CN101322735A - Optimizing method for attenuation processing of Tripterygium wilfordii and related formulation - Google Patents
Optimizing method for attenuation processing of Tripterygium wilfordii and related formulation Download PDFInfo
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Abstract
The invention discloses two optimization methods for attenuated processing of thunder god vine and the relevant preparations thereof. The optimization method (1) is that under the optimized condition, the traditional Chinese medicine thunder god vine is processed with salt water (or processed with acid) and processed with the acid (or processed with the salt water), or acid solution containing chloride ion is used for attenuation; the optimization method (2) comprises the following steps: under the optimized condition, thunder god vine extract, or the further purified product of the thunder god vine extract, or thunder god vine preparation is processed with the salt water (or processed with the acid) and processed with the acid (or processed with the salt water), the detailed method is described in the specifications. The methods have the advantages that the relevant preparations of the thunder god vine with the relatively low toxicity are obtained by the optimization of processing method, the medicinal resources of herb, root and bark, stem leaf parts of the thunder god vine with strong toxicity are utilized better, and the curative effect of thunder god vine preparations is strengthened. The methods of the invention simultaneously have the advantages of simple operation, low cost and being easy for popularization. The relevant preparations of the invention are used for treating rheumatoid arthritis, chronic nephritis, hepatitis, thrombopenic purpura and various dermatoarthritis, and can be used for resisting generation, anti-tumor, resisting rejection in the body after organ transplantation, etc.
Description
Technical field
The present invention relates to natural drug extraction, purification and authentication method, particularly the method for modifying of Chinese medicine Radix Tripterygii Wilfordii and extract thereof.
Background technology
Radix Tripterygii Wilfordii general reference Celastraceae tripterygium plant, in homemade four kinds, wherein have three kinds to be used as medicine, promptly Radix Tripterygii Wilfordii (Triptergiumwilfordii Hook.f.) originates in provinces and regions such as Zhejiang, Anhui, Hunan, Fujian, mainly in area, the middle and lower reach of Yangtze River; Tripterygium hypoglaucum (T.hypoglaucum Hutch) originates in the Yangtze river basin and southwest; Tripterygium regelii Spragus et Takeda (T.regelii Spragnc etTakcda) claims black climing again, mainly originates in northeast and Japan.Radix Tripterygii Wilfordii is recorded the earliest in Shennong's Herbal, clinical for many years treatment rheumatoid arthritis, chronic nephritis, hepatitis, thrombocytopenic purpura and the various dermatosis of being widely used in.The research of the aspect such as repelling at present after antifertility, antitumor, anti-organ transplantation in the body has also obtained bigger progress, is a kind of rising medicine.Since reported first in 1936 gets terpenoid pigment trypterygine from Radix Tripterygii Wilfordii root extraction separation, from tripterygium plant, isolated more than 100 kind of composition, wherein Diterpenes and alkaloids are main active component.
The Radix Tripterygii Wilfordii Herb is all poisonous, if improper use, period in a medicine often takes place with toxicity.Zoopery shows that the most responsive target organ of Radix Tripterygii Wilfordii toxicity is a gastronintestinal system, and hemopoietic system and reproductive system are the most common with digestive tract reaction.Epoxy diterpene-kind compound Radix Tripterygii Wilfordii lactone alcohol (I) also be main toxic component, and effective dose is almost suitable with toxicity dose for the main effective ingredient of treatment rheumatoid arthritis, and this hinders the further popularization of Chinese medicine Radix Tripterygii Wilfordii aspect clinical practice greatly.The important epoxy diterpene-kind compound of another one in the Radix Tripterygii Wilfordii is tripchlorolide (II), the pharmacologically active The selection result shows, II has antiinflammatory, immunosuppressant and the antifertility activity of I, and tiring is about 100～200 times of I, and has been proved no mutagenic action.Chemistry is related based on having between Compound I I and the I, utilizes the higher I of relative amount to realize that II's is semi-synthetic in laboratory.But the content of Compound I in former plant is not high yet, and this has just limited the application of semisynthesis greatly, and it is extremely low by former plant extract yield to add Compound I I, and its raw material sources have become a vital problem.
Experimentation shows that the active constituent content in the Radix Tripterygii Wilfordii leaf is higher than rhizome, but can't directly be used as medicine because toxicity is big.Though the long-time decoction of slow fire can reduce the toxicity of Radix Tripterygii Wilfordii effectively, destroyed because of many effective ingredient, its curative effect also descends to some extent.Therefore can utilize the attenuation synergistic technology of optimization that the I in tripterygium wilfordii and the extract thereof is carried out structure of modification, to obtain the Radix Tripterygii Wilfordii medicine of high-efficiency low-toxicity.
