阅读说明：本技术 雪莲黄酮类组合物在制备治疗关节炎疾病的药物中的用途 (Application of saussurea involucrate flavonoid composition in preparation of medicine for treating arthritis diseases ) 是由 徐宁 万志超 于 2019-11-29 设计创作，主要内容包括：本发明涉及雪莲黄酮类组合物在制备治疗关节炎疾病的药物制剂中的用途,所述雪莲黄酮类组合物包含四种组分,所述四种组分分别为：芦丁、肉豆蔻醚、异槲皮苷、木犀草素-7-o-葡萄糖苷。本发明通过大量的动物实验,证实了本发明提供的黄酮类组合物的药效活性,其可以用于制备各类临床应用的解痛镇痛类药物和关节炎治疗药物,相比于直接应用雪莲制备的中成药制剂来说,其具有药效活性高、临床使用剂量小、见效快、副作用低等效果。药物剂型可以采用常规的片剂或胶囊剂,也可以根据药物组分的特性,制备成吸收率更高、或储存性更佳的一些药物新剂型。(The invention relates to an application of a snow lotus flavonoid composition in preparing a medicinal preparation for treating arthritis diseases, wherein the snow lotus flavonoid composition comprises four components: rutin, myristicin, isoquercitrin, and luteolin-7-o-glucoside. The invention proves the pharmacodynamic activity of the flavonoid composition through a large number of animal experiments, can be used for preparing various pain-relieving and pain-relieving medicines for clinical application and arthritis treatment medicines, and has the effects of high pharmacodynamic activity, small clinical dosage, quick response, low side effect and the like compared with a Chinese patent medicine preparation prepared by directly applying saussurea involucrate. The medicament dosage form can adopt conventional tablets or capsules, and can also be prepared into novel medicament dosage forms with higher absorption rate or better storage property according to the characteristics of the medicament components.)

1. The application of the saussurea involucrate flavonoid composition in preparing the medicinal preparation for treating the arthritis disease is characterized in that the composition comprises four components: rutin, myristicin, isoquercitrin, and luteolin-7-o-glucoside.

2. The use according to claim 1, wherein the four components are present in the composition in the following weight ratios:

rutin: 28-32; myristicin: 0.8-1.2; isoquercitrin: 1.8-2.2; luteolin-7-o-glucoside: 6.8-7.2.

3. Use according to claim 1 or 2, wherein the four components are present in the composition in the following weight ratios:

rutin: 30, of a nitrogen-containing gas; myristicin: 1; isoquercitrin: 2; luteolin-7-o-glucoside: 7.

4. the use according to any one of claims 1 to 3, wherein the composition is extracted from saussurea involucrate.

5. Use according to claim 1, wherein the pharmaceutical formulation is a tablet.

6. Use according to claim 1, wherein the pharmaceutical formulation is a capsule.

Technical Field

The invention relates to the technical field of compositions with medical application, in particular to a snow lotus flavonoid composition and application thereof.

Background

Snow lotus (the name of Saussurea involucrata (kar. et Kir.) Sch. -Bip.) has the shape similar to lotus flower, so the name of snow lotus, which is called snow lotus for short, is obtained. Is perennial herb of saussurea of Compositae, and has a height of 15-35 cm. The rhizome is thick and the neck is left with most of the brown leaves. Thick and strong stem without hair. Dense leaves, basal leaves and cauline leaves without stems, oval leaves or oval ovate leaves; the most upper part is in the shape of leaf bud, membranous and light yellow, and surrounds the general inflorescence, and sharp teeth are arranged on the edge. 10-20 head inflorescences, and spherical total inflorescences are densely arranged at the top of the stem. The involucre is hemispherical and has the diameter of 1 cm; 3-4 layers of bract, purple brown at edge or all. The floret is purple. The slim fruit is oblong. The crown hair is white. The flower and fruit period is 7-9 months. It is distributed in the alpine region of many places in China, and Russia and Kazakhstan are also distributed. Grow in rock cracks, stone walls and gravel beaches near high mountain snow lines, and have an altitude of 2400 + 4000 meters. Tianshan produced the most and the best quality. Can be used as a medicine and also has certain ornamental value.

