CN108703993B

CN108703993B - Preparation process of RongjinNiantong prescription

Info

Publication number: CN108703993B
Application number: CN201810899834.9A
Authority: CN
Inventors: 吴广文; 许文; 陈俊; 许惠凤; 王丽丽
Original assignee: Fujian University of Traditional Chinese Medicine
Current assignee: Fujian University of Traditional Chinese Medicine
Priority date: 2018-08-08
Filing date: 2018-08-08
Publication date: 2021-08-13
Anticipated expiration: 2038-08-08
Also published as: CN108703993A

Abstract

The invention provides a preparation method of a pharmaceutical composition (a 'Rongjinniantong prescription') for treating arthritis, which comprises the following steps: a. weighing the following raw material medicines in parts by weight: 10-14 parts of achyranthes bidentata, 4-8 parts of angelica sinensis, 6-12 parts of radix angelicae pubescentis, 4-8 parts of notopterygium root, 4-8 parts of divaricate saposhnikovia root and 4-8 parts of liquorice; b. adding 8-12 times of 50-90% ethanol, soaking for 20-40 min, heating to 80-85 ℃, extracting for 1-2 h for 1-3 times, filtering, and concentrating to obtain the product. The novel preparation process of the recipe for the tongbing Niantong adopts ethanol with polarity weaker than water as an extraction solution, although the extraction range of polar substances is smaller than that of water, and small molecular substances such as alkaloid and the like are mainly extracted, the extraction range of nonpolar substances is wider than that of water, and the extraction rate is high; the prepared medicine is used for resisting inflammation and easing pain, can reduce the content of IL-1 beta, IL-6 and TNF-alpha and improve the content of IL-10, particularly the adjusting effect of the alcohol extract on inflammatory factors is better than that of a water extract, the curative effect of the alcohol extract is better than that of a control group, and a new choice is provided for the use of a clinical recipe for treating tenderness.

Description

Preparation process of RongjinNiantong prescription

Technical Field

The invention relates to a preparation process of a recipe for treating pain.

Background

The recipe of RongjinNiantong is derived from a high-frequency and core medicine prescription for treating bone rheumatism (osteoarthritis) in Qinggong prescription integration of Chencove Ji yard of national medical specialist, and comprises 6 traditional Chinese medicines of achyranthes root, Chinese angelica, pubescent angelica root, incised notopterygium rhizome, divaricate saposhnikovia root and liquoric root. In the formula, achyranthes root is used as monarch drug for tonifying liver and kidney to strengthen muscles and bones, and achyranthes root is used as monarch drug for promoting blood circulation and relaxing the joints and tendons of limbs. The angelica is used as a minister to nourish blood and regulate blood and to nourish the blood vessels of muscles and bones. Du Huo is good at treating wind and removing chronic arthralgia; notopterygii rhizoma has effects of dispelling pathogenic wind and removing dampness, benefiting articulation, and relieving pain; the three medicines are matched to eliminate the arthralgia of the whole body, so that the effects of dispelling wind-damp and stopping the pain of the arthralgia are taken as adjuvant medicines. Licorice root, radix Glycyrrhizae is good at treating spasm of limbs, relieving spasm and alleviating pain, and is used as a guiding drug for harmonizing various herbs. The medicines have the effects of tonifying liver and kidney, strengthening muscles and bones, dispelling wind-damp and relieving arthralgia, and can play a role in treating the etiology and pathogenesis of knee osteoarthritis.

The research shows that: the Rongjinnitong prescription can effectively relieve clinical symptoms and physical signs of a patient with knee osteoarthritis [ ZhudingYu, Linmunan, Wanning, and the like ] the Rongjinnitong prescription can be used for treating the knee osteoarthritis, and the clinical curative effect observation [ A ] is compiled in the twenty-fourth annual meeting of Chinese and western medicine combined orthopedics and traumatology academic society of China [ C ] 2017 ]; the method is characterized in that a computer simulation method based on compound-target interaction is applied, active ingredients and action targets of the recipe for treating osteoarthritis are clarified, the recipe for treating osteoarthritis not only can inhibit inflammatory factor expression and relieve pain, but also can achieve the effect of protecting cartilage at multiple targets in aspects of inhibiting catabolism, stimulating anabolism, inhibiting synthesis of eicosanoids and the like [ Zhengchun pine, Vanshanbiao, leaf mustards and the like ]. 20-24. ]; in addition, computer simulation has also explained the effect of broad spectrum of the recipe of sinkiang bugle from the chemical space perspective, wherein the liver and kidney tonifying and bone and muscle strengthening drugs have the effect of stimulating proteoglycan synthesis in articular cartilage, and the rheumatism and arthralgia relieving drugs have the effects of anti-inflammation and analgesia [ zhengchun pine, leaf mustard, qianlong, etc. ] the effects of liver and kidney tonifying and muscle and bone strengthening of the recipe of sinkiang bugle and rheumatism and arthralgia relieving [ J ]. rheumatism and arthritis, 2018, 7 (2): 33-36. ]. Therefore, the recipe of the sinew-tendering pain has better clinical curative effect on the knee osteoarthritis, and the active ingredients of the recipe have the effect of multi-target-point synergistic treatment on the knee osteoarthritis.