In addition, the research of Radix Tripterygii Wilfordii pharmaceutical dosage form at present mainly concentrates on increases medicine stability, dissolubility, reduction toxic and side effects aspect, but the toxicity that should reduce this medicine in the exploitation novel form improves again or keeps curative effect just very difficult.The present invention is at first by at first reducing the toxicity of Radix Tripterygii Wilfordii by optimization with different in the past concocting methods to the present invention, develop the relevant dosage form of Radix Tripterygii Wilfordii medicine on this basis, to making full use of this drug resource, the suitable crowd and the therapeutic domain that satisfy clinical application and expansion Radix Tripterygii Wilfordii preparation are significant.
Summary of the invention
The objective of the invention is to remedy the deficiencies in the prior art part, and the related preparations that a kind of cost is low, concocting method that can effectively reduce the toxic optimization of Chinese medicine Radix Tripterygii Wilfordii also prepares low toxicity on this basis is provided.Radix Tripterygii Wilfordii refers to Radix Tripterygii Wilfordii herb or part.
This method principle is: under optimized conditions, under the concentration of both having optimized and pH, directly by processed with salt (or processed with acid) and processed with acid (or processed with salt), or use the acid solution of chloride ion-containing to handle Radix Tripterygii Wilfordii, or Radix Tripterygii Wilfordii extractum or Radix Tripterygii Wilfordii the extractum product or the Radix Tripterygii Wilfordii preparation that are further purified, physicochemical property according to main activity, toxic component, make its main active, also be that main toxic component structure changes, the toxicity of Radix Tripterygii Wilfordii reduces like this, promotes its industrialization as drug developments such as antiinflammatory, antitumor.
The object of the invention can realize by following measure: under concentration of optimizing and pH value condition, by processed with salt (or processed with acid) and processed with acid (or processed with salt), or use the acid solution of chloride ion-containing to handle Radix Tripterygii Wilfordii, perhaps with Radix Tripterygii Wilfordii (herb or part) or the extractum that makes from Radix Tripterygii Wilfordii, with the extracting of middle polarity organic solvent, extract is crossed chromatography purification earlier, remove pigment and little polar impurity part, with the active part of collecting, pass through processed with salt (or processed with acid) and processed with acid (or processed with salt) again, or use the acid solution processing of chloride ion-containing to make its attenuation.At last by processed with salt (or processed with acid) and the acid solution processing Radix Tripterygii Wilfordii of processed with acid (or processed with salt) or use chloride ion-containing or extractum and middle polarity extractive with organic solvent or the Radix Tripterygii Wilfordii preparation that makes from Radix Tripterygii Wilfordii.
The preparation of concrete concocting method of the present invention and related preparations is carried out according to the following steps:
Directly handle the Chinese medicine Radix Tripterygii Wilfordii by the acid solution of processed with salt (or processed with acid) and processed with acid (or processed with salt) or use chloride ion-containing, make its attenuation, and preparation decoct, tincture, syrup, capsule, mixture, pill (drop pill, injection moulding ball etc.), electuary, slow release (microcapsule) sheet and alcohol (water) extractum; Or carry out according to the following steps;
The first step: Radix Tripterygii Wilfordii is used solvent extraction, and sucking filtration reclaims solvent, gets extractum;
Second step: with first step gained Radix Tripterygii Wilfordii extractum, use organic solvent extraction, concentrate; Concentrate is through chromatography purification, and method comprises column chromatography, thin layer chromatography, and one or more of methods such as liquid chromatograph get diterpene-kind compound;
The 3rd step: intermediate product that obtains in Radix Tripterygii Wilfordii extractum and the purge process thereof or the terpenoid behind the purification, under optimized conditions, by processed with salt (or processed with acid) and processed with acid (or processed with salt), or the acid solution of use chloride ion, in temperature range of optimizing and immersion treatment in the processing time, make its attenuation, and preparations such as preparation decoct, tincture, syrup, capsule, mixture, pill (drop pill, injection moulding ball etc.), electuary, slow release (microcapsule) sheet and alcohol (water) extractum; Or
The Radix Tripterygii Wilfordii preparation is by processed with salt (or processed with acid) and processed with acid (or processed with salt), or the acid solution of use chloride ion-containing, in temperature range of optimizing and immersion treatment in the processing time, make its attenuation, reuse organic solvent extraction, washing, must concentrate modified product, and preparations such as preparation decoct, tincture, syrup, capsule, mixture, pill (drop pill, injection moulding ball etc.), electuary, slow release (microcapsule) sheet and alcohol (water) extractum; Or
The Radix Tripterygii Wilfordii preparation is used earlier solvent extraction, again by processed with salt (or processed with acid) and processed with acid (or processed with salt), or use contains the acid solution of finite concentration chloride ion, in temperature range of optimizing and immersion treatment in the processing time, make its attenuation, reuse organic solvent extraction, washing must concentrate modified product, and preparations such as preparation decoct, tincture, syrup, capsule, mixture, pill (drop pill, injection moulding ball etc.), electuary, slow release (microcapsule) sheet and alcohol (water) extractum.
