CN101780124A - Capparis acutifolia sweet exact and preparation method and application thereof - Google Patents

Abstract

The invention discloses an extract of a thousand miles alone and its preparation method and application. The extract of the plant is taken from the rhizome of the plant, crushed, heated to reflux, cold soaked or leaked for extraction, concentrated under reduced pressure to obtain a crude extract, which is obtained by extracting the crude extract with chloroform, which can be applied to the preparation of neuropathic neuropathic Pain, treatment of inflammatory pain, treatment of cancer pain, treatment of diabetes pain, treatment of rheumatism pain, treatment of pain from bruises, treatment of sore throat, treatment of toothache or treatment of abdominal pain drugs, has good analgesic and anti-inflammatory activities , It has obvious effect on Freund's complete adjuvant arthritis. The preparation process of the invention has the characteristics of low cost and easy industrial production.

Description

A kind of Lonely Thousand Miles Extract and its preparation method and application

technical field

The invention belongs to the technical field of medicines, and in particular relates to an extract of a thousand miles alone, its preparation method and its application in the preparation of anti-inflammatory, analgesic and anti-rheumatic arthritis drugs.

Background technique

It is the dry root of Capparisacutifolia Sweet [C.membranacea Gardn.et Champ.], a plant of Capparaceae (Capparaceae). Alias button button (Guangdong milk source), membranous leaf mallet fruit vine, Duhulong, black snake, black hook banyan (Hainan), etc., produced in Guangdong, Guangxi, Jiangxi (Dayu), Fujian (Shaxian, Chong'an) , Yanping), Hunan (Yizhang), Taiwan; born in low-altitude shrubs, forests, wilderness or hillside roadsides. This product is a folk herbal medicine in southern my country. Flat in nature, bitter in the mouth, astringent, poisonous; Function promoting blood circulation and dispelling blood stasis, relieving spasm and relieving pain. It is used to treat rheumatic arthralgia, muscle and bone discomfort, sore throat, and abdominal pain; the root or leaves are used externally to treat sores, furuncles, swollen toxins, and traumatic injuries.

At present, the clinical application of Lonely Thousand Miles is mostly simple processing and use, which can be taken orally or externally; crude extraction preparations have complex components, and generally have the shortcomings of being thick, large, and black. Defects such as difficult control and unstable therapeutic effect. Under the guidance of pharmacodynamics, it is urgent to screen the effective parts of Lonely Thousand Miles and carry out applied research. However, there is no report on the extraction process and optimal extraction conditions of the effective parts of Lonely Thousand Miles. Research reports on anti-inflammatory and anti-rheumatic pharmacology.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies in the prior art and provide a kind of extract of a thousand miles alone.

Another object of the present invention is to provide a preparation method of the extract.

Another object of the present invention is to provide the application of the extract of the thousand miles alone.

The purpose of the present invention is achieved through the following technical solutions:

Provided is an extract of the solitary plant, which is obtained by taking the rhizome of the plant climax, pulverizing it, heating to reflux, cold soaking or seepage extraction, concentrating under reduced pressure to obtain a crude extract, and extracting it with chloroform.

The present invention provides the preparation method of described Alone Thousand Miles extract, comprises the following steps:

(1) Dried rhizomes of Duxingqianli are crushed into coarse powder;

(2) using ethanol extraction, reflux extraction or ultrasonic extraction with a volume ratio concentration of 70% to 95% of the rhizome powder of the Rhizome of Lone Flower; combine the extracts and concentrate under reduced pressure to obtain the extract;

Or use ethanol with a volume ratio concentration of 70% to 95% to leak and extract the rhizome powder of Duxingqianli; combine the extracts, concentrate under reduced pressure to obtain the extract;

(3) After the extract obtained in step (2) is purified by column chromatography, the effective part of the independent journey can be obtained: the chloroform part or the ethyl acetate part;

Alternatively, the extract obtained in step (3) is degreased with petroleum ether, extracted with chloroform, refluxed with chloroform or ultrasonically extracted with chloroform, and the extract is decompressed to recover chloroform to obtain a thick paste, which is the effective part of a thousand miles alone.

Step (2) If ethanol extraction is used, the amount of ethanol is preferably added according to 8 to 20 times the weight of the rhizome powder of Lone Walking Thousand Miles; it is preferably extracted 3 times, each time for 1 to 7 days;

Step (2) If ethanol is used for reflux extraction, the amount of ethanol is preferably added according to 8 to 20 times the weight of the rhizome powder of Lone Walking Thousand Miles; it is preferably extracted 2 to 3 times, each time for 1 to 6 hours;

Or the ethanol ultrasonic extraction described in step (2), the addition of ethanol is preferably added according to 8 to 20 times the weight of the rhizome powder of Lone Walking Thousand Miles, and the ultrasonic extraction is 2 to 3 times, 1 to 6 hours each time;

Or the ethanol seepage extraction described in step (2), preferably at a flow rate of 1 to 3 ml/sec, until the seepage is lighter in color.