Application No.: 201710284659.8, title of the invention: the invention discloses a recipe of longjinNiantong and a preparation method and application thereof. The pharmaceutical composition is a preparation prepared from the following raw material medicines in parts by weight: 10-14 parts of achyranthes bidentata, 4-8 parts of angelica sinensis, 6-12 parts of radix angelicae pubescentis, 4-8 parts of notopterygium root, 4-8 parts of divaricate saposhnikovia root and 4-8 parts of liquorice. The invention discloses a water extraction process, wherein water is a polar solvent, mainly extracts hydrophilic components, has wide extraction range but poor selectivity, is easy to extract macromolecular substances such as polysaccharide, protein and the like, brings difficulty to identification and preparation, is easy to mildew and is difficult to store.

Disclosure of Invention

In order to solve the technical problem, the invention discloses a novel preparation process of a prescription for treating tenderness.

The invention provides a preparation method of a pharmaceutical composition for treating arthritis, which comprises the following steps:

a. weighing the following raw material medicines in parts by weight:

10-14 parts of achyranthes bidentata, 4-8 parts of angelica sinensis, 6-12 parts of radix angelicae pubescentis, 4-8 parts of notopterygium root, 4-8 parts of divaricate saposhnikovia root and 4-8 parts of liquorice;

b. adding 8-12 times of 50-90% ethanol, soaking for 20-40 min, heating to 80-85 ℃, extracting for 1-2 h for 1-3 times, filtering, and concentrating to obtain the product.

Further preferably, the weight ratio of the raw material medicines in the step a is as follows:

12 parts of achyranthes root, 6 parts of angelica, 9 parts of pubescent angelica root, 6 parts of notopterygium root, 6 parts of divaricate saposhnikovia root and 6 parts of liquorice.

Wherein, the adding amount of the ethanol in the step b is 10 times of the adding amount of the ethanol.

Wherein the ethanol concentration is 70% ethanol.

Wherein the dipping time is 30 min.

Wherein the heating temperature is 80 ℃.

Wherein the extraction time is 2 h.

Wherein, the extraction times are 3 times.

The novel preparation process of the recipe for tendering the tenderness adopts ethanol with polarity weaker than water as an extraction solution, although the extraction range of polar substances is smaller than that of water, small molecular substances such as alkaloid and the like are mainly extracted, the extraction range of nonpolar substances is wider than that of water, and the extraction rate is high; the prepared medicine is used for resisting inflammation and relieving pain, can reduce the content of IL-1 beta, IL-6 and TNF-alpha and improve the content of IL-10, particularly the adjusting effect of the alcohol extract on inflammatory factors is better than that of a water extract, the curative effect of the alcohol extract is better than that of a control group, and a new choice is provided for the use of a clinical recipe for treating the pain in the sinews.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 shows the content detection of ecdysteroid, ferulic acid, cimicidin glycoside, 5-O-methylvisammioside and glycyrrhizic acid in the compound formula

FIG. 2 shows the content detection of osthole, isoimperatorin and notopterygium alcohol in the compound recipe

FIG. 3 Effect of ethanol concentration on Compound extraction

FIG. 4 Effect of solvent-doubling on Compound extraction

FIG. 5 Effect of extraction time on response value of Fufang extraction results

FIG. 6 Effect of extraction frequency on Compound extraction

FIG. 7 Effect of different intervention groups on IL-1. beta. content in synovial tissue

FIG. 8 Effect of different intervention groups on IL-6 content in synovial tissue

FIG. 9 Effect of different intervention groups on TNF-. alpha.content in synovial tissue

FIG. 10 Effect of different intervention groups on IL-10 content in synovial tissue

FIG. 11 Effect of different intervention groups on IL-1. beta. content in serum

FIG. 12 Effect of different intervention groups on IL-6 content in serum

FIG. 13 Effect of different intervention groups on TNF-. alpha.content in serum

FIG. 14 Effect of different intervention groups on IL-10 content in serum

Detailed Description

Example 1 preparation of the drug extract of the present invention

Accurately weighing 12g of achyranthes bidentata, 9g of radix angelicae pubescentis, 6g of angelica sinensis, 6g of notopterygium root, 6g of radix sileris and 6g of liquorice, placing the weighed materials in a round-bottom flask, fixing the extraction temperature at 80 ℃, extracting by 10 times, soaking the materials in 70% of ethanol solvent for 30min, performing reflux extraction for 1.5h, performing suction filtration, concentrating to a constant volume of 500mL, taking a concentrated solution, diluting the concentrated solution by 5 times with 50% acetonitrile, and performing filtration membrane and sample injection.

