CN103405494B - Bauhinia championii n-butyl alcohol extract and preparation method and application thereof - Google Patents

Abstract

The invention provides a bauhinia championii n-butyl alcohol extract, which contains beta-sitosterol with the weight percentage not less than 0.1%. The invention also provides a preparation method and application of the bauhinia championii n-butyl alcohol extract. The n-butyl alcohol part of the bauhinia championii is used for treating rheumatoid arthritis, has definite drug effect, controllable quality and stable preparation method, is suitable for large-scale production, and provides a new choice for clinic.

Description

A kind of n-butanol extract of asparagus vine and its preparation method and application

technical field

The invention relates to ethyl acetate extract and n-butanol extract of astragalus and its preparation method and application, which belong to the field of medicine.

Background technique

Longxuteng is the vine of Bauhinia championii Benth., also known as Jiulongteng and Meihuarugudan. It is mainly distributed in Fujian, Jiangxi, Guangxi, Zhejiang, etc., and has the effect of dispelling rheumatism and promoting blood qi. Modern pharmacological studies have proved that the active ingredients of Astragalus chinensis have the functions of expelling wind and dampness, promoting blood circulation and relieving pain, invigorating spleen and regulating qi, and are used to treat rheumatoid arthritis, waist and leg pain, bruises, stomach pain, and malnutrition in children. According to the prescription of Huang Jingang, a well-known doctor in southern Fujian, Kangmeifu Burn Ointment with Longxuteng as the main ingredient has a good clinical effect on treating burns and scalds. The chemical constituents of Longxuteng medicinal materials are mainly flavonoids, cyanogenic glycosides, gallic acid and so on.

Literature HONG Z F, ZHENG H Y, XU W, et al. Study on resistance inflammation and analgesic effect of Kangmeifu Burns Ointment (KBO) [J]. Chin J Tradit Med SciTechnol (China Traditional Chinese Medicine Science and Technology), 2007,14(4):261 -262. HONG Z F, ZHENG H Y, WANG R G, et al. Study on antibacterial activity of Kangmeifu Burns Ointment (KBO) [J]. Chin J Microecol (Chinese Journal of Microecology), 2007,19(2): 177-178.] reported that the flavonoids in Astragalus have analgesic and anti-inflammatory effects. This document only mentions that the crude total flavonoids have analgesic and anti-inflammatory effects, and does not involve specific compounds. There are also literature reports to study the chemical constituents in the vine, such as: Bai Haiyun, et al., Research on the chemical constituents of the vine (I), Chinese Journal of Traditional Chinese Medicine, Volume 30, No. 1, January 2005, reported that the ethanol extract 5 compounds were isolated and identified in the plant. The extraction and separation methods are as follows: take 7Kg of Jiulong rattan, crush it and percolate with 95% industrial ethanol for 3 times. The extract is suspended in water and extracted with petroleum ether, ethyl acetate and n-butanol in turn. The ethyl acetate part was separated by repeated silica gel, macroporous resin, Sephadex and reversed phase silica gel column chromatography to obtain 5 compounds.

Bai Haiyun et al. Research on the chemical constituents of Jiulongteng (II), developed by the Institute of Natural Products, 2004, Vol.16NO.4, isolated gallic acid, sitosterol, carotene, quercetin, bayberry from the effective part of ethyl acetate Glycosides, epicatechin, 5, 6, 7, 3', 4', 5'-hexamethoxyflavone.

Contents of the invention

The technical solution of the present invention is to provide a kind of ethyl acetate extract and n-butanol extract of asparagus vine. Another technical solution of the present invention is to provide the preparation method and application of the extract.

The invention provides an extract of ethyl acetate of astragalus, which contains not less than 0.05% by weight of quercetin and not less than 0.04% by weight of myricetin.

Further preferably, it contains 0.05-1.50% by weight of quercetin and 0.04-1.25% by weight of myricetin.

Even more preferably, it contains compounds I, II, III, IV, V.

Astragalus ethyl acetate extract of the present invention is prepared by the following steps:

Take the medicinal material of asparagus vine, add 40-95% ethanol to heat and reflux for extraction, recover the ethanol from the extract until it has no alcohol smell, extract the ethanol extract with petroleum ether to obtain a petroleum ether layer and a water layer, add chloroform to the water layer to obtain a chloroform layer and the water layer; the water layer was extracted with ethyl acetate to obtain an ethyl acetate extract, which was recovered and dried to obtain the ethyl acetate extract of Astragalus chinensis.

