CN101167816A - Ranunculoside and its preparation method and application - Google Patents

Abstract

The invention discloses ranunculosides, which are extracted from whole herb of ranunculus and contain 60% to 90% of triterpene saponins. The invention also provides a preparation method and application of the ranunculosides. At the same time, it provides the application of the total ranunculoside in the preparation of cardiovascular drugs, the application in the preparation of anti-myocardial hypertrophy drugs, drugs for relaxing blood vessel smooth muscle, drugs for inhibiting heart action, analgesic and anti-inflammatory drugs, and the like. Further research on ranunculosides is expected to discover new cardiovascular and analgesic and anti-inflammatory drugs with high selectivity, strong activity, and less toxic and side effects, which will lay a solid foundation for the wide application of ranunculosides in medicine and has important Potential industrial development value and a major contribution to human research on new natural medicines.

Description

Ranunculoside and its preparation method and application

technical field

The invention relates to the technical field of medicine, in particular to ranunculosides, a ranunculus whole herb extract, a preparation method thereof and an application in the field of medicine.

Background technique

Diseases such as cardiovascular disease and pain inflammation are common clinical diseases. Myocardial hypertrophy is a target organ response to chronic pressure or volume overload, which can lead to decreased myocardial flexibility, cardiac systolic insufficiency, serious cardiovascular events, and increased mortality and disability rates;

Inflammation is a defense response of living tissue with vascular system to injury factors, and its local clinical features are redness, swelling, heat, pain and dysfunction.

Although there are many therapeutic drugs for the above diseases, finding new drugs in this area is still a current and future research hotspot based on the curative effect and adverse reactions.

Buttercup, also known as cichlid grass, duck foot board, wild celery, mountain pepper, tiger claw grass, hairy celery, blister grass, grows on the edge of hills or low ravines, paddy fields or wet grass. The Dictionary of Traditional Chinese Medicine records that buttercup is used in the folk to treat various inflammatory pains such as migraine, stomach pain, rheumatic joint pain, toothache, etc., indicating that buttercup contains some or some kind of anti-inflammatory and analgesic substances. Researching it as a new drug and applying it clinically has broad application prospects and social and economic benefits.

Contents of the invention

An object of the present invention is to provide a natural plant extract total glycosides of Ranunculus (TGOR), through the present invention, the effective part of Ranunculus medicinal action can be clearly obtained.

Another object of the present invention is to provide a simple and feasible preparation method of the ranunculoside.

Another object of the present invention is to provide the application of ranunculoside in the field of medicine, especially in the preparation of cardiovascular drugs or analgesic and anti-inflammatory drugs.

The technical solution of the present invention is to provide a kind of ranunculus total glycosides (TGOR), which is obtained by extracting ranunculus whole grass as a raw material, and contains 60% to 80% of triterpene saponins.

The invention provides a preparation method of the ranunculoside, comprising the following steps:

(1) The whole ranunculus herb is dried and chopped, extracted with water or different concentrations of ethanol or methanol by reflux or percolation with different concentrations of ethanol or methanol, filtered, combined extracts, recovered solvent under reduced pressure, and concentrated;

(2) Add an equal volume of ethyl acetate to the concentrated solution of (1) for extraction; remove the lower aqueous layer and evaporate the solvent to obtain a paste;

(3) The paste is dissolved with a certain amount of water, adsorbed by macroporous resin, first washed with distilled water to clear the resin, then eluted with 50-95% ethanol or methanol, concentrated, and evaporated to dryness to obtain paste ranunculoside product .

The extraction in step (1) uses water or 10-95% ethanol or methanol, and the ethanol or methanol aqueous solution is extracted 2-3 times according to the weight ratio of the ranunculus whole herb being 7:1.

The present invention also provides the application of the total ranunculosides in the preparation of cardiovascular drugs, especially in the preparation of anti-cardiac hypertrophy drugs, in the preparation of vascular smooth muscle relaxation drugs, and in the preparation of heart-inhibiting drugs.

The present invention also provides the application of the total ranunculosides in the preparation of analgesic and anti-inflammatory drugs, which can be used for the treatment of diseases such as frozen shoulder and rheumatoid arthritis. The pharmaceutical composition of the total ranunculosides of the present invention can be a tablet , granules, capsules, pills, dropping pills, effervescent tablets, ointments, syrups, injections, oral liquids, mixtures, sustained-release preparations, controlled-release preparations or targeted preparations.