The acid solution of the above chloride ion-containing, acid solution are selected from acid solutions such as hydrochloric acid, sulphuric acid, acetic acid, nitric acid, acetic acid, carbonic acid, phosphoric acid, oxalic acid, Fluohydric acid., citric acid, perchloric acid; The solution of chloride ion-containing is selected from the solution of various hydrochlorates such as sodium chloride, potassium chloride, calcium chloride, magnesium chloride, ammonium chloride, and the concentration of optimization is that 0.001% (weight ratio) is to saturated; The optimization pH value scope of used chloride ion-containing acid solution is-2.0-6.0; The temperature ranges of optimizing is-20 ℃-50 ℃; The processing time of optimizing is more than 5 minutes.
Optimization method of the present invention is compared with prior art has following advantage: the inventive method is easy and simple to handle, with low cost, prove through pharmacological evaluation, the extractum that Radix Tripterygii Wilfordii (herb or part), Radix Tripterygii Wilfordii make, the product behind Radix Tripterygii Wilfordii or the extractum purification, the Radix Tripterygii Wilfordii preparation is through processed with salt (or processed with acid) and processed with acid (or processed with salt), or use contains the acid solution of finite concentration chloride ion, under optimization pH value and temperature, handle certain hour, its toxicity is obviously descended, and drug effect is still remarkable.
Concrete embodiment:
Enumerate 16 embodiment below, the present invention is further specified, but the present invention is not only limited to these embodiment.The present invention is a material with tripterygium plant-Radix Tripterygii Wilfordii, Tripterygium hypoglaucum and the Tripterygium regelii Spragus et Takeda etc. of China's abundant, and the basic physicochemical property according to its main activity, toxic component by special method of modifying, reduces its toxicity at last.
Embodiment 1:
Take by weighing Radix Tripterygii Wilfordii 200g, soak, in-20 ℃ of refrigerators, placed 5 minutes simultaneously with concentrated hydrochloric acid.Take out back chloroform extraction 3 times, each 300ml, extracting solution washes with water to neutrality, the reuse anhydrous Na
2SO
4Drying obtains concentrate 5.68g behind the concentrating under reduced pressure, the preparation preparation.Add the 20mL water dissolution, filter the back and add potassium sorbate 2g, preparation decoct, mixture; Or add hypromellose (HPMC) mix homogeneously, and make soft material as wetting agent in the molten ethanol of citric acid, granulate, drying, granulate adds the magnesium stearate mixing, tabletting; Or dry, grind the back and cross 400 mesh sieves, add the 0.8mg stearic acid after getting 40mg and 10mg sucrose mix homogeneously, No. 5 capsulae vacuuses of packing into seal, and prepare capsule; Or add water, and add simple syrup, add the glycerol hydrotropy, the preparation syrup.
Embodiment 2:
Take by weighing Radix Tripterygii Wilfordii 200g,, in 4 ℃ of refrigerators, placed 48 hours simultaneously with the hydrochloric acid solution immersion of pH6.0.Take out back chloroform extraction 3 times, each 300ml, extracting solution washes with water to neutrality, the reuse anhydrous Na
2SO
4Drying obtains concentrate 5.37g behind the concentrating under reduced pressure, the preparation preparation.The related preparations preparation method is with embodiment 1.
Embodiment 3:
Take by weighing Radix Tripterygii Wilfordii 200g,, placed 48 hours at 50 ℃ simultaneously with the hydrochloric acid solution immersion of pH6.0.Take out back chloroform extraction 3 times, each 300ml, extracting solution washes with water to neutrality, the reuse anhydrous Na
2SO
4Drying obtains concentrate 5.62g behind the concentrating under reduced pressure, the preparation preparation.The related preparations preparation method is with embodiment 1.