Wherein, described ethanol is the ethanol that concentration is 80%.

The present invention also provides a kind of method for preparing described asparagus ethyl acetate extract, comprises the steps:

Take the medicinal material of asparagus vine, add 40-95% ethanol to heat and reflux for extraction, recover the ethanol from the extract until it has no alcohol smell, extract the ethanol extract with petroleum ether to obtain a petroleum ether layer and a water layer, add chloroform to the water layer to obtain a chloroform layer and the water layer; the water layer was extracted with ethyl acetate to obtain an ethyl acetate extract, which was recovered and dried to obtain the ethyl acetate extract of Astragalus chinensis.

The present invention also provides the application of the extract of ethyl acetate of asparagus in preparing medicines for analgesia, anti-inflammation and anti-rheumatoid arthritis.

The invention provides a n-butanol extract of asparagus vine, which contains not less than 0.1% by weight of β-sitosterol.

Wherein, it contains 0.1-0.85% by weight of β-sitosterol.

Furthermore, it contains compounds V and VI.

Astragalus n-butanol extract of the present invention is prepared by the following method:

Take the medicinal material of asparagus vine, add 40-95% ethanol to heat and reflux for extraction, recover the ethanol from the extract until it has no alcohol smell, extract the ethanol extract with petroleum ether to obtain a petroleum ether layer and a water layer, add chloroform to the water layer to obtain a chloroform layer and the water layer; the water layer is extracted with ethyl acetate to obtain the ethyl acetate layer and the water layer; the water layer is extracted with n-butanol to obtain the n-butanol extract, which is recovered and dried to obtain the n-butanol extract of Astragalus . Further preferably, the ethanol is 80% ethanol.

The present invention also provides a kind of method for preparing described Asparagus chinensis n-butanol extract, it comprises the steps:

Take the medicinal material of asparagus vine, add 40-95% ethanol to heat and reflux for extraction, recover the ethanol from the extract until it has no alcohol smell, extract the ethanol extract with petroleum ether to obtain a petroleum ether layer and a water layer, add chloroform to the water layer to obtain a chloroform layer and the water layer; the water layer is extracted with ethyl acetate to obtain the ethyl acetate layer and the water layer; the water layer is extracted with n-butanol to obtain the n-butanol extract, which is recovered and dried to obtain the n-butanol extract of Astragalus .

The invention also provides the use of the n-butanol extract of the asparagus vine in the preparation of analgesic, anti-inflammation and anti-rheumatoid arthritis drugs.

The ethyl acetate part and the n-butanol part of the Chinese herbal medicine are used for treating rheumatoid arthritis, and the medicinal effect is clear, the quality is controllable, the preparation method is stable, the preparation method is suitable for mass production, and a new clinical choice is provided.

Description of drawings

Figure 1 Structural formula of β-sitosterol

Figure 2 Structural formula of carotin

Figure 3 Quercetin structural formula diagram

Figure 4 Structural formula of compound Ⅵ

Figure 5 General state of rats

Figure 6 Normal control group

Figure 7 model group

Figure 8 tripterygium glycosides group

Figure 9 ethyl acetate low dose group

Figure 10 ethyl acetate middle dose group

Figure 11 ethyl acetate high dose group

Figure 12 n-butanol low dose group

Figure 13 n-butanol medium dose group

Figure 14 n-butanol high dose group

detailed description

Example 1 Separation and purification of the components of the ethyl acetate part and n-butanol part of Astragalus

1. Extraction and separation

Take 1000g of Longxuteng medicinal material, add 80% ethanol to heat and reflux for extraction three times, each time for 1.5h, filter, combine the ethanol solution, recover the ethanol until it has no alcohol smell, and obtain the alcohol extraction extract. Add 2000ml of water to suspend the extract, add petroleum ether to extract 5 times, 1000ml/time, combine the petroleum ether extract, recycle, and obtain an oily substance; add chloroform to the water layer and extract 5 times, 1000ml/time, combine the chloroform extract, Recover and dry to obtain extract; Add ethyl acetate to extract the water layer 5 times, 1000ml/time, combine ethyl acetate extract, recover, dry to obtain extract; Add n-butanol to extract 5 times, 1000ml Each time, the n-butanol extract was combined, recovered, dried to obtain an extract.