Example 2 preparation of the drug extract of the present invention

Accurately weighing 12g of achyranthes bidentata, 9g of radix angelicae pubescentis, 6g of angelica sinensis, 6g of notopterygium root, 6g of radix sileris and 6g of liquorice, placing the weighed materials in a round-bottom flask, fixing the extraction temperature at 80 ℃, extracting by 10 times, soaking the materials in 70% of ethanol solvent for 30min, performing reflux extraction for 2h, performing suction filtration, concentrating to a constant volume of 500mL, taking a concentrated solution, diluting the concentrated solution by 5 times with 50% acetonitrile, and performing filtration membrane and sample injection.

Example 3 preparation of the drug extract of the present invention

Accurately weighing 10g of achyranthes bidentata, 6g of radix angelicae pubescentis, 4g of angelica sinensis, 4g of notopterygium root, 4g of radix sileris and 4g of liquorice, placing the weighed materials in a round-bottom flask, fixing the extraction temperature at 85 ℃, extracting by 8 times, soaking the materials in an ethanol solvent by 50 percent for 20min, performing reflux extraction for 1h, performing suction filtration, concentrating to a constant volume of 500mL, taking a concentrated solution, diluting the concentrated solution by 5 times with 50 percent acetonitrile, and performing filtration membrane and sample injection.

Example 4 preparation of the drug extract of the present invention

Accurately weighing 14g of achyranthes bidentata, 12g of radix angelicae pubescentis, 8g of angelica sinensis, 8g of notopterygium root, 8g of radix sileris and 8g of liquorice, placing the weighed materials in a round-bottom flask, fixing the extraction temperature at 85 ℃, extracting by 10 times, soaking the materials in 90% of ethanol solvent for 40min, performing reflux extraction for 2h, performing suction filtration, concentrating to a constant volume of 500mL, taking a concentrated solution, diluting the concentrated solution by 5 times with 50% acetonitrile, and performing filtration membrane and sample injection.

Example 5 optimized process for preparing recipe of longjinNiantong

1 materials of the experiment

1.1 sources of medicinal materials and reference substances

The reference beta-ecdysterone (batch No. 111638-. The medicinal achyranthes root is from Xiamen Yanlefu pharmacy Co., Ltd (batch No. 171122).

The reference osthole (batch No. 111820-201504, content 98.88%), and columbic acid angelate (batch No. 111583-201605, content 98.6%) were purchased from the China institute for food and drug assay. The radix angelicae pubescentis is purchased from the Lu Chaoxi pharmaceutical industry (batch number: 1708030352).

The reference ferulic acid (batch No. 110773-201614, content 99.0%) was purchased from the institute of food and drug testing, China. The Angelica sinensis was purchased from Biotech Inc. of Richctang, Fuzhou (batch No.: 195201701).

The control product of the 5-O-methylvisammioside (batch No.: 111522-201712, 96.2%) was purchased from the institute of food and drug assay (batch No.: 111523-201610, 96.1%). Ledebouriella seseloides is purchased from Biotech Inc., of Relaichuntang, Fuzhou (batch No.: 197201702).

The reference ammonium glycyrrhizinate (batch: 110731) -201619 with 93.0% content) was purchased from China institute for food and drug testing. The medicinal material licorice is purchased from Anhui synergetic patent pharmaceutical industry Co., Ltd. (batch number: 17082004).

The control notopterygium alcohol (batch No.: 111820-201504, content 98.88%) was purchased from the institute of food and drug testing. Notopterygium incisum is purchased from Tianji pharmaceutical Co., Ltd (batch No. 170301) in Bozhou city.

1.2 instruments

One hundred thousand analytical balance model CPA225D (sydows scientific instruments ltd), SHB-III circulating water multi-purpose vacuum pump (zheng changchengchi industries and trade ltd), LX-07A multi-function pulverizer (shanghai jiangxin science ltd), HH-Z digital display constant temperature water bath (changzhou china electric appliances), Agilent TechnOlOgies 1260 high performance liquid chromatograph (Agilent), KQ-500E desktop ultrasonic cleaner (kunshan ultrasonic instruments ltd), Milli-Q ultra pure water instrument (millipoore). Chromatographic grade acetonitrile and methanol (Merck, Germany), chromatographic grade formic acid (Shanghai Allantin reagents, Inc.), the remaining reagents were analytical grade.

2 methods and results

2.1 chromatographic conditions

2.1.1 chromatographic conditions

Measuring the components of achyranthes, angelica, radix sileris and liquorice, wherein the achyranthes detects ecdysteroid, the angelica detects ferulic acid, the radix sileris detects cimicifuga glycoside and 5-O-methylvisammioside, and the liquorice detects glycyrrhizic acid.