Embodiment 4:
Take by weighing Radix Tripterygii Wilfordii 200g, after 2 hours, add perchloric acid and regulate pH-2.0, in 4 ℃ of refrigerators, placed 48 hours simultaneously with the immersion of NaCl saturated solution.Take out back chloroform extraction 3 times, each 300ml, extracting solution washes with water to neutrality, the reuse anhydrous Na
2SO
4Drying obtains concentrate 5.72g behind the concentrating under reduced pressure, the preparation preparation.The related preparations preparation method is with embodiment 1.
Embodiment 5:
Take by weighing Radix Tripterygii Wilfordii 200g, after 2 hours, add sulphuric acid and regulate pH6.0, placed 48 hours at 4 ℃ simultaneously with the immersion of NaCl saturated solution.Take out back chloroform extraction 3 times, each 300ml, extracting solution washes with water to neutrality, the reuse anhydrous Na
2SO
4Drying obtains concentrate 3.05g behind the concentrating under reduced pressure, the preparation preparation.The related preparations preparation is with method embodiment 1.
Embodiment 6:
Take by weighing Radix Tripterygii Wilfordii 200g, after 2 hours, add 0.001%NaCl solution, regulate pH-2.0, in 4 ℃ of refrigerators, placed 48 hours simultaneously with the perchloric acid solution immersion.Take out back chloroform extraction 3 times, each 300ml, extracting solution washes with water to neutrality, the reuse anhydrous Na
2SO
4Drying obtains concentrate 5.11g behind the concentrating under reduced pressure, the preparation preparation.The related preparations preparation method is with embodiment 1.
Embodiment 7:
Take by weighing Radix Tripterygii Wilfordii 200g, after 2 hours, add 0.001%NaCl solution, regulate pH6.0, in 4 ℃ of refrigerators, placed 48 hours simultaneously with the sulfuric acid solution immersion.Take out back chloroform extraction 3 times, each 300ml, extracting solution washes with water to neutrality, the reuse anhydrous Na
2SO
4Drying obtains concentrate 4.86g behind the concentrating under reduced pressure, the preparation preparation.The related preparations preparation method is with embodiment 1.
Embodiment 8:
Take by weighing Radix Tripterygii Wilfordii 200g, soak, regulate pH-2.0, placed 48 hours at 4 ℃ simultaneously with the 0.001%NaCl-perchloric acid solution.Take out back chloroform extraction 3 times, each 300ml, extracting solution washes with water to neutrality, the reuse anhydrous Na
2SO
4Drying obtains concentrate 5.34g behind the concentrating under reduced pressure, the preparation preparation.The related preparations preparation method is with embodiment 1.
Embodiment 9:
Take by weighing Radix Tripterygii Wilfordii 200g, soak, regulate pH6.0, placed 48 hours at 4 ℃ simultaneously with saturated NaCl-sulfuric acid solution.Take out back chloroform extraction 3 times, each 300ml, extracting solution washes with water to neutrality, the reuse anhydrous Na
2SO
4Drying obtains concentrate 5.12g behind the concentrating under reduced pressure, the preparation preparation.The related preparations preparation method is with embodiment 1.
Embodiment 10:
The first step: take by weighing Radix Tripterygii Wilfordii 650g, add 800ml95% ethanol, reflux, extract, 3 times, each 6 hours, filter, merge ethanol liquid, decompression recycling ethanol gets total composition extractum 12.0g.
Second step: take by weighing Radix Tripterygii Wilfordii 130g, add the 300ml ethyl acetate, reflux, extract, 3 times, each 6 hours, filter, combined ethyl acetate liquid, the reclaim under reduced pressure ethyl acetate must be mainly the extractum 1.56g of middle polarity chemical compound.Extractum 6.0g with the first step obtains adds the 25ml ethyl acetate, supersound extraction 3 times, and each 45min filters, combined ethyl acetate liquid, the reclaim under reduced pressure ethyl acetate gets extractum 1.31g.With 1.56g extractum, after the amount of ethyl acetate dissolving, admix the 4.4g neutral alumina, peroxidating aluminum post, discard ethyl acetate: petroleum ether (1: 9) eluent 250ml, collect ethyl acetate: the eluent 450ml of petroleum ether (1: 1), concentrating under reduced pressure gets sample A.1.31g extractum with after the amount of ethyl acetate dissolving, is admixed the 3.2g neutral alumina, and peroxidating aluminum post discards ethyl acetate: petroleum ether (1: 9) eluent 250ml, collect ethyl acetate: the eluent 450ml of petroleum ether (1: 1), concentrating under reduced pressure gets sample B.