The snow lotus herb has warm nature, sweet and bitter taste, enters liver, spleen and kidney meridians, has the functions of dispelling cold, strengthening yang, regulating menstruation and stopping bleeding, and is used for treating impotence, weak waist and knees, irregular menstruation, metrorrhagia and metrostaxis, leukorrhagia, rheumatic arthritis, traumatic hemorrhage and other diseases. Has effects in dredging channels, promoting blood circulation, expelling cold, removing dampness, stopping bleeding, relieving swelling, and removing toxic materials. Snow lotus herb contains protein, amino acid, flavonoid, alkaloid, etc. However, how to generate and take effect of the unique drug effect is always a difficult point and a blind point in the research field.

Disclosure of Invention

The snow lotus herb flavonoid composition takes snow lotus herb as a research object, deeply researches main pharmacodynamic components of snow lotus herb, successfully discovers the most important pharmacodynamic components and compatibility of the snow lotus herb, and provides the snow lotus herb flavonoid composition and the application thereof on the basis.

The property of the saussurea involucrate adopted by the invention is observed by naked eyes as follows: the stem is upright and hairless. Basal leaves are not seen; the leaf is oblong or oval, 7-20 cm long, 3-6 cm wide, and has serrate edge and glandular hair on both sides; the cauline leaves have no stems; the uppermost part is in the shape of bract, membranous, yellow, oblong or oval long round, 16 cm long, 7 cm wide, blunt top, with serrate edge, and the two sides are surrounded by short mollissima and glandular hair, which surround the total inflorescence. 13 capitulums, spherical total inflorescences with no small pedicel or short small pedicel at the stem tip. The involucre is hemispherical, and the diameter is 1-1.5 cm; involucre 4 layers, oval outer layer, elliptical middle layer, and linear inner layer. The top of all bracts is sharp, the edge is dark purple, and the outside is short, soft and glandular hair. The small flower is blue-purple, the length of the small flower is 1.8 cm, the length of the tube part is 8 mm, and the length of the eave part is 10 mm. 2 layers of crown hair, light brown and short outer layer; coarse, 5 mm long, inner layer long, feather-like, 1.2 cm long.

Saussurea involucrate has strong medicinal action, but the chemical components of saussurea involucrate are complex, and the most main medicinal components are the difficulty of research. The known chemical components of the saussurea involucrate comprise flavonoids, alkaloids, sesquiterpenes, volatile oil and the like, wherein the flavonoids are the main chemical components of the saussurea involucrate, have obvious biological activities of eliminating superoxide anion free radicals, hydroxyl free radicals and resisting oxidation of NADH peroxidase, and have stronger effects of resisting oxidation, radiation and aging and the like. Therefore, the invention firstly extracts and analyzes the flavonoid substances in the saussurea involucrate. After long-time complex extraction, analysis and pharmacodynamic tests, four flavonoid components extracted from saussurea involucrate are innovatively found to have strong pharmacodynamic activity, and are respectively: rutin, myristicin, isoquercitrin, and luteolin-7-o-glucoside.

Meanwhile, when the extracted components of the saussurea involucrate flavonoid are analyzed, the weight compatibility of the four components is rutin: 28-32; myristicin: 0.8-1.2; isoquercitrin: 1.8-2.2; luteolin-7-o-glucoside: 6.8-7.2, the combination has the strongest pharmacodynamic activity, and more preferably, the compatibility is as follows: rutin: 30, of a nitrogen-containing gas; myristicin: 1; isoquercitrin: 2; luteolin-7-o-glucoside: 7.

after the types and the compatibility of the flavonoid components of the saussurea involucrate are obtained, the extraction method of the components can be carried out by adopting known extraction methods of various traditional Chinese medicine extracts, wherein one preferable extraction method is as follows:

the method comprises the following steps:

(1) alcohol extraction: pulverizing whole plant of saussurea involucrata, adding 10 times of 70% ethanol, heating and reflux-extracting for 3 times, each time for 1h, filtering, mixing extractive solutions, recovering solvent under reduced pressure, and concentrating to 1/50 of original solution volume as sample solution;

(2) fine extraction: sampling the sample solution, loading the pretreated resin column, and eluting, wherein the elution step comprises: washing the resin column with water for 3BV to remove impurities, and then eluting with 15% ethanol for 8BV to remove impurities, wherein the elution flow rate is 3 BV/h; finally eluting with 40% ethanol for 6BV at a flow rate of 3BV/h, and respectively collecting 40% of eluent of 1-2BV, 40% of eluent of 3-4BV and 40% of eluent of 5-6 BV;

(3) freeze-drying: mixing the collected eluates, recovering solvent under reduced pressure, and lyophilizing to obtain powder.

