CN116726117A - Traditional Chinese medicine composition for resisting gouty arthritis - Google Patents
Traditional Chinese medicine composition for resisting gouty arthritis Download PDFInfo
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- CN116726117A CN116726117A CN202310974318.9A CN202310974318A CN116726117A CN 116726117 A CN116726117 A CN 116726117A CN 202310974318 A CN202310974318 A CN 202310974318A CN 116726117 A CN116726117 A CN 116726117A
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
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- A61K36/07—Basidiomycota, e.g. Cryptococcus
- A61K36/076—Poria
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K36/185—Magnoliopsida (dicotyledons)
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- A61K36/51—Gentianaceae (Gentian family)
- A61K36/515—Gentiana
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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)
- A61K36/88—Liliopsida (monocotyledons)
- A61K36/896—Liliaceae (Lily family), e.g. daylily, plantain lily, Hyacinth or narcissus
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K36/18—Magnoliophyta (angiosperms)
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- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
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Abstract
The invention provides a traditional Chinese medicine composition for resisting gouty arthritis. The composition comprises the following raw materials: chicory, poria cocos, smilax glabra, pagodatree flower bud and gentiana macrophylla, and provides a new medicine choice for removing uric acid crystals of joints and further resisting gouty arthritis. The traditional Chinese medicine composition can obviously reduce the crystallization of uric acid in joints, reduce the uric acid level of joint fluid, obviously improve the swelling degree of quail joints in gouty arthritis, reduce the interleukin-1 beta level of inflammatory factors in serum, has no influence on liver and kidney functions, and has the pharmacological action of resisting gouty arthritis.
Description
Technical Field
The invention relates to a traditional Chinese medicine composition, in particular to a novel traditional Chinese medicine composition for removing joint uric acid crystals and resisting gouty arthritis by compatibility of chicory, tuckahoe, glabrous greenbrier rhizome, pagodatree flower bud and gentiana macrophylla.
Background
Gouty arthritis is a progressive, disabling metabolic disease closely related to uric acid crystallization, and also has a tendency to develop and younger worldwide. Currently common drugs for gouty arthritis include non-steroidal anti-inflammatory drugs (NSAIDs), colchicine, and glucocorticoids. Among them, NSAIDs block Prostaglandin (PGE) production mainly by inhibiting Cyclooxygenase (COX) activity, colchicine reduces inflammatory substances production mainly by preventing neutrophil chemotaxis, and glucocorticoids exert anti-inflammatory and analgesic effects mainly by inhibiting arachidonic acid release. Although the medicine has definite action target points and stronger pharmacological activity, the medicine is mostly limited to the intervention of a single pathological stage, and is clinically used together to meet the comprehensive treatment requirement of gout. This not only increases the medication burden on the patient, but also potentially creates a safety risk for multi-drug combinations.
At present, the research on the acute gouty arthritis is temporarily free from radical medicines, mainly anti-inflammatory and analgesic medicines, but most medicines have side effects such as gastrointestinal reactions and the like, and the treatment effect is affected. The traditional operation for treating gouty arthritis is large in general operation traumas, the pathological change degree in the joint cannot be accurately judged, and meanwhile, the traditional operation is very easy to infect, has more adverse reactions and complications and has poor clinical effect when being used for operation treatment. The general consensus of the medical science of Chinese hyperuricemia related diseases indicates that 'gout is the occurrence of urate crystal deposition of hyperuricemia patients, which leads to gouty arthritis, uric acid nephropathy and kidney stones', corrects the limitation recognition that gout is only defined as inflammatory joint diseases, and defines the pathological characteristics of gout of urate deposition. This also indicates that the pathogenesis of gouty arthritis is that uric acid crystals appear at joints, and how to remove the uric acid crystals of joints to resist gouty arthritis is also a problem which needs to be solved in clinic at present.
The traditional Chinese medicine has advantages in preventing and treating advanced diseases related to uric acid metabolic disorders such as gouty arthritis and the like. The composition can remove joint uric acid crystallization, further play a role in resisting gouty arthritis, invigorate spleen, remove dampness and turbid, promote joint circulation and regulate uric acid metabolism; poria and radix Gentianae Marcrophyllae with effects of dispelling pathogenic wind, removing arthralgia, and intervening in gouty inflammation swelling and pain; the pagodatree flower bud has the functions of clearing heat and cooling blood, diminishing inflammation and relieving pain. The whole formula has the effects of strengthening spleen, eliminating dampness, eliminating turbid pathogen, reducing acid, reducing swelling and relieving pain. Modern researches can be reported in the literature of partial medicines for preventing and treating gouty arthritis, however, the research report of the medicine combination for removing the uric acid crystals of joints to prevent gouty arthritis is not yet known at present.
In addition, the chicory, the poria cocos and the pagodatree flower bud in the traditional Chinese medicine compound combination have the characteristics of dual purposes of medicine and food, and are high in safety. Wherein, chicory, tuckahoe and pagodatree flower bud are all varieties collected in the article list which is published by the Ministry of health and is both food and medicine, and rhizoma smilacis glabrae is a variety collected in the article list which can be used for health food.
Disclosure of Invention
The invention aims to provide a traditional Chinese medicine composition for removing joint uric acid crystals and resisting gouty arthritis, and provides a new medicine choice for preventing and treating gouty arthritis.
As one aspect of the invention, the invention provides a traditional Chinese medicine composition for treating gouty arthritis, which is prepared from the following raw materials: chicory, tuckahoe, rhizoma smilacis glabrae, pagodatree flower bud and gentiana macrophylla.
Preferably, the traditional Chinese medicine composition is prepared from the following raw materials: 6-18 parts of chicory, 5-15 parts of poria cocos, 10-60 parts of rhizoma smilacis glabrae, 5-10 parts of pagodatree flower bud and 3-10 parts of gentiana macrophylla.
Further preferably, the traditional Chinese medicine composition is prepared from the following raw materials: 6-10 parts of chicory, 10-15 parts of poria cocos, 20-40 parts of rhizoma smilacis glabrae, 5-10 parts of pagodatree flower bud and 3-10 parts of gentiana macrophylla.
Most preferably, the traditional Chinese medicine composition is prepared from the following raw materials: 9 parts of chicory, 10 parts of poria cocos, 30 parts of smilax glabra, 6 parts of pagodatree flower bud and 9 parts of gentiana macrophylla.
In the above technical scheme, the traditional Chinese medicine composition can be any form formed by or prepared from the above raw materials, and comprises the following components: the raw materials are respectively crushed and then mixed to form a composition; or the composition is obtained by mixing the raw materials and crushing the mixture; or mixing the above raw materials, extracting by conventional extraction method to obtain extract, purifying to obtain effective components, and preparing into conventional oral dosage form by conventional preparation process.
The conventional extraction method comprises soaking extraction, decocting extraction, reflux extraction, percolation extraction, ultrasonic extraction, microwave extraction, etc.; the extraction solvent comprises water or conventional organic solvents such as ethanol, methanol, ethyl acetate, petroleum ether, isopropanol, etc.; the refining and purifying process comprises extraction, column chromatography separation, high performance liquid chromatography separation and the like.
The conventional oral dosage forms comprise tablets, capsules, granules, pills, powder and oral liquid. The preparation of the dosage form requires the addition of common pharmaceutically acceptable auxiliary materials, including: fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, flavoring agents, preservatives, matrices, and the like. The filler comprises: starch, pregelatinized starch, lactose, mannitol, chitin, microcrystalline cellulose, sucrose, and the like; the disintegrating agent comprises: starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, crosslinked polyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, crosslinked sodium carboxymethyl cellulose, and the like; the lubricant comprises: magnesium stearate, sodium lauryl sulfate, talc, silica, and the like; the suspending agent comprises: polyvinylpyrrolidone, microcrystalline cellulose, sucrose, agar, hydroxypropyl methylcellulose, and the like; the binder includes starch slurry, polyvinylpyrrolidone, hydroxypropyl methylcellulose, etc.