The 3rd step: sample A and sample B soaked with the Sal saturated solution respectively add vinegar after 2 hours and regulate pH1-2, handled 36 hours at 4 ℃, under the room temperature with ethyl acetate extraction 3 times, each 200ml, extracting solution washes with water to neutrality, the reuse anhydrous Na
2SO
4Drying adds 10 times of amount ethanol behind the concentrating under reduced pressure, stir, and transfers to pH 5-6, leaves standstill, and wait to precipitate and draw supernatant fully, after 65 ℃ of decompression recycling ethanols concentrate, the preparation preparation.The related preparations preparation method is with embodiment 1.
Embodiment 11:
10 of leaf of Radix Tripterygii Wilfordii total esters sheets, grind the back and extract three times (50mL * 3) with the ethyl acetate Soxhlet, extracting solution concentrates the back and adds sulphuric acid adjusting pH1-2 after 2 hours with saturated NaCl solution soaking, handled 36 hours at 4 ℃, use ethyl acetate extraction 3 times under the room temperature, each 200ml, extracting solution washes with water to neutrality, the reuse anhydrous Na
2SO
4Drying, behind the concentrating under reduced pressure, the preparation preparation.The related preparations preparation method is with embodiment 1.
Embodiment 12:
15 of LEIGONGTENG JIAONANGJI, remove the hungry area softgel shell after, use saturated NH
4The Cl solution soaking added vinegar after 3 hours, handled 36 hours at 4 ℃, used chloroform extraction 3 times under the room temperature, each 20ml, and extracting solution washes with water to neutrality, the reuse anhydrous Na
2SO
4Drying, behind the concentrating under reduced pressure, the preparation preparation.The related preparations preparation method is with embodiment 1.
Embodiment 13:
LEIGONGTENG TANGJIANGJI 500mL, with chloroform extraction three times (3 * 200mL), concentrating under reduced pressure behind the merge extractive liquid,, concentrate adds acetic acid with saturated NaCl solution soaking after 3 hours, handled 36 hours at 0 ℃, three times (3 * 100mL), extracting solution washes with water to neutrality, the reuse anhydrous Na with chloroform extraction under the room temperature
2SO
4Drying, behind the concentrating under reduced pressure, the preparation preparation.The related preparations preparation method is with embodiment 1.
Embodiment 14:
10 of Glucosidorum Tripterygll Totorums grind the back with ethyl acetate extraction three times (50mL * 3), use saturated CaCl after extracting solution concentrates
2Solution soaking adds nitric acid and regulates pH1-2 after 2 hours, handled 36 hours at 4 ℃, uses ethyl acetate extraction 3 times under the room temperature, each 200ml, and extracting solution washes with water to neutrality, the reuse anhydrous Na
2SO
4Drying, behind the concentrating under reduced pressure, the preparation preparation.The related preparations preparation method is with embodiment 1.
Embodiment 15:
30 of LEIGONGTENG DIWANJI are used saturated NH
4The Cl solution soaking added vinegar after 3 hours, regulated pH1-2, handled 36 hours at 4 ℃, used chloroform extraction 3 times under the room temperature, each 20ml, and extracting solution washes with water to neutrality, the reuse anhydrous Na
2SO
4Drying, behind the concentrating under reduced pressure, the preparation preparation.The related preparations preparation method is with embodiment 1.
Embodiment 16:
LEIGONGTENG CHONGJI 15g uses saturated MgCl
2Solution soaking added phosphoric acid after 3 hours, regulated pH1-2, handled 36 hours at 4 ℃, used ethyl acetate extraction 3 times under the room temperature, each 20ml, and extracting solution washes with water to neutrality, the reuse anhydrous Na
2SO
4Drying, behind the concentrating under reduced pressure, the preparation preparation.The related preparations preparation method is with embodiment 1.
Enumerate the experiment of acute toxicity testing and antiinflammatory below, effect of the present invention is further specified, but the present invention is not only limited to these embodiment.
One, acute toxicity testing
1 experiment material
1.1 medicine is not concocted the powder of Radix Tripterygii Wilfordii extractum preparation; The powder of the low toxicity Radix Tripterygii Wilfordii extractum preparation that new method is concocted adds 0.5%CMC-Na solution and is made into suspension, and pH 6.0.