The resin column in the step (2) adopts AB-8 macroporous resin, and the pretreatment step comprises the following steps: soaking the column in absolute ethyl alcohol for 24h, filling the column in a wet method, eluting the column with the ethanol at the flow rate of about 2.5BV/h until the eluent is not turbid after being added with water with the volume of 1:5, and finally washing the column with deionized water at the same flow rate of about 5BV until the eluent has no alcohol smell for later use. The BV refers to the volume of resin loaded in the resin column, called bed volume or bed volume (bed volume), abbreviated BV.

The invention proves the pharmacodynamic activity of the flavonoid composition through a large number of animal experiments, can be used for preparing various pain-relieving and pain-relieving medicines for clinical application and arthritis treatment medicines, and has the effects of high pharmacodynamic activity, small clinical dosage, quick response, low side effect and the like compared with a Chinese patent medicine preparation prepared by directly applying saussurea involucrate. The medicament dosage form can adopt conventional tablets or capsules, and can also be prepared into novel medicament dosage forms with higher absorption rate or better storage property according to the characteristics of the medicament components.

Drawings

FIG. 1 is a BPI diagram (good repeatability of two times of sample injection) of saussurea involucrata purified substance in positive ion mode;

FIG. 2 is a BPI diagram (good repeatability of two times of sample injection) of saussurea involucrata purified substance in negative ion mode;

FIG. 3 is a BPI plot in positive ion mode;

FIG. 4 shows the mice administered by oral gavage;

FIG. 5 is the tail vein administration of mice;

FIG. 6 is intraperitoneal administration of mice;

FIG. 7 is a mouse writhing;

FIG. 8 is a photograph of toe swelling;

fig. 9 is a photograph of toe swelling measurements.

Detailed Description

Embodiments of the present invention are described below with reference to the drawings. Elements and features depicted in one drawing or one embodiment of the invention may be combined with elements and features shown in one or more other drawings or embodiments. It should be noted that the figures and description omit representation and description of components or processes that are not relevant to the present invention and that are known to those of ordinary skill in the art for the sake of clarity.

Example 1 extraction of flavonoids from saussurea involucrata

1.1 alcohol extraction of saussurea involucrata medicinal material

Taking 6 saussurea involucrata medicinal materials, weighing the following materials before and after powdering: no. 1: 28.5g, 28.3 g; no. 2: 16.3g, 16.5 g; no. 3: 13.3g, 13.5 g; no. 4: 15.6g, 15.7 g; no. 5: 23.1g, 23.2 g; no. 6: 14.6g and 14.7 g. And (3) the total powder is as follows: extracting with 10 times of 70% ethanol under reflux for 3 times (1 hr each time), filtering with 8 layers of gauze, and mixing extractive solutions.

1.2 preparing the sample loading solution

Recovering solvent from the extractive solution under reduced pressure to obtain about 70mL of sample solution.

2 fine lifting

2.1 AB-8 macroporous resin (95% ethanol soaked for 24h) is selected, the inner diameter of the column is about 2.5cm, and the ratio of the diameter to the height is 1: 10. Soaking the macroporous resin in absolute ethyl alcohol for 24h, and then filling the column with a wet method, wherein a layer of cotton is required to be plugged at the bottom of the resin column before filling the column, and the column is soaked in absolute ethyl alcohol. Eluting with ethanol at about 2.5BV/h until the eluate plus five times water (1:5) is clear of turbidity. The eluent is washed by deionized water with the same flow rate of about 5BV until the eluent has no alcohol smell for standby.

2.2, the water-washed liquid level is leveled, and then the sample is loaded.