The present invention also simultaneously provides another preparation method of the extract of a thousand miles alone, comprising the following steps:

(A) the rhizome of Duxingqianli is dried and pulverized into coarse powder;

(B) using 60-80% ethanol to reflux to extract the rhizome powder of Lonely Thousand Miles, combine the extracts, concentrate under reduced pressure to obtain the extract;

(C) After acidifying (preferably acidifying to a pH value of 2 to 3) with 1 to 2% (volume ratio concentration) of dilute acid water (hydrochloric acid or sulfuric acid, etc.), the acid water liquid is adjusted to a pH value of 9 to 9 with ammonia water. 11. Chloroform extraction, the thick paste obtained by recovering chloroform under reduced pressure is the effective part of Lone Walking Thousand Miles.

The amount of ethanol added in step (B) is 8 to 20 times the weight of the rhizome powder of Lone Walking Thousand Miles, and the extraction is performed 3 times, each time for 1 to 6 hours.

The present invention successfully extracts anti-inflammatory, analgesic and anti-arthritic effective parts from the plant. The raw material is pulverized and then heated to reflux, cold soaked or leaked to extract, and concentrated under reduced pressure to obtain a crude extract, which is sequentially extracted with petroleum ether, chloroform, and acetic acid. Ethyl ester, n-butanol, acetone, water, etc. are extracted to obtain different parts and undergo pharmacological rough screening. In the present invention, the chloroform parts are subjected to qualitative reactions in test tubes of mercuric potassium iodide, bismuth potassium iodide, and silicotungstic acid alkaloid indicators. , all showed a positive reaction, indicating that the effective part of Duxingqianli prepared by the present invention is the total alkaloid component. After systematic anti-inflammatory and analgesic and anti-Freund's complete adjuvant arthritis experiments, its anti-inflammatory, analgesic, anti-adjuvant The effect of drug-induced arthritis is obvious, which proves that it has significant pharmacological effects, and can be used to prepare drugs for anti-inflammatory and analgesic, anti-rheumatic arthritis, rheumatoid arthritis, gout, and frozen shoulder, providing a new choice for clinical medication .

Therefore, the present invention also provides the application of the extract of Duxingqianli in the preparation of anti-inflammatory and analgesic drugs, anti-rheumatic arthritis, rheumatoid arthritis, gout, and frozen shoulder.

Preferably, the extract of the lone traveler can be applied to the preparation of medicines for treating neuropathic pain, medicine for treating inflammatory pain, medicine for treating cancer pain, medicine for treating pain in diabetes, medicine for treating rheumatism pain, pain from bruises, etc. , sore throat, toothache, abdominal pain (especially spasmodic pain) and other drugs.

For the above application, the preferred dose is 0.5-200 mg/kg body weight.

The effective part can be prepared into various pharmaceutical preparations by adding pharmaceutically acceptable excipients, and the preparation forms include oral tablets, capsules, pills, granules, powders, oral liquids or emulsions; or external lotions, ointments, cataplasms elixirs, patches or gels.

The beneficial effects of the present invention are:

The present invention fully extracts, concentrates, and separates the effective components in the medicinal material of the lone thousand miles, and the obtained effective part (the extract of the lone thousand miles) accounts for about 0.2 to 1.5% of the weight of the medicinal material, has remarkable pharmacological effects, and can Anti-inflammatory analgesic, anti-rheumatoid arthritis.

The effective part obtained by the present invention is better than the crude extract used as medicine, and is convenient to be made into various preparations, providing a new choice for clinical medicine.

The method of the invention is reasonable in design and has the characteristics of simple process, low cost and low energy consumption.

Description of drawings

Figure 1 Structural diagram of the ankle joint of model group rats under endoscopic light microscope

Figure 2 Lone Travel Thousand Miles Effective Part High-dose Group on the structure diagram of rat ankle joint cavity light microscope

Figure 3 Lone Walking Thousand Miles effective part of the middle dose group on the rat ankle cavity light microscope structure diagram

Figure 4 Lone Travel Thousand Miles effective part small dose group on rat ankle cavity light microscope structure diagram

Fig. 5 Positive control group to rat ankle arthroscopy structural diagram

Detailed ways

The present invention will be described in further detail below in conjunction with the accompanying drawings and specific embodiments. Through a large number of experiments, the technical solution of the present invention is obtained. For the summary, see the aforementioned content of the invention. The preferred schemes of the parameters such as the amount of ethanol added, the number of times of extraction and the time in the extraction of ethanol extraction, reflux extraction, ultrasonic extraction, and seepage extraction are different. The examples are described in detail, but do not limit the scope of the present invention.