1.2 (body weight 18～22g) ♂ ♀ dual-purposes are provided by Hebei province's Experimental Animal Center animal cleaning level kunming mice.Animal feeding in the dustless environment of cleaning, 18～22 ℃ of temperature, relative humidity 55%, illumination 12h, feedstuff and bedding and padding are provided by Hebei province's Experimental Animal Center.
2 methods
Oral LD
50Determine the dosage range of oral administration by trial test, between agent than being 1: 0.75-0.85, administration volume are 0.2ml/10g.110 of mices, male and female half and half are divided into 11 groups at random by body weight, 10 every group: the normal saline group, do not concoct Radix Tripterygii Wilfordii and concoct each 5 various dose group of Radix Tripterygii Wilfordii, at one night of fasting before the experiment, freely drink water.1 gastric infusion is measured oral LD by " new drug preclinical study guide "
50, fasting 4h after the administration tightly observes reaction and death condition after the mice administration, and observes mice activity, gait, feed, two day by day and just wait situation, and record dead animal number is observed dead mice important organ and is changed, and observes continuously 7 days.
Two, antiinflammatory experiment
1 material
1.1 animal and grouping
((body weight 150 ± 20g), ♂ ♀ dual-purpose provides by Hebei province's Experimental Animal Center a cleaning level kunming mice with the Wistar rat for body weight 18～22g).Animal feeding in the dustless environment of cleaning, 18～22 ℃ of temperature, relative humidity 55%, illumination 12h, feedstuff and bedding and padding are provided by Hebei province's Experimental Animal Center.Be divided into 6 groups at random: normal saline matched group (model group), prednisone positive controls, do not concoct Radix Tripterygii Wilfordii group, concoct three dosage groups of the high, medium and low dosage of Radix Tripterygii Wilfordii, 10 every group.Provide by Hebei province's Experimental Animal Center.
1.2 medicine
Do not concoct the powder of Radix Tripterygii Wilfordii extractum preparation; The powder of the low toxicity Radix Tripterygii Wilfordii extractum preparation that new method is concocted adds 0.5%CMC-Na solution and is made into suspension, and pH 6.0.
1.3 reagent
Dimethylbenzene (analytical pure, the prosperous chemical materials in Tianjin factory); 0.9%NaCl injection (Siyao Co., Ltd., Shijiazhuang); Evans Blue (U.S. Sigma, E-2129); Injection penicillin (North China pharmaceutical Co. Ltd, lot number: 00408040).
2 methods
2.1 dimethylbenzene induced mice ear inflammation test
60 kunming mices are divided into 6 groups at random, adopt not isoconcentration medicinal liquid gastric infusion of isometric(al), 0.2ml/10g body weight, every day 1 time, for three days on end, after the last administration 1 hour, dimethylbenzene 0.1ml evenly is applied to the mouse right ear auricle, and left ear is a blank, and 2h post-tensioning neck is put to death, with the operation basal part of the ear of having one's hair trimmed left and right sides ear is cut, with diameter be the gmm card punch about
1.3 reagent
Auricle is laid at the same position of ear, weighs on the analytical balance, as the swelling degree, and calculates suppression ratio with the difference of left and right sides auricle weight.
2.2 mouse peritoneal capillary permeability experiment
Get the kunming mice of 60 male and female half and half, grouping, administration are all with 2.1.Give Mus tail iV1% azovan blue 0.1ml/10g after irritating stomach 30min on the 3rd day, and ip 1% acetic acid 0.2mL/ immediately.20min post-tensioning neck is put to death, and with 6ml normal saline gradation flushing abdominal cavity, in centrifuge tube, adjusts final volume to 10mL, the centrifugal 5min of 4000rpm with suction pipe sucking-off flushing liquor.Get supernatant and measure optical density (OD) value in 22PC spectrophotometer 590nm place, and calculate suppression ratio.
2.3 cotton balls brings out the experiment of mice granuloma
Make 70 50mg cotton balls, volume is consistent as far as possible, in order to avoid influence the granuloma hypertrophy.Cotton balls is put into 121 ℃ of sterilizations of test tube 30min.Get 60 kunming mices and be divided into 6 groups.Mice is put into super-clean bench,, fix its extremity on the Mus plate with pin with the light anaesthesia of ether ball, from the center to periphery sterilization twice, take off iodine with iodine tincture with 75% ethanol, treat that ethanol is dried after, cut the 1cm osculum with operating scissors from ventrimeson, use ophthalmology tweezer separate skin gently.With the groin place that another ophthalmology tweezer is implanted mice one side with cotton balls, sew up then.Postoperative mouse peritoneal injection penicillin is in case infect.Postoperative was irritated stomach, successive administration 7 days the same day.Administration 1 time again in the 8th day is put to death mice, and is separated the granuloma ball, rejects fatty tissue, in 70 ℃ of oven dry 12 hours, weighs, and its weight deducts the raw cotton ball weight, and to be granuloma heavy, with the analysis that takes statistics of the granuloma weight of every g body weight.
