BV purified water, and eluting by using 2BV 10-95% ethanol in sequence to obtain the total organic acid.
Example 2: identification and confirmation of active ingredients of Valerianaceae plant and its ethyl acetate extract
1. Experimental Material
1.1 Experimental reagent
Sample preparation: taking 1g of the medicinal powder prepared from Patrinia villosa and Patrinia scabiosaefolia in example 1, adding 50mL of 85% methanol, performing ultrasonic treatment (250W,40KHz) for 30min, filtering with 0.45 μm filter membrane, diluting the filtrate by 20 times, and filtering with 0.22 μm microporous filter membrane; separately collecting ethyl acetate part extracts of Patrinia villosa and Patrinia scabiosaefolia, 0.0331g and 0.0338g, adding 85% methanol 5mL, ultrasonic treating (250W,40KHz) for 30min, filtering with 0.22 μm filter membrane, diluting the filtrate by 10 times, and filtering with 0.22 μm microporous filter membrane.
Reagent testing: methanol, acetonitrile (chromatografic, Fisher Scientific, Fairlawn, NJ, USA), formic acid (chromatografic, Sigma-Aldrich co.ltd, St Louis, MO, USA), distilled water (drochen), and the rest of the reagents were analytically pure.
1.2 Experimental instruments
An ultra-high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometer (AB SCIEX Triple TOF 5600+, Foster City, CA) is provided with an electrospray ion source (ESI), an analysis TF1.6 and a peakview1.2 data processing system (Sciex corporation, USA), an LC-30A ultra-high performance liquid chromatograph (Shimadzu corporation), and is specifically configured as two LC-30AD high-pressure infusion pumps, a CBM-20A system controller, an SIL-30AC automatic sample injector, a DUG-20A5 online degasser, a CTO-30A column incubator, a KQ-500DE numerical control ultrasonic cleaner (Kunshan ultrasonic instruments, Inc.), an electronic balance 210S (Sartorius corporation), and a pulverizer (BP, Yongyao pharmaceutical machinery, Inc., the Regan City.
2 method of experiment
2.1 liquid phase conditions
Using Acquity UPLC BEH C18Column (100 mm. times.2.1 mm, 1.7 μm, Waters Co.), column temperature 40 deg.C, volume flow 0.3mL/min, sample size 2 μ L, mobile phase 0.1% formic acid water solution (phase A) -acetonitrile (phase B). Phase B example varied over time: 0-10min, 5% -15%; 15% -75% for 10-40 min; 75-85% for 40-50 min; 85-90% for 50-55 min; 55-60min, 90-5%; this ratio was then maintained and equilibrated for 5 min.
2.2 Mass Spectrometry conditions
Positive and negative ion scanning modes, using electrospray ion source (ESI), air curtain gas (CUR): 35psi, atomizing gas (GS1) and assist gas (GS 2): both 50psi, temperature: the scanning range is 50-1500 Da at 500 ℃, the data acquisition time is 60min, and the scanning mode is the scanning of the parent ion triggered daughter ions (TOF/MS-IDA-MS)2) In the negative ion mode: spraying voltage: 4500V, declustering voltage (DP): 100V, impact energy (CE): -50eV, Collision Energy Superposition (CES): 15eV, in positive ion mode: spraying voltage: 5500V, declustering voltage (DP): 100V, Collision Energy (CE): 50eV, Collision Energy Superposition (CES): 15 eV.
2.3 data analysis
The data obtained were analyzed by using the software analyze TF1.6 and Peakview 1.2. Extracting and screening the obtained target ions to check the integrity of the peak shapes of the target ions and confirm the accurate retention time of the target ions; within the tolerance of 0.03min retention time, multiple characteristic excimer ions from 1 compound will be matched and combined, and other possible above characteristic ions will be confirmed by XIC Manager extraction, thereby integrating various adducted ion information to lock target compounds. And from multiple target compounds screened at the same time point, removing false positive screening results of the source fragment ions by carefully examining the molecular formula/relative molecular mass relationships between each other. Finally, the target compound is preliminarily identified by combining the daughter ion information provided in the IDA Explorer function in the software.
In order to improve the screening accuracy and simplify the task of the subsequent identification process, the main ions detected in the ion scan type were screened using IDA Explorer with an intensity exceeding 20000cps in the positive ion mode and 15000cps in the negative ion mode.