Embodiment 1 The preparation of the effective part of a thousand miles alone (i.e. the extract of a thousand miles alone)

Get about 2 kilograms of the crushed Lone Walking Thousand Miles (rhizome), add 12 times the amount (weight: volume) of 95% ethanol to extract for 7 days, filter the residue and then use 10 times the amount of 95% alcohol to extract it for 5 days, and filter the residue again Extract with 10 times the amount of 95% ethanol for 3 days, combine the filtrates, recover the ethanol under reduced pressure, and obtain the extract. The extract is mixed with 60g of silica gel and put on a silica gel column, eluting with petroleum ether, chloroform, ethyl acetate, n-butanol and acetone respectively, and recovering the solvent under reduced pressure, the obtained chloroform parts and ethyl acetate parts are respectively 5.8g and 6.1 g.

Example 2 The preparation of the effective fraction of traveling thousands of miles alone

Take about 2 kg of crushed Lone Walking Thousand Miles (rhizome), add 12 times the amount of 85% ethanol and sonicate (100KHZ) 3 times, each time for 45 minutes, to obtain about 86.2 g of extract. After the extract was mixed well with 60g of silica gel, it was extracted 3 times with petroleum ether, chloroform, ethyl acetate, n-butanol, and acetone under reflux, each time with 10 times the amount of solvent, and extracted for 1 hour; the solvent was recovered under reduced pressure, and the obtained chloroform part , ethyl acetate parts were 9.2g and 10.9g.

Example 3 The preparation of the effective part of traveling thousands of miles alone

Take about 2 kilograms of crushed Lone Walker (rhizome), add 12 times the amount of 85% ethanol to reflux and extract 3 times, respectively for 2 hours, 1 hour, and 1 hour, to obtain about 95.2 g of extract. After the extract was mixed well with 60g of silica gel, it was extracted 3 times with petroleum ether, chloroform, ethyl acetate, n-butanol, and acetone under reflux, each time with 10 times the amount of solvent, and extracted for 1 hour; the solvent was recovered under reduced pressure, and the obtained chloroform part , ethyl acetate parts were 11.1g and 12.6g, respectively.

Example 4 Preparation of effective fractions of traveling thousands of miles alone

Take about 2 kg of the crushed Lone Walking Thousand Miles (rhizome), add 95% ethanol to extract by leakage, the flow rate is 1-3ml/min, until the color of the extract is light and there is no alkaloid color reaction, recover the ethanol, and obtain about 91.3g of extract . After degreasing petroleum ether, suspend in water, then extract with chloroform, ethyl acetate, n-butanol, and acetone respectively, and concentrate the obtained extracts to obtain 6.3 g of chloroform fraction and 7.2 g of ethyl acetate fraction, respectively.

Example 5 The preparation of the effective part of traveling thousands of miles alone

Dried rhizomes of Lone Walking Thousand Miles were crushed, and 20, 15, and 10 times the amount of 60-80% (volume ratio concentration) ethanol was added to reflux extraction for 3 times, and the extraction time was 2, 1.5, and 1.5 hours respectively. The combined extracts were concentrated under reduced pressure to The extract is acidified with 1-2% dilute acid water (hydrochloric acid, sulfuric acid or nitric acid, etc.), the pH value of the acid water solution is adjusted to 9 with ammonia water, extracted with chloroform, and the thick paste obtained by recovering chloroform under reduced pressure is the chloroform part of Lone Walking Thousand Miles 5.46g.

The chloroform parts prepared in Examples 1-5 were subjected to qualitative reactions in test tubes of mercuric potassium iodide, bismuth potassium iodide, and silicotungstic acid alkaloid indicators, all of which showed positive reactions.

Embodiment 6 smear preparation

Weigh 120g of polyvinyl alcohol (preferably 60g each of PVA05-88 and 17-88), wet it with 10g of glycerin and add it to 300ml of distilled water to soak and swell. In addition, take 50g of the effective fraction (chloroform fraction) obtained by the method of any one of the above 1~5 examples and add it together with 30g of benzoic acid into 500ml of ethanol and stir until the medicine is basically dissolved or uniformly suspended, and then slowly add In the polyvinyl alcohol liquid, stir as it is added, stir well and then filter.

Embodiment 7 ointment preparation

Weigh 100g of stearic acid and melt it on a water bath, add 20g of lanolin, 120g of white vaseline and heat it to 80°C in a water bath, and add 50g of the effective part obtained by the method of any one of the above-mentioned 1 to 5 examples; add 120g of glycerin and Heat 500ml of distilled water to 90°C, then add 10g of sodium lauryl sulfate and 1g of ethylparaben to dissolve into the water phase, then slowly pour the water phase into the oil phase, and stir until it condenses to obtain Creamy ointment.