The part of ethyl acetate obtained by this method is calculated according to the extract, and the yield should not be less than 2%, and the content of quercetin and myricetin should not be less than 0.05% and 0.04%, respectively.

The n-butanol part of Asparagus chinensis obtained by this method is calculated according to the extract, and the yield should not be less than 6%, and the content of β-sitosterol should not be less than 0.1%.

The specific measurement method is:

The content of quercetin and myricetin in the ethyl acetate extract of Asparagus chinensis was determined by HPLC. The chromatographic conditions are acetonitrile (A)-0.05% phosphoric acid solution (B), the gradient elution program is 0-5min from 20% to 30% acetonitrile, 5-10min from 30% to 50%, and then keep for 20min; the flow rate is 1.0ml /min; the measurement wavelength is 370nm; the column temperature is 30°C.

The experiment was repeated three times, and the content of quercetin obtained by measuring the extract by this method was 0.062%, 0.064%, and 0.065%, respectively, and the content of myricetin was respectively 0.051%, 0.053%, and 0.055%, and the average content was respectively 0.064%, 0.053%. %.

HPLC method was used to determine the content of β-sitosterol in the n-butanol extract of Asparagus chinensis. The chromatographic conditions are acetonitrile (A)-water (B) (96:4); the flow rate is 1.0ml/min; the measurement wavelength is 210nm; the column temperature is 30°C.

The experiment was repeated three times, and the content of β-sitosterol in the extract was determined by this method to be 0.364%, 0.363%, and 0.363%, respectively. The average content is 0.363%.

The ethyl acetate part was mixed with crude silica gel (160-200 mesh) 1:1, added to a silica gel column wet-packed with dichloromethane, eluted with dichloromethane-methanol gradient, and the eluate was collected in equal volumes, and the fractions were After TLC detection and recombination, repeated separation and purification of silica gel, polyamide, SephadexLH-20 and ODS, compounds Ⅰ, Ⅱ, Ⅲ, Ⅳ, Ⅴ were obtained.

The n-butanol part was dissolved in hot water, and the water-insoluble part was mixed with crude silica gel (160-200 mesh) 1:1, added to a silica gel column wet-packed with dichloromethane, and eluted with dichloromethane-methanol gradient, etc. The eluate was collected by volume, and the fractions were detected by TLC and then combined. After repeated separation and purification of silica gel, polyamide, Sephadex LH-20, and ODS, compounds V and VI were obtained.

2. Structure Identification

Compound Ⅰ: white needle crystal (acetone), mp141-142°C. 5% sulfuric acid-ethanol solution is heated to purple, and vanillin-concentrated sulfuric acid is purple. IR shows absorption bands at 3432, 2936, 2860, 1461, 1381, 1054, 959, and 800cm ^-1 , and the matching degree with β-sitosterol in the Sigma Biological Sample Library is over 90%. Under three different development conditions of petroleum ether-ethyl acetate, petroleum ether-acetone, and dichloromethane-acetone, its Rf value is consistent with that of sitosterol, and the mixing melting point does not decrease. Consistent with the literature, it was identified as β-sitosterol, and its structural formula is shown in Figure 1.

Compound Ⅱ: white powder, mp288～289℃. IR shows absorption bands at 3397, 2959, 2933, 2869, 1463, 1379, 1073, 1024cm ^-1 , and the matching degree with the carotene in the Aldrich Condensed PhaseSample Library is over 90%. Developed on a silica gel thin-layer plate with dichloromethane-acetone, dichloromethane-methanol, dichloromethane-methanol-water and other systems, all of which are single-point, the Rf value is consistent with the carotin standard, and the mixed melting point does not drop , so it was identified as carotin, and its structural formula is shown in Figure 2.

Compound Ⅲ: white flaky crystal (petroleum ether-ethyl acetate), mp65～66℃. IR shows that there are absorption bands at 2917, 2848, 1472, 1061, and 719 cm ^-1 , and the matching degree with triacane in the Aldrich CondensedPhase Sample Library is over 90%, and different solvent systems are used on silica gel thin layer plates Expanded, all are single points, and its Rf value is consistent with the standard product of triaconane, so it is identified as n-triaconane.

Compound Ⅳ: white flaky solid (petroleum ether-ethyl acetate), mp97～98℃. In IR, there are absorption bands at 2917, 2849, 1742, 1472, 1363, 1239, 1179cm ^-1 , and the matching degree with the hexacontane in the Aldrich Condensed Phase Sample Library is over 90%. It was developed with different solvent systems, all of which were single-point, so it was identified as n-hexacontane.