3. Results of the experiment
Identifying and analyzing chemical components of the patrinia plants and the ethyl acetate extracts thereof by using an UPLC-QTOF-MS/MS technology. By collecting a total ion flow diagram (figure 1) of patrinia villosa juss, patrinia villosa juss and ethyl acetate extracts thereof in a positive and negative ion mode, accurate relative molecular mass, mass spectrum fragment structure information and a chromatographic retention rule of each chromatographic peak in MS are obtained; analyzing by using analysis TF1.6 software; through a database and a database retrieval tool which are established in the previous period, the target compound is identified and confirmed by combining with the XIC Manager function in peakview software, 164 compounds are identified from the patrinia villosa and the patrinia scabiosaefolia, and most chemical components are found in the patrinia scabiosaefolia and the patrinia scabiosaefolia for the first time (see table 1); a total of 84 compounds (shown in Table 2) were identified from the ethyl acetate fraction, including saikoside d, betulinic acid, ecliptin A, oleanolic acid, saikoside a, alpha-hederagenin, buddlein IV, protocatechuic acid, chenodeoxycholic acid, protocatechualdehyde, 3, 4-dicaffeoylquinic acid, kaempferide, isorhamnetin, naringin dihydrochalcone, quercetin, raddeanin A, madecassic acid, aesculetin, 4, 5-dicaffeoylquinic acid, 2, 5-dicaffeoylquinic acid, isochlorogenic acid, neochlorogenic acid, liquidambaric acid, polygalacturone VI, asiatic acid, sanguisorbin II, triptolide, asperuloside, 8-epinuic acid, liriopsiloside A, belamcandin, scopolamine, p-coumarin, cinnamic acid, isoflavanonone, isovalerolactone, madecanolic acid, isovalerolactone, isovaler, Quinic acid and resveratrol are not reported, and the anti-atherosclerosis efficacy of the compounds is not reported.
Example 3: animal experiments
1. Experimental Material
1.1 Experimental reagent
Sample preparation: taking the water extract, ethanol extract, petroleum ether extract, dichloromethane extract, ethyl acetate extract, n-butanol extract, total saponin extract, total flavone extract and total organic acid extract prepared from the patrinia villosa and patrinia scabiosaefolia in example 1 respectively, and adding 0.5% CMC-Na before use to prepare a suspension as a test solution; positive control: atorvastatin (Guangzhou poplar leaf Biotechnology Co., Ltd.) and Tongxinluo capsule (Shijiazhu Ling pharmaceutical Co., Ltd.) were prepared into solutions just before use.
Reagent testing: mouse maintenance feed (Jiangsumeidisen biopharmaceutical, Inc.); high-fat high-cholesterol diet (1.25% cholesterol, 20% lard, 78.75% basal diet); saturated oil red O staining solution (Solarbio), hematoxylin staining solution, eosin staining solution (Nanjing Technology Co., Ltd.), absolute ethyl alcohol, xylene, isopropyl alcohol, CMC-Na (Xilonga chemical Co., Ltd.), embedded paraffin (Rebs), aqueous block, neutral gum block (Shi Ming Dynasty), ultrasonic coupling agent (West district Techner Co., Ltd.), chloral hydrate, paraformaldehyde (Shanghai Aladdin Biotechnology Co., Ltd.), Phosphate Buffer Solution (PBS) (Solarbio)
1.2 Experimental instruments
RE5205 rotary evaporator (Shanghai Yangrong biochemical apparatus factory), electronic balance (Aohaus apparatus Shanghai Co., Ltd.), water bath (Shanghai Yangrong biochemical apparatus factory), body microscope (Leica), tissue embedding machine (Leica EG 1150C), tissue slicer (Leica VT 1000P), tissue roast machine (Leica HI1220), tissue slice spreading machine (Leica HI1210), automatic dyeing machine (Leica ST5020 Multistainer), automatic dehydrator (Leica TP1020), five-person sharing microscope (Leica multiview), visual sonic Vevo2100 (Toronto, Canada)
1.3 Experimental animals
SPF-class ApoE-/-C57BL/6 male mouse, weighing 18-22g, Beijing Wittingle laboratory animal technology Limited, license SCXK (Jing) 2016-0011. All animals in the experiment are raised in animal barriers of the experimental animal science and technology center of the university of traditional Chinese medicine in Jiangxi, one week before the experiment is an adaptation period, food and drinking water are freely taken, and the feed and the drinking water are completely the same in the adaptation period.