Embodiment 8 plaster preparation

Take the raw rubber, wash it, cut it into strips of appropriate size, masticate it into a mesh film in a rubber mixer, immerse it in an appropriate amount of solvent gasoline, soak it until it is completely swollen into a gel, and move it into a paste barrel and stir for 3 After ~4 hours, add vaseline, lanolin, rosin, zinc oxide, etc. in turn to make a matrix according to the conventional method, then add 5 ~ 50 g of the effective part of the lone thousand miles obtained by the method of any one of the above-mentioned 1 ~ 5 embodiments, continue to stir for 3 hours, wait for When it has become a uniform paste, it can be obtained after coating, cutting, lining and packaging.

Embodiment 9 gel preparation

Take an appropriate amount of Carbomer 980 and wet it with 10ml of propylene glycol, gradually slowly stir and add 90ml of water to make it fully swell; then add 1% sodium benzoate solution dissolved in 10ml of water; Grind and suspend 5-50 g of the effective part of Qianli with 90 ml of propylene glycol, then add it to the colloid, stir evenly, and add triethanolamine dropwise under stirring to obtain a gel.

Embodiment 10 cataplasm preparation

Ultrez 10(卡伯母，市购)撒在去离子水15ml中，低速搅拌30min，得到A相溶液；称取独行千里有效部位浸膏0.1～20g，加入少量水，加入10ml甘油、透皮促进剂，不断搅拌，水浴加热使熔化得B相；高岭土、二氧化钛混合均匀，再与聚丙烯酸钠NP700混合均匀，加入剩余甘油润湿，研匀，将熔化的稠膏加入，研匀；山梨醇、亚硫酸氢钠、EDTA用28ml去离子水溶解，逐渐缓慢加入到B相中；甘羟铝、柠檬酸溶解于18ml水中，为C相；搅拌中将C相透明溶液加入B相，再将A相加入B、C相的混合物中。研磨均匀后涂布在无纺布上，室温下放置过夜，干燥后，与保护层复合，切割成6×8cm²包装即可。1~20g of

Sprinkle Ultrez 10 (Cabomu, commercially available) in 15ml of deionized water, stir at a low speed for 30min to obtain a phase A solution; weigh 0.1-20g of the extract from the effective part of Duxingqianli, add a small amount of water, add 10ml of glycerin, and a skin penetration enhancer , continuously stirred, heated in a water bath to melt to obtain phase B; kaolin, titanium dioxide mixed evenly, then mixed with sodium polyacrylate NP700 evenly, added the remaining glycerin to moisten, grinded evenly, added the melted thick paste, grinded evenly; Sodium bisulfate and EDTA were dissolved in 28ml of deionized water, and gradually added to phase B; aluminum glycolate and citric acid were dissolved in 18ml of water, which was phase C; while stirring, add the transparent solution of phase C to phase B, and then add phase A Add to phase B and C mixture. Grind it evenly and spread it on the non-woven fabric, leave it overnight at room temperature, after drying, compound it with the protective layer, cut it into 6×8cm ² and pack it.

Embodiment 11 patch preparation

Weigh 1-30g of polyvinyl alcohol 17-88, add distilled water to soak overnight, after it is fully swollen, reflux in water bath until dissolved, and set aside. Precisely weigh 0.1-20g of the active part extract of Duxingqianli, add 10-30ml of absolute ethanol, 5-20ml of glycerin, and 800.1-1ml of Tween, heat and dissolve properly, add polyvinyl alcohol 17-88 glue while it is hot, and stir slowly Mix evenly, coat and dry, that is to say.

Example 12 tablet

Take 50g of the effective part of Duxingqianli obtained by the method of any one of the above-mentioned 1 to 5, pass through an 80-mesh sieve after crushing, mix with 120g of starch, 20g of lactose, and 10g of sodium carboxymethyl starch, add 8% starch slurry in an appropriate amount to make soft Material, cross 20 mesh sieves to make wet granules, after drying at 60°C for granulation, add 0.8% magnesium stearate of dry granule weight as lubricant, press into 1000 plain tablets, and coat with film to get final product.

Embodiment 13 capsules

After taking by weighing 120g of starch, 20g of dextrin, and 10g of sodium carboxymethyl starch, and mixing them evenly, slowly add the medicine (take 50g of the effective part of a thousand miles obtained by the method of any one of the above-mentioned 1 to 5 embodiments, add an appropriate amount of 95% ethanol, and melt in a water bath liquid), made into soft materials, passed through a 20-mesh sieve to make wet granules, dried at 60°C for granulation, and then filled with No. 2 capsule shells.

Embodiment 14 pill preparation

Weigh 50 g of the effective part of Duxingqianli obtained by the method of any one of the above 1 to 5 examples, crush it and pass it through an 80-mesh sieve, and use an appropriate amount of honey as a binder to form a pill.

