CN102654488A

CN102654488A - Method for detecting dripping pills of Chinese medicine composite with function of removing heat to cool blood and promoting blood circulation to stop pains

Info

Publication number: CN102654488A
Application number: CN2012101389959A
Authority: CN
Inventors: 付立家; 付建家
Original assignee: Beijing Asia East Bio Pharmaceutical Co Ltd
Current assignee: Beijing Asia East Bio Pharmaceutical Co Ltd
Priority date: 2009-04-10
Filing date: 2009-04-10
Publication date: 2012-09-05

Abstract

The invention discloses a method for detecting dripping pills of a Chinese medicine composite with the function of removing heat to cool blood and promoting blood circulation to stop pains. The composite comprises the following components in parts by weight: 14.08-25.12 parts of tree peony bark, 22.4-44.8 parts of rhizoma chuanxiong and 1.6-2.7 parts of borneol. The Chinese medicine composite has good effects of removing heat to cool blood and promoting blood circulation to stop pains, shows obvious analgesic and spasmolytic effects and has good treatment effects for obstruction of qi in the chest and cardiodynia. The quality detecting method of the Chinese medicine composite preparation provided by the invention has the advantages of good specificity, high stability, good reproducibility and high precision, thereby being more suitable for industrial production and really ensuring safety, effectiveness and reliability of clinical medication.

Description

A kind of clearing heat and cooling blood, the detection method of promoting blood circulation and stopping pain Chinese medicine composition pill

The present invention is for dividing an application, and the original bill application number is 200910081762.8, and the original bill applying date is on April 10th, 2009, and the original bill name is called a kind of clearing heat and cooling blood, promoting blood circulation and stopping pain Chinese medicine composition and preparation, detection method.

Technical field

The present invention relates to a kind of detection method of Chinese medicine composition pill, particularly a kind of clearing heat and cooling blood, the detection method of the Chinese medicine composition pill of promoting blood circulation and stopping pain.

Background technology

Along with the development of society, social competition and pressure that people face are increasing, have seriously influenced people's healthy and quality of life.The angiocardiopathy that is called " human health first killer " has become Chinese first cause of death, and coronary heart disease is exactly wherein topmost a kind of heart disease.According to statistics, per 100 Chinese more than 40 years old 4-7 people is just arranged is patients with coronary heart disease.

Present composition preparation has clearing heat and cooling blood, and the effect of promoting blood circulation and stopping pain is through years of researches and clinical practice; Prescription is perfect, special effect, have no side effect, and the partial heat type is light, the moderate chest impediment and cardialgia being used to treat, the pain dysphoria with smothery sensation of holding concurrently; The tongue fur look yellow, has better curative effect.

Summary of the invention

First purpose of the present invention is to provide a kind of clearing heat and cooling blood, the Chinese medicine composition of promoting blood circulation and stopping pain; Second purpose of the present invention is to provide the preparation method of this Chinese medicinal composition preparation.The 3rd purpose of the present invention is to provide the quality determining method of this Chinese medicinal composition preparation.

The present invention seeks to realize through following technical scheme:

A kind of clearing heat and cooling blood of the present invention, the raw material of the Chinese medicinal composition preparation of promoting blood circulation and stopping pain consists of:

Moutan bark 14.08-25.12 weight portion, Ligusticum wallichii 22.4-44.8 weight portion, borneol 1.6-2.7 weight portion.

A kind of clearing heat and cooling blood of the present invention, the raw material composition of the Chinese medicinal composition preparation of promoting blood circulation and stopping pain is preferably:

Moutan bark 20.1 weight portions, Ligusticum wallichii 33.6 weight portions, borneol 2.18 weight portions.