The beneficial effects of the present invention are:

(1) The present invention provides a new natural plant extract, and the identification and analysis of feasible quality control aspects have been carried out.

(2) The extraction process of total ranunculoside is provided, the method is simple, safe and easy, and the cost is low.

(3) The present invention has studied the medical use of ranunculosides, and further research on said ranunculosides is expected to find novel cardiovascular and analgesic and anti-inflammatory drugs with high selectivity, strong activity, and small toxic and side effects, which is ranunculosides It has laid a solid foundation for its wide application in medicine, has important potential industrialization development value, and has made great contributions to human research on new natural medicines.

Description of drawings

Figure 1 Schematic diagram of TLC of ranunculosides

Figure 2 Morphology of cardiomyocytes (HE staining)

Figure 3 Inhibitory effect of ranunculosides on myocardial hypertrophy

Figure 4 Inhibitory effect of ranunculosides on isolated frog hearts

Figure 5 The effect of total ranunculosides against NE-induced vascular ring contraction

Figure 6 Comparison of the inhibition rate of ranunculosides on the contraction of vascular rings induced by NE

Figure 7 Inhibitory effect of ranunculosides on xylene-induced ear swelling in mice

Figure 8 Effects of ranunculosides on paw swelling induced by carrageen in rats

Figure 9 Effects of ranunculosides on the pain model of rats induced by hot plate stimulation

Figure 10 Effect of total ranunculosides on the pain model of mice induced by acetic acid stimulation

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

Example 1

Preparation, composition identification and content determination of ranunculosides

1. Preparation

(1) 2kg of the whole ranunculus herb was dried and chopped, extracted three times with 14kg of water or 10-95% ethanol or methanol under reflux or percolation, filtered through an 80-mesh sieve, combined the extracts, recovered the solvent under reduced pressure, and concentrated to 1.05kg.

(2) Add ethyl acetate equal to the volume of the concentrated solution to the concentrated solution of (1) and extract 4 times until the ethyl acetate layer is light green, remove the lower aqueous layer and evaporate the solvent to obtain 200 g of paste;

(3) 200g paste is adsorbed by D101 macroporous resin, washed with 50% ethanol to 95% methanol, concentrated with 5-10L of alcohol, evaporated to dryness under normal pressure or reduced pressure by conventional methods, to obtain buttercup buttercup The total glycosides product is 100g, and the dry weight of the effective parts of the total ranunculosides in the whole herb of ranunculus is about 5%.

2. Composition identification

A. Chemical identification: Libermann-Burchard reaction: take a small amount of the above paste ranunculoside product, put it on a white porcelain plate, dissolve it with 1ml glacial acetic acid, add 1ml acetic anhydride-concentrated sulfuric acid reagent (19:1), mix well, and the color The change is yellow→brown→tan, and the change is fast. Indicates that it contains triterpenoid saponins.

B. TLC identification: Sample treatment: Weigh 7.5g of the above paste ranunculoside product, and use 200-300 mesh normal phase silica gel column chromatography, CHCl ₃ -CH ₃ OH (3:1-1:1) gradient For elution, collect tubes 51-58, evaporate the solvent to dryness, add 20% sulfuric acid aqueous solution to hydrolyze at 110°C for 3 hours, remove and cool, extract with chloroform three times, and remove the lower chloroform layer.

Thin-layer chromatography plate: silica gel-0.3% CMC-Na (1:2.5) plate, dry at room temperature, activate at 105°C for 30min;

Developing agent: petroleum ether-ethyl acetate (1:1);

Ultraviolet lamp observation: the wavelength is 254nm or 365nm;

Color developer: concentrated sulfuric acid-vanillin. The results are shown in Figure 1.

3. Content determination

In this experiment, the content of ranunculus total saponins was determined by spectrophotometry. Oleanolic acid was used as the reference substance. Precisely weigh an appropriate amount of the extract and put it into a 10ml stoppered test tube, add 0.2ml of newly prepared 5% vanillin glacial acetic acid solution and 0.8ml of perchloric acid, keep warm in a 70°C water bath for 15min, and immediately place it in ice water to cool for 5min. Add 5ml of glacial acetic acid, shake well and let stand for 10min. Except that no oleanolic acid or ranunculoside was added to the blank tube, the rest of the reagents were the same. According to the spectrophotometric method [Chinese Pharmacopoeia (2005 edition) one] experiment, measure the absorbance A immediately at the wavelength of 560nm. By plotting A (absorbance) against the concentration C, the regression equation is A=4.5245×10 ^-2 C+2.032×10 ^-3 , r=0.9975. The linear range is 6.0mg/L～18.0mg/L. The methodological investigation complied with the regulations. In this paper, the colorimetric method is used to determine the content of saponins, which is simple, fast and cheap. According to this method, the total ranunculoside content is determined to be between 60% and 80%.