2.4 carrageenin is induced rat ankle swelling experiment
Get 60 of the healthy male Wistar rats of body weight (150 ± 20) g, be divided into 6 groups at random, 10 every group, adopt not isoconcentration medicinal liquid gastric infusion of isometric(al), every day 1 time, successive administration 7d.40min after the last administration, the right back sufficient pad subcutaneous injection 10g/L of portion carrageenin 0.1ml causes inflammation rat, respectively cause scorching before and cause scorching back 0.5,1,2,4,6h, measure rat with the capillary tube volumetric method and cause 0.5cm place, scorching limb ankle joint below volume, with causing inflammation front and back difference as the swelling degree.
2.5 rat assist agent arthritis (adjuvant arthritis, AA) model
2.5.1 the model preparation adds the bacillus calmette-guerin vaccine (80 ℃ of water-bath 1h) of deactivation in the aseptic liquid paraffin, the Freund Freund's complete adjuvant is made in emulsifying.The concentration of bacillus calmette-guerin vaccine is 10g/L, and 0.1ml causes inflammation in the left back toes intradermal injection of every Mus Freund Freund's complete adjuvant.
2.5.2 observation index
Get 60 of Wistar rats, ♂, grouping is the same.Cause scorching preceding 3d ig administration, before causing inflammation, cause scorching back 6h, 12h, 18h and 24h cause scorching forward and backward rat with the detection of capillary tube volumetric method and cause scorching parapodum swelling, to observe AA rat constitutional pathological changes; Cause scorching back 12d and begin the ig administration, and detect the non-scorching parapodum swelling that causes, per 4 days 1 time, so that difference is the swelling degree before and after scorching.Carry out joint scoring (0～4) simultaneously: 0: do not have redness, 1: sufficient little toe redness and swelling of joints, 2: toe joint redness, the sufficient sole of the foot are all red and swollen, and 3: ankle joint is following all red and swollen, and 4: comprise ankle joint, whole red and swollen; Except that causing scorching side, the integration sum of tripodia is the joint total mark, is up to 12 fens, with score value AA rat polyarthritis is assessed, to observe AA rat Secondary cases pathological changes.
Overall merit
Show by pharmacological evaluation, with compare without Radix Tripterygii Wilfordii of concocting or product or the Radix Tripterygii Wilfordii preparation behind Radix Tripterygii Wilfordii extractum or the Radix Tripterygii Wilfordii purification, Radix Tripterygii Wilfordii of concocting with new method or product or the Radix Tripterygii Wilfordii preparation behind Radix Tripterygii Wilfordii extractum or the Radix Tripterygii Wilfordii purification, its toxicity obviously descends, LD
50By becoming (931.10 ± 0.02) mg/kg, (840.10 ± 0.02) mg/kg by original (279.4 ± 0.02) mg/kg, (252.0 ± 0.02) mg/kg respectively.It is evident in efficacy that product of the present invention is used for the treatment of rheumatoid arthritis.In the research of aspects such as treatment chronic nephritis, hepatitis, thrombocytopenic purpura and various dermatoarthritises, also obtained bigger progress at present.