2.3 washing the resin column with water for 3BV to remove impurities, and then eluting with 15% ethanol for 8BV to remove impurities, wherein the elution flow rate is 3 BV/h. Finally eluting with 40% ethanol for 6BV at a flow rate of 3BV/h, and collecting 40% of eluent of 1-2BV, 40% of eluent of 3-4BV and 40% of eluent of 5-6 BV.

And 2.4, decompressing the eluent, recovering the solvent, and freeze-drying by using a freeze dryer to obtain a powdery product.

Example 2 analytical characterization of the powdered product obtained in example 1

1. Instruments and reagents

Waters acquisition UPLC Class H ultra high performance liquid chromatograph (Waters corporation, USA), Waters G2Q-TOF/MS high resolution mass spectrometer (Waters corporation, USA), Waters acquisition UPLC BEH C18 chromatographic column (2.1mm × 100mm, 1.7 μm) (Waters corporation, USA); data processing software 4.1 MassLynx; acetonitrile (mass spectrum grade, Fisher Scientific); drochen ultrapure water (guangzhou drochen food & beverage limited)); formic acid (quality spectrum grade, Fisher Scientific Co., Ltd.)

2 method

2.1 preparation of the solution

Preparation of a test solution: precisely weighing 0.02054g of the total extract, placing in a 10mL volumetric flask, adding 40% ethanol to a constant volume, mixing uniformly, centrifuging at a high speed of 12000r/min for 15min, and taking supernatant to obtain a test solution.

2.2 conditions of analysis

A chromatographic column: WatersAcquity UPLC BEH C18(2.1 mm. times.100 mm, 1.7 μm); the detection wavelength is 360 nm; the injection volume was 2. mu.L. Mobile phase: the A phase is 0.1% acetonitrile formate, the B phase is 0.1% formic acid water, gradient elution is adopted, and the specific elution conditions are as shown in the following table.

Chromatographic conditions are as follows: flow rate: 0.3 mL/min; column temperature: 30 deg.C

Time (min)	A (0.1% formic acid acetonitrile)%	B (0.1% formic acid water)%
			0	5	95
4	9	91
			10	13	87
20	16	84
			30	17	83
35	28	72
			40	95	5
43	5	95

Mass spectrometry conditions, using ESI ion source MSE positive ion mode: the ion source temperature is 120 ℃; the temperature of desolventizing gas is 400 ℃; the desolventizing air flow rate is 600L/h; the flow rate of the gas in the taper hole is 50L/h; the taper hole voltage is 40V; a capillary tube with a voltage of 3.0kV high-energy channel; the collision voltage is 20-75 eV. MSE scan mode: the mass scanning range m/z is 50-1200; the scan time was 0.2 s. To ensure accurate mass determination, mass correction during data collection using leucine-enkephalin as an external standard can produce fragment ions M/z 556.2771[ M + H ] +. Data acquisition and analysis were performed using MassLynx V4.1 software.

3 results and analysis

The BPI diagram of the saussurea involucrata purified product in positive ion mode is shown in figure 1, and the BPI diagram of the saussurea involucrata purified product in negative ion mode is shown in figure 2.

The chromatogram in positive ion mode was resolved, see FIG. 3.

The positive ion mode analysis results are shown in table 1:

example 3 Components and compatibility of saussurea involucrate flavonoid composition

The purified saussurea involucrate obtained by the extraction method mainly contains 4 flavonoid substances which are respectively shown in the following steps: rutin: myristicin: isoquercitrin: luteolin-7-o-glucoside, wherein the weight ratio of luteolin to glucoside is 30:1: 2: 7.

example 4 efficacy test (analgesic effect) of flavonoid composition of example 3

The pharmacodynamics of the flavonoid composition of example 3 was evaluated based on the therapeutic effect of the flavonoid composition of example 3 on an acetic acid-induced mouse pain model.

The method comprises the step of randomly dividing mice into 5 groups, namely a model control group (7 mice), a positive drug control group, a flavonoid composition high-dose group in example 3, a flavonoid composition medium-dose group in example 3 and a flavonoid composition low-dose group in example 3, wherein 9 mice are respectively divided into 9 mice, and a flavonoid composition tail vein administration group (7 mice) in example 3 is respectively adopted. Divided into groups and administered on the same day, 1 time per day for 3 days, and the control group is administered with physiological saline for intragastric administration under the same conditions. After 1 hour of the last administration, each mouse was intraperitoneally injected with 0.2ml of 1% acetic acid (glacial acetic acid), and the number of times of typical writhing of each mouse was observed within 20 minutes, and the results were counted by using a comparative t-test between groups.