2 method of experiment
2.1 construction of pathological model of Atherosclerosis
Selecting male ApoE with SPF grade of 6-8 weeks and weight of 18-22g-/-After the mice are fed with the high-fat and high-cholesterol feed for 8 weeks, the pathological model is evaluated by living observation of relevant indexes of atherosclerosis of the mice, HE staining of liver tissue sections of the mice and aortic oil red O staining of the mice through an ultrasonic imaging system of the mice, and the pathological model has significant difference (P) with a blank group<0.05) the model can be considered successful.
2.2 grouping and administration
Mice molded for 8 weeks were randomly divided into 9 groups: positive control groups are respectively administered with 6.0mg/kg.d atorvastatin and 1.5g/kg.d heart-meridian-dredging capsules by gastric perfusion, each extract administration group is respectively administered with 150g crude drug/kg.d petroleum ether, dichloromethane, ethyl acetate and n-butanol extracts by gastric perfusion, a water extraction part and an ethanol extraction part are respectively administered with 15g crude drug/kg.d extract by gastric perfusion, a normal control group is administered with a control solvent CMC-Na according to 0.1ml/10g body weight and is administered at the same time every day for 8 weeks, the normal control group is fed with a mouse maintenance feed during administration, and the rest groups are fed with high-fat and high-cholesterol feed. The body weight of the mice was measured periodically every week to adjust the dose administered.
2.3 body weight determination
One day before the mice began to be administered by gavage (week 0), the mice in each group were fasted for 8h without water deprivation, and the fasting body weights were respectively weighed and recorded. During the administration treatment, the fasting body weight was weighed 1 time per week, and the body weight change of the mice was observed throughout the administration period.
2.4 Observation of ultrasound indicators
The Visul sonic Vevo2100 instrument is used for carrying out living body ultrasound on the mice, and the aortic arch innominate vascular stenosis rate, plaque area, liver lipid accumulation, carotid plaque area and blood flow resistance coefficient of the mice of different groups are observed.
2.5 Collection of samples
2.5.1 obtaining the blood vessel
The heart was found under a stereomicroscope. Removing peripheral fat to expose pulmonary artery and aortic arch, removing pulmonary artery and then finding carotid artery upwards along three branches of aortic arch, and picking up with forceps and cutting. After the aortic arch is found, the heart is firstly cut off, and then the aortic arch is continuously separated downwards along the vertebral column until the whole blood vessel is taken off and soaked in 4% PFA fixing solution.
2.5.2 liver selection
After cardiac perfusion is finished, the left lobe of the liver is taken down, the edge of the left lobe is cut off, the left lobe is cut into 5mm multiplied by 2mm blocks, the blocks are placed into a tissue embedding box, and the blocks are soaked in 4% PFA after information such as tissue name, group, number, date and the like is marked.
2.6 data processing
All data analyses were performed using Graphpad Prism 8.0 data analysis software. All data are expressed as Mean ± standard deviation (Mean ± SD). Comparisons between groups were made using a one-way analysis of variance (ANOVE) method with P <0.05 as a criterion for statistical significance of the differences.
3 results of the experiment
3.1 Atherosclerosis model construction evaluation
The ultrasonic imaging system of the living small animal can dynamically observe the change of arterial blood vessels under the non-invasive condition, and the Vevo2100 ultrasonic instrument is adopted to evaluate ApoE-/-Development of pathological models of AS in mice.
3.1.1 carotid and aortic arch ultrasound results
ApoE-/-The atherosclerotic plaques in mice are mostly present in the branch points of blood vessels, aorta, aortic root, major branches of the aortic arch, and carotid arteries. For ApoE at the same time-/-The mouse carotid atherosclerotic plaque area, aortic arch innominate vascular stenosis rate and carotid artery blood flow resistance coefficient (RI) are monitored, and the progress of atherosclerotic lesion can be dynamically observed in a living body. The results of the ultrasonic experiments show (see table 3) that the carotid artery blood flow resistance coefficient (RI) of the mice in the model group is obviously increased (P) compared with the normal control group<0.05), the area ratio of carotid plaque and the aortic arch innominate vascular stenosis rate are obviously higher than that of a normal control group (P)<0.01)。
Table 3 changes in the mouse carotid atherosclerotic plaque area, aortic arch innominate vascular stenosis rate, and carotid artery blood flow resistance coefficient (RI) (mean ± SD, n ═ 6) after feeding with high-fat high-cholesterol diet
Compared to the model set: p <0.01, P < 0.05.