A chromatographic column: thermo BETASIL C18 Analytical column (4.6 mm. times.250 mm, 5 μm), mobile phase: acetonitrile (A) -0.1% formic acid (B), gradient elution (0-5 min, 18.0% A → 18.0% A; 5-35 min, 18.0% A → 30.0% A; 35-45 min, 30.0% A → 55.0% A; 45-54 min, 55.0% A → 92.0% A; 54-54.5 min, 92.0% A → 12.0% A; 54.5-60 min, 12.0% A → 12.0% A), column temperature: 35 ℃, detection wavelength: 237nm and 317 nm; flow rate: 1.0mL/min, and a sample size of 10. mu.L. The chromatogram of the complex is shown in FIG. 1.

2.1.2 chromatographic conditions

Determining the components of radix Angelicae Pubescentis and Notopterygii rhizoma, wherein radix Angelicae Pubescentis is used for determining osthole, and Notopterygii rhizoma is used for determining isoimperatorin and Notopterygii rhizoma alcohol.

A chromatographic column: thermo BETASIL C18 Analytical column (4.6 mm. times.250 mm, 5 μm), mobile phase: acetonitrile (A) -water (B), gradient elution (0-10 min, 49% A → 49% A; 10-18 min, 49% A → 55% A; 18-25 min, 55% A → 70% A; 25-42 min, 70% A → 85% A; 42-45 min, 85% A → 90% A; 45-45.5 min, 90% A → 40% A; 45.5-50 min, 40% A → 40% A), column temperature: 35 ℃, detection wavelength: 310nm, flow rate: 1.0mL/min, and a sample size of 10. mu.L. See FIG. 2

2.2 preparation of the solution

2.2.1 preparation of control solutions

Precisely weighing beta-ecdysterone, ferulic acid, notopterygium alcohol, isoimperatorin, 5-O-methylvisammol glycoside, glycyrrhizic acid, and reference substances of osthole such as 5.15mg, 4.58mg, 5.14mg, 5.48mg, 5.73mg, 4.92mg, 5.00mg and 3.02mg, respectively placing the other components in a 5mL volumetric flask, dissolving ferulic acid and glycyrrhizic acid with 70% methanol and diluting to scale, dissolving the other components with methanol and diluting to scale, and shaking up to obtain reference solutions with mass concentrations of 1.03mg/mL beta-ecdysterone, 0.916mg/mL ferulic acid, 1.028mg/mL notopterygium alcohol, 1.096mg/mL isoimperatorin, 1.146mg/mL 5-O-methylvisammol glycoside, 0.984mg/mL glycyrrhizic acid, 1.000mg/mL osthole and 604 μ g/mL of rubusoside. Taking the reference substance solution, preparing a mixed reference substance solution according to the content determination item of the Chinese pharmacopoeia external standard one-point method: diluting with 70% methanol to obtain mixed solution of beta-ecdysterone 0.103mg/mL, ferulic acid 18.32 μ g/mL, linarin 60.4 μ g/mL, 5-O-methyl visammoside 57.3 μ g/mL, glycyrrhizic acid 196.8 μ g/mL, for determining components of Achyranthis radix, radix Angelicae sinensis, radix Saposhnikoviae and Glycyrrhrizae radix, wherein Achyranthis radix detects ecdysteroid, radix Angelicae sinensis detects ferulic acid, radix Saposhnikoviae detects linarin and 5-O-methyl visammoside, and Glycyrrhrizae radix detects glycyrrhizic acid; diluting with 70% methanol to obtain mixed solution of Notopterygii rhizoma alcohol 51.4 μ g/mL, osthole 150 μ g/mL and isoimperatorin 27.4 μ g/mL for determining radix Angelicae Pubescentis and Notopterygii rhizoma components, wherein radix Angelicae Pubescentis is used for determining osthole, and Notopterygii rhizoma is used for determining isoimperatorin and Notopterygii rhizoma alcohol.

2.2.2 preparation of test solutions

Alcohol extraction: accurately weighing 12g of achyranthes bidentata, 9g of radix angelicae pubescentis, 6g of angelica sinensis, 6g of notopterygium root, 6g of radix sileris and 6g of liquorice, placing the weighed materials in a round-bottom flask, fixing the extraction temperature at 80 ℃, extracting by 10 times, soaking the materials in 70% of ethanol solvent for 30min, performing reflux extraction for 1.5h, performing suction filtration, concentrating to a constant volume of 500mL, taking a concentrated solution, diluting the concentrated solution by 5 times with 50% acetonitrile, and performing filtration membrane and sample injection.

Water extraction: accurately weighing 12g of achyranthes bidentata, 9g of radix angelicae pubescentis, 6g of angelica sinensis, 6g of notopterygium root, 6g of radix sileris and 6g of liquorice, placing the weighed materials in a round-bottom flask, fixing the extraction temperature at 80 ℃, extracting by 10 times, soaking for 30min, performing reflux extraction for 1.5h, adding 95% ethanol to ensure that the ethanol concentration is 70%, performing alcohol precipitation overnight, performing suction filtration, concentrating to a constant volume of 500mL, taking a concentrated solution, diluting by 5 times with 50% acetonitrile, performing filtration membrane, and injecting samples.