The result shows that the flavonoid composition in the example 3 can obviously reduce the writhing frequency of mice when being applied to the model experiment of the mice pain caused by acetic acid, and has good dose-effect relationship dependence.

The conclusion is that the flavonoid composition in the example 3 has obvious analgesic effect.

1 test Material

1.1 test animals

BALB/c mice (SPF grade), Experimental animals technology, Inc. of Wei Tony Hua, Beijing. Production license of experimental animal: SCXK (Jing) 2016-: days 42-62, males 62, animal certification number: no. 1100111911022372.

1.2 drugs and reagents

1.2.1 flavonoid composition of example 3, light earthy yellow powder, dry stored away from the sun, dosed orally by gavage in the test at 60mg/kg, 30mg/kg, 15mg/kg, dissolved in physiological saline, dosed by volume: 0.1ml/10 g.

1.2.2 positive drugs, diclofenac sodium sustained-release tablets (trade name: hibilin), produced by Beijing Nouhua pharmaceutical industry Co., Ltd., national drug standard H10980297, dry storage in dark, light pink triangular film coated tablets, 75 mg/tablet, the dosage in the experiment is: 5 mg/kg. Dissolving with normal saline, and administering via oral gavage.

1.2.3 solvent: sodium chloride injection, specification: 250ml 2.25g, property: liquid, storage conditions: and (4) sealing and preserving and packaging: soft-packed bag, approved document No.: h13023201, production unit: shijiazhuang four drugs Co Ltd

1.2.4 modeling medicine: acetic acid (glacial acetic acid) (analytical purity content 99.5%) with the English name of acetic acid, molecular formula of C2H4O 2; CH3COOH, molecular weight 60.05 (international atomic weight in 1985), density 1.0492 boiling point 117.9 ℃, CAS number 64-19-7 Tianjin Cheng Yuan Chemicals, Inc

1.3 test apparatus

Electronic balance Sartorius, model: BSA 3202S-CW;

electronic balance, METTLER-TOLEDO, model: LE 204;

small animal body weight balance, shanghai yuping scientific instruments ltd, model: YP 1002; a liquid transfer device: eppendorf (eduncut);

IVC mouse rearing cage, Suzhou teaching cage products

High definition imaging System, Canon EOS 5D

2. Experimental methods

2.1 preparation of acetic acid-induced pain model in mice

12 Balb/c mice are purchased, acetic acid (glacial acetic acid) solution is prepared by using physiological saline, the concentration is respectively set to be 0.6%, 0.7%, 0.8% and 1%, 3 mice are respectively injected into each concentration, each mouse is subjected to intraperitoneal injection administration by 0.2ml, a pre-experiment is constructed as a model, the pre-experiment is immediately observed after administration, the frequency of typical body twisting of each mouse within 20 minutes after administration is recorded by using a video camera, and the administration dosage with proper body twisting frequency is found out. The results of preliminary experiments show that 0.7% of the administered dose is the most appropriate dose.

2.2 Experimental method for model drug effect of acetic acid induced pain in mice

50 Balb/c mice were selected, and randomly divided into 7 model negative control groups (given physiological saline) and 7 positive drug control groups (5mg/kg) according to body weight, 9 mice each group, three lavage dose groups (60mg/kg dose group, 30mg/kg dose group, 15mg/kg dose group) of the flavonoid composition of test drug example 3, 9 mice each group, and 7 groups of the flavonoid composition of test drug example 3 by tail vein injection at 30mg/kg dose. The administration frequency of each group is 1 time per day for 3 days, and the control group is administered with physiological saline under the same condition. After 1 hour from the last administration, 0.7% acetic acid (glacial acetic acid) was intraperitoneally injected into each mouse at a dose of 0.2 ml/mouse, and the number of times of the occurrence of typical writhing in each mouse was observed within 20 minutes, and the writhing inhibition rate was calculated.