The traditional Chinese medicine composition can be added in the form of raw materials, and can also be added in the form of an extract or prepared into particles. Therefore, as another aspect of the present application, the present application further provides a Chinese medicinal composition for treating gouty arthritis, which is prepared from the following raw materials: 6-18 parts of chicory extract, 5-15 parts of poria cocos extract, 10-60 parts of rhizoma smilacis glabrae extract, 5-10 parts of pagodatree flower bud extract and 3-10 parts of gentiana macrophylla extract; wherein the extracts are water extracts of the raw materials respectively or are granules prepared from the water extracts according to a conventional process.
As another aspect of the application, the application provides application of the traditional Chinese medicine composition in preparing a medicine for removing joint uric acid crystals and resisting gouty arthritis.
Pharmacological researches show that the traditional Chinese medicine composition with different concentrations can obviously reduce the crystallization of joint uric acid, reduce the uric acid level of joint fluid, obviously improve the swelling degree of quail joints in gouty arthritis, reduce the interleukin-1 beta level of inflammatory factors in serum, has no influence on liver and kidney functions, and has the pharmacological effect of resisting gouty arthritis.
Compared with the prior art, the invention has the following beneficial effects: the invention provides a new medicine choice for preventing and treating gouty arthritis, the traditional Chinese medicine composition can obviously reduce the crystallization of joint uric acid, experimental researches show that each prescription does not have the effect after the prescription is disassembled, which indicates that the specific compatibility of the raw material medicines of each traditional Chinese medicine composition generates a synergistic effect and has a specific technical effect in the aspect of removing the crystallization of joint uric acid and resisting gouty arthritis. The traditional Chinese medicine composition has the advantages of simple medicine taste, medicine and food containing two medicines, rich medicine sources and high safety, and has better clinical application prospect and economic utilization value.
Drawings
Fig. 1 is a flowchart of the method for extracting the Chinese medicinal composition.
FIG. 2 is a diagram showing uric acid crystals of quail joints of each group in example 2; wherein 2A is normal group joint flushing fluid, 2B is positive control group suspension, 2C is model group joint flushing fluid, 2D is high-dose group joint flushing fluid of chrysanthemum indicum, 2E is medium-dose group joint flushing fluid of chrysanthemum indicum, and 2F is low-dose group joint flushing fluid of chrysanthemum indicum.
FIG. 3 is a graph showing uric acid levels of joint fluid of quails in each group in example 2.
Fig. 4 is a graph showing the swelling degree of the left wrist joint at each time point of each group of quails in example 2.
Fig. 5 is a graph showing the swelling degree of the right wrist joint at each time point of each group of quails in example 2.
Fig. 6 is a graph showing the swelling degree of the left paw joint at each time point of each group of quails in example 2.
Fig. 7 is a graph showing the swelling degree of the joints of the right paw of each group of quails at each time point in example 2.
Fig. 8 is a graph showing the inhibition rate of left wrist joint swelling at each time point of each group of quails in example 2.
Fig. 9 is a graph showing the inhibition rate of swelling of the right wrist joint at each time point of each group of quails in example 2.
Fig. 10 is a graph showing the inhibition rate of swelling of the left paw joint at each time point of each group of quails in example 2.
Fig. 11 is a graph showing the inhibition rate of swelling of the joints of the right paw of the quails at each time point in example 2.
FIG. 12 is a morphology of quail joints of model group in example 2; wherein 12A is a side morphology and 12B and 12C are front morphologies.
Fig. 13 is a morphology of quail paw appearance of each group in example 2, wherein 13A is a model group (left) versus a normal group (right), 13B is a high-dose group (left) versus a model group (right), 13C is a medium-dose group (left) versus a model group (right), and 13D is a low-dose group (left) versus a model group (right).
FIG. 14 is a graph showing IL-1β levels of quail serum for each group in example 2.
FIG. 15 is a chart showing the pathological changes of hematoxylin-eosin staining of joint tissues of quails in each group in example 2.
FIG. 16 is a graph of quail serum AST, ALT activity for each group of example 2.
FIG. 17 is a graph showing the serum CRE and BUN content of quail in example 2.
Detailed Description
The present invention will be further described with reference to the following specific embodiments, which are, however, not limited to the following embodiments.
Example 1 formulation for anti gouty arthritis preferred study
1. The purpose of the experiment is as follows: the pharmacological actions of different traditional Chinese medicine compositions for reducing uric acid and resisting gouty arthritis are explored, and the effective prescription is optimized.
2. Experimental materials
2.1 experiment System
2.1.1 experimental animals: kunming mice, males, body weight 28+ -2 g, total 80.
2.1.2 animal sources: s Bei Fu (beijing) biotechnology limited, experimental unit use license number: SYXK (jing) 2020-0033, license number: SCXK (jing) 2019-0010, experimental animal ethical number: BUCM-4-2023030901-1033 ].
2.1.3 feeding conditions: experimental animal barrier system of Beijing university of Chinese medicine, room temperature 20-25deg.C, relative humidity 35-75%, and standard illumination for 12 h.
2.1.4 feed: and Bei Fu biotechnology limited.
2.2 Experimental drugs and reagents
And (3) a molding agent: MSU, sigma, lot number: BCBQ2650V;
experimental decoction pieces:
positive control drug: colchicine, kunyaku group Co., ltd., 210603-01.
Solvent: deionized water
Reagent:
0.9% sodium chloride injection, shijia four-medicine Co., ltd., lot number: 2208102011;
general tissue fixative, marten schwander biotechnology limited, lot number: g1101-500ML; chloral hydrate, shanghai Minlin Biochemical technology Co., ltd., lot: c12204202
2.3 detection kit
Mouse interleukin-1 beta (IL-I beta) ELISA kit, jiangsu Kort Biotech Co., ltd., lot: 20230240;
creatinine (CRE) assay kit, south kyo institute of biotechnology, lot number: 20230129;
urea Nitrogen (BUN) assay kit, south-kyo institute of biotechnology, lot number: 20230129;
glutamic Pyruvic Transaminase (GPT) kit, nanjing built institute of biological engineering, lot number: 20230111;
glutamic-oxaloacetic transaminase (GOT) kit, south kyo institute of biotechnology, lot number: 20230111.
2.4 laboratory apparatus and devices
HH-2 digital display constant temperature water bath, nangsu Jiujinta Ind. Nanghua instruments manufacturing Co., ltd;
DH5-3 low-speed desk-top centrifuge, beijing era North centrifuge Co., ltd;
MTW120 Lemei electronic balance, shenzhen Mobil electronic Co., ltd;
TLE303E/02 electronic balance, metrele-Torisuo instruments (Shanghai) Co., ltd;
KQ5200E type ultrasonic cleaner, kunshan ultrasonic instruments Co., ltd;
SPECROstar Nano microplate reader, BMG, germany;
reichert Histo STAT paraffin tissue microtome, AO corporation, usa;
olmpus BX53 microscope, DP72CCD camera, olympin bas japan;
RE-501 rotary evaporator, beijing shentai Wei industry instruments and equipment Co., ltd;
D2F-6050ABF type electric heating vacuum drying oven, shanghai Kun Tian laboratory instruments Co., ltd
3. Experimental method
3.1 Experimental design basis
The experimental scheme and the administration dosage are determined by referring to the pharmaceutical research technical guidelines (trial) of each stage of the research of the new traditional Chinese medicine and the earlier-stage research results of the laboratory issued by the national drug administration.