3.1.2 hepatic ultrasonography results
Under model B, for ApoE-/-Carrying out ultrasonic imaging on the liver of the mouse, and calculating the ultrasonic echo attenuation coefficient of the liver to carry out ApoE-/-The accumulation of liver fat in mice was evaluated. The ultrasound results showed (see Table 4) that ApoE was administered-/-After the mice are fed with the high-fat and high-cholesterol feed for eight weeks, the liver ultrasonic echo attenuation coefficient of the mice is obviously greater than that of the normal control group mice fed with the common feed, and the condition of fat accumulation in the liver is prompted.
Table 4 change in the liver echo attenuation coefficient of mice after feeding high fat and high cholesterol diet (mean ± SD, n ═ 6)
Compared to the model set: p <0.01, P < 0.05.
3.1.3 mouse Heart index (CI)
Mice were weighed for body weight and heart mass and the Cardiac Index (CI) was calculated. As shown in table 5, the heart index of the model group mice was significantly higher than that of the blank control group (. about.p <0.05) compared to the blank control group.
Table 5 change in heart index of mice after feeding high fat and high cholesterol diet (mean ± SD, n ═ 6)
Compared to the model set: p <0.01, P < 0.05.
3.1.4 aortic plaque oil Red O staining
The statistical results of the plaque areas of the two groups are shown in Table 6, and the results show that the model group and the blank control group have significant difference (n is 6, p is <0.05)
Table 6 effect of aortic plaque in mice after feeding high fat and high cholesterol diet (mean ± SD, n ═ 6)
Compared to the model set: p <0.01, P < 0.05.
The above results show that ApoE is administered-/-After the mice are fed with the high-fat and high-cholesterol feed for 8 weeks, the aorta of the mice is formed with atherosclerotic plaques, the resistance coefficient of carotid artery blood flow is increased, the cardiac index is increased, and the liver has fat accumulation, which indicates that the model building of the atherosclerosis pathological model is successful.
3.2 Effect of Patrinia plants or different extracts thereof on atherosclerotic disease mice
3.2.1 Effect on body weight
All mice were fed normal diet water, and 9 groups of ApoE were fed on normal diet and high-fat and high-cholesterol diet-/-The mouse has no abnormal urine and feces, the weight of the mouse increases week by week, and the administration mode of intragastric administration can be well tolerated. The results show that the weight growth amplitude of the mice in the model group is the largest, the weight growth of the mice in the administration group is slower than that of the mice in the model group, and the weight growth amplitude of the mice in the normal group is the smallest; wherein the model group has a very significant difference (P) compared with the normal group<0.01); the body weight of the patrinia villosa and patrinia villosa ethyl acetate fraction group after 5 weeks of administration was significantly different from that of the model group compared with each administration group (P)<0.05), indicating that the ethyl acetate part has obvious regulation and control effect on the weight gain of the atherosclerosis mouse. The results are shown in tables 7 and 8.
TABLE 7 influence of Patrinia villosa juss on the body weight of mice model of atherosclerosis (mean + -SD, n ═ 6)
Comparison with normal control group: # P <0.01, # P < 0.05; compared to the model set: p <0.01, P <0.05
TABLE 8 influence of Patrinia scabiosaefolia Each site on the weight of atherosclerosis model mice (mean + -SD, n ═ 6)
Comparison with normal control group: # P <0.01, # P < 0.05; compared to the model set: p <0.01, P <0.05
3.2.2 carotid and aortic arch ultrasound results
After 8 weeks of administration, compared with the model group, the atorvastatin group has very significant differences (P <0.01) in carotid artery and aortic arch innominate vascular atherosclerotic plaque area, aortic arch innominate vascular stenosis rate and carotid artery blood flow resistance coefficient (RI); the area and the stenosis rate of the aortic arch innominate vascular atherosclerotic plaque of the heart dredging group have significant difference (P <0.05), and the area and the RI of the carotid plaque have very significant difference (P < 0.01); the area and RI of the carotid atherosclerotic plaque of the patrinia villosa and patrinia villosa ethyl acetate part group are remarkably different (P <0.01), and the area and stenosis rate of the aortic arch innominate vascular atherosclerotic plaque are remarkably different (P < 0.05); the carotid plaque area ratio and RI of the patrinia villosa petroleum ether group, the dichloromethane group, the n-butanol group, the water group and the ethanol group are all significantly different (P < 0.05); the carotid plaque area ratio, RI, aortic arch innominate vascular plaque area and stenosis rate of the patrinia villosa dichloromethane group have significant differences (P <0.05), the carotid plaque area ratio and RI of the petroleum ether group have significant differences (P <0.05), and the carotid plaque area ratio, RI and aortic arch innominate vascular stenosis rate of the n-butanol group, the water group and the ethanol group have significant differences (P < 0.05).