2.2.3 sample determination

According to the method under the item 2.1, the retention time of each component in the sample is determined according to the retention time of the reference substance, the peak area of each component is obtained by integration, the peak area of the reference substance and the concentration of the reference substance are substituted, the concentration of the sample is calculated, and the content of each component in each sample is calculated.

2.3 alcohol extraction Process

2.3.1 initial examination of ethanol volume fraction

Respectively selecting 30%, 50%, 70% and 90% ethanol as extraction solvents, wherein the extraction times are 10 times, performing reflux extraction for 1 time and 1.5 hours to obtain extract, measuring the contents of beta-ecdysterone, ferulic acid, cnidium lactone, cimicidin, notopterygium alcohol, isoimperatin, 5-O-methylvisammioside and glycyrrhizic acid of the Senecio liana according to the preparation method of a test product under the item '2.2.2' and the chromatographic method under the item '2.1', and inspecting the influence of the ethanol extraction solvents on the extraction of the Senecio liana, wherein the response value of the extraction result of the Senecio liana is not obviously changed between 30% and 50% ethanol concentration, but the ethanol concentration is increased from 50% to 70% ethanol concentration, the response value of the extraction result is increased, and the response value is decreased when the ethanol concentration of 90% is reached. In general, the 70% ethanol ratio response value is the highest, so that in the orthogonal experiment, 50%, 70%, and 90% are selected as the levels for selecting the ethanol concentration in the orthogonal experiment.

2.3.2 preliminary examination of the amount of extraction

Respectively selecting ethanol with extraction times of 5, 10, 15 and 20, 70% as extraction solvents, reflux-extracting for 1 time and 1.5 hours to obtain extract, measuring the contents of beta-ecdysterone, ferulic acid, cnidium lactone, primordin, notopterygium alcohol, isoimperatorin, 5-O-methylvisammioside and glycyrrhizic acid of the recipe of the sinergic pain according to the preparation method of the test product under the item '2.2.2' and the chromatographic method under the item '2.1', wherein the response value of the extraction result of the recipe of the sinergic pain is increased along with the increase of the material-liquid ratio when the result is shown in a figure 4, 5-10 times, and 15 times and 20 times are obviously lower than 10 times, so 8 times, 10 times and 12 times are selected as the level of the times of orthogonal experiment.

2.3.3 preliminary examination of extraction time

Extracting for 1, 1.5, 2 and 2.5h respectively, extracting with 70% ethanol as extraction solvent with extraction multiple of 10 times, reflux-extracting for 1 time to obtain extractive solution, measuring contents of beta-ecdysterone, ferulic acid, cnidium lactone, cimicidin, notopterygium alcohol, isoimperatin, 5-O-methylvisammioside, and glycyrrhizic acid of Nippon reineckea according to the preparation method of the test sample under item "2.2.2" and the chromatography under item "2.1", and examining the influence of the extraction time in alcohol extraction on the response value of the Nippon reineckea extraction result. As a result, as shown in FIG. 5, the response value gradually increases as the extraction time increases, but the response value tends to decrease when the extraction time is 2h to 2.5h, while saving the extraction time is considered. Finally, 1h, 1.5h and 2h are selected as orthogonal test investigation indexes.

2.3.4 preliminary examination of extraction times

Extracting with 70% ethanol as extraction solvent at a material-to-liquid ratio of 1: 10 under reflux for 1, 2, 3, and 4 times respectively, extracting for 1.5 hr to obtain extractive solution, measuring contents of beta-ecdysterone, ferulic acid, cnidium lactone, cimicidin, notopterygium alcohol, isoimperatin, 5-O-methylvisammioside, and glycyrrhizic acid of Nippon reineckea according to the preparation method of the test sample under item "2.2.2" and the chromatography under item "2.1", and examining the influence of the reflux extraction frequency of ethanol extraction on the response value of the Nippon reineckea extraction result. The results are shown in fig. 6, the number of extractions was lower than 2, 3, and 4 at 1, and the equilibrium was reached when the extraction reached 2. Therefore, 1, 2 and 3 times were selected as the index for examining the number of times of extraction in the orthogonal test.

2.3.5 alcohol extraction Process orthogonal experiment

According to relevant literature reports and previous experimental results, the content of beta-ecdysterone, ferulic acid, osthole, cimicin glycoside, notopterygium alcohol, isoimperatorin, 5-O-methylvisammioside and glycyrrhizic acid is taken as an investigation index, the extraction process score adopts a total scoring method, and the calculation method comprises the following steps: total score ═ Σ (extraction rate of each component/maximum value of extraction rate of each component). An orthogonal test of the alcohol extraction process of the recipe of the pain in the soft-mouth is arranged by adopting an L16(45) orthogonal table, 4 factors of the volume fraction, the extraction time, the extraction frequency and the extraction time of the ethanol are examined, and the result is subjected to variance analysis and significance test to determine the optimal extraction process of the recipe of the pain in the soft-mouth. The factor level table, orthogonal test schedule table, and analysis of variance table are shown in tables 1, 2, and 3 below.