Compound V: yellow needle crystal (chloroform-methanol), mp>300°C. Hydrochloric acid - magnesium powder reaction was positive. IR shows absorption bands at 2918, 2849, 1664, 1610, 1521, 1450, 1381, 1318, 1261, 1198, 1167, 1013cm ^-1 , and the matching degree of quercetin in HR Aldrich Aldehydes and Ketones library is up to More than 90%, the 1H-NMR data is consistent with the quercetin data reported in the literature ^[7] . And it was developed on a silica gel thin-layer plate with different solvent systems, all of which were single-point, and the Rf value was consistent with that of the quercetin reference substance, and the melting point of the mixture did not decrease. Therefore, it was identified as quercetin, and its structural formula is shown in Figure 3.

Compound VI: Yellow granular crystals (chloroform-methanol), mp>280°C. IR shows absorption bands at 3409, 1647, 1611, 1576, 1458, 1293, 1257, 1168, 1119 cm-1. After identification by hydrogen spectrum and mass spectrum, the molecular weight of the compound is deduced to be 326. The preliminary deduced structure is shown in Figure 4.

Prove the beneficial effect of the present invention below by concrete pharmacodynamics test.

Drugs and reagents:

Tripterygium glycosides tablets, produced by Huangshi Feiyun Pharmaceutical Co., Ltd. (approval number: Z42021212);

The medicine of the present invention (Ethyl acetate extract and n-butanol extract of Asparagus chinensis prepared in Example 1); the low dose of the medicine of the present invention is 500 mg/mL of the part containing ethyl acetate or n-butanol, and the middle dose is 500 mg/mL of the part containing The ethyl acetate or n-butanol fraction is 250 mg/mL, and the high dose is 125 mg/mL for the ethyl acetate or n-butanol fraction.

Carrageenan (carrageenan) was provided by the Fujian Provincial Drug Control Institute, made up 1% with normal saline, and placed for 2 days before use. Sodium sulfide: Shanghai Tongya Chemical Technology Development Co., Ltd., batch number: 051212. Xylene: AR, Shanghai Xingda Chemical Reagent Factory, batch number: 20050203. Diethyl ether: AR, Shanghai Jiuyi Chemical Reagent Co., Ltd., lot number: 20050401. Bovine type II collagen, complete Freund's adjuvant, produced by SIGMA Company of the United States, was purchased from Beijing Braid Biotechnology Co., Ltd. TNF-α, IL-1 (ELISA method), immunoglobulin (IgG, IgM) kits: purchased from Wuhan Boster Bioengineering Co., Ltd.

Anti-inflammatory experiment of test example 1 medicine of the present invention

1 Influence on mouse auricle swelling caused by xylene: 80 20 ± 2g gram mice of body weight are randomly divided into 8 groups according to body weight: low-dose drug group of the present invention, middle-dose drug group of the present invention, high-dose the present invention Invention drug group, positive control group (tripterygium glycosides tablets), model control group (white petrolatum). The mice in the 8 groups were given intragastric administration once a day for 3 consecutive days. 30 minutes after the last drug application, 0.1ml/mouse of xylene was applied evenly on the same place on both sides of the right ear, and the left ear was used as a control, and they were killed 4 hours later. , cut off both ears immediately, and use a 8mm diameter puncher to take the same part of the ear piece, weigh it on a semi-automatic analytical balance of 1/10,000, and use (right ear weight-left ear weight)/left ear weight as the swelling rate, Take (average swelling degree of model group-average swelling degree of administration group)/average swelling degree of model group as swelling inhibition rate. The degree of swelling in the control group and the treatment group was processed and analyzed using SPSS statistical software, and the difference between the averages was analyzed by one-way analysis of variance, as shown in Table 1.

Table 1 The effect of the drug group of the present invention on the inflammation induced by xylene in mice (n=10)

Compared with the model control group, each administration group has significant difference (P<0.01, P<0.05), and three kinds of dosages of the present invention's medicine group are all greater than 20% to the inhibitory rate of xylene-induced inflammation, prompting the present invention's medicine group It has inhibitory effect on mouse auricle swelling induced by xylene. And a dose-effect relationship.