This indicates that the above extracts of the patrinia family all have the effect of inhibiting the progression of atherosclerotic disease, with the ethyl acetate fraction having the most significant effect. The results are shown in tables 9 and 10.
TABLE 9 influence of Patrinia villosa on atherosclerotic plaque area of carotid artery and aortic arch innominate blood vessel, aortic arch innominate blood vessel stenosis rate and carotid artery blood flow resistance coefficient (RI) (mean + -SD, n is 6)
Comparison with normal control group: # P <0.01, # P < 0.05; compared to the model set: p <0.01, P <0.05
TABLE 10 influence of Patrinia scabiosaefolia Each site on atherosclerotic plaque area of carotid artery and aortic arch innominate blood vessel, aortic arch innominate blood vessel stenosis rate and carotid artery blood flow resistance coefficient (RI) (mean + -SD, n ═ 6)
Comparison with normal control group: # P <0.01, # P < 0.05; compared to the model set: p <0.01, P <0.05
3.2.3 liver ultrasound and liver tissue HE staining results
After 8 weeks of administration, the ultrasound echo attenuation coefficients of the liver of mice in the atorvastatin group, the cardiovascular group and the ethyl acetate group were significantly different (P <0.05) in each administration group compared with the model group. By carrying out HE staining on liver tissues, finding that the liver cells of a blank group of mice are orderly arranged, the structure is clear and visible, the cell nucleus is positioned in the middle of the cell, only little vesicular steatosis is generated, and inflammatory cell infiltration is not generated; the mouse hepatocyte cell nucleus of the model group is positioned in the middle of the cell, a large amount of vacuoles and lipid droplets are gathered around the nucleus, and large-vesicular steatosis, small-vesicular steatosis and hepatocyte balloon-like degeneration exist, wherein small-sized foam-like degeneration is taken as the main factor, and infiltration of inflammatory cells can be seen; the arrangement of the liver cells of the mice at the atorvastatin group, the cardiovascular dredging group and the ethyl acetate part is regular, the cell swelling is obviously reduced, the cell steatosis is obviously reduced, and a small amount of inflammatory cells are infiltrated. This indicates that the ethyl acetate site has a significant inhibitory effect on liver fat accumulation. The results are shown in Table 11 and Table 12 and FIGS. 2 and 3.
TABLE 11 influence of Patrinia villosa juss on the ultrasonic echo attenuation coefficient of the liver of the mouse model of atherosclerosis (mean + -SD, n is 6)
Comparison with normal control group:##P<0.01，#P<0.05; compared to the model set: p<0.01，*P<0.05
Table 12 influence of Patrinia scabiosaefolia FIq on ultrasonic echo attenuation coefficient of mouse liver in atherosclerosis model (mean ± SD, n ═ 6)
Comparison with normal control group:##P<0.01，#P<0.05; compared to the model set: p<0.01，*P<0.05
3.2.4 aortic vascular oil Red O staining results
After 8 weeks of administration, the aortic plaque area ratios of the atorvastatin group, the vein relaxing group and the ethyl acetate part group were very significantly different (P <0.01) and the aortic plaque area ratios of the n-butanol part, the water extraction part, the ethanol extraction part, the dichloromethane part and the petroleum ether part of patrinia villosa and patrinia villosa were significantly different (P <0.05) in each administration group compared with the model group. This indicates that the above extract of the Valerianaceae plant has an inhibitory effect on the formation of atherosclerotic plaque, wherein the ethyl acetate fraction, the water extract fraction, the ethanol extract fraction, the n-butanol fraction, the methylene chloride fraction and the petroleum ether fraction have significant effects. The results are shown in tables 13 and 14.
TABLE 13 influence of Patrinia villosa Each site on aortic plaque in atherosclerosis model mice (mean + -SD, n ═ 6)
Comparison with normal control group: # P <0.01, # P < 0.05; compared to the model set: p <0.01, P <0.05
TABLE 14 Effect of Patrinia scabiosaefolia Each site on aortic plaque in atherosclerosis model mice (mean + -SD, n ═ 6)
Comparison with normal control group: # P <0.01, # P < 0.05; compared to the model set: p <0.01, P <0.05
3.2.5 Cardiac Index (CI) results
After 8 weeks of administration, the CI of the mice in the model group is obviously increased (P <0.001) compared with that in the normal control group, and the heart indexes of the atorvastatin group, the cardiovascular dredging group and the ethyl acetate part administration group are obviously different (P <0.05) compared with the model group in each administration group, which indicates that the ethyl acetate part of the patrinia plant has a treatment effect on heart related diseases caused by the atherosclerotic diseases.