Example 2

Anti-cardiac hypertrophy effect experiment of the present invention

1. Drugs

Angiotensin II, Sigma, lot number: 125K51011

DMEM dry powder, Gibco company, batch number: 1324945

Fetal bovine serum, Hyclone company, batch number: 20060404

Trypsin, Beijing Dingguo, lot number: DH355-2

Type II collagenase, Invitrogen, lot number: DH071

Penicillin, Sigma, lot number: DH231-1

Streptomycin, Sigma, lot number: DH325

5-BrdU, Roche, lot number: 280879

Sulforhodamine B, Sigma-Aldrich, Lot: 3520-42-1

Trichloroacetic acid, Genebase, lot number: G2365

All other reagents were domestic analytically pure

2. Instrument

_CO2 incubator, Haier Company;

Microplate reader 850, Biotech laboratories;

Biological inverted microscope XSP-15C, Shanghai Changfang Optical Instrument Co., Ltd.;

Low-speed centrifuge LD4-2A, Beijing Medical Centrifuge Factory;

Vertical pressure steam sterilizer YXQ-LS-30S II, Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory.

3. Isolation and Culture of Cardiomyocytes

Use 75% alcohol to sterilize the newborn SD suckling mice for 1-4 days, take out their hearts in a sterile workbench, put them into pre-cooled D-Hanks solution, and remove the lungs, large blood vessels and peduncle tissues around the heart. Then transfer the heart to another pre-cooled D-Hanks plate, squeeze the heart, and wash away the blood cells. Then all the hearts were transferred into a 25ml beaker with a self-made magnetic stirrer, and the hearts were chopped into sand grains. Add 0.125% trypsin: 0.08% = 1:1 collagenase digestion solution, digest at room temperature for 6 minutes, stop digestion with calf serum, let stand for a while, and suck off the supernatant. Add an equal volume of the above-mentioned collagenase digestion solution to continue digestion, and collect the supernatant. After continuous digestion for several times, all supernatants were collected and centrifuged at 1000rpm for 8min. Discard the supernatant, resuspend the cells with D-Hanks solution, and centrifuge at 1000rpm for 8min. Discard the supernatant. Collect the precipitate, suspend it with 20% FBS, pass it through a 120-mesh cell sieve, inoculate it into a culture bottle and culture it for 2 hours, suck out the supernatant, adjust the cell density to 1×10 ⁵ ~4×10 ⁵ cells/ml, and add a final concentration of 0.1 mmol/l of BrdU. After the cells were cultured for 72 hours, they were replaced with 5% FBS for 24 hours to perform the following experiments.

4. Construction of cardiac hypertrophy model

build conditions

4.1 The influence of the three factors of cell density, angiotensin Ang II concentration and drug intervention time on hypertrophic cardiomyocytes was examined by orthogonal experimental design method, and the optimal conditions for the construction of cardiomyocyte hypertrophy model were determined. The experiment was repeated twice.

Table 1 Factor level table

4.2 Cell grouping

The cells were seeded into 96 wells, divided into 9 groups, and treated according to the method in Table 2.

Table 2 L ₉ (3 ³ )

4.3 Index detection

Cardiomyocytes are highly differentiated terminal cells with contractile function, generally unable to proliferate, accompanied by an increase in volume or length, but not in number. Cardiac hypertrophy is the response of cardiomyocytes and interstitial cells to neurohumoral factors or mechanical stretch, which include angiotensin II (angiotensin II, Ang II), endothelin-1 (endothelin-1, ET -1), catecholamine transmitters, insulin-like growth factor-1 (insulin-like growth factor-1), tumor necrosis factor-α. These factors are all positive regulatory factors for the occurrence and development of cardiac hypertrophy. Their corresponding receptors have become the main target molecules for the prevention and treatment of myocardial hypertrophy. It has been reported in the literature that the binding of angiotensin II to its type I receptor (AT ₁ ) can directly induce cardiomyocyte hypertrophy. Cardiomyocyte hypertrophy manifests as an increase in volume morphologically, and internally as an increase in total protein content, protein synthesis, and DNA synthesis in the cell.