Claims (8)
1, the optimization method of attenuation processing of Tripterygium wilfordii and related preparations is characterized in that
(1), under the concentration conditions of optimizing, the product or the Radix Tripterygii Wilfordii preparation that are further purified by processed with salt (or processed with acid) and processed with acid (or processed with salt) Chinese medicine Radix Tripterygii Wilfordii or Radix Tripterygii Wilfordii extractum or Radix Tripterygii Wilfordii extractum, or the acid solution of the chloride ion-containing of the pH value scope of use optimization, in the temperature range of optimizing with soak in the processing time, make its attenuation, and preparation decoct, tincture, syrup, capsule, mixture, pill (drop pill, injection moulding ball etc.), electuary, slow release (microcapsule) sheet and alcohol (water) extractum; Or
(2), carry out according to the following steps:
The first step: Radix Tripterygii Wilfordii is used solvent extraction, and sucking filtration reclaims solvent, gets extractum;
Second step: with first step gained Radix Tripterygii Wilfordii extractum, use organic solvent extraction, concentrate; Concentrate is through chromatography purification, and method comprises column chromatography, thin layer chromatography, and one or more of methods such as liquid chromatograph get diterpene-kind compound;
The 3rd step: intermediate product that obtains in Radix Tripterygii Wilfordii extractum and the purge process thereof or the terpenoid behind the purification are by processed with salt (or processed with acid) and processed with acid (or processed with salt), or the acid solution of the certain density chloride ion-containing of the pH value scope of use optimization, in the temperature range of optimizing with soak in the processing time, make its attenuation, and preparations such as preparation decoct, tincture, syrup, capsule, mixture, pill (drop pill, injection moulding ball etc.), electuary, slow release (microcapsule) sheet and alcohol (water) extractum.Or
(3), the process of preparing Chinese medicine of Radix Tripterygii Wilfordii preparation:
The Radix Tripterygii Wilfordii preparation is by processed with salt (or processed with acid) and processed with acid (or processed with salt), or the acid solution of the certain density chloride ion-containing of the pH value scope of use optimization, in the temperature range of optimizing, soak in the processing time of optimizing, make its attenuation, reuse solvent extraction, washing must concentrate modifier, and preparation decoct, tincture, syrup, capsule, mixture, pill (drop pill, injection moulding ball etc.), electuary, slow release (microcapsule) sheet and alcohol (water) extractum; Or
The Radix Tripterygii Wilfordii preparation is used earlier solvent extraction, again by processed with salt (or processed with acid) and processed with acid (or processed with salt), or the acid solution of the certain density chloride ion-containing of the pH value scope of use optimization, in the temperature range of optimizing, soak in the processing time of optimizing, make its attenuation, reuse organic solvent extraction, washing, must concentrate modifier, and preparation decoct, tincture, syrup, capsule, mixture, pill (drop pill, injection moulding ball etc.), electuary, slow release (microcapsule) sheet and alcohol (water) extractum.
2, concocting method according to claim 1 is characterized in that described Radix Tripterygii Wilfordii is meant Radix Tripterygii Wilfordii herb or part.
3, concocting method according to claim 1 is characterized in that described salt is Sal and other hydrochlorates, and described acid comprises vinegar, acetic acid, perchloric acid, sulphuric acid, nitric acid, acetic acid, hydrochloric acid, carbonic acid, phosphoric acid, oxalic acid, Fluohydric acid., citric acid etc.
4, be used for the treatment of rheumatoid arthritis, chronic nephritis, hepatitis, thrombocytopenic purpura and various dermatoarthritis by the low toxicity Radix Tripterygii Wilfordii herb of the described new method process of preparing Chinese medicine of claim 1 or the related preparations of part preparation, and the medicine or the health promoting product that repel in the body behind the antifertility, antitumor, anti-organ transplantation, and other control fast slow releasing pharmaceutical such as solid dispersion medicament, medicine and health product such as cyclodextrin inclusion compound medicament.
5, the temperature range by the described optimization of claim 1 is-20 ℃-50 ℃.
6, the pH value scope by the described optimization of claim 1 is-2.0-6.0.
7, be more than 5 minutes by the described optimization process of claim 1 time.
8, be 0.001% to saturated by the concentration of the saline solution of the described chloride ion-containing of claim 1.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104306426A (en) * | 2014-11-06 | 2015-01-28 | 江西中医药大学 | Processing method of thunder god vine |
| CN110241247A (en) * | 2019-07-12 | 2019-09-17 | 华润三九医药股份有限公司 | A kind of molecular labeling, primer pair and discrimination method identified for tripterygium wilfordii |
-
2007
- 2007-06-11 CN CNA2007101108136A patent/CN101322735A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104306426A (en) * | 2014-11-06 | 2015-01-28 | 江西中医药大学 | Processing method of thunder god vine |
| CN104306426B (en) * | 2014-11-06 | 2017-06-20 | 江西中医药大学 | A kind of concocting method of thunder godvine |
| CN110241247A (en) * | 2019-07-12 | 2019-09-17 | 华润三九医药股份有限公司 | A kind of molecular labeling, primer pair and discrimination method identified for tripterygium wilfordii |
| CN110241247B (en) * | 2019-07-12 | 2023-06-20 | 华润三九医药股份有限公司 | Molecular marker and primer pair for identifying tripterygium wilfordii medicinal materials and identification method |
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