A kind of clearing heat and cooling blood of the invention described above, the preparation method of the Chinese medicinal composition preparation of promoting blood circulation and stopping pain is:

Moutan bark is ground into meal, and percolation extracts, and makes solvent with 60-100% ethanol, and cold soaking 12-48 hour, slowly diacolation was collected percolate, reclaims ethanol; Rhizoma Chuanxiong power is broken into fragment, extracting in water volatile oil 1-3 hour, and the WS after the distillation inclines and; Dregs of a decoction boiling 0.5-3 hour again filter, and filtrating merges with the above-mentioned WS; Being condensed into relative density is the clear cream of 1.1-1.4 (70 ℃), adds ethanol and makes the alcohol amount of containing be 30-70%, stirs; Left standstill 12-48 hour, and drew supernatant, reclaim ethanol; Merge moutan bark and Ligusticum wallichii soup, continue to be evaporated to thick paste; According to common process; Process clinical or pharmaceutically acceptable formulation, include but not limited to dripping pill, tablet, capsule, powder, soft capsule, honeyed bolus, pill, granule, soft extract with bee honey agent, sustained release preparation, quick releasing formulation, controlled release preparation, oral liquid, ejection preparation or external preparation;

A kind of clearing heat and cooling blood of the present invention, the preparation method of the Chinese medicine composition pill of promoting blood circulation and stopping pain is preferably:

Moutan bark is ground into meal, and percolation extracts, and makes solvent with 90% ethanol, cold soaking 24 hours, and slowly diacolation is collected percolate, reclaims ethanol; Rhizoma Chuanxiong power is broken into fragment, extracting in water volatile oil 2 hours, and the WS after the distillation inclines and; Dregs of a decoction boiling 1 hour again filters, and filtrating merges with the above-mentioned WS; Being condensed into relative density is the clear cream of 1.20～1.25 (70 ℃), adds ethanol and makes that to contain alcohol amount be 50%, stirs; Left standstill 24 hours, and drew supernatant, reclaim ethanol; Merge moutan bark and Ligusticum wallichii soup, continue to be evaporated to thick paste (about 6.6～6.8g); Taking polyethylene glycol 6000 31.2g in addition, heating, treat whole fusions after, add above-mentioned thick paste, rhizoma chuanxiong volatile oil and borneol, stir, from top to bottom, splash in the whiteruss, with the dripping pill drop that is shaped to the greatest extent and the erasing liquor paraffin body, process 1000 balls, promptly get.

A kind of clearing heat and cooling blood of the present invention, the preparation method of the Chinese medicinal composition granules of promoting blood circulation and stopping pain is preferably: above three flavors, moutan bark is ground into meal; According to the percolation under liquid extract and the extract item, make solvent with 90% ethanol, cold soaking 24 hours; Slowly diacolation is collected percolate, reclaims ethanol.Rhizoma Chuanxiong power is broken into fragment, extracting in water volatile oil 2 hours, and the WS after the distillation inclines and; Dregs of a decoction boiling 1 hour again filters, and filtrating merges with the above-mentioned WS; Being condensed into relative density is the clear cream of 1.20～1.25 (70 ℃), adds ethanol and makes that to contain alcohol amount be 50%, stirs; Left standstill 24 hours, and drew supernatant, reclaim ethanol.Merge moutan bark and Ligusticum wallichii soup, continue to be evaporated to thick paste (about 6.6～6.8g).Add Icing Sugar and dextrin, mixing, drying is granulated, and promptly gets.

A kind of clearing heat and cooling blood of the present invention, the preparation method of the Chinese medicinal composition capsules agent of promoting blood circulation and stopping pain is preferably: above three flavors, moutan bark is ground into meal; According to the percolation under liquid extract and the extract item, make solvent with 90% ethanol, cold soaking 24 hours; Slowly diacolation is collected percolate, reclaims ethanol.Rhizoma Chuanxiong power is broken into fragment, extracting in water volatile oil 2 hours, and the WS after the distillation inclines and; Dregs of a decoction boiling 1 hour again filters, and filtrating merges with the above-mentioned WS; Being condensed into relative density is the clear cream of 1.20～1.25 (70 ℃), adds ethanol and makes that to contain alcohol amount be 50%, stirs; Left standstill 24 hours, and drew supernatant, reclaim ethanol.Merge moutan bark and Ligusticum wallichii soup, continue to be evaporated to thick paste (about 6.6～6.8g).Add Icing Sugar and dextrin, mixing, drying is granulated, and is encapsulated, promptly gets.