Amino acids such as leucine, phenylalanine, lysine, and valine are essential amino acids for human body to synthesize proteins, so ³ H-leucine or ¹⁴ C-phenylalanine is incorporated into cardiomyocytes cultured in vitro Cardiomyocyte protein synthesis can be investigated. In general, the amount of incorporation is positively correlated with the amount of protein synthesis in cardiomyocytes. Use a liquid scintillation counter to measure the radioactivity of ³ H, ¹⁴ C and other low-energy β-rays to indirectly reflect the amount of protein synthesis in cardiomyocytes.

The DNA structure contains four bases: adenine, guanine, thymine and cytosine. Thymine is a unique base of DNA and an essential substance for DNA synthesis. The depletion of thymine indicates the activity of DNA synthesis, and the incorporation of ³ H-TDR can be used to quantify the DNA synthesis of cardiomyocytes.

Traditional staining methods such as Coomassie brilliant blue method, biuret method and lorry-phenol reagent method can measure the protein content of cells, and these methods can be used to quantify the total protein in hypertrophic cardiomyocytes. Sulphorhodamine B (SRB) is a protein-binding dye that can be combined with basic amino acids in cell proteins fixed by TCA to develop color, showing a bright pink color, and the absorbance is measured with an enzyme-linked immunosorbent assay , which provides a sensitive value for the protein content of the cell. The SRB method avoids the use of radioactive elements. Compared with the MTT method, the experimental method has its outstanding advantages: ① The time required for the operation is shorter, TCA fixes the cells for 1 hour, and SRB staining only takes 10-30 minutes, while the cells in the MTT method add MTT In the future, it will take another 4 hours; ② The SRB method does not have to complete all the operations within a fixed period of time. The cells can be stored after being fixed with TCA or combined with SRB, and the OD value is still very stable when it is tested again at an appropriate time. The MTT method must complete all operations at one time; ③ The SRB method is more sensitive than the MTT method. If there are only 150 cells in a well, the SRB method can detect them, but the MTT method cannot. Compared with the traditional Coomassie brilliant blue method, biuret method and lorry-phenol reagent method, the operation method is simpler and the sensitivity is higher.

Accordingly, this experiment stained cardiomyocytes with SRB to determine the total protein content in the cells.

After the cells have been cultured for a certain period of time, take out the 96-well plate, suck away the medium, add 50ul of 50% trichloroacetic acid to each well, fix at 4°C for 30min, discard the trichloroacetic acid, wash 5 times with deionized water, and place it at room temperature for 24 After 1 hour, let the water in the culture well evaporate to dryness, then stain with 70ul 0.4% SRB at room temperature for 20min, pour off the SRB, wash 5 times with 1% acetic acid, and place it at room temperature for 24 hours. After the water in the culture well evaporates to dryness, Add 200ul 10mmol/LUnbuffered Tris-base to each well, shake on a plate shaker for 10min, and then use a microplate reader to detect the absorbance in each well at 492nm, see Table 3.

Absorption ratio values of 9 test groups in table 3

test number AngII concentration (A) Cell Density (B) Culture time (C) Absorbance Mean 123456789 111222333 123123123 123231312 0.26950.47260.37020.27150.37600.39420.23070.52030.5023 T1T2T3R×3 1.11231.04171.27630.2346 0.77171.39191.26670.6206 1.18401.24640.97690.2695

The intuitive analysis of the above test results shows that the primary and secondary factors are judged by the R value, and the cell density is the main factor, followed by the time of drug addition and the concentration of Ang II. The optimal condition is A ₃ B ₂ C ₂ , that is, the concentration of Ang II is 10 ^-6 mol/L, the cell density is 2×10 ⁵ /ml, and the incubation time is 48 hours. This combination does not appear in the orthogonal table. In order to prove its reliability, the test is carried out according to the condition of A ₃ B ₂ C _2. The absorbance value obtained by the test result is: 0.5433, which is higher than the absorbance value obtained by any group of tests in Table 3. large, proving that the obtained optimal conditions are reliable.

4.4 Morphological comparison of HE staining

Cell grouping:

The cells were cultured in a petri dish with a diameter of 3.5 cm and 2 × 2 cm slides placed at the bottom, and 1 ml of cell suspension was added to each petri dish, and they were divided into three groups: (1) normal group: no drug treatment; (2) ) model group: add Ang II for incubation, the final concentration is 10 ^-6 mol/L; (3) drug group: add Ang II and losatan at the same time, the final concentration is 10 ^-6 mol/l, and at the same time add the test drug for intervention 48 hours.