3.2 preparation of test drugs
3.2.1 extract of Juling prescription
The chrysanthemum and poria cocos formula comprises the following components: 9g of chicory, 10g of tuckahoe, 30g of glabrous greenbrier rhizome, 6g of pagodatree flower bud and 9g of gentiana macrophylla;
the preparation method of the chrysanthemum and poria cocos formula extract comprises the following steps: weighing appropriate amount of medicinal materials according to the prescription proportion of the chrysanthemum and poria, adding 12 times of deionized water, soaking for 30min, extracting for 1.5h by adopting a decoction method, collecting the extract, repeating for 3 times, and combining the extracts. Evaporating and concentrating in a rotary evaporator to obtain compound water decoction concentrate containing crude drug content of 1mg/ml, steaming in water bath at 80deg.C until no fluidity, drying in vacuum drying oven at 80deg.C for 7 days, taking out, standing at room temperature, and pulverizing to obtain compound extract powder.
3.2.2 preparation of extract from formula (de-gentiana macrophylla), extract from formula (de-pagodatree flower bud), extract from formula (de-tuckahoe), extract from formula (de-smilax glabra)
Extract of gentiana macrophylla radix prescription: removing radix Gentianae Marcrophyllae in the chrysanthemum and poria cocos prescription based on the chrysanthemum and poria cocos prescription, and extracting other medicinal materials according to the original proportion of the chrysanthemum and poria cocos prescription and the preparation process; extract of the formulation of removing pagodatree flower bud: removing flos Sophorae Immaturus in the chrysanthemum and poria prescription, and extracting other medicinal materials according to the original proportion of the chrysanthemum and poria prescription and the preparation process; poria cocos formula extract: removing Poria in the chrysanthemum and poria cocos formula, and extracting other medicinal materials according to the original proportion of the chrysanthemum and poria cocos formula and the preparation process; rhizoma smilacis glabrae prescription extract: removing rhizoma Smilacis Glabrae in the chrysanthemum and poria prescription, and extracting other medicinal materials according to the original proportion of the chrysanthemum and poria prescription and the preparation process.
3.2.3 colchicine solution
Colchicine is selected as a positive control drug in the experiment. When in use, a proper amount of colchicine is taken and dissolved in deionized water to be prepared into corresponding dosage.
3.3 grouping and dosing amount:
the experiment was performed in 8 groups. The mice are respectively a normal group, a model group, a colchicine group, a chrysanthemum and poria cocos formula group, a gentiana macrophylla group, a pagodatree flower bud group, a poria cocos group and a glabrous greenbrier rhizome group, and 10 Kunming mice are arranged in each group. Each of the administration groups was administered prophylactically and therapeutically by gastric lavage, and the amounts of the crude drugs in each group were converted in equal proportions to give the doses shown in table 1.
Table 1 design of dosing for mice of each group during the experiment
| Group of | Medicament | Dosage for administration | Stomach filling volume (ml/100 g) |
| Normal group | —— | —— | 1 |
| Model group | —— | —— | 1 |
| Colchicine group | Colchicine | 1mg/kg | 1 |
| Chrysanthemum and poria cocos square group | Chrysanthemum and poria cocos prescription | 12.86g/kg | 1 |
| Gentiana macrophylla removing group | Gentiana macrophylla removing prescription | 11.05g/kg | 1 |
| Sophora flower bud removing group | Sophora flower bud removing prescription | 11.65g/kg | 1 |
| Poria removing group | Poria removing prescription | 10.85g/kg | 1 |
| Rhizoma smilacis glabrae removing group | Rhizoma smilacis glabrae removing prescription | 6.83g/kg | 1 |
3.4 method of administration:
route of administration: gastric administration (consistent with the human clinical intended route of administration).
Dosing volume: 1ml/100g.
Dosing time: the stomach was irrigated 1 time per body weight in the morning each day.
Drug administration period: the administration was continued for 7 days.
3.5 preparation of test drug: at present, a certain amount of the tested medicine is weighed and added into quantitative aqueous solution for standby after ultrasonic mixing.
3.6 preparation and administration of the Molding agent: at present, a certain amount of MSU crystals are taken and irradiated with proper physiological saline for 6 hours under ultraviolet light, and then MSU is dissolved in sterile physiological saline to prepare MSU suspension with the concentration of 50 mg/ml.
3.7 molding method: 1h after the 5d administration of the experiment, 25. Mu.l of MSU suspension was injected into the ankle cavity of the mice in the model group and each administration group, and 25. Mu.l of 0.9% sterile physiological saline was injected into the ankle cavity of the mice in the normal group, using a 1ml sterile insulin syringe. Ankle injections were normalized to the success of the injection with the bulge on the opposite side of the articular cavity.
3.8 sample collection and handling: mice were measured for foot thickness, ankle circumference before (0 h) and 4h, 8h, 12h, 24h, 48h after MSU injection, and scored for inflammation. The material was taken at experiment 7 d. Collecting eyeball blood, separating serum for detection of biochemical indexes, and preserving at-20deg.C; taking joint tissue, adding 4% paraformaldehyde for fixation, performing decalcification treatment with 10% formic acid solution after fixation for 48 hours, embedding paraffin, slicing (thickness of 4 μm), spreading, and fishing out slices overnight at 37 ℃.
3.9 detection index
3.9.1 general status index: ankle swelling, foot thickness swelling, and inflammation score.
1) Ankle/foot thickness swelling: the circumference and foot thickness of the ankle joint of the mice tested before (0 h) and after (4 h, 8h, 12h, 24h and 48 h) MSU injection were measured with a vernier caliper and repeated three times. The swelling degree at each time point was calculated according to formula (1).
2) Inflammation scoring
The inflammation index scoring criteria were as follows: 0 minutes, normal; 2 minutes, the skin erythema of the joint to be tested, mild swelling and visible bony marks; 4, the tested joint is obviously red and swollen, the osseous mark disappears, but the swelling is limited to the joint part; and 6 minutes, the limbs except the joints to be tested are swelled.
3.9.2 inflammation-related index: interleukin-1 beta (IL-1 beta) levels, and the kit detects serum IL-1 beta levels.
3.9.3 renal function related indicators: serum creatinine (SCre), urea nitrogen (BUN) levels, and the kit detects serum Cre, BUN levels.
3.9.4 liver function related index, serum glutamic pyruvic transaminase (ALT) and glutamic oxaloacetic transaminase (AST), and the kit detects serum ALT and AST levels.
4 statistics and analysis of results
Statistics using SPSS27.0 softwareAnalysis, data employed mean ± standard deviationAs shown, the data comparison between the groups is based on whether each group is normal or not, a one-way anova or a Kruskal-Wallis H nonparametric test is selected. The comparison between normal distribution data sets is based on whether the variance is uniform or not, and LSD test or Dunnett's T test is selected; the non-normal distribution data is selected from the Kruskal-Wallis H test average rank multiple comparison, and P is used<A difference of 0.05 is statistically significant.
5. Results
5.1 General state index
5.1.1 ankle swelling degree
The ankle swelling degree was calculated in this experiment by the formula (1).
Model mice had significantly increased ankle swelling levels (P < 0.01) at each observation time point compared to the normal group.
Compared with the model group, the ankle joint swelling degree of the colchicine group mice is obviously reduced (P < 0.05) 24h and 48h after molding, and no obvious difference exists between the rest time points; the ankle joint swelling degree of the chrysanthemum and poria cocos compound mice is obviously reduced (P is less than 0.05) 24h and 48h after molding, and no obvious difference exists in other time points; the ankle joint swelling degree of the gentiana macrophylla group-removed mice is obviously reduced (P is less than 0.05) 24h and 48h after molding, and no obvious difference exists in the rest time points; the ankle joint swelling degree of the mice without the pagodatree flower bud group is obviously reduced (P is less than 0.05) 24 hours after molding, and the rest time points have no obvious difference; the ankle joint swelling degree of the mice with the poria cocos removed and the smilax glabra removed has no obvious difference at all observation points.