The present invention provides the quality determining method of this Chinese medicinal composition preparation, and the method comprising the steps of:

A, assay: chromatographic condition and system suitability test are stationary phase with the carbowax-20M, coating concentration 10%, carrier Chromaxorbw (Aw-Dmcs); Column temperature 170-200 ℃.

Correction factor is measured, and it is an amount of to get eugenol, adds ethyl acetate and processes the solution that every 1ml contains 10-40 μ g, shakes up, as inner mark solution; Other gets the about 50-100mg of borneol reference substance, the about 2-12mg of Paeonol reference substance, and accurate the title, decide, and puts in the 10-50ml measuring bottle; Add the ethyl acetate dissolving and be diluted to scale, shake up, the accurate 2-10ml that draws, the accurate inner mark solution 0.5-1.5ml that adds; Shake up, draw 2～10 μ l, inject gas chromatograph, the calculation correction factor.Determination method: get present composition preparation, mixing is got about 1g, and accurate the title decides, and puts in the separating funnel; Add water 5-20ml, jolting constantly makes dissolving, extracts 3-5 time with ethyl acetate, merges extract, puts in the 10-50ml measuring bottle; Add ethyl acetate to scale, shake up, the accurate 2-10ml that draws, the accurate inner mark solution 0.2-1.2ml that adds shakes up; Draw 2～8 μ l, inject gas chromatograph is measured, and promptly gets; Contain moutan bark in Paeonol (C9H10O3), must not be less than 0.26%; Contain borneol (C10H18O) and must not be less than 4.8%.

B, discriminating: get present composition preparation 0.5-2g, grind, add water 10ml stirring and make dissolving, with sherwood oil (30～60 ℃), extract 2-3 time, each 5-20ml merges sherwood oil liquid, puts the tepidarium Back stroke to 1-3ml, as need testing solution; Other gets the Paeonol reference substance, adds ethanol and processes the solution that per 1 ml contains 0.5-3mg, as reference substance solution.Drawing above-mentioned two kinds of solution 1-10 μ l, put respectively on same silica gel g thin-layer plate, is developping agent with the cyclohexane-ethyl acetate mixed solvent of (2-4:1) ratio; Launch, take out, dry; Spray is with 5% ferric trichloride ethanolic solution, and it is clear that hot blast blows to the spot colour developing.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on show the spot of same color.

C, discriminating: get present composition preparation 0.5-5g, porphyrize, the 5-20ml that adds diethyl ether filters, and filtrating is as need testing solution.Other gets Ligusticum wallichii control medicinal material 0.5-5g, adds ethanol 1-10ml, and sonicated 1-15 minute, filter, filtrating is as control medicinal material solution.According to the thin-layered chromatography test, draw each 2-20 μ l of above-mentioned two kinds of solution, put in same silica G F respectively ₂₅₄On the thin layer plate, be developping agent, launch, take out, dry with the cyclohexane-ethyl acetate mixed solvent of (2-4:1) ratio.Put under the ultraviolet lamp (365nm) and inspect.In the test sample chromatogram, with the corresponding position of Ligusticum wallichii control medicinal material chromatogram on, show the spot of same color.

D, discriminating: get the borneol reference substance, add ethanol and process the solution that per 1 ml contains 1mg, as reference substance solution.Get present composition preparation 0.5-2g, grind, add water 10ml stirring and make dissolving, with sherwood oil (30～60 ℃), extract 2-3 time, each 5-20ml merges sherwood oil liquid, puts the tepidarium Back stroke to 1-3ml, as need testing solution; Drawing reference substance solution and each 1-20 μ l of need testing solution, put respectively on same silica gel g thin-layer plate, is developping agent with sherwood oil (60～90 ℃) the ethyl acetate mixed solvent of 13-25:2-4 ratio; Launch, take out, dry; Spray is with 5% vanillic aldehyde sulfuric acid solution, about 5 minutes of 105 ℃ of bakings.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color.