HE staining of the cells, photomicrograph, see Figure 2, the HE staining results of SD neonatal rat cardiomyocytes (×100) shown in Figure 2, A normal cardiomyocytes, B hypertrophic myocardium induced by angiotensin II Cells, C hypertrophic cardiomyocytes were treated with losatan for 48 hours.

From the results of HE staining, it can be seen that the surface area of hypertrophic cardiomyocytes induced by angiotensin II (Figure B) was significantly larger than that of the normal group, and the surface area of hypertrophic cardiomyocytes was basically restored to the normal level after being intervened by losatan for 48 hours (Figure C) ).

5. Carry out anti-cardiomyocyte hypertrophy drug experiments according to the above experimental conditions

5.1 Grouping:

Cardiomyocytes were seeded and cultured in a 96-well plate. After 72 hours, the culture medium was replaced with 5% FBS and cultured for 24 hours. The cells were divided into groups, and each group had 8 replicate wells. Drug intervention: (1) normal group, add an equal volume of DMEM; (2) hypertrophy model group: the final concentration of Ang II is 10 ^-6 mol/l; (3) losatan group: add Ang II and losatan at the same time, the final concentration is 10 ^-6 mol/l; (4) the present invention Ranunculosides group: the final concentration of Ang II was 10 ^-6 mol/l, the final concentration of ranunculosides was 50 μg/ml, and the drug was added for 48 hours.

5.2 Indicator detection:

In this experiment, the SRB method was used to examine the total protein content in each group of cardiomyocytes, and the specific method was the same as that described in 4. Construction of the cardiac hypertrophy model. To observe the effect of ranunculosides on hypertrophic cardiomyocytes. According to the orthogonal experiment design and HE staining detection, the optimal conditions for hypertrophy model construction were determined, that is, cell concentration: 2×10 ⁵ /ml, final concentration of Ang II: 10 ^-6 mol/L, drug intervention time: 48 hours. Based on this condition, the experiment was divided into normal cardiomyocyte group, hypertrophy model group, losartan treatment group, and drug intervention group. The results showed that ranunculosides had a significant anti-cardiac hypertrophy effect, which was equivalent to that of the losartan group.

5.3 Experimental results:

After the cells in each group were incubated with 10 ^-6 mol/l Ang II for 48 hours, the OD value obtained by the SRB method was significantly larger than that of the normal group, indicating that the total protein content of the cells was increased after being induced by Ang II, that is, cardiomyocytes after 10 ^-6 After incubation with mol/l Ang II for 48 hours, the cells became hypertrophic. The results of the experiment show that 50 μg/ml ranunculosides can significantly inhibit myocardial hypertrophy, compared with the model group, p<0.01. The experimental results are shown in Figure 3.

Eight replicate wells were set up for each group of experiments, and the experiment was repeated more than 3 times. The figure shows a representative result. The data are represented by mean ± standard deviation, ^** p<0.01 means that there is a significant difference compared with the Ang II group.

Example 3

Ranunculosides Inhibit Cardiac Effect Experiment

1 Apparatus, Drugs and Animals

1.1 Instrument

Biological signal acquisition system (MS4000U), Guangzhou Longfeida Technology Co., Ltd. 10g tension transducer, Beijing Xinhang Xingye Technology and Trade Co., Ltd.

1.2 Drugs

Propranol Hydrochloride Tablets, Shaanxi Yongshou Pharmaceutical Co., Ltd., batch number: 20051028 Isoproterenol Hydrochloride Injection, Shanghai Hefeng Pharmaceutical Co., Ltd., batch number: 5E20004

1.3 Animals

Frogs, about 60-90g, for both ♀♂ and 30 pieces, provided by Guangzhou Suibei Animal Center;

2 Experimental methods

2.1 Frog heart intubation:

Refer to the existing experimental method to make an isolated heart specimen of Stuart, then clamp the apex of the heart with a frog heart clip, connect the biological signal acquisition system through the tension transducer, add 1ml of Ren's solution to each cannula, and measure a period of normal heartbeat curves, and perform tests in groups as follows.

2.2 Experimental grouping

2.2.1 The effect of ranunculosides on the basal tension of isolated frog hearts:

Add 10 μg/ml propranolol, 1 μg/ml and 10 μg/ml total ranunculosides to the cannula respectively, trace the effect curve, calculate the ratio of the shrinkage amplitude before and after adding the drug, and the shrinkage amplitude after adding the drug/basic shrinkage amplitude, using D/F said, see Table 4.