TABLE 1 level table for alcohol extraction factors of orthogonal test

TABLE 3 analysis of variance of alcohol extraction orthogonal test results

Note: f_0.05(2，2)＝19。

As can be seen from the equation difference analysis in tables 1 to 3, the B factor with the minimum range is used as an error term to perform variance analysis, in the alcohol extraction process, the four factors of the extraction solvent, the extraction times and the extraction time have the largest influence on the total score, and the influence factors influencing the total score of the recipe for pain caused by sinew are respectively in the following order: c is more than A and more than D is more than B, and the optimal extraction process is A₂B₃C₃D₂Namely, the extraction time is 2 hours, the extraction is carried out for 3 times, and 70% ethanol is used.

The beneficial effects of the present invention are demonstrated by specific efficacy tests below.

Test example 1 anti-inflammatory action study of the recipe for RongjinNiantong of the present invention

The cell factors IL-1 beta, IL-6, TNF-alpha and IL-10 are selected as indexes to compare the anti-inflammatory and analgesic effects of the alcohol extract and the water extract.

The main pathological change in Osteoarthritis (OA) is cartilage degeneration, manifested by fibrosis, fractures, defects, exposure of the subchondral bone plate. The maintenance of articular cartilage structure and function depends on the coordination of both chondrocytes and matrix, while the normal synthesis and breakdown of cartilage matrix depends on the regulation of cytokines. Cytokines are common in inflammatory responses, IL-1 β, IL-6, TNF- α are typical inflammatory factors, and IL-10 is effective against inflammatory factors. These cytokines are closely related to OA cartilage matrix structure and function, the physiological state of chondrocytes, and even affect the structure and function of synovium, subchondral bone.

1 materials of the experiment

1.1 animals 2 months old clean grade SD rats 30 from Shanghai Slek laboratory animals Co., Ltd, with an animal license number SCXK (Shanghai) 2012-0002. The animal feed is bred in the experimental animal center of Fujian Chinese medicine university, and the experimental scheme accords with the approval of the ethical committee of medical animal experiments.

1.2 Instrument Elx-800 microplate reader (Bio-Tek, USA).

1.3 reagent IL-1 beta detection kit (Nanjing Pistaving biology company, Cat: MD6744), IL-6 detection kit (Nanjing Pistaving biology company, Cat: MD6746), TNF-alpha detection kit (Nanjing Pistaving biology company, Cat: MD6987), IL-10 detection kit (Nanjing Pistaving biology company, Cat: MD 6920).

2 method of experiment

2.1 modeling and grouping 30 rats were randomly divided into 5 groups by a random number table method, namely a normal group, a model group, a control group, a water extract group (water extraction group) of the recipe of tenacisum and an alcohol extract group (alcohol extraction group) (prepared in example 2). Except for the normal group, rats in other groups adopt 10 percent chloral hydrate to carry out intraperitoneal injection anesthesia every 0.3ml/100g and then carry out improved Hulth surgery method to establish a knee OA model (Hultha, Lindbergl, Telhag H. Experimental osteoarthardritis in rabbit. preliminary report [ J ]. ActaOrthop Scand, 1970, 41 (5): 522. cake 530.; Liu Xiang, Li West sea, West Jiang Tao. Experimental study for duplicating knee osteoarthritis by the improved Hulth modeling method [ J ]. Chinese and Western medicine combination journal, 2005, 25 (12): 1104).

2.2 intervention method rat dose was converted from equivalent dose ratio table (Huang-Duhan, Huang-Xiao-Hui, Cheng-Yang, etc.. equivalent dose conversion between animals and humans in pharmacological tests [ J]Chinese clinical pharmacology and therapeutics, 2004, 9 (9): 1069-1072.), the control group is given diclofenac sodium sustained release tablets according to the weight of 7.5 mg.kg^-1·d^-1The equivalent dose of the water extract and the alcohol extract of the recipe is 4.5g kg^-1·d^-1By the amount ofAnd (4) performing intragastric administration, and administering equal-dose physiological saline for the model group and the normal group. The gavage is performed for 1 time every day and continuously for 6 weeks.

2.3 after the last gastric lavage of the materials is finished, 10 percent chloral hydrate is used for carrying out intraperitoneal injection anesthesia according to the proportion of 0.3ml/100g, the abdominal cavity is opened, and the abdominal aorta is sampled for blood. And opening the joint cavity, cutting the synovial tissue of the rat joint, putting the synovial tissue into a 1.5mL centrifuge tube, putting the centrifuge tube into a liquid nitrogen tank, and storing the centrifuge tube for later use. After the arterial blood is kept stand for 2h, the arterial blood is centrifuged for 15min at 3000r/min, and serum is collected in a 5mL centrifuge tube and is rapidly placed in a refrigerator at minus 80 ℃ for detection.