2.3 photograph of Experimental Process

FIG. 4 shows the administration of the drug by oral gavage, FIG. 5 shows the administration of the drug by tail vein injection, FIG. 6 shows the administration of the drug by intraperitoneal injection, and FIG. 7 shows the writhing of the mouse (manifestations: reaction of belly indent, trunk hind limb stretch, hip lift, etc.).

3. Results of the experiment

3.1 statistical methods

The data is processed by adopting an SPSS13.0 statistical software package, and the data of each group are subjected to addition and subtraction of standard deviation by meanRepresents; the results were compared using an interclass comparison t test, P<0.05 is statistically significant, P<0.01 is that the difference has significant statistical significance

3.2 statistical results

TABLE 2 Effect on acetic acid-induced pain model in mice

Comparison with model control group: p < 0.05; p <0.01

Table 2 the results show: after acetic acid injection, the number of writhing of mice in each administration group is obviously less than that of a model control group, and the writhing number of the mice in each administration group is obviously different from that of the model control group (P <0.05 and P < 0.01).

4 conclusion

In the acetic acid induced mouse pain model drug effect experiment, the flavonoid composition high, medium and low dose groups and the tail vein middle dose group in the example 3 have obvious pain formation effects, and each administration group can greatly reduce the body writhing times of mice and show good dose-effect correlation.

Example 5 efficacy test (arthritis therapeutic effect) of flavonoid composition of example 3

The efficacy of the flavonoid composition of example 3 was evaluated on the basis of the therapeutic effect of the flavonoid composition of example 3 on a freund's complete adjuvant (CFA) arthritis-causing mouse model.

Method, mice are injected with 30 mu L of Freund's complete adjuvant into the skin of the left hind toe at one time to establish an adjuvant arthritis mouse model. The success of the model building is judged by the obvious swelling of the model building side, the contralateral side and the foot sole of the front paw, the affected joint deformity, the inconvenient movement and the obvious reduction of the physical quality of the mouse. After 24h of molding, mice which failed in molding were removed, and all mice which succeeded in molding were randomly divided into 5 groups, which were respectively a model control group (7 mice), a positive drug control group, a flavonoid composition high dose group of example 3, a flavonoid composition medium dose group of example 3, and a flavonoid composition low dose group of example 3, 9 mice per group, and a flavonoid composition tail vein administration group of example 3 (7 mice). Divided into groups and administered on the same day, 1 time per day for 10 days, and the control group is administered with physiological saline for intragastric administration under the same conditions. The animal status was recorded, toe swelling thickness was measured with a vernier caliper, data was recorded, and results were counted using a comparative t-test between groups.

The result shows that the flavonoid composition in the example 3 can obviously reduce the joint swelling degree of mice when being applied to adjuvant arthritis mouse model experiments, and has good dose-effect relationship dependence.

The conclusion is that the flavonoid composition in the example 3 has obvious effect of reducing the joint swelling.

1 test Material

1.1 test animals

BALB/c mice (SPF grade), Schbefu (Beijing) Biotechnology, Inc. Production license of experimental animal: SCXK (Jing) 2016-: days 42-56, 50 males, animal certification number: no. 1103241911021019.

1.2 drugs and reagents

1.2.1 flavonoid composition of example 3, yellow-white powder, dry stored in the dark, dosed orally by gavage at 60mg/kg, 30mg/kg, 15mg/kg in the test, dissolved in physiological saline, dosed by the volume: 0.1ml/10 g.

1.2.2 positive drugs, diclofenac sodium sustained-release tablets (trade name: hibilin), produced by Beijing Nouhua pharmaceutical industry Co., Ltd., national drug standard H10980297, dry storage in dark, light pink triangular film coated tablets, 75 mg/tablet, the dosage in the experiment is: 5 mg/kg. Dissolving with normal saline, and administering via oral gavage.