2.2.2 Inhibitory effect of ranunculosides on isolated frog hearts

Add 10 μg/ml isoproterenol (Iso) solution to Ren’s solution, and after the effect is obvious, add 10 μg/ml propranolol, 1 μg/ml or 10 μg/ml total ranunculoside respectively, and draw the effect curve, Calculate the ratio of shrinkage before and after dosing, the shrinkage after dosing/shrinkage after adding Iso, expressed in D/I, see Table 4.

)Table 4 The inhibitory effect of ranunculosides on isolated frog hearts (n=8,

)

group Final concentration (μg/ml) D/F D/I propranolol ranunculosides ranunculosides 10110 0.506±0.0710.898±0.058 ^** 0.836±0.115 ^** 0.712±0.0980.919±0.058 ^** 0.885±0.056 ^**

Note: ^** , compared with propranolol group, p<0.01

2.2.3 Data processing

The experimental data are represented by (x±s), and the t-test is used for statistical processing, and P<0.05 is taken as the boundary value of significant difference.

3 Experimental results The experimental results are shown in Figure 4.

4 Conclusion

This experiment shows that 1μg/ml and 10μg/ml ranunculosides can reduce the basal tension of the isolated frog heart, and can resist the strengthening of cardiac contractility induced by isoproterenol.

Example 4

Experiment of the effect of ranunculosides of the present invention on vascular rings

1 Apparatus, Drugs and Animals

1.1 Instrument

Biological signal acquisition system, Medlab-U/4C501 type, Nanjing Meiyi Technology Co., Ltd. 10g tension transducer, Beijing Xinhang Xingye Technology and Trade Co., Ltd.

Super Thermostat 501, Shanghai Experimental Instrument Factory

1.2 Drugs

Norepinephrine Bitartrate Injection (NE), Tianjin Jinyao Amino Acid Co., Ltd., batch number: 0503221

Acetylcholine chloride, Shanghai Sanaisi Reagent Co., Ltd., batch number: 20030620 Phentolamine Mesylate Injection (Phe), Xudonghaipu, batch number: 060201

1.3 Animals

SD rats, SPF grade, about 200g, male and female, 30 rats, provided by Guangdong Medical Experimental Animal Center, license number: SCXK (Guangdong) 2003-0002 Yuejian Zhengzi 2006A015

2. Experimental method

2.1 Experimental grouping:

2.1.1 The effect of ranunculosides on NE-induced vascular ring contraction

Under the circulation of oxygen to the blood vessels, a preload of 1.5 g was applied, and after 1.5 hours of equilibrium, norepinephrine was added with a final concentration of 10 ^-6 mol/l to cause the vasoconstriction to plateau. The experiments were divided into four groups: (1) Blank Control group, add normal saline 0.1ml; (2) ranunculoside low dose group, its final concentration is 1mg/ml; (3) ranunculoside high dose group, its final concentration is 2mg/ml; (4) phenytoside In the Lamin group, the final concentration was 5×10 ^-6 mol/L. The results are shown in Figure 5.

In order to quantitatively investigate the effect of ranunculus against NE causing vascular ring contraction, the inhibition rate of the test drug against NE causing vascular ring contraction is calculated according to the formula, inhibition rate (%)=(the tension of the vascular ring after adding NE-adding the test drug Vascular tension)/vascular ring tension after adding NE. The results are shown in Figure 6. Figure 6 shows that ranunculosides with a final concentration of 1 mg/ml and 2 mg/ml significantly inhibited the vasoconstriction induced by norepinephrine, and the inhibition rate was obviously dose-dependent.

Example 5

Experiment of anti-inflammatory and analgesic effects of ranunculosides of the present invention

The paw swelling model induced by carrageen in rats and the ear swelling model in mice induced by xylene are both classic inflammatory models, with telangiectasia and capillary hyperpermeability as the main symptoms. The writhing reaction of mice induced by glacial acetic acid is indirectly caused by acute inflammation in the peritoneal cavity, which increases the levels of PGE and PGF in the peritoneal fluid, thereby producing a peripheral pain response; and the animal pain model caused by thermal stimulation is a central pain model .

1 Drugs, instruments and animals

1.1 Drugs

Dexamethasone (DEX), Zhejiang Xianju Pharmaceutical Co., Ltd., batch number: 060105 Rotundine Sulfate Injection (Rotundine), Guangdong Boluo Pioneer Pharmaceutical Group Co., Ltd., batch number: 060104

Other reagents: Xylene (analytical grade), produced by the 90662 Factory of the Chinese People's Liberation Army.