Embodiment 15 Granule preparation

Weigh 300g of powdered sugar, 50g of lactose, and 150g of dextrin and mix well, then slowly add the drug (take 5g of the effective part of a thousand miles obtained by the method of any one of the above 1 to 5 embodiments, add 95% ethanol in an appropriate amount of water bath to melt into a liquid), Made into soft materials, passed through a 14-mesh sieve to make wet granules, dried at 60°C with a 12-mesh sieve for granulation, and packed separately (each bag weighs 5g), and then packed.

Embodiment 16 powder preparation

Take 5g of the effective part of Duxingqianli obtained by the method of any one of the above-mentioned 1-5 examples, add 100g of starch, mix evenly, pack 1g per pack, and pack it.

Embodiment 17 emulsion preparation

Dry 5-50g of the effective part extract of Duxingqianli and pass through a 120-mesh sieve into fine powder, add 110ml of soybean oil and mix evenly, add soy lecithin solution (add 11g of soy lecithin to 18ml of glycerin, add about 50ml of water, grind and dilute to 250ml with distilled water), Add distilled water to 1000ml, homogenize twice with a colloid mill, and obtain.

Example 18 The analgesic experiment of the extract of Lone Walking Thousand Miles

Animals: ICR mice (provided by the Animal Center of Guangzhou University of Traditional Chinese Medicine), female, weighing 20.34±1.43g.

Drugs and reagents: effective parts of Duxingqianli (prepared with 0.5% carboxymethylcellulose (CMC) for suspension); aspirin tablets (Shijiazhuang Pharmaceutical Co., Ltd., specification 0.3g/tablet, with 0.5% CMC before use) Suspension); 0.5% CMC solution.

(1) Twisting method: 50 mice were adaptively fed for three days, randomly divided into 5 groups according to body weight, and fed with chloroform parts (converted into medicinal materials) of 40g/kg (small dose group) and 80g/kg respectively. kg (medium dose group), 120g/kg (high dose group), aspirin 200mg/kg (positive control group), 0.5% CMC solution (blank control group), administered continuously for 5 days. Fasted for 16 hours before the experiment, free to drink water, 30 minutes after intragastric administration, 0.2ml of 0.6% glacial acetic acid solution was injected intraperitoneally, the number of writhing times of the mice within 40 minutes was recorded, and the t-test between groups was performed. The results are shown in Table 1:

Table 1 The effect of effective fractions on the number of times of writhing in mice (x±SD, n=10)

Note: Compared with the control group, *P<0.01; △P<0.05

The experimental results showed that, compared with the blank group, both the aspirin group and the high-dose group could significantly reduce the number of mice writhing (P<0.01); the large, medium and low-dose groups could all reduce the number of mice writhing (P<0.05) .

(2) Hot plate method: 50 mice (primary screening pain threshold is 10-30s), adaptively fed for three days, randomly divided into 5 groups according to body weight, and fed with 40 g of chloroform parts (converted into medicinal materials) /kg (low-dose group), 80g/kg (middle-dose group), 120g/kg (high-dose group), aspirin 200mg/kg (positive control group), 0.5% CMC solution (blank control group), administered continuously for 5 sky. 40 minutes after the last administration, the pain threshold was measured again. Those whose pain threshold exceeds 60s are counted as 60s. The pain threshold after taking the medicine is subtracted from the pain threshold before taking the medicine to get the pain threshold extension time after taking the medicine. The results are shown in Table 2:

Table 2 The effect of the effective parts of Lone Walking Thousand Miles on the pain domain of mice (x±SD, n=10)

Note: Compared with the control group, *P<0.01

The results showed that compared with the blank group, the aspirin group, high-dose, and medium-dose groups could significantly increase the hot-plate pain threshold of mice; the low-dose group could not significantly improve the hot-plate pain threshold of mice. The results of analgesic experiment show that the effective part of Duxingqianli has obvious analgesic effect at high and medium doses.

Example 19 Anti-inflammation experiment of the effective part of traveling thousands of miles alone

1. Anti-inflammatory model caused by xylene-induced ear swelling in mice

Animals: ICR mice (provided by the Animal Center of Guangzhou University of Traditional Chinese Medicine) are male and female, with a body weight of 20.31±1.66 g.

Equipment: mouse gavage device, BP-211D electronic analytical balance (Beijing Sartorius Balance Co., Ltd.), 50μL adjustable micro sampler, 6mm hole punch

Drugs and reagents: effective parts of Duxingqianli (prepared with 0.5% carboxymethylcellulose (CMC) for suspension); aspirin tablets (Shijiazhuang Pharmaceutical Co., Ltd., specification 0.3g/tablet, with 0.5% CMC before use) Suspension); 0.5% CMC solution.