2.4ELISA assay rat sera were removed and thawed at low temperature for experiments. Precisely weighing 0.1g of synovial tissue, adding 1mL of phosphate buffer solution, ultrasonically crushing at low temperature, fully shaking, centrifuging at 12000r/min, and taking supernatant for experiments. Preparing a standard substance according to a gradient dilution method, designing coordinates of a sample loading hole, respectively adding the standard substance, quality control and a sample, uniformly shaking, incubating in an oven at 37 ℃ for 2 hours, washing a plate for 3 times by using an automatic plate washing instrument, adding an antibody, and incubating in the oven at 37 ℃. And (3) adding the enzyme conjugate for incubation after the plate is washed by an automatic plate washer, adding a color developing agent, adding a stop solution to stop the reaction, and measuring the OD value by using a 450nm wavelength of an enzyme-labeling instrument. The concentrations of IL-1 beta, IL-6, TNF-alpha and IL-10 were calculated for each sample.

3 results

3.1 Effect of Each intervention group on inflammatory cytokines in synovial tissue

3.1.1 Effect of different intervention groups on IL-1 β content in synovial tissue

Compared with the normal group, the IL-1 beta content in the model group is obviously increased, and the difference is statistically significant (P is 0.000). Compared with the model group, the content of the IL-1 beta in the control group, the water extraction group and the alcohol extraction group is reduced, and the differences have statistical significance (P is 0.000, and P is 0.000), which indicates that the control group, the water extraction and the alcohol extraction can effectively regulate the content of the IL-1 beta. Compared with the water extraction group, the content of IL-1 beta in the alcohol extraction group is reduced more obviously, and the difference has statistical significance (P is 0.000), which shows that the curative effect of the alcohol extract on adjusting IL-1 beta is better than that of the water extract. Compared with the control group, the content of the IL-1 beta in the alcohol extract group is obviously reduced, and the difference has statistical significance (P is 0.000), so that the curative effect of the alcohol extract on regulating the IL-1 beta is better than that of the control group. See fig. 7.

Note: p < 0.05, P < 0.01 compared to normal; in comparison with the set of models,^▲▲P＜0.05，^▲p is less than 0.01; compared with the water extraction group,^★★P＜0.05，^★p is less than 0.01; compared with the control group, the compound of the formula,^□□P＜0.05，^□P＜0.01。

3.1.2 Effect of different intervention groups on IL-6 content in synovial tissue

Compared with the normal group, the IL-6 content in the model group is obviously increased, and the difference is statistically significant (P is 0.000). Compared with the model group, the content of IL-6 in the control group, the water extraction group and the alcohol extraction group is reduced, and the differences have statistical significance (P is 0.000, and P is 0.000), which indicates that the control group, the water extract and the alcohol extract can effectively regulate the content of IL-6. Compared with the water extract group, the IL-6 in the alcohol extract group is reduced more obviously, and the difference has statistical significance (P is 0.000), which shows that the curative effect of the alcohol extract on adjusting the IL-6 is better than that of the water extract. Compared with the control group, the content of IL-6 in the alcohol extract group is obviously reduced, and the difference has statistical significance (P is 0.000), which shows that the curative effect of the alcohol extract on adjusting IL-6 is better than that of the control group. See fig. 8.

3.1.3 Effect of different intervention groups on TNF- α content in synovial tissue

Compared with the normal group, the content of TNF-alpha in the model group is obviously increased, and the difference is statistically significant (P is 0.000). Compared with the model group, the content of the TNF-alpha in the control group, the water extraction group and the alcohol extraction group is reduced, and the differences have statistical significance (P is 0.000, and P is 0.000), which shows that the control group, the water extract and the alcohol extract can effectively regulate the content of the TNF-alpha. Compared with the water extraction group, the content of the TNF-alpha in the alcohol extraction group is obviously reduced, and the difference has statistical significance (P is 0.000), which shows that the curative effect of the alcohol extraction for regulating the TNF-alpha is better than that of the water extraction. Compared with the control group, the content of the TNF-alpha in the alcohol extract group is obviously reduced, and the difference has statistical significance (P is 0.000), which shows that the curative effect of the alcohol extract on regulating the TNF-alpha is better than that of the control group. See fig. 9.

3.1.4 Effect of different intervention groups on IL-10 levels in synovial tissue

Compared with the normal group, the IL-10 content in the model group is obviously reduced, and the difference is statistically significant (P is 0.000). Compared with the model group, the IL-10 content of the control group, the water extraction group and the alcohol extraction group is increased, and the differences have statistical significance (P is 0.000, and P is 0.000), which indicates that the IL-10 content of the control group, the water extraction and the alcohol extraction can be effectively adjusted. Compared with the water extract group, the content of IL-10 in the alcohol extract group is obviously increased, and the difference has statistical significance (P is 0.000), which shows that the curative effect of the alcohol extract on adjusting IL-10 is better than that of the water extract. Compared with the control group, the content of IL-10 in the alcohol extract group is obviously increased, and the difference has statistical significance (P is 0.000), which shows that the curative effect of the alcohol extract on adjusting IL-10 is better than that of the control group. See fig. 10.