Above-mentioned method of quality control preferably includes following assay:

Chromatographic condition and system suitability test: with the carbowax-20M is stationary phase, coating concentration 10%, carrier Chromaxorbw (Aw-Dmcs); Column temperature 170-200 ℃.Number of theoretical plate is pressed the eugenol peak and is calculated, and should be not less than 5000.The degree of separation at Paeonol, borneol peak and eugenol peak should be greater than 2.

Correction factor is measured: it is an amount of to get eugenol, adds ethyl acetate and processes the solution that every 1ml contains 20 μ g, shakes up, as inner mark solution; Other gets the about 70mg of borneol reference substance, the about 8mg of Paeonol reference substance, and accurate the title, decide, and puts in the 25ml measuring bottle; Add the ethyl acetate dissolving and be diluted to scale, shake up, the accurate 5ml that draws, the accurate inner mark solution 1ml that adds; Shake up, draw 2～4 μ l, inject gas chromatograph, the calculation correction factor.

Determination method is got present composition preparation, and mixing is got about 1g, and accurate the title decides, and puts in the separating funnel; Add water 10ml, jolting constantly makes dissolving, extracts (8,5,5,5ml) 4 times with ethyl acetate, merges extract, puts in the 25ml measuring bottle; Add ethyl acetate to scale, shake up, the accurate 5ml that draws, the accurate inner mark solution 1ml that adds shakes up; Draw 2～4 μ l, inject gas chromatograph is measured, and promptly gets.

Preferably include following differential method in the above-mentioned quality determining method:

The moutan bark thin layer is differentiated: get present composition preparation content 1g, grind, add water 10ml stirring and make dissolving, with sherwood oil (30～60 ℃), extract 2 times, each 10ml merges sherwood oil liquid, puts the tepidarium Back stroke to 2ml, as need testing solution.Other gets the Paeonol reference substance, adds ethanol and processes the solution that per 1 ml contains 1mg, as reference substance solution.Drawing above-mentioned two kinds of solution, 5 μ l, put respectively on same silica gel g thin-layer plate, is developping agent with cyclohexane-ethyl acetate (3:1), launches, and takes out, and dries, and spray is with 5% ferric trichloride ethanolic solution, and it is clear that hot blast blows to the spot colour developing.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on show the spot of same color.

The Ligusticum wallichii thin layer is differentiated: get present composition preparation content 2g, and porphyrize, the 10ml that adds diethyl ether filters, and filtrating is as need testing solution.Other gets Ligusticum wallichii control medicinal material 1g, adds ethanol 5ml, and sonicated 5 minutes filters, and filtrating is as control medicinal material solution.Draw each 10 μ l of above-mentioned two kinds of solution, put in same silica G F respectively ₂₅₄On the thin layer plate, be developping agent, launch, take out, dry with cyclohexane-ethyl acetate (3:1).Put under the ultraviolet lamp (365nm) and inspect.In the test sample chromatogram, with the corresponding position of Ligusticum wallichii control medicinal material chromatogram on, show the spot of same color.

The borneol thin layer is differentiated: get present composition preparation content 1g, grind, add water 10ml stirring and make dissolving, with sherwood oil (30～60 ℃), extract 2 times, each 10ml merges sherwood oil liquid, puts tepidarium Back stroke to 2 ml, as need testing solution.Get the borneol reference substance, add ethanol and process the solution that per 1 ml contains 1mg, as reference substance solution.Drawing reference substance solution and moutan bark and differentiate each the 5 μ l of need testing solution under the item, put respectively on same silica gel g thin-layer plate, is developping agent with sherwood oil (60～90 ℃) ethyl acetates (17:3); Launch, take out, dry; Spray is with 5% vanillic aldehyde sulfuric acid solution, about 5 minutes of 105 ℃ of bakings.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color.