2. Effects on carrageenan-induced paw swelling of rats: 80 SD rats with a body weight of 150 ± 10 g were randomly divided into 8 groups according to body weight: model group, low, medium and high dose groups of the present invention, and Lei Gong Rattan polyglycoside tablet group. Straighten the right hind limb of the rat, and subcutaneously inject 0.1 mL of 1% carrageenan from the middle of the sole of the foot. 0.5h, 1h, 1.5h, 2h, 4h, 6h after injection respectively measure the thickness of the swollen limbs with a vernier caliper, and calculate the swelling rate with (after causing inflammation-causing the thickness of the front sole of the foot)/causing the thickness of the front sole of the inflammation×100%, Calculate the inhibition rate with (the average swelling rate of the model control group-the average swelling rate of the experimental group)/the average swelling rate of the model control group×100%. The data processing of each group is processed and analyzed using SPSS statistical software, and the difference between the averages is analyzed by one-way analysis of variance. , the results are shown in Table 2.

Table 2 Effect of the pharmaceutical composition of the present invention on the inhibition rate of paw swelling caused by carrageenan in rats (n=10)

The results showed that there were significant differences (P<0.05) between each drug group and the model group at 1h to 6h after inflammation, suggesting that each drug group had an inhibitory effect on paw swelling of rats induced by carrageenan. Three doses of the medicine group of the present invention, 1h inhibition rate just reaches more than 30% after causing inflammation; Reach peak when 1h, its high-dose group and tripterygium glycosides tablet group compare difference to have very significant significance (P<0 .O1). The anti-inflammatory duration of the low-dose medicine group of the present invention reaches 4h, and the middle and high doses reach more than 6h, and single-factor analysis of variance shows when 4h and 6h, low, middle and high doses have no significant difference with Tripterygium wilfordii Polyglycoside Tablets (P> 0.05).

Test Example 2 The analgesic experiment of the medicine of the present invention (the influence on the pain caused by hot plate in female mice)

Take female mice with a body weight of 20 ± 2 g, and measure 80 mice whose pain threshold is within 5s-30s on a hot plate analgesia instrument before the test according to the hot plate method, and randomly divide them into 8 groups according to body weight: blank control group, The medicine group of the present invention is a low, middle and high dose group, and a tripterygium glycosides tablet group. The observation index was the occurrence of hind paw licking reaction. The above drugs were administered to each mouse respectively, 0.2mL/mouse. After 0.5h, measure the pain threshold according to the above method, then administer the drug according to the previous method, and measure the pain threshold at 1h, 1.5h, and 2h. Compare the change of pain threshold before and after administration, and calculate the percentage increase of pain threshold according to (after administration-average pain threshold before administration)/average pain threshold before administration×100%. The data of each group was processed and analyzed using SPSS statistical software, and the difference between the means was analyzed by one-way analysis of variance. The results are shown in Table 3.

Table 3 Effects of Drugs on Pain Threshold and Pain Threshold Raising Rate of Hot Plate in Mice (n=10)

The results show that the pharmaceutical composition of the present invention has significant difference (P<0.05) compared with the model control group, and still has analgesic effect in 2h. All three dosages of the pharmaceutical composition of the present invention can obviously improve the pain threshold of mice. Tip: The pharmaceutical composition of the present invention has a significant analgesic effect on heat-induced pain, and the analgesic effect lasts for more than 2 hours.

Test example 3 the influence of medicine of the present invention on arthritis

Grouping and modeling: Wistar rats (male), with an average body weight of (200±20) g, were adaptively fed for 1 week, under uniform light, in a low-temperature environment, free to drink water, and free to eat solid feed. They were randomly divided into 9 groups, namely normal group (A), model group (B), tripterygium glycosides group (C), low-dose ethyl acetate group (D), medium-dose ethyl acetate group (E), ethyl acetate group Ester high-dose group (F), n-butanol low-dose group (G), n-butanol middle-dose group (H), n-butanol high-dose group (I), 10 rats in each group. Mix complete Freund's adjuvant (CFA) and 2mg/ml bovine type II collagen emulsion in equal volumes to make collagen emulsion. Except for the normal group, each rat is intradermally injected with 0.2mg of collagen at the base of the tail. After the initial immunization On the first day, intragastric administration was started in groups, once a day. The second immunization was carried out on the 7th day. In addition to the normal group, each rat was intradermally injected with 0.1 mg of collagen emulsion (incomplete Freund's adjuvant (IFA) mixed with 2 mg/ml of bovine type II collagen emulsion in equal volume) at the root of the tail. , made into collagen emulsion). The normal group and the model group were given 9.0g/L sodium chloride injection 10ml/kg every day; the high-dose group was given 500mg/kg by intragastric administration, the medium-dose group was given 250mg/kg by intragastric administration daily, and the low-dose group was given intragastric administration daily 125mg/kg was given, and the tripterygium glycosides group was given 6mg/kg daily. All animals were administered continuously for 28 days. The volume of the bilateral hind paws of the rats was measured 1 day before modeling by the volumetric method, and the volume of the bilateral hind paws of the rats was measured every 4 days after the inflammation was induced, and the volume value was recorded, which was used as the base for evaluating the intensity of the anti-inflammatory effect.