3.2 Effect of various intervention groups on inflammatory cytokines in serum

3.2.1 Effect of different intervention groups on IL-1. beta. content in serum

Compared with the normal group, the IL-1 beta content in the model group is obviously increased, and the difference is statistically significant (P is 0.000). Compared with the model group, the content of the IL-1 beta in the control group, the water extraction group and the alcohol extraction group is reduced, and the differences have statistical significance (P is 0.000, and P is 0.000), which indicates that the control group, the water extraction and the alcohol extraction can effectively regulate the content of the IL-1 beta. Compared with the water extract group, the content of IL-1 beta in the alcohol extract group is obviously reduced, and the difference has statistical significance (P is 0.000), which shows that the curative effect of the alcohol extract on regulating IL-1 beta is better than that of the water extract. Compared with the control group, the content of the IL-1 beta in the alcohol extract group is reduced, and the difference has statistical significance (P is 0.004), which shows that the curative effect of the alcohol extract on adjusting the IL-1 beta is better than that of the control group. See fig. 11.

3.2.2 Effect of different intervention groups on IL-6 content in serum

Compared with the normal group, the IL-6 content in the model group is obviously increased, and the difference is statistically significant (P is 0.000). Compared with the model group, the IL-6 expression levels of the control group, the water extraction group and the alcohol extraction group are all reduced, and the differences have statistical significance (P is 0.000, and P is 0.000), which indicates that the control group, the water extraction and the alcohol extraction can effectively regulate the IL-6 content. Compared with the water extraction group, the content of IL-6 in the alcohol extraction group is obviously reduced, and the difference has statistical significance (P is 0.000), which shows that the curative effect of the alcohol extraction for regulating IL-6 is better than that of the water extraction. Compared with the control group, the content of IL-6 in the alcohol extract group is reduced, and the difference has statistical significance (P is 0.001), which shows that the curative effect of the alcohol extract on adjusting IL-6 is better than that of the control group. See fig. 12.

3.2.3 Effect of different intervention groups on TNF-alpha content in serum

Compared with the normal group, the content of TNF-alpha in the model group is obviously increased, and the difference is statistically significant (P is 0.000). Compared with the model group, the content of the TNF-alpha in the control group, the water extraction group and the alcohol extraction group is reduced, and the differences have statistical significance (P is 0.000, and P is 0.000), which shows that the control group, the water extract and the alcohol extract can effectively regulate the content of the TNF-alpha. Compared with the water extraction group, the content of the TNF-alpha in the alcohol extraction group is obviously reduced, and the difference has statistical significance (P is 0.000), which shows that the curative effect of the alcohol extraction for regulating the TNF-alpha is better than that of the water extraction. Compared with the control group, the content of the TNF-alpha in the alcohol extract group is obviously reduced, and the difference has statistical significance (P is 0.000), which shows that the curative effect of the alcohol extract on regulating the TNF-alpha is better than that of the control group. See fig. 13.

3.2.4 Effect of different intervention groups on IL-10 content in serum

Compared with the normal group, the IL-10 content in the model group is obviously reduced, and the difference is statistically significant (P is 0.000). Compared with the model group, the IL-10 content of the control group, the water extraction group and the alcohol extraction group is increased, and the differences have statistical significance (P is 0.000, and P is 0.000), which indicates that the IL-10 content of the control group, the water extraction and the alcohol extraction can be effectively adjusted. Compared with the water extract group, the content of IL-10 in the alcohol extract group is obviously increased, and the difference has statistical significance (P is 0.000), which shows that the curative effect of the alcohol extract on adjusting IL-10 is better than that of the water extract. Compared with the control group, the content of IL-10 in the alcohol extract group is obviously increased, and the difference has statistical significance (P is 0.000), which shows that the curative effect of the alcohol extract on adjusting IL-10 is better than that of the control group. See fig. 14.

and (4) conclusion:

the alcohol extract and the water extract can effectively reduce the content of IL-1 beta, IL-6 and TNF-alpha and improve the content of IL-10, and particularly the adjusting effect of the alcohol extract on the inflammatory factors is better than that of the water extract, and the curative effect of the alcohol extract is better than that of a control group.

Claims

1. A preparation method of a pharmaceutical composition for treating arthritis is characterized by comprising the following steps: the method comprises the following steps:

a. weighing the following raw material medicines in parts by weight:

b. adding 10 times of 70% ethanol, soaking for 30min, heating to 80 deg.C, extracting for 2 hr for 3 times, filtering, and concentrating.

2. The method of claim 1, wherein: the weight ratio of the raw material medicines in the step a is as follows: 12 parts of achyranthes root, 6 parts of angelica, 9 parts of pubescent angelica root, 6 parts of notopterygium root, 6 parts of divaricate saposhnikovia root and 6 parts of liquorice.