TABLE 15 influence of Patrinia villosa Each site on the mouse CI model atherosclerosis (mean + -SD, n ═ 6)
Comparison with normal control group:##P<0.01，#P<0.05; compared to the model set: p<0.01，*P<0.05
TABLE 16 Effect of Patrinia scabiosaefolia Each site on the atherosclerosis model mouse CI (mean + -SD, n ═ 6)
Comparison with normal control group:##P<0.01，#P<0.05; compared to the model set: p<0.01，*P<0.05
The experimental results show that the ethyl acetate part can well reduce the formation of vascular plaques of the atherosclerosis model mouse, has a good regulating effect on the blood lipid disorder condition of the mouse, improves the weight change and the heart function of the model mouse to a certain extent, and well improves the liver lipid accumulation of the atherosclerosis model mouse; in addition, the petroleum ether, the dichloromethane part, the n-butanol part, the water part and the ethanol part have the same pharmacological action on an atherosclerosis model mouse, but the effect is not as obvious and comprehensive as the effect of the ethyl acetate part. Therefore, by combining the experimental results, effective parts of the patrinia plants for resisting the atherosclerosis disease are screened out to be petroleum ether parts, ethyl acetate parts, dichloromethane parts, n-butanol parts, water parts and ethanol parts.
Example 4: cell experiments
1. Experimental Material
1.1 Experimental reagent
Sample preparation: the effective parts of the total saponins, the total flavonoids and the total organic acids prepared from the patrinia plants in the example 1 are respectively taken and added with 0.5 percent of CMC-Na before use to prepare suspension as a test solution. Saikosaponin d, betulinic acid, ecliptin A, oleanolic acid, saikosaponin a, a-hederin, buddlein saponin, protocatechuic acid, chenodeoxycholic acid, protocatechuic aldehyde, 3, 4-dicaffeoylquinic acid, kaempferide, isorhamnetin, naringin dihydrochalcone, quercetin, radixol A and madecassic acid are purchased from China pharmaceutical and biological product certification institute, and are prepared into suspension before use, and the suspension passes through a 0.22 micron filter membrane.
Reagent testing: superoxide dismutase (SOD), Malondialdehyde (MDA) test kit (Nanjing institute of bioengineering), DMEM culture medium (Wuhan doctor Ded bioengineering Co., Ltd.), PBS buffer (0.01 mmol. L-1, pH 7.2-7.4, Beijing Solebao Tech Co., Ltd.), Australia newborn fetal bovine serum (FBS, Jiangsu Enmoassai Biotech Co., Ltd.), trypsin digestive juice (containing trypsin 0.25%, Beijing Solebao Tech Co., Ltd.), ox-LDL (Beijing Solebao Tech Co., Ltd.).
1.2 Experimental instruments
371 type CO2Incubator (Thermo corporation, usa); model 550 full-automatic enzyme labeling machine (Rapim Corp., USA); CKX41 type inverted fluorescence microscope (Olympus, japan); model 5417D bench-top high speed centrifuge (Eppendorf, Germany).
1.3 cell lines
HUVECs were purchased from the cell resource center of the institute of basic medicine, national academy of medical sciences.
2 method of experiment
2.1 cell culture
HUVECs cells were seeded in DMEM medium containing 10% fetal bovine serum and 1% double antibody at 37 deg.C with 5% CO2Culturing under the condition, changing the liquid every 2d on average, carrying out 1 passage every 2-3 d, and carrying out subculture to logarithmic phase for later use.
2.2 cell grouping and intervention
The cultured HUVECs cells are divided into a normal group, a model group, a total saponin group, a total flavone group and a total organic acid group. The normal group is only cultured by DMEM complete medium without any medicine; the model group was given 100 mg/Lox-LDL induced endothelial cells for 48 h; adding 100mg/L of ox-LDL to the ox-LDL + total saponin group, the ox-LDL + total flavone group and the ox-LDL + total organic acid group respectively to act for 24h, then continuing acting for 24h by using the low-medium-high-solubility total saponin, the total flavone and the total organic acid, and detecting each index after finishing the culture.