Sample collection and index detection: Before and after modeling, the thickness of paw swelling and body weight were measured; before treatment and after treatment, the degree of arthritis swelling was calculated; after treatment on the 28th day, the abdominal aorta of all animals was taken The blood was divided into drying tubes, and the drying tubes were placed in 3000 rpm/centrifugation for 8 minutes, and the supernatant was taken to separate the serum, and the serum TNF-α, interleukin 6 (IL-6), The content of interleukin 8 (IL-8) and interleukin 10 (IL-10).

Statistical method: SPSS15.0 data statistical package was used for data processing, and the experimental data was used express. Measurement data were analyzed using paired t-test within group and analysis of variance between groups.

1 Observation of the general state of the rats is shown in Figure 5. After the rats in the model group were sensitized with collagen emulsion and immunized, their mental state, diet, and activity were reduced. scab. About 3 days after the second immunization, the rats began to develop symptoms. The toes and ankle joints of the model rats began to swell, and gradually aggravated. After treatment, the above-mentioned symptoms were obviously alleviated.

2 Body weight of rats: see Table 4 for the results. Except the drug group, the body weight of the rats in other groups increased slowly, and the body weight began to decrease during the 14th and 21st days, and then increased slowly.

The impact of each group on the body weight of the rats in table 4

Note: Compared with the normal group, ^# P﹤0.05, ^## P﹤0.01; compared with the model group, ^* P﹤0.05, ^** P﹤0.01;

3 Histopathological observation: the pathology of the ankle joints of animals in each group showed that the synovial lining of the normal control group (A) was 1-2 layers thick, the cells were arranged neatly, and there was no inflammatory cell infiltration and angiogenesis , the articular cartilage is smooth and undamaged. In the CIA model group (B), the synoviocytes of the rats proliferated significantly, and the arrangement was disordered. The cells in the synovial lining layer proliferated from the original 1-3 layers to 5-6 layers or more, accompanied by cellulose exudation and collagen fiber deposition. , a large number of spindle-forming fibrous cells can be seen, the synovium is thickened, the synovial tissue shows different degrees of mushroom-like or papillary hyperplasia, the small blood vessels in the subsynovial layer increase, pannus forms, the tissue is loose, edema, and there are a large number of inflammatory cells Exudation, some sections can see the erosive growth of the synovial membrane towards the cartilage surface. The low-dose ethyl acetate group (D) had moderate hyperplasia and inflammatory cell infiltration in the synovial tissue, with pannus formation and a small amount of cartilage destruction; the medium-dose ethyl acetate group (E) showed moderate synovial tissue hyperplasia and a small amount of inflammatory Cell infiltration without obvious destruction of cartilage and bone tissue; in the high-dose ethyl acetate group (F) the synovium was only mildly hyperplastic, with regular cell morphology, a small amount of inflammatory cell infiltration, no typical pannus formation, smooth cartilage surface, and no cartilage destruction and bone erosion. The lesion changes in the low-dose group were less than those in the model group, but the degree of lesion relief was not as good as that in the high- and middle-dose groups. The pharmaceutical composition of the present invention reduces the deformation and destruction of the ankle joint, the abnormal hyperplasia of synovial tissue, and the infiltration of inflammatory cells in a dose-dependent manner (see Figures 6-14).

4 Joint swelling degree of rats: the results are shown in Table 5. After treatment, the treatment groups and the tripterygium glycosides group were lower than the model group (P<0.01 or P<0.05), and the high-dose group was lower than the other groups (P<0.01 or P<0.05).