1.2.3 solvent: sodium chloride injection, specification: 250ml 2.25g, property: liquid, storage conditions: and (4) sealing and preserving and packaging: soft-packed bag, approved document No.: h13023201, production unit: shijiazhuang four drugs Co Ltd

1.3 test apparatus

Electronic balance Sartorius, model: BSA 3202S-CW;

electronic balance, METTLER-TOLEDO, model: LE 204;

small animal body weight balance, shanghai yuping scientific instruments ltd, model: YP 1002; a liquid transfer device: eppendorf (eduncut);

IVC mouse rearing cage, Suzhou teaching cage products

High definition imaging System, Canon EOS 5D

2. Experimental methods

2.1 preparation of adjuvant arthritis mouse model

Mice were injected subcutaneously with 30 μ L of freund's complete adjuvant in one injection in the left hind toe to establish an adjuvant arthritis mouse model. The success of the model making is judged by the obvious swelling of the side, the contralateral side and the foot sole of the front paw of the model making of the mouse, the affected joint deformity, the inconvenient movement and the obvious reduction of the physical quality

2.2 adjuvant arthritis mouse model drug effect experimental method

After random grouping, the test drug was divided into 7 (saline administration), a positive drug control group (5mg/kg), 9 per group, three gavage dose groups (60mg/kg dose group, 30mg/kg dose group, 15mg/kg dose group) of test drug, 9 per group, and 7 tail vein injection groups of test drug at 30mg/kg dose. The administration frequency of each group is 1 time per day for 10 days, and the control group is administered with physiological saline under the same condition. The state of the animals was observed periodically, and the swelling thickness of the toes of the mice was measured with a vernier caliper. The photograph of toe swelling is shown in fig. 8, and the photograph of toe swelling measurement is shown in fig. 9.

3. Results of the experiment

3.1 statistical methods

The data is processed by adopting an SPSS13.0 statistical software package, and the data of each group are subjected to addition and subtraction of standard deviation by meanRepresents; the results were compared using an interclass comparison t test, P<A difference of 0.05 is statistically significant.

3.2 statistical results

TABLE 3 Effect on adjuvant arthritis mouse model

Group of	Dosage (mg/kg)	Animal number (only)	Swelling and proliferation rate%
				Model control group	-	7	13.23±12.81
Drug X high dose	60	9	-2.17±14.59*
				Drug X Medium dose	30	9	0.08.±6.52*
Drug X Medium dose injectionShooting device	30	7	4.31.±10.54
				Drug X Low dose	15	9	6.45±13.34
Positive drug	5	9	6.41±5.57

Comparison with model control group: p <0.05

Table 3 the results show: the dose groups all inhibit the toe swelling of arthritis mice to different degrees, wherein the dose group of drug X and the high dose group of drug X have significant difference compared with the model control group (P < 0.05).

4 conclusion

In an adjuvant arthritis mouse model efficacy experiment, the flavonoid composition high, medium and low dose groups and the medium dose injection group in the embodiment 3 have the effect of inhibiting toe swelling of mice and show good dose-effect correlation, wherein the compound high dose group and the compound medium dose group have significance.

Example 6

To further prove the effect of the snow lotus flavonoid composition, in example 6, based on the compatibility of the components obtained in example 3, four commercially available compounds are adopted to compound the composition in the application, and then a pharmacodynamic test is performed, wherein the test process is consistent with the test processes of examples 4 and 5, and is not repeated here, and the compounding ratio and the test results are shown in the following table:

TABLE 4 composition Components and compounding ratios (by weight)

Components	Rutin	Myristicin	Isoquercitrin	Luteolin-7-o-glucoside
					Compounding ratio	30	1	2	7

TABLE 5 Effect on acetic acid-induced pain model in mice

Comparison with model control group: p < 0.05; p <0.01

TABLE 6 Effect on adjuvant arthritis mouse model

Group of	Dosage (mg/kg)	Animal number (only)	Swelling and proliferation rate%
				Model control group	-	7	14.45±11.80
Drug X high dose	60	9	-1.40±11.37*
				Drug X Medium dose	30	9	-2.45±8.99**
Drug X Medium dose injection	30	7	-2.58±10.96*
				Drug X Low dose	15	9	-1.67±11.83*
Positive drug	5	9	-0.05±7.99*

Comparison with model control group: p <0.05

As can be seen from tables 5 and 6, the flavonoid composition directly compounded by the four compounds still has good analgesic effect and arthritis treatment effect.

Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the disclosure of the present invention, processes, machines, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized according to the present invention. Accordingly, the appended claims are intended to include within their scope such processes, devices, means, methods, or steps.