2.3 determination of SOD and MDA in cell supernatant
Taking HUVECs in logarithmic growth phase, digesting with trypsin, and adjusting cell density to 1 × 104Inoculating 2mL of the extract per well into a 24-well plate, inoculating 6 groups of multiple wells in each group, and adding culture medium after adherence to prepare the extract with final mass concentration of 25, 50 and 100 mg.L－1Total saponins, 20, 40, 80 mg.L－1Total Flavonoids, 30, 60, 120 mg.L－1Adding PBS with equal volume into total organic acid, blank group and model group, incubating for 48h, adding ox-LDL with final concentration of 100mg/L, adding PBS into blank group, culturing for 8h in incubator, collecting cell supernatant, and determining SOD and MDA content according to kit instruction.
2.4 determination of cell viability by different monomers
HUVECs cultured in the test are divided into a blank control group, a model group and a low-medium dose administration group. The culture medium of the control group is conventional DMEM solution + 5% serum, the culture medium of the other groups is conventional DMEM solution + 5% serum +100mg/L ox-LDL and is performed under the randomization condition, the grouped cultured cells are inoculated in a 24-well plate, and the experimental intervention treatment is performed after the cells grow to 90% confluence. The above groups were cultured for 24 hours, and the cell viability was measured by MTT method.
2.5 statistical analysis
All data analyses were performed using Graphpad Prism 8.0 data analysis software. All data are expressed as Mean ± standard deviation (Mean ± SD). Comparisons between groups were made using a one-way analysis of variance (ANOVE) method with P <0.05 as a criterion for statistical significance of the differences.
3. Results
3.1 measurement of SOD and MDA in cell supernatant
After cells are subjected to ox-LDL damage modeling, the MDA content of a model group is remarkably increased (P is less than 0.01), and the SOD activity is remarkably reduced (P is less than 0.01). Compared with a model group, the medicine dry prognosis of the total saponins, the total flavonoids and the total organic acids of the patrinia plant can obviously reduce the content of MDA (malondialdehyde) and can improve the SOD activity (superoxide dismutase) activity (P is less than 0.01 and less than 0.05), and experiments show that the total saponins, the total flavonoids and the total organic acids of the patrinia plant have a protection effect on HUVECs damaged by oxidative stress.
TABLE 1 influence of effective fractions of Patrinia villosa on oxidative stress injury-related indices in ox-LDL injured HUVECs (mean + -SD, n ═ 6)
Comparison with normal control group: # P <0.01, # P < 0.05; compared to the model set: p <0.01, P <0.05
TABLE 2 influence of effective fractions of Patrinia scabiosaefolia on oxidative stress injury-related indices in ox-LDL injured HUVECs (mean + -SD, n ═ 6)
Comparison with normal control group: # P <0.01, # P < 0.05; compared to the model set: p <0.01, P <0.05
3.2 cell viability assay
As shown in the results in Table 3, after treatment with Ox-LDL, the survival activity of vascular endothelial cells in the model group is obviously lower than that in the blank control group, the survival activities of vascular endothelial cells in the low, medium and high dose groups of the administered monomer are all higher than that in the model group, and the survival activity of vascular endothelial cells is increased along with the increase of the administered concentration
TABLE 3 influence of Patriniaceae plant active monomers on oxidative stress injury-related indices in ox-LDL injured HUVECs (mean + -SD, n ═ 6)
Comparison with normal control group: # P <0.01, # P < 0.05; compared to the model set: p <0.01, P <0.05
From the above experimental results, it can be known that the total saponins, total flavonoids, total organic acids and active monomer components of the patrinia plant have a good protective effect on HUVECs damaged by oxidative stress.
TABLE 1 UPLC-QTOF-MS/MS identification results of compounds in Patrinia villosa and Patrinia scabiosaefolia
TABLE 2 identification of compounds in Patrinia scabiosaefolia and Patrinia scabiosaefolia ethyl acetate extraction sites and their response values
Claims (12)
1. A herba Patriniae extract with anti-atherosclerosis effect is characterized in that: is extract of Patrinia villosa and/or Patrinia scabiosaefolia.
2. The patrinia extract according to claim 1, wherein: the extract is water extract or ethanol extract.
3. The patrinia extract according to claim 1, wherein: the extract is prepared by respectively extracting ethanol extract with petroleum ether, dichloromethane, ethyl acetate and n-butanol to obtain petroleum ether extract, dichloromethane extract, ethyl acetate extract and n-butanol extract.
4. The Patrinia extract according to any one of claims 1 to 3, wherein: the main effective substances in the extract are total saponins, total flavonoids and total organic acids.