Above-mentioned quality determining method Chinese medicinal composition preparation preferred feedstock consists of: the pill of moutan bark 20.1g, Ligusticum wallichii 33.6g, borneol 2.18g; And through the preparation of following method: moutan bark is ground into meal, and percolation extracts, and makes solvent with 90% ethanol; Cold soaking 24 hours; Slowly diacolation is collected percolate, reclaims ethanol.Rhizoma Chuanxiong power is broken into fragment, extracting in water volatile oil 2 hours, and the WS after the distillation inclines and; Dregs of a decoction boiling 1 hour again filters, and filtrating merges with the above-mentioned WS; Being condensed into relative density is the clear cream of 1.20～1.25 (70 ℃), adds ethanol and makes that to contain alcohol amount be 50%, stirs; Left standstill 24 hours, and drew supernatant, reclaim ethanol.Merge moutan bark and Ligusticum wallichii soup, continue to be evaporated to thick paste (about 6.6～6.8g).Taking polyethylene glycol 6000 31.2g in addition, heating, treat whole fusions after, add above-mentioned thick paste, rhizoma chuanxiong volatile oil and borneol, stir, from top to bottom, splash in the whiteruss, with the dripping pill drop that is shaped to the greatest extent and the erasing liquor paraffin body, process 1000 balls, promptly get.

Chinese medicine composition of the present invention has good clearing heat and cooling blood, the effect of promoting blood circulation and stopping pain, and present composition preparation all shows significant analgesia, spasmolysis effect.

The quality determining method of Chinese medicinal composition preparation of the present invention; Warp experiment proof: content assaying method of the present invention detects good, the stable height of specificity, favorable reproducibility, precision height; Be fit to industrialized production more, really guaranteed safety of clinical administration, effective, reliable.

Following experimental example and embodiment are used to further specify but are not limited to the present invention.

The preparation that all experimental examples of the present invention are selected for use is the pill of the present invention that makes according to the embodiment of the invention 1, but experimental result of the present invention is not limited in this pill.

Experimental example 1: pharmacodynamic experiment

The pharmacological action of present composition preparation analgesia spasmolysis

1, test material:

Present composition preparation number draws in comminutor and grinds, with 30-40 ℃ of dissolved in distilled water to desired concn, rotundin; Oxytocin; Acetylcholine bromide; Barium chloride.

The hot plate pain threshold detector, Physiological Experiment is used appearance more.

Kunming mouse.18-22g, the male and female dual-purpose.Rabbit 2-3kg all is a healthy animal.

2, test method and result:

2.1 analgesic experiment

2.1.1 acetate is caused the influence of writhing response:

38 of mouse are divided four groups at random, and every group of male and female half and half are fed to adapt in 2 days to encircle and filled out.Respectively organize mouse before the test and irritate stomach (ig) distilled water respectively, present composition preparation 0.24g/kg, 0.48g/kg and rotundin 45mg/kg capacity are the 0.1ml/lOg body weight.1h lumbar injection 0.6% acetate 0.2ml/ only observes the interior animal of 15min immediately and turns round the body number of times behind the medicine.Each medicine of result's (table 1) all can be significantly with highly significant inhibition acetate due to writhing response, fast-acting heart disease curing two dose groups are asked no significant difference, but with the rotundin ratio notable difference are arranged.The analgesic activity of explanation present composition preparation in this model is not as good as rotundin.

Table 1 present composition preparation is to the influence of acetate writhing response

Compare with distilled water ^*P＜0.05, ^*P＜0.01, ^{* *}P＜0.001

Compare with present composition preparation ^{△ △ △}P＜0.001

2.1.2 the electricity thorn is drawn the influence of pain

Mouse is some; Use and many two acusectors that link to each other with appearance of Physiological Experiment. thrust respectively apart from the lcm of mouse tail root subcutaneous (stimulation parameter. frequency 60 times/second; Continuous wave), slowly rotate the magnitude of voltage knob and gradually strengthen voltage, when mouse occur first trachyphonia the magnitude of voltage index of having a pain; Every mouse is surveyed three times, averages.If magnitude of voltage is greater than 1.5 or less than 0.25 volt of person's rejecting.Get 40 of qualified mouse, divide equally four groups, every group of male and female half and half, ig heats up in a steamer water successively, present composition preparation 0.48g/kg and 0.96g/kg, rotundin 45mg/kg.Measure behind the medicine 60,120min is the value of closing bitterly.Heavy