Compared with the chrysanthemum and poria cocos formula group, the ankle joint swelling degree of the rat with gentiana macrophylla removed group has no obvious difference at all observation points; the ankle joint swelling degree of the mice without the pagodatree flower bud group is obviously increased (P is less than 0.05) for 4h and 48h after molding, and the rest time points have no obvious difference; the ankle joint swelling degree of the mice with the Poria groups is obviously increased (P < 0.05) 48h after molding, and the rest time points have no obvious difference; the ankle joint swelling degree of the glabrous greenbrier rhizome group-removed mice is obviously increased (P < 0.05) 48 hours after molding, and no obvious difference exists between the rest time points. See table 2 below.
Table 2 sets ofAnkle swelling (%,n＝10)
note that: p <0.05, P compared to normal group<0.01; compared with the model group, the #P is less than 0.05; compared with the chrysanthemum and poria cocos prescription group, & P＜0.05。
5.1.2 foot thickness swelling degree
The experiment calculates the foot thickness swelling degree with formula (2).
The model group mice had significantly elevated foot thickness swelling levels (P <0.05 or P < 0.01) at each observation time point compared to the normal group.
Compared with the model group, the colchicine group mice have no significant difference in foot thickness swelling degree at each observation time point; the foot thickness swelling degree of the chrysanthemum and poria cocos compound mice is obviously reduced (P is smaller than 0.05) after molding for 4 hours, and no obvious difference exists at other time points; the foot thickness swelling degree of the pagodatree flower bud group-removed mice is obviously increased (P is less than 0.01) after 12 hours of molding, and no obvious difference exists at other time points; the foot thickness swelling degree of the mice with gentiana macrophylla group, glabrous greenbrier rhizome group and glabrous greenbrier rhizome group is not significantly different at each observation time point.
Compared with the chrysanthemum and poria cocos formula group, the gentiana macrophylla removing group has no significant difference at all observation time points; the pagodatree flower bud group is obviously increased (P is less than 0.05) 48 hours after molding, and the rest time points have no obvious difference; the tuckahoe group is obviously increased 8h and 48h (P is less than 0.05 or P is less than 0.01) after molding, and no obvious difference exists in other time points; the group of smilax glabra removed has no significant difference at each observation time point. See table 3.
Table 3 mice of each group had a thick enough swelling (%,n＝10)
note that: p < 0.05, P compared to normal group<0.01; compared with the model group, the # P is less than 0.05, and the # P is less than 0.01; compared with the chrysanthemum and poria cocos prescription group, & P＜0.05， && P＜0.01。
5.1.3 mice inflammation score
The model group mice had significantly elevated inflammation scores (P < 0.01) at each observation time point compared to the normal group.
Compared with the model group, the inflammation score of the colchicine group mice is obviously reduced (P is less than 0.05) after 12 hours of molding, and no obvious difference exists at other time points; the inflammatory scores of the mice in the chrysanthemum-poria prescription group are obviously reduced (P is less than 0.05 or P is less than 0.01) for 4h and 12h after molding, and no obvious difference exists in other time points; the mice with the gentiana macrophylla group, the pagodatree flower bud group, the poria cocos group and the smilax glabra group have no significant difference in inflammation scores at all observation time points.
Compared with the chrysanthemum and poria prescription group, the inflammation scores of the mice without gentiana macrophylla group have no significant difference at all observation time points; the inflammation scores of the mice without the pagodatree flower bud group are obviously increased (P is less than 0.05) after 12 hours of molding, and no obvious difference exists in other time points; the inflammation scores of the mice with the Poria cocos removed are obviously increased (P is less than 0.05) after 12 hours of molding, and no obvious difference exists in other time points; the inflammation scores of the smilax glabra group mice are obviously increased (P is less than 0.05) 12h after molding, and no obvious difference exists between the other time points. See table 4.
TABLE 4 inflammation score for each time point for each group of micen＝10)
Note that: p <, compared to the normal group0.05，**P<0.01; compared with the model group, the # P is less than 0.05, and the # P is less than 0.01; compared with the chrysanthemum and poria cocos prescription group, & P＜0.05， && P＜0.01。
5.2 mouse serum interleukin-1 beta (IL-1 beta) levels
The serum IL-1β levels were significantly elevated (P < 0.01) in the mice of the model group compared to the normal group.
The serum IL-1β levels were significantly reduced (P < 0.01 or P < 0.05) in mice from each of the dosing groups compared to the model group.
Compared with the chrysanthemum and poria prescription group, the serum IL-1 beta level of the mice with the gentiana macrophylla group, the poria cocos group and the smilax glabra group is obviously increased (P is less than 0.01 or P is less than 0.05), and the serum IL-1 beta level of the mice with the pagodatree flower bud group is reduced in trend but has no obvious difference. See table 5.
Table 5 serum IL-1 beta content (ng/L,)
note that: p < 0.05, P compared to normal group<0.01; compared with the model group, the # P is less than 0.05, and the # P is less than 0.01; compared with the chrysanthemum and poria cocos prescription group, & P＜0.05。
5.3 pathological changes in ankle joints in mice
The pathological section results show that the ankle joint structure of the normal group mice is clear, the joint surface is smooth, and the synovial tissue has no obvious pathological change; the model group mice show remarkable hyperplasia of ankle synovial tissue accompanied by massive inflammatory cell infiltration; the ankle joint structure of the colchicine group mice is clear, and the synovial tissue has no obvious pathological change; the ankle joint structure of the chrysanthemum-poria cocos formula mice is clear, the joint surface is smooth, and the synovium tissue has no obvious pathological change; the ankle joint of the rat with the gentiana macrophylla group has clear structure, the joint surface is smooth, and a small amount of hyperplasia of synovial tissue occurs; the ankle joint structure of the mice with the pagodatree flower bud group removed is clear, and the synovial tissue has little hyperplasia; the mouse ankle synovial tissue from the Poria group shows a small amount of hyperplasia with a small amount of inflammatory cell infiltration; the mouse ankle synovial tissue from the glabrous greenbrier rhizome group showed a small amount of hyperplasia with a small amount of inflammatory cell infiltration.
5.4 serum creatinine (Cre), urea Nitrogen (BUN) levels
Compared with the normal group, the serum Cre and BUN levels of the model group have no significant difference; there was no significant difference in Cre, BUN levels in serum from each dosing group.
5.5 glutamic-pyruvic transaminase (ALT) and glutamic-oxaloacetic transaminase (AST) expression levels
Compared with a normal group, the activity of serum ALT and AST of mice in a model group is not obviously different 48 hours after the model is built; the mouse serum ALT and AST activities of colchicine group, chrysanthemum and poria formula group, gentiana macrophylla group, pagodatree flower bud group, poria cocos group and smilax glabra group have no significant difference.
Discussion 6
The chrysanthemum and poria cocos compound prescription is Zhang Bing professor crystallization of a drug experience prescription for treating arthralgia, acute and chronic gouty arthritis, and is combined with the study of the subject group for more than 20 years, and the whole prescription has the effects of dredging collaterals and relieving pain, clearing heat and resolving phlegm, and strengthening spleen and eliminating dampness. The aim of this study was to observe and compare the effect of the Juling prescription and its prescription on the mouse model of acute gouty arthritis.
In the overall animal characterization, the experiment uses the ankle joint swelling degree and the foot thickness swelling degree of the mice as main evaluation indexes and uses the inflammation score as an auxiliary evaluation index.
At each observation time point of the experiment, ankle swelling, foot thickness swelling, inflammation score, serum IL-1β levels were all significantly elevated (P < 0.05 or P < 0.01) in the model group mice compared to the normal group.
Serum IL-1β levels were significantly reduced (P < 0.05 or P < 0.01) in mice from each of the dosing groups compared to the model group; the ankle joint swelling degree of colchicine mice is obviously reduced (P is less than 0.05) 24h and 48h after molding, the inflammation score is obviously reduced (P is less than 0.05) 12h after molding, and all indexes of the rest time points have no obvious difference; the ankle joint swelling degree of the chrysanthemum-poria cocos square group mice is obviously reduced (P is smaller than 0.05) 24h and 48h after molding, the foot thickness swelling degree is obviously reduced (P is smaller than 0.05) 4h after molding, the inflammation score is obviously reduced (P is smaller than 0.05 or P is smaller than 0.01) 4h and 12h after molding, and all indexes of the rest time points have no obvious difference; the ankle joint swelling of the mice with gentiana macrophylla groups is obviously reduced (P is less than 0.05) 24h and 48h after molding, and all indexes of the rest time points have no obvious difference; the ankle joint swelling degree of the mice without the pagodatree flower bud group is obviously reduced (P is less than 0.05) 24 hours after molding, the foot thickness swelling degree is obviously increased (P is less than 0.05) 12 hours after molding, and all indexes of the rest time points have no obvious difference; the other indexes of the mice with the poria cocos removed and the smilax glabra removed have no obvious difference at all observation time points of the experiment.
Compared with the chrysanthemum and poria prescription, the regulating and controlling effects of the gentiana macrophylla removing group, the poria cocos removing group and the smilax glabra removing group on the serum IL-1 beta level of mice are reduced (P is less than 0.05 or P is less than 0.01), and no obvious difference exists between the sophora flower bud removing groups; the other indexes of the gentiana macrophylla-removed mice have no obvious difference at all observation time points of the experiment. Ankle joint swelling of mice with pagodatree flower bud groups is obviously higher than Yu Ju poria square group (P < 0.05) after molding for 4 hours and 48 hours, foot thickness swelling degree is obviously higher than Yu Ju poria square group (P < 0.05) after molding for 48 hours, and inflammation score is obviously higher than Yu Ju poria square group (P < 0.05) after molding for 12 hours; the ankle joint swelling of the mice with the tuckahoe group is obviously higher than Yu Ju tuckahoe square group (P is less than 0.05) 48 hours after molding, the foot thickness swelling degree is obviously higher than Yu Ju tuckahoe square group (P is less than 0.05 or P is less than 0.01) 8 hours after molding, and the inflammation score is obviously higher than Yu Ju tuckahoe square group (P is less than 0.05) 12 hours after molding; the ankle joint of the glabrous greenbrier rhizome group removed mice is obviously higher than Yu Ju tuckahoe square group (P is less than 0.05) after 48 hours of molding; the inflammation score is significantly higher than Yu Ju poria cocos formula group (P is less than 0.05) in 12 hours after molding, and all indexes of the rest time points have no significant difference;
compared with the normal group, the model group mice show remarkable hyperplasia of ankle synovial tissue accompanied by massive inflammatory cell infiltration. Compared with the model group, the colchicine group and the chrysanthemum and poria square group have clear ankle joint structure, and the synovium tissue has no obvious pathological change; the ankle joint structure of the mice with the gentiana macrophylla group, the pagodatree flower bud group, the poria cocos group and the smilax glabra group is clear, and the synovium tissue has a small amount of hyperplasia.
During the experiment period, the CRE, BUN, ALT, AST level in the serum of each group of experimental animals has no significant difference, which indicates that the traditional Chinese medicine test object has no obvious influence on the liver and kidney functions of mice.
The above results indicate that: colchicine is used as a clinically common anti-gout drug, and the colchicine is used as a positive drug to exert an anti-inflammatory effect in the experiment, so that the establishment of the acute gouty arthritis mouse model is proved to be successful. The chrysanthemum and poria cocos formula plays a good role in inhibiting the ankle joint swelling degree and the foot thickness swelling degree of the mice, and each formula can also inhibit the ankle joint swelling degree and the foot thickness swelling degree of the mice. The IL-1 beta results in serum prove that the chrysanthemum indicum prescription and all the prescription have better effect of resisting acute gouty arthritis, the anti-gout effect of the chrysanthemum and poria cocos formula is obviously better than that of each formula.
In conclusion, the chrysanthemum and poria cocos formula and all the disassembly formulas thereof can improve the acute gouty arthritis induced by MSU of mice, and the chrysanthemum and poria cocos formula has the best curative effect, so that the result that the traditional Chinese medicine compound is the synergistic effect of all the components is reflected.
EXAMPLE 2 study of the efficacy of Chinese medicinal composition against gouty arthritis
1. The purpose of the experiment is as follows: based on the embodiment 1, the pharmacological action of the traditional Chinese medicine composition for resisting gouty arthritis is explored, and a new medicine choice is provided for preventing and treating gouty arthritis.
2. Experimental method
2.1 grouping of animals
72 quails of 40-day-old Di-Fa-ke quails are selected and fed adaptively for 3d, and are randomly divided into 6 groups according to the mass, namely a normal group, a model group, a benzbromarone group (20.00 mg/kg), a high-dose group of the extract of the inula japonica (corresponding to 10g/kg of crude drug), a medium-dose group of the extract of the inula japonica (corresponding to 7.5g/kg of crude drug) and a low-dose group of the extract of the inula japonica (corresponding to 3.75g/kg of crude drug), wherein each group is 12.
2.2 modeling
Experiments 1-50 d, the model group and each administration group are given high purine feed (basal feed: yeast extract powder is 8:2); experiments 51-83 d, model groups and administration groups were given high protein and high calcium feed (basal feed: yeast extract powder: bone meal 5:2:3) in combination with 10% fructose water (limit of 15 mL/d). During the experiment, the normal group was given sufficient basal feed, clear water. Controlling the room temperature of the animal house to be 20-25 ℃ and the humidity to be 40-60%.
2.3 preparation method of composition (extract of Chrysanthemum Indicum)
The formula comprises the following components: 9g of chicory, 10g of tuckahoe, 30g of glabrous greenbrier rhizome, 6g of pagodatree flower bud and 9g of gentiana macrophylla;
the preparation method comprises the following steps: adding deionized water with a feed liquid ratio of 12 times, soaking for 30min, extracting for 1.5 hr by decocting method, collecting extractive solution, repeating for 3 times, and mixing extractive solutions. Evaporating and concentrating to concentration of 10g/kg, 7.5g/kg and 3.75g/kg in a rotary evaporator, and storing at 4deg.C for use. The specific operation flow is shown in figure 1.
2.4 experimental dosing
The high, medium and low dosage groups of the composition have the administration concentrations of 10g/kg, 7.5g/kg and 3.75g/kg respectively. The experiment selects benzbromarone as a uric acid-reducing positive control drug. When in use, a proper amount of benzbromarone is dissolved in deionized water to be prepared into corresponding dosage. The administration concentration of the benzbromarone is 20mg/kg. The administration mode is intragastric administration, and the normal group and the model group are filled with the ultrapure water with the same volume of the intragastric administration every day.
2.5 materials selection
Taking 1.0-1.5 mL of the jugular vein of each group of quails, and taking the blood without taking water for 12 hours after fasting. The serum sample is centrifugated for 10min at normal temperature of 3500r/min, and the supernatant is used for detecting serum biochemical indexes. And respectively flushing the left wrist joint cavities of the quails by adopting 0.2mL of physiological saline, coating the obtained wrist joint flushing liquid on a glass slide, and observing uric acid crystals in the wrist joint flushing liquid under a polarized light microscope. And collecting joint tissues of quails of each group, performing HE staining, and observing inflammatory infiltration.
2.6 Joint swelling index Collection
Experiments 50d, 60d, 70d and 80d, the circumferences of the left and right wrist joints and the left and right claw joints of each group of quails were measured by using a flexible ruler. Taking the measurement result of the experiment 50d as the circumference of the wrist and jaw joint at the beginning of the experiment, calculating the swelling degree of the wrist and jaw joint of each group of quail of the experiment 60d, 70d and 80d according to the formula (1), and calculating the swelling inhibition rate of the wrist and jaw joint of each group of quail of the experiment 60d, 70d and 80d according to the formula (2).
2.7 Joint flushing fluid Collection and treatment
In experiment 83d, the left wrist joint cavities of all groups of quails are respectively washed by adopting 0.2mL of physiological saline, the obtained wrist joint washing liquid is coated on a glass slide, and uric acid crystals in the wrist joint washing liquid are observed under a polarized light microscope.
2.8 joint histopathological observations
Paraffin sections were prepared from 10% formalin-fixed joint tissue and stained with hematoxylin eosin. An inverted microscope was used to collect images for histopathological observation.
3. Data analysis
Statistical analysis was performed using GraphPad prism7.0 software, and data were taken as mean ± standard deviation The data comparison between groups is expressed by selecting one-factor analysis of variance or Kruskal-Wallis anecdotal test according to normal and variance of each group, and the comparison between groups is based on variance of each group and is selected by Dunnett-t test or Dunnett's T3 test according to variance of each group, and P is calculated by<A difference of 0.05 is statistically significant.
4. Main instruments and equipment (see Table 6 below)
TABLE 6 Main instruments and apparatus
| Name of the name | Model number | Manufacturer' s |
| Electromagnetic oven | HY-221 | Guangdong Hemisphere Industry Group Co. |
| Rotary evaporator | RE-501 | Beijing shentai equipment company |
| Circulating water type multipurpose vacuum pump | SHB-III | Beijing shentai equipment company |
| Electronic balance | TLE303E | Mettler Toledo instruments (Shanghai) Co.,Ltd. |
| Desk type centrifugal machine | DT5-3 type | Beijing era North centrifuge Co., ltd |
| Freezing embedding machine | KH-BL | HUBEI XIAOGAN KUOHAI MEDICAL TECHNOLOGY Co.,Ltd. |
| Paraffin tissue slicer | Reichert Histo STAT | AO Co Ltd |
| Microscope | Olmpus BX53 | Orinbas of JapanCompany (Corp) |
| Camera with camera body | DP72CCD | Orinbas of Japan |
| Electrothermal blowing drying box | 101-1AB type | TIANJIN TAISITE INSTRUMENT Co.,Ltd. |
| Enzyme label instrument | sunrise | TeCAN company Switzerland |
| Water bath kettle | HH-1 high-end type | Jintan city and west sense, laboratory instrumentation factory |
5. Main reagents and drugs (see Table 7 below)
TABLE 7 Main reagents and drugs
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6. Results
6.1 Effect of Chinese medicinal composition on Joint uric acid crystallization
A 0.375% sodium urate suspension was prepared and applied to a glass slide as a positive control, and the positive control and the quail wrist joint washes of each group were observed under a 200-fold polarized light microscope. The results are shown in FIG. 2.
Under the black visual field, needle-shaped urate crystals are not seen in the normal group. In contrast to the normal group, the positive control suspensions were seen as bright, blue or yellow urate crystals, mostly clustered into "spherical" clusters, with large needles scattered around.
Compared with the normal group, the model group is mostly scattered, bright, blue or yellow urate crystals, the shape of the model group is small needle-shaped with big head and small tail or sharp two ends, the model group is occasionally aggregated into a feather-shaped crystal cluster, and the length of a single needle crystal is about 1-20 mu m.
In contrast to the model group, the high (10 g/kg), medium (7.5 g/kg), low (3.75 g/kg) dose groups of the chrysanthemum indicum showed little scattering distribution of needle-like urate crystals, and little bundles of needle-like crystals arranged approximately in parallel, or bundles of needle-like crystals crossed and overlapped to form an X-shaped crystal bundle, and no aggregation of the crystal clusters was found.
6.2 uric acid levels in joint fluid
The joint cavities of quails were rinsed with 0.2ml of physiological saline, ankle fluids of each group were collected, and uric acid levels of joint fluids of each group were measured. The results are shown in FIG. 3. The joint fluid uric acid levels of model group quails were significantly elevated compared to normal group (P < 0.01).
Compared with the model group, the high (10 g/kg) and medium (7.5 g/kg) dosage group of the poria extract has significantly reduced uric acid level (P < 0.05) of quail joint fluid.
6.3 joint swelling degree
6.3.1 left wrist joint swelling degree
The left wrist joint swelling degree was calculated in this experiment by formula (1). The results are shown in Table 8 and FIG. 4. Experiments 60d, 70d, 80d showed a significant increase in swelling of the left wrist joint of the model group quail (P < 0.01) compared to the normal group.
Compared with the model group, the swelling degree of the left wrist joint of the quail is obviously reduced (P <0.05 or P < 0.01) in the group with high (10 g/kg), medium (7.5 g/kg) and low (3.75 g/kg) dosage of the poria extract in the experiment 70 d.
Table 8 the left wrist joint swelling degree (%, n=12, )/>
And (3) injection: p <0.01 compared to normal group; compared to the model group, #p <0.05, #p <0.01.
6.3.2 degree of swelling of the right wrist
The present experiment calculates the degree of swelling of the right wrist joint with equation (1). The results are shown in Table 9 and FIG. 5. Experiments 60d, 70d, 80d, model group quail right wrist joint swelling increased significantly compared to normal group (P < 0.01).
Compared with the model group, in the experiment 70d, the swelling degree of the right wrist joint of the quail in the dosage group (7.5 g/kg) in the chrysanthemum indicum extract is obviously reduced (P < 0.05), and the swelling degree of the right wrist joint of the quail in the dosage group (3.75 g/kg) in the chrysanthemum indicum extract is high (10 g/kg) and the swelling degree of the right wrist joint of the quail in the dosage group is reduced (P=0.059 and P=0.052); experiment 80d, the swelling degree of the right wrist joint of quail in high (10 g/kg) dose group of the chrysanthemum indicum extract has a reduced tendency (P=0.092).
Table 9 the swelling degree (%, n=12,)
note that: p <0.01 compared to normal group; compared to the model group, #p <0.05.
6.3.3 left paw joint swelling degree
The present experiment calculates the left paw joint swelling degree with formula (1). See table 10, fig. 6. Compared with the normal group, the swelling degree of the joints of the left paw of the quail in the model group is increased in the experiment 60d (P=0.069); experiments 70d, 80d, model group quails had significantly increased left paw joint swelling (P <0.05 or P < 0.01).
Compared with the model group, the swelling degree of the left paw joint of the quail in the experiment 60d, the benzbromarone group and the chrysanthemum indicum extract low (3.75 g/kg) dosage group has a reduced tendency (P=0.076, P=0.064); experiment 70d, the swelling degree of the joints of the left foot paw of quail in a benzbromarone group and a chrysanthemum indicum (7.5 g/kg) dosage group is obviously reduced (P < 0.05); experiment 80d, benzbromarone group, juling high (10 g/kg), medium (7.5 g/kg), low (3.75 g/kg) dose group left paw joint swelling degree was significantly reduced (P <0.05 or P < 0.01).
Table 10 the swelling degree (%, n=12,)/>
note that: p <0.05, < P <0.01 compared to normal group; compared to the model group, #p <0.05, #p <0.01.
6.3.4 degree of swelling of the right paw joint
The present experiment calculates the degree of swelling of the right paw joint with formula (1). The results are shown in Table 11 and FIG. 7. Experiment 80d, model group quail right paw joint swelling was significantly increased (P < 0.01) compared to normal group.
Compared with the model group, the swelling degree of the joints of the right paw of the quail in the experiment 80d, the tribenuron-methyl group, the chrysanthemum indicum high (10 g/kg), the middle (7.5 g/kg) and the low (3.75 g/kg) dose group is obviously reduced (P < 0.01).
Table 11 the swelling degree (%, n=12,)
note that: p compared to normal group <0.01; in contrast to the set of models, ## P<0.01。
6.3.5 inhibition rate of left wrist joint swelling
The experiment calculates the left wrist joint swelling inhibition rate by the formula (2). The results are shown in Table 12 and FIG. 8. In experiment 60d, the highest inhibition rate of the swelling degree of the left wrist joint of the quail in the dosage group (7.5 g/kg) in the poria cocos extract is 65.77%, and the dosage groups of low (3.75 g/kg) and high (10 g/kg) are sequentially reduced, but are higher than those of the tribenuron-methyl group.
In experiment 70d, the inhibition rate of the swelling degree of the left wrist joint of the quail in the dosage group of 7.5g/kg in the extract of the chrysanthemum indicum is 77.95 percent at the highest, the dosages of the extract of the chrysanthemum indicum are equivalent to each other in high (10 g/kg) and low (3.75 g/kg) dosages, but are higher than that of the tribenuron-methyl group,
experiment 80d, the highest inhibition rate of swelling degree of left wrist joint of quail in dosage group (7.5 g/kg) in the extract of the chrysanthemum indicum is 24.61%, and the dosage group (7.5 g/kg) and the dosage group (3.75 g/kg) are weaker in the second time of the tribromoron group.
Table 12 inhibition of left wrist joint swelling (%, n=12) for each time point of quail administration group
6.3.6 inhibition rate of swelling of Right wrist joint
The present experiment calculates the right wrist joint swelling inhibition rate by formula (2). The results are shown in Table 8 and FIG. 9. Experiment 60d, the inhibition rate of the swelling degree of the right wrist joint of the quail in the high (10 g/kg), medium (7.5 g/kg) and low (3.75 g/kg) dosage groups is higher than that of the phenylbromarone group, and the highest inhibition rate of the swelling degree of the left wrist joint of the quail in the high (10 g/kg) dosage group is 28.86%.
In experiment 70d, the inhibition rate of the swelling degree of the right wrist joint of the quail in the high (10 g/kg), medium (7.5 g/kg) and low (3.75 g/kg) dosage groups is higher than that of the phenylbromarone group, and the highest inhibition rate of the swelling degree of the left wrist joint of the quail in the medium (7.5 g/kg) dosage group is 57.87%.
In experiment 80d, the inhibition rate of the swelling degree of the right wrist joint of the quail in high (10 g/kg), medium (7.5 g/kg) and low (3.75 g/kg) dosage groups is higher than that of the phenylbromarone group, and the inhibition rate of the swelling degree of the left wrist joint of the quail in high (10 g/kg) dosage groups is 28.40% at most.
Table 8 inhibition of swelling of the right wrist at each time point (%, n=12) for each group of quails
6.3.7 inhibition rate of left paw joint swelling
The experiment calculates the inhibition rate of left paw joint swelling by the formula (2). The results are shown in Table 9 and FIG. 10. Experiment 60d, the highest inhibition rate of the swelling degree of the left paw joint of the quail in the low-dose (3.75 g/kg) group of the chrysanthemum indicum extract is 36.73 percent higher than that of the tribenuron-methyl group.
Experiment 70d, the highest inhibition rate of the swelling degree of the left paw joint of the quail in the dosage group (7.5 g/kg) of the chrysanthemum indicum extract is 34.23% higher than that of the benzbromarone group.
Experiment 80d, the inhibition rate of the swelling degree of the left paw joint of the quail in the dosage group (7.5 g/kg) in the chrysanthemum indicum extract is equal to that of the benzbromarone group, and the inhibition rate is 38.66% and 39.78% respectively.
Table 9 inhibition of left paw joint swelling (%, n=12) for each time point of quail group
6.3.8 inhibition rate of swelling of the joints of the right foot paw
The experiment calculates the inhibition rate of the swelling of the joints of the right paw by the formula (2). The results are shown in Table 10 and FIG. 11. Experiment 60d, the highest inhibition rate of the swelling degree of the right foot paw joint of quail in a dosage group (7.5 g/kg) in the poria cocos extract is 32.59%, the high (10 g/kg) dosage group of the poria cocos extract is equivalent to the tribenuron-methyl, the swelling degree of the low (3.75 g/kg) dosage group is the lowest, and the dosage groups are 28.81% and 28.97% respectively.
Experiment 70d, the highest inhibition rate of the swelling degree of the right foot paw joint of the quail in the dosage group (7.5 g/kg) of the chrysanthemum indicum extract is 32.82 percent, which is higher than that of the benzbromarone group, and the dosage group with high (10 g/kg) of the chrysanthemum indicum extract is equivalent to the dosage group with low (3.75 g/kg) of the chrysanthemum indicum extract, which are 25.52 percent and 27.63 percent respectively.
Experiment 80d, the low (3.75 g/kg) dosage group of the poria extract is equivalent to that of the benzbromarone group, namely 50.19 percent and 52.03 percent, and the medium and low dosage groups are lower.
Table 10 inhibition of right paw joint swelling (%, n=12) for each time point of quail group
6.4 appearance and shape changes of wrist joints and feet paws
During the experiment, the wrist joint and the paw of the quail are normal, and the movement is flexible. The model group had a different degree of swelling and local erythema at the wrist joint compared to the normal group; obvious swelling or a plurality of swelling nodules (tophus) with different sizes can be seen at the joints of the feet, and the swelling of the feet of the individual quails is silted, purulent and crumbled, as shown in fig. 12.
Compared with the model group, the high, medium and low dosage group quail of the chrysanthemum indicum extract has slight swelling of the wrist joint and the paw joint, as shown in figure 13.
6.5 inflammatory factor expression levels
The results are shown in FIG. 14. Compared with the normal group, the serum IL-1 beta level of the quail in the model group is obviously increased in the experiment 83 d; compared with the model group, the serum IL-1 beta level of the quail in the high, medium and low dose group of the chrysanthemum indicum in the experiment 83d is obviously reduced (P < 0.01).
6.6 pathological observation of joint tissue
The results are shown in FIG. 15. The HE staining of the quail wrist joint tissue can be seen, the normal quail wrist joint tissue structure is clear, the joint surface is smooth and flat, and the synovial cells are loose in arrangement and normal in shape; the model group wrist joint tissue can be seen as synovial fibrous tissue hyperplasia, pannus formation, and massive inflammatory cell infiltration. Compared with the model group, the high-dose group of the chrysanthemum and the middle-dose group of the chrysanthemum and the poria have obviously reduced inflammatory cell infiltration and reduced synovial fibrous tissue proliferation.
6.7 liver and kidney function index
6.7.1 serum AST, ALT activity
The results are shown in Table 11 and FIG. 16. Compared with the normal group, the experimental group 83d has no obvious difference in quail serum AST and ALT activities.
Compared with the model group, in the experiment 83d, the quail serum AST activity of the high-dose (10 g/kg) of the poria extract has a reduced trend but no significant difference (P=0.052), and the quail serum AST and ALT activity of the other administration groups have no significant difference.
Table 11 quail serum AST, ALT viability (U/L, n=12,)/>
6.7.2 serum CRE, BUN content
The results are shown in Table 12 and FIG. 17. Compared with the normal group, the experiment 83d shows that the serum CRE content of the model group quail has no significant difference, the serum BUN content is significantly increased (P < 0.05), and the serum CRE content and the BUN content of the administration group quail have no significant difference.
Compared with the model group, in the experiment 83d, the CRE content of the quail serum of the group with high chrysanthemum and poria (10 g/kg) dosage is obviously reduced (P is less than 0.05), and the CRE content of the quail serum of the other group with administration is not obviously different; the serum BUN content of the quail in the group with high (10 g/kg), medium (7.5 g/kg) and low (3.75 g/kg) dosage of the chrysanthemum is obviously reduced (P < 0.01), the chrysanthemum is dose-dependent, and the serum BUN content of the quail in the group with the benzbromarone is not obviously different.
Table 12 quail serum CRE and BUN content for each group (n=12,)
note that: compared with normal group, P<0.05; in contrast to the set of models, # P<0.05， ## P<0.01。
7. analysis of results
The study was conducted on the use of the pharmaceutical composition to remove the uric acid crystals of joints against gouty arthritis. The results show that the high, medium and low dosage groups of the traditional Chinese medicine composition can obviously reduce the uric acid crystallization of quail joints of a model group, reduce the uric acid level of joint fluid, obviously improve the swelling degree of the quail joints of gouty arthritis, reduce the interleukin-1 beta level of inflammatory factors in serum, and define the efficacy of the traditional Chinese medicine composition in removing the uric acid crystallization of the joints and resisting gouty arthritis.
In the case of joint uric acid crystals, the experiment shows that needle-shaped, bright and blue-yellow joint uric acid crystals are visible in the wrist joint flushing liquid of the model group quails through microscopic observation of the wrist joint flushing liquid of each group of quails by polarized light. The uric acid crystal in the joint synovial fluid is used as a gold index for clinically diagnosing gouty arthritis, and can effectively prove the modeling success of a gouty arthritis model. The experimental result shows that uric acid crystals deposited in the model group quail wrist joint flushing liquid are in the shape of scattered needles or gathered into feathers, and have the unique needle-shaped, blue/yellow and strong negative birefringence morphology and optical characteristics of uric acid crystals under polarized light.
In the condition of inflammatory expression, the experiment uses joint swelling degree as a main evaluation index and joint appearance form as an auxiliary evaluation index, and discovers the typical inflammatory manifestation of swelling and redness when the local wrist and paw joints of the model group quail have acute attacks of gouty arthritis. The joint swelling results show that the swelling of the left and right wrist joints of the quails in the model group is significantly increased at different time points compared with the model group. The joint appearance morphology observation shows that compared with the joints with normal morphology in a normal group, the toe joints of the model group quail can see a plurality of swelling nodules with different sizes, and the swelling parts of the severe quail can see the crumbling and pus flowing; the wrist joint is also seen as a clear swelling, and the skin at the swollen site is reddish purple.
The experiment evaluates the anti-gout efficacy of the poria cocos extract in terms of the swelling degree of the wrist joint, the swelling degree of the paw joint and the crystal shape of uric acid deposited in joint flushing liquid. First, in the case of anti-joint urate deposition: compared with the uric acid salt crystal clusters scattered or gathered into feather shape in the model group quail wrist joint flushing liquid, the uric acid salt crystals in the high, medium and low dosage group quail wrist joint flushing liquid of the chrysanthemum and poria extract are scattered, single needle crystals or crystal bundles which are arranged in parallel or overlapped in an X shape in two-three directions. Second, in the case of inflammation due to anti-articular urate deposition: the chrysanthemum indicum extract can obviously reduce the swelling degree of the wrist joint and the paw joint under the dosage of 3.75-10 g/kg, which indicates that the chrysanthemum indicum extract has better effect of resisting gouty arthritis.
The research results show that the traditional Chinese medicine composition has obvious effect of resisting gouty arthritis, the efficacy of the traditional Chinese medicine composition is possibly related to removing joint uric acid crystals and improving joint injury of model animals, and a new medicine choice is provided for preventing and treating gouty arthritis.
Example 3 Chinese herbal Compound composition and method of preparing the same
The traditional Chinese medicine composition is prepared by extracting the following raw materials in water: 9g of chicory, 10g of tuckahoe, 30g of rhizoma smilacis glabrae, 6g of pagodatree flower bud and 9g of gentiana macrophylla.
The extraction method comprises the following steps: s1, adding deionized water with the mass ratio of 12 times according to the weight parts, extracting for 1h by adopting a decoction method, collecting an extracting solution, repeating for 3 times, and combining the extracting solutions;
s2, concentrating: concentrating the obtained mixed liquid medicine under reduced pressure at-0.09 Mpa and 80deg.C to obtain soft extract;
s3, preparing medicinal powder: drying the obtained soft extract under reduced pressure of-0.09 Mpa at 80deg.C to obtain dry extract; pulverizing the dry extract, and sieving with 60 mesh sieve to obtain Chinese medicinal composition.
Auxiliary materials: lactose; starch; dextrin; sorbitol; mannitol (mannitol)
The dry extract powder of the traditional Chinese medicine composition is added with a proper amount of auxiliary materials and prepared into the dosage forms of granules, capsules or oral liquid by a conventional method.
Example 4 Chinese herbal Compound composition and method for preparing the same
The traditional Chinese medicine composition is prepared by extracting the following raw materials in water: 7g of chicory, 13g of tuckahoe, 20g of rhizoma smilacis glabrae, 9g of pagodatree flower bud and 5g of gentiana macrophylla.
The extraction method is the same as in example 3
Auxiliary materials: lactose; starch; dextrin; sorbitol; mannitol (mannitol)
The dry extract powder of the traditional Chinese medicine composition is added with a proper amount of auxiliary materials and prepared into the dosage forms of granules, capsules or oral liquid by a conventional method.
Example 5 Chinese herbal Compound composition and method for preparing the same
The traditional Chinese medicine composition is prepared by extracting the following raw materials in water: 15g of chicory, 6g of tuckahoe, 50g of rhizoma smilacis glabrae, 5g of pagodatree flower bud and 8g of gentiana macrophylla.
The extraction method is the same as in example 3
Auxiliary materials: lactose; starch; dextrin; sorbitol; mannitol (mannitol)
The dry extract powder of the traditional Chinese medicine composition is added with a proper amount of auxiliary materials and prepared into the dosage forms of granules, capsules or oral liquid by a conventional method.
Claims (10)
1. The traditional Chinese medicine composition for treating gouty arthritis is characterized by being prepared from the following raw materials: chicory, tuckahoe, rhizoma smilacis glabrae, pagodatree flower bud and gentiana macrophylla.
2. The traditional Chinese medicine composition according to claim 1, wherein the traditional Chinese medicine composition is prepared from the following raw materials: 6-18 parts of chicory, 5-15 parts of poria cocos, 10-60 parts of rhizoma smilacis glabrae, 5-10 parts of pagodatree flower bud and 3-10 parts of gentiana macrophylla.
3. The traditional Chinese medicine composition according to claim 2, wherein the traditional Chinese medicine composition is prepared from the following raw materials: 6-10 parts of chicory, 10-15 parts of poria cocos, 20-40 parts of rhizoma smilacis glabrae, 5-10 parts of pagodatree flower bud and 3-10 parts of gentiana macrophylla.
4. The traditional Chinese medicine composition according to claim 3, wherein the traditional Chinese medicine composition is prepared from the following raw materials: 9 parts of chicory, 10 parts of poria cocos, 30 parts of smilax glabra, 6 parts of pagodatree flower bud and 9 parts of gentiana macrophylla.
5. The traditional Chinese medicine composition according to any one of claims 1 to 4, wherein the traditional Chinese medicine composition is: the raw materials are respectively crushed and then mixed to form a composition; or, the composition is obtained by crushing the mixed raw materials; or mixing the above raw materials, extracting by conventional extraction method to obtain extract, purifying to obtain effective components, and preparing into conventional oral dosage form by conventional preparation process.
6. The traditional Chinese medicine composition according to claim 5, wherein the conventional oral dosage forms comprise tablets, capsules, granules, pills, powders and oral liquids.
7. The method for preparing a Chinese medicinal composition according to any one of claims 1 to 4, wherein the method comprises the steps of: s1, taking all the raw materials according to a proportion, and adding water for extraction;
s2, concentrating the extracting solution into thick paste;
s3, drying the thick paste under reduced pressure to obtain dry extract; pulverizing, and sieving to obtain Chinese medicinal composition.
8. The use of a traditional Chinese medicine composition according to any one of claims 1-4 in the preparation of a medicament for treating gouty arthritis.
9. The use of a Chinese medicinal composition according to any one of claims 1-4 in the preparation of a medicament for removing joint uric acid crystals.
10. The use of a Chinese medicinal composition according to any one of claims 1-4 in the manufacture of a medicament for inhibiting the inflammatory response of gouty arthritis.
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