5. The Patrinia extract according to any one of claims 1 to 3, wherein: the effective substances are saikosaponin d, betulinic acid, ecliptin A, oleanolic acid, saikosaponin a, alpha-hederin, buddlein IV, protocatechuic acid, chenodeoxycholic acid, protocatechualdehyde, 3, 4-dicaffeoylquinic acid, kaempferide, isorhamnetin, naringin dihydrochalcone, quercetin, raddeanin A, madecassic acid and aesculetin, 4, 5-dicaffeoylquinic acid, 2, 5-dicaffeoylquinic acid, isochlorogenic acid, neochlorogenic acid, liquidambaric acid, polygala root kobushiki VI, asiatic acid, sanguisorbin II, triptolide A, asperuloside, 8-epinux vomica acid, methyl ophiopogon root dihydrohomoisoflavonoid A, belamcanda rhizome glycoside, scopoletin, p-coumarin, cinnamic acid, quinic acid and acetylated resveratrol.
6. The patrinia extract according to claim 2, wherein: the preparation of the water extract comprises the following steps: pulverizing patrinia, then decocting with 8-10 times of water for 1-3 times, each time for 0.5-1.5 hours, and filtering to obtain filtrate which is the aqueous extract of patrinia.
7. The Patrinia extract according to any one of claims 1 to 3, wherein: the preparation method of the ethanol extract comprises the following steps: pulverizing patrinia, heating and refluxing the smashed patrinia for 2 times by 8-10 times of 40-100% ethanol, extracting for 1-2.5 hours each time, filtering to obtain primary filtrate, and recovering ethanol under reduced pressure to obtain ethanol extract, namely the ethanol extract.
8. The patrinia extract according to claim 3, wherein the process of extracting with petroleum ether, dichloromethane, ethyl acetate and n-butanol comprises the steps of: preparing ethanol extract according to the method of claim 7, adding appropriate amount of water, suspending, extracting with petroleum ether, dichloromethane, ethyl acetate, and n-butanol for 3 times respectively to obtain extracts corresponding to each part, and recovering solvent to obtain petroleum ether extract, dichloromethane extract, ethyl acetate extract, and n-butanol extract, respectively.
9. The patrinia extract according to claim 4, wherein: the preparation process of the patrinia extract comprises the following steps:
(1) the preparation of the total saponins comprises the following steps: drying whole herbal materials of the patrinia, crushing into coarse powder, heating and refluxing for 3 times by 10-20 times of 40-95% ethanol for 1-3 h each time, filtering to obtain primary filtrate, and recovering ethanol under reduced pressure to obtain ethanol extract; and (3) respectively taking pretreated HPD-100, HPD-300, D101, X-5 and AB-8 macroporous resin, loading the macroporous resin on a column by a wet method, adding 0.20-1.00 g/mL of extracting solution, sampling according to the medicinal material quantity/resin volume ratio of 0.75g/mL and the volume flow of 1-4 BV/h, removing impurities by 1.5-3.5 BV of water, and eluting by 5BV 15-95% of ethanol to obtain the total saponins.
(2) The preparation of the total flavone comprises the following steps: the ethanol extract of the Patrinia family is prepared in accordance with (1) in claim 9. After the macroporous adsorption resin is pretreated, the column is filled by a wet method, 1g of extract is taken, the extract is precisely weighed, a solution with the concentration of 3mg/ml is prepared by distilled water and is loaded on the column, deionized water is used for washing until effluent is colorless, elution is sequentially carried out at the flow rate of 1BV/h according to 10 to 75 percent ethanol, collection and drying are carried out, and the total flavone is obtained.
(3) Preparation of the organic acid: the ethanol extract of the Patrinia family is prepared in accordance with (1) in claim 9. Taking 5 types of pretreated HPD-100, HPD-600, D101, NKA-9 and AB-8 macroporous resin, loading the macroporous resin on a column by a wet method, wherein the volume of a column bed is 30mL, the volume ratio of medicinal materials to the volume of the resin is 1g/mL, the pH value is 3.0, and the flow rate is 2BV/h, loading the macroporous resin, then quickly leaching by 1BV of purified water, and eluting by 2BV 10-95% ethanol in sequence to obtain the total organic acid.
10. Use of the patrinia extract according to any one of claims 1 to 9 for the preparation of a health product or a medicament for preventing or treating atherosclerosis.
11. The use of claim 10, wherein: the atherosclerosis related diseases comprise diseases caused by high-fat and high-cholesterol diet, and systemic or local diseases directly or indirectly after the atherosclerosis diseases.
12. The use of claim 11, wherein: the atherosclerosis related diseases include nonalcoholic fatty liver disease, heart function reduction and heart structure change caused by atherosclerosis diseases.
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