The influence of each group of table 5 on rat joint swelling degree

Effects of each group on swelling degree of right hind paw of CIA rats

Note: Compared with the blank group, ^# P﹤0.05, ^## P﹤0.01; compared with the model group, ^△ P﹤0.05, ^△△ P﹤0.01;

5 Contents of TNF-α, interleukin 6 (IL-6), interleukin 8 (IL-8) and interleukin 10 (IL-10) in rat serum: the results are shown in Table 6. Compared with the normal group, the serum IL-6, IL-8 and TNF-α in the model group were significantly increased (P<0.01), and the ethyl acetate and n-butanol low, middle, high and tripterygium glycoside groups were all acceptable. Down-regulate the content of IL-6, IL-8 and TNF-α in the serum of CIA rats, and there were significant differences among the ethyl acetate groups (P<0.05). There were differences in IL-6, IL-8 and TNF-α between the ethyl acetate high group and the ethyl acetate low group (P<0.05).

Table 6 Comparison of IL-6, IL-8 and TNF-α content in serum of rats in each group

Note: Compared with the normal group, ^* P﹤0.05, ^** P﹤0.01; compared with the model group, ^△ P﹤0.05, ^△△ P﹤0.01; compared with the low-dose group, ^# P<0.05.

3 Discussion

Anti-inflammatory experimental studies have shown that both ethyl acetate and n-butanol pharmaceutical compositions can significantly reduce mouse ear swelling caused by xylene, reduce carrageenan-induced rat toe swelling, and have good anti-inflammatory effects .

Analgesic experimental research shows that in the hot plate method, the three dosages of the pharmaceutical composition of the present invention are significantly different from those before administration, and can obviously prolong the pain response latency of mice, and the analgesic effect increases with the increase of dosage. Enhanced, effective analgesic effect lasts for more than 2 hours.

At the same time, the pharmaceutical composition of the invention can effectively treat type II collagen arthritis, indicating that it has a good therapeutic effect on rheumatoid arthritis.

Claims (8)

1. a bauhinia championii n-butyl alcohol extract, is characterized in that: the weight percentage that it contains cupreol must not be less than 0.1%, and described bauhinia championii n-butyl alcohol extract is prepared from by following steps:

Get Caulis Bauhiniae Championii medical material, add 40-95% alcohol heating reflux and extract, extracting solution reclaims ethanol extremely without alcohol taste, alcohol extract petroleum ether extraction, and obtain petroleum ether layer and water layer, water layer adds chloroform extraction, obtains chloroform layer and water layer; Aqueous layer with ethyl acetate extracts, and obtains ethyl acetate layer and water layer; Water layer adds n-butanol extraction, obtains n-butanol extracting liquid, reclaims, and dries, obtains bauhinia championii n-butyl alcohol extract.

2. bauhinia championii n-butyl alcohol extract according to claim 1, is characterized in that: the weight percentage that it contains cupreol is 0.1-1.1%.

3. bauhinia championii n-butyl alcohol extract according to claim 1 and 2, is characterized in that: it contain Quercetin,

4. bauhinia championii n-butyl alcohol extract according to claim 1, is characterized in that: described ethanol to be concentration be 80% ethanol.

5. prepare a method for bauhinia championii n-butyl alcohol extract according to claim 1, it comprises the steps:

Get Caulis Bauhiniae Championii medical material, add 40-95% alcohol heating reflux and extract, extracting solution reclaims ethanol extremely without alcohol taste, alcohol extract petroleum ether extraction, and obtain petroleum ether layer and water layer, water layer adds chloroform extraction, obtains chloroform layer and water layer; Aqueous layer with ethyl acetate extracts, and obtains ethyl acetate layer and water layer; Water layer adds n-butanol extraction, obtains n-butanol extracting liquid, reclaims, and dries, obtains bauhinia championii n-butyl alcohol extract.

6. the purposes of bauhinia championii n-butyl alcohol extract according to claim 1 in the medicine preparing antalgic and inflammation relieving, resisting rheumatoid arthritis.

7. treat a pharmaceutical composition for Rheumatoid arthritis, it is active component by bauhinia championii n-butyl alcohol extract according to claim 1, adds pharmaceutically acceptable adjuvant or complementary composition is prepared into pharmaceutically conventional preparation.

8. the pharmaceutical composition for the treatment of rheumatoid arthritis according to claim 7, is characterized in that: described preparation is oral formulations.