50 mice were adaptively fed for three days, randomly divided into 5 groups according to body weight, 10 in each group, and fed with the effective part (converted into medicinal material) 40g/kg (small dose group), 80g/kg (medium dose group) and 10 mice in each group. dose group), 120g/kg (high dose group), aspirin 200mg/kg (positive control group), 0.5% CMC solution (blank control group), once a day for 5 consecutive days. One hour after the last administration, each mouse was coated with 0.03ml of xylene on both sides of the left ear to cause inflammation. After 30 minutes, the animal was killed by dislocation. The round ears at the place were weighed with an electronic analytical balance, and the swelling degree and anti-inflammatory inhibition rate were calculated. The results are shown in Table 3.

Swelling degree = left ear piece weight - right ear piece weight

Anti-inflammatory inhibition rate (R)=(average swelling degree of control group-average swelling degree of administration group)/average swelling degree of control group×100%

Table 3 The effect of the effective parts of Lone Traveling Thousand Miles on the ear swelling of mice caused by xylene (x±SD, n=10)

Note: Compared with the control group, *P<0.05, △P<0.01; compared with the aspirin group, ☆P<0.05

From the experimental results, it can be seen that compared with the control group, the large, medium and small dose groups of the effective part of Lone Walking Thousand Miles all showed different degrees of inhibition of the swelling of the ear shells of mice, and the effect tended to increase with the increase of the dose. The high dose group had extremely There was a significant difference (P<0.01), and there was a significant difference between the medium and small dose groups (P<0.05). Compared with the aspirin group, there was a significant difference in the high-dose group (P<0.05), and there was no significant difference in the medium-dose and low-dose groups.

2. Inflammation model of mouse toe swelling induced by egg white

Fifty mice, half male and half male, were randomly divided into 5 groups, 10 mice in each group. Feed the effective parts of Lone Walking Thousand Miles (converted into the amount of medicinal material) 40g/kg (low dose group), 80g/kg (medium dose group), 120g/kg (high dose group), aspirin 200mg/kg (positive control group), 0.5 % CMC solution (blank control group), administered once a day for 5 consecutive days. One hour after the last administration, 0.03 ml of egg white was intradermally injected into the left hind paw of the mice, and the volume of the right hind paw was measured before and 1 h and 2 h after the injection. Increased percentage of paw swelling (%)=(right hind paw volume after inflammation-right hind paw volume before inflammation)/right hind paw volume before inflammation×100%. The experimental results are shown in Table 4.

Table 4 The effect of the effective parts of Lone Walking Thousand Miles on mouse toe swelling caused by egg white (x ± SD)

Note: Compared with the control group, △P<0.01; compared with the aspirin group, ☆P<0.01

The experimental results showed that compared with the blank control group, the aspirin group and the administration group all showed a significant effect of inhibiting inflammation and swelling at 2 hours (P<0.01); compared with the aspirin group, there was also a significant difference between the high-dose group and the aspirin group.

3. Mouse peritoneal capillary permeability experiment

Fifty mice, half male and half male, were randomly divided into 5 groups, 10 mice in each group. Feed the effective parts of Lone Walking Thousand Miles (converted into the amount of medicinal material) 40g/kg (low dose group), 80g/kg (medium dose group), 120g/kg (high dose group), aspirin 200mg/kg (positive control group), 0.5 % CMC solution (blank control group), administered once a day for 5 consecutive days. One hour after the last administration, take Evans blue saline solution, inject it into the tail vein with 0.1ml/10g, and immediately inject 0.2ml of 0.6% glacial acetic acid intraperitoneally. After 20 minutes, the spine was cut off, the abdominal cavity was cut open with scissors, washed repeatedly with 5ml of normal saline, the washing liquid was collected in a centrifuge tube, centrifuged at 1000r/min for 5 minutes to take the filtrate, and the filtrate was measured with a UV2300 spectrophotometer. The OD value was measured at a wavelength of 590nm, and the results are shown in Table 5.

Table 5 The influence (x ± SD) of the effective fraction of the lone travel thousands of miles on the capillary permeability of mice

Note: Compared with the control group, △P<0.01

The experimental results show that the effective part of Lone Walking Thousand Miles has a good anti-inflammatory effect.

Example 20 Therapeutic Effects of Effective Parts of Lonely Traveling Thousand Miles on Adjuvant Arthritis in Rats

In this experiment, a rat adjuvant arthritis (AA) model was established with Freund's complete adjuvant (FAC), and the thickness of the paws of the rats was measured with a vernier caliper. , to observe the anti-inflammatory effect of the effective part of Lonely Thousand Miles.

Drugs: effective parts of Duxingqianli, Zhengqing Fengtongning Tablets (Xi'an Lijun Group)

Reagent: Freund's Adjuvant Complete (FAC) product of SIGMA, USA.