1.2 Instruments

FA2104S Electronic Analytical Balance, Shanghai Balance Instrument Factory

Electronic platform scale, super thermostat (501 type) produced by Guangdong Zhongshan Xinheji Technology Development Co., Ltd., Shanghai Experimental Instrument Factory

1.3 Animals

SPF grade SD rats were provided by Guangdong Provincial Medical Experimental Animal Center, license number: SCXK (Guangdong) 2003-002, Guangdong Jianzheng Zi 2005A010; body weight 150g-160g. SPF-grade NIH mice were provided by the Guangdong Provincial Medical Experimental Animal Center, license number: SCXK (Guangdong) 2003-002, Guangdong Jianzheng Zi 2004A018; weight 18-22g, male and female in half. SPF-grade NIH mice were provided by the Guangdong Medical Experimental Animal Center, license number: SCXK (Guangdong) 2003-002, Guangdong Jianzheng Zi 2005A012; body weight 18-22g, male and female in half.

2 Experimental methods

2.1 Effects of ranunculosides on ear swelling in mice induced by xylene

Fifty NIH mice, half male and half male, were randomly divided into 5 groups with 10 mice in each group. That is, normal saline group, low, medium and high dose groups of total ranunculoside, namely 200mg, 400mg, 800mg/kg, dexamethasone group, positive, the total dose for each adult is 30mg, and the adult body weight is calculated as 60kg, and the mice are as follows: 20 times the dose for adults, that is, 30mg×20/60=10mg/kg. Dosing once a day for 3 consecutive days, 1 hour after the last administration, the right ear of mice in each group was coated with xylene 0.05ml/only, and the left ear was used as a control. Cut off the same parts of both ears, weigh them separately with an electronic balance, and calculate the swelling rate of the right ear according to the following formula: swelling rate of the right ear (%)=(weight of the right ear piece-weight of the left ear piece)/weight of the left ear piece ×100%. The results are shown in Figure 7.

2.2 Effects of ranunculosides on carrageen-induced paw swelling in rats

Get 50 SD rats, half male and half male, randomly divided into 5 groups, i.e. normal saline group, three groups of low, medium and high doses of total ranunculoside, i.e. 400mg, 200mg, 100mg/kg, dexamethasone group, positive, The total dose for each adult is 30 mg, and the adult body weight is calculated as 60 kg. Rats are given 10 times the dose for adults, that is, 30 mg×10/60=5 mg/kg, once a day. For 3 consecutive days, 2 hours after the last administration, first measure the normal ankle joint circumference of the right hindlimb of each mouse with a soft tape measure, and inject 0.1ml/ of 1% carrageenan suspension subcutaneously at the right hindfoot of the rat respectively, Inflammation, then measure the circumference of the ankle joint at 1 hour, 2 hours, 4 hours, and 6 hours respectively, record the results, and calculate the change of foot circumference according to the formula

Foot circumference change = ankle joint circumference after inflammation - ankle joint circumference before inflammation

Calculate the change in circumference of each mouse's foot. The results are shown in Figure 8, which shows that 200mg/kg and 400mg/kg ranunculosides can significantly inhibit rat paw swelling, suggesting that ranunculosides have good anti-inflammatory effects.

2.3 Effects of ranunculosides on the pain response of mice induced by hot plate stimulation

Adjust the temperature of the super constant temperature water bath at 55°C ± 0.5°C, and preheat the hot plate for 10 minutes. Take 18-22g female mice, put one on the hot plate each time, and the number of seconds required for the mice to lick their hind paws after being placed on the hot plate is taken as the pain threshold of the mouse. Those who lick the hind feet for less than 5 seconds or more than 30 seconds or jump, discard them. Qualified 50 mice were randomly divided into 5 groups, i.e. normal saline group, low, medium and high dose groups of total ranunculoside, i.e. 200mg, 400mg, 800mg/kg, rotundine group, positive, adult per person total The dose is 90 mg, the adult body weight is calculated as 60 kg, and the mouse is given 20 times the adult dose, that is, 90 mg×20/60=30 mg/kg. The mice in each experimental group were administered once a day, and the control group was given the same amount of normal saline for three consecutive times. The pain threshold of the mice was measured 60 minutes after the last administration. If there was still no response within 60 seconds, the mice were taken out to avoid burns. The pain threshold is calculated in 60 seconds. The results are shown in Figure 9, which shows that 200mg/kg, 400mg/kg and 800mg/kg ranunculosides can significantly inhibit the pain response induced by 55°C hot plate stimulation, and the analgesic effect is equivalent to that of rotundine.