Animals: SD rats, male, weighing 180-200 g. Provided by the Experimental Animal Center of Guangzhou University of Traditional Chinese Medicine.

Instrument: A℃S-1EAS electronic balance (accuracy 0.5g) product of Beijing Feimusi Technology Development Co., Ltd.

50 rats were fed adaptively for three days, and were randomly divided into 5 groups according to their body weight. 120g/kg (high-dose group), Zhengqing Fengtongning Tablets group, model group, after continuous administration for 2 days. On the third day, after measuring the normal foot thickness with a vernier caliper, inject 0.1ml of Freund's complete adjuvant intradermally into the right hind toe of each rat, and measure the swelling degree of the inflammatory hind foot at 6h, 18h, 24h, 3d, and 5d respectively , and at 10d, 14d, 18d, 22d, and 26d, the thickness of the left hind toe (non-inflammatory foot) was measured respectively.

Taking the degree of paw swelling as an index, SPSS13.0 statistical software package was used for one-way analysis of variance. The results of acute and secondary inflammation experiments are shown in Table 6 and Table 7, respectively.

Table 6. Acute inflammation test results of effective parts of Lone Walking Thousand Miles on rats (right foot swelling) (x±SD, n=10)

Note: Compared with the model group: P>0.05*; P<0.01***; 0.05>P>0.01**

From the experimental results, it can be known that acute inflammation occurs within 24 hours. Compared with the model group, each dose of the administration group and the positive control group have obvious anti-inflammatory effects on it. After 24 hours, it was the formation period of secondary inflammation. Although each administration group had a certain inhibitory effect on the formation of secondary inflammation, it was not particularly obvious. After 3 days, the anti-inflammatory effects of the administration group and the positive control group were weak. It can be seen that the high, medium and low dose groups of the effective part of Duxingqianli all have inhibitory effect on the acute inflammation of adjuvant arthritis in rats.

Table 7 The experimental results of the effective parts of Lonely Thousand Miles on secondary inflammation in rats (swelling degree of left foot) (x±SD, n=10)

Note: Compared with the model group: P<0.01***; 0.05>P>0.01**

From the experimental results in Table 7, it can be seen that within 18 days is the formation period of secondary inflammation, and the drug has a certain inhibitory effect on the formation of secondary inflammation, and the effect is relatively mild and insignificant. After 18 days, secondary inflammation formed, and the anti-inflammatory effect of the drug began to be effective. Compared with the blank group, the high-dose and middle-dose groups and the positive control group all had obvious anti-inflammatory effects. There was an extremely significant difference (P<0.01***) in the high-dose group at 26 days, and a significant difference between the middle-dose group and the positive control group (P<0.05).

Example 21 Effect of the effective part of Duxingqianli on the pathological changes of adjuvant arthritis in rats 1 Tissue fixation and slicing methods

Material collection, fixation and decalcification: after the 26th day of the drug efficacy experiment, the rats were killed, and the hind limbs were cut off at 15 mm above the ankle joint, soaked and fixed in 10% neutral formalin for 48 hours, and then decalcified with formic acid Soak for 4 weeks to decalcify.

Rinse: Rinse with running water for 24 hours to remove the formic acid decalcification solution.

Dehydration: dehydration with series concentration of ethanol step by step, 70% ethanol for 30min, 80% ethanol for 30min, 90% ethanol for 30min, 95% ethanol for 30min, absolute ethanol I for 60min, absolute ethanol II for 30min.

Transparent: xylene I 20min, xylene II observation until the tissue is transparent.

Wax immersion: The transparent tissue was immediately placed in paraffin wax melted in an oven in advance, paraffin wax I 60min, paraffin wax II 60min.

Embedding: Place the tissue in an embedding plate filled with melted paraffin, and wait for the paraffin to solidify into a block.

Slicing, patching and baking: Cut the wax block into 6 μm thin slices with a paraffin microtome, stick the wax slice on a glass slide coated with protein glycerin, and store it at room temperature for staining after overnight in an incubator at 45°C.

2HE staining steps

Put the dried slices into xylene I, xylene II, and xylene III for 5 minutes each to dewax; transfer them into absolute ethanol I, absolute ethanol II, 95% ethanol, 90% ethanol, 80% ethanol, and 70% ethanol in sequence , 60% ethanol, 50% ethanol, and rehydration in distilled water, each for 5 minutes; hematoxylin staining for 5 minutes; tap water and distilled water for 3 times; 1% hydrochloric acid ethanol color separation for 5 seconds; tap water for 3 times, ammonia alkalization 10 seconds; wash 3 times with distilled water; stain with 0.5% eosin alcohol for 13 minutes; transfer into 90% ethanol, 95% ethanol, absolute ethanol I for 0.5 minutes each; absolute alcohol II for 5 minutes; xylene I, xylene II, xylene III each for 5 minutes; finally seal the slide with neutral gum.

Experimental results:

On the 26th day after inflammation, AA rat ankle tissue slices were observed under a microscope. In the model group, inflammatory cell infiltration in the synovium was seen, the synovial tissue was swollen, and a small amount of fibrous tissue exuded; the synovial cells proliferated to a certain extent, arranged Irregular; subsynovial telangiectasia, vascular lumen filled with inflammatory cells. See attached drawing 1.

Microscopically, the synovial tissue showed less inflammatory cells, less swollen synovial tissue and no obvious exudation of fibrous tissue in each administration group; synovial cell hyperplasia but lower than that in the model group; synovial capillary endoluminal inflammation There were few sex cells, as shown in accompanying drawings 2 to 4, and the results of the positive control group were shown in accompanying drawing 5.

Claims (10)

1. an extract of a solitary plant, which is characterized in that the rhizome of a plant is taken, pulverized, heated to reflux, cold soaked or leaked to extract, and concentrated under reduced pressure to obtain a crude extract, which is obtained by extracting the crude extract with chloroform. 2. a preparation method of the described Lone Walking Thousand Miles extract of claim 1, is characterized in that comprising the following steps: (1) The rhizome of Duxingqianli is dried and crushed into coarse powder; (2) using ethanol extraction with a volume ratio concentration of 95%, reflux extraction or ultrasonic extraction of the rhizome powder of Rhizome of Lone Flower; combine the extracts and concentrate under reduced pressure to obtain the extract; Or use ethanol with a volume ratio concentration of 70% to 95% to leak and extract the rhizome powder of Duxingqianli; combine the extracts, concentrate under reduced pressure to obtain the extract; (3) After the extract obtained in step (2) is purified by column chromatography, the effective part of traveling thousands of miles is obtained: the chloroform part; Alternatively, the extract obtained in step (3) is degreased with petroleum ether, extracted with chloroform, refluxed with chloroform or ultrasonically extracted with chloroform, and the extract is recovered under reduced pressure to obtain a thick extract, which is the Lone Walker Thousand Miles extract. 3. The preparation method according to claim 2, characterized in that the amount of ethanol used in the ethanol leaching of step (2) is added according to 8 to 20 times the weight of the rhizome powder of Lone Walking Thousand Miles; preferably leaching 3 times, 1 day to 7 days each time; Or the ethanol reflux extraction described in step (2), the amount of ethanol added is added according to the amount of 8 to 20 times the weight of the rhizome powder of Lonely Thousand Miles; preferably extracted 2 to 3 times, each time for 1 to 6 hours; Or the ethanol ultrasonic extraction described in step (2), the addition of ethanol is added according to the amount of 8 to 20 times of the weight of the rhizome powder of Lonely Thousand Miles, and the ultrasonic extraction is 2 to 3 times, each time for 1 to 6 hours; Or the flow rate of the ethanol leakage extraction described in step (2) is 1-3ml/sec. 4. a preparation method of the described Lone Walking Thousand Miles extract of claim 1, is characterized in that comprising the following steps: (A) the dry rhizome of Duxingqianli is pulverized; (B) using 60-80% ethanol to reflux to extract the rhizome powder of Lonely Thousand Miles, combine the extracts, concentrate under reduced pressure to obtain the extract; (C) After acidifying with dilute acid water, adjust the pH value of the acid water solution to 9-11 with ammonia water, extract with chloroform, and recover the chloroform under reduced pressure to obtain a thick paste, which is the effective part of Lone Walking Thousand Miles. 5. The preparation method according to claim 2, characterized in that the amount of ethanol added in step (B) is 8 to 20 times the weight of the rhizome powder of Lone Walker Thousand Miles, and the extraction is performed 3 times, each time for 1 to 6 hours. 6. the application of the described Lone Walking Thousand Miles extract of claim 1, is characterized in that being applied to the medicine of preparation anti-inflammatory analgesia, anti-rheumatic arthritis, treatment of rheumatoid arthritis, treatment of gout or periarthritis of shoulder. 7. The application according to claim 6, characterized in that it is applied to prepare and treat neuropathic pain, treat inflammatory pain, treat cancer pain, treat diabetes pain, treat rheumatism pain, treat traumatic injury pain, treat sore throat Pain, toothache, or abdominal pain medication. 8. The application according to claim 6 or 7, characterized in that it is applied to the preparation of medicines for the treatment of spastic pain. 9 . The application according to claim 6 or 7 , characterized in that the application dose of the extract of the solitary thousand miles is a single dose of 0.5-200 mg/kg body weight. 10. The application according to claim 6, characterized in that the excipients allowed by the pharmacy are prepared into oral tablets, capsules, pills, granules, powders, oral liquids or emulsions with the extract of the solitary traveler ; or prepared as an external lotion, ointment, poultice, patch or gel.

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