2.4 Effects of total ranunculosides on the writhing response of mice induced by acetic acid stimulation

Take 50 NIH mice, half male and half female, and randomly divide them into 5 groups, i.e. normal saline group, three groups of low, medium and high doses of total ranunculoside, namely 200mg, 400mg, 800mg/kg, rotundine group, 30mg/kg kg. The mice in each experimental group were administered once a day, and the control group was given the same amount of normal saline for three consecutive times. One hour after the last administration, each mouse was intraperitoneally injected with 0.6% acetic acid 0.2ml/only, and the mice were observed within 15 minutes. Writhing times, and calculate the analgesic percentage of each drug:

Calculate the percentage of analgesia for each mouse. The results are listed in Table 7 and Figure 10.

Table 7 The effect of ranunculosides on the pain model induced by acetic acid stimulation

group no Dose (mg/kg) Number of twists within 15′ Analgesic percentage (%) Normal saline rotundine ranunculosides ranunculosides ranunculosides   1010101010 Equal volume 30200400800 28.5±10.99.0±10.26 ^*** 15.5±5.4 ^*** 6.4±4.6 ^*** 8.8±6.7 ^*** 68.445.647.776.8

Compared with normal saline group: ***, p<0.01; T test

Table 7 shows that ranunculosides have a significant inhibitory effect on the writhing response of mice caused by acetic acid, and Figure 10 shows that: 200mg/kg, 400mg/kg and 800mg/kg ranunculosides significantly inhibit the pain response induced by acetic acid stimulation , and dose-dependent.

2.5 Statistical analysis

表示，并采用T检验进行统计学处理The experimental data are all used

Indicated, and the T test was used for statistical processing

3 Experimental results

The results showed that three dose groups of ranunculosides, 200mg/kg, 400mg/kg and 800mg/kg, all had significant inhibitory effects on xylene-induced ear swelling in mice, and there was a dose-dependence. Two dose groups of 200mg/kg and 400mg/kg of ranunculosides had a significant inhibitory effect on carrageen-induced paw swelling in rats, suggesting that ranunculosides have good anti-inflammatory effects. Ranunculosides 200mg/kg, 400mg/kg and 800mg/kg three dose groups significantly antagonized the pain response induced by thermal stimulation (dose-dependent) and acetic acid, suggesting that ranunculosides may have antagonism on peripheral and central pain effect.

Claims (8)

1. a Herba Ranunculi Japonici total glycosides is that the raw material extraction obtains with the Herba Ranunculi Japonici herb, it is characterized in that containing 60%～90% triterpene saponin constituents.

2. the preparation method of a Herba Ranunculi Japonici total glycosides is characterized in that may further comprise the steps:

(1) the Herba Ranunculi Japonici herb dries chopping, and 60%～95% alcohol reflux percolation extracts, filter, and merge extractive liquid,, decompression and solvent recovery concentrates;

(2) in (1) concentrated solution, add and the concentrated solution equal volume of ethyl acetate, take off a layer water layer solvent evaporated, get paste;

(3) paste is through macroporous resin adsorption, and after first water washed down, reuse 50%～80% ethanol or methanol-eluted fractions concentrated, and solvent evaporated gets paste Herba Ranunculi Japonici total glycosides product.

3. the preparation method of Herba Ranunculi Japonici total glycosides according to claim 2 is characterized in that the described extraction of step (1) adopts 10%～95% ethanol, ethanol water according to Herba Ranunculi Japonici herb weight ratio be 7: 1 consumption, extract 2～3 times.

4. the application of the described Herba Ranunculi Japonici total glycosides of claim 1 is characterized in that the application aspect the preparation cardiovascular drugs.

5. according to the application of the described Herba Ranunculi Japonici total glycosides of claim 4, it is characterized in that the application aspect preparation resisting cardiac hypertrophy medicine.

6. according to the application of the described Herba Ranunculi Japonici total glycosides of claim 4, it is characterized in that the application aspect preparation vasodilator smooth muscle medicine.

7. according to the application of the described Herba Ranunculi Japonici total glycosides of claim 4, it is characterized in that the application aspect preparation inhibition heart drugs with function.

8. the application of the described Herba Ranunculi Japonici total glycosides of claim 1 is characterized in that the application at preparation antalgic anti-inflammatory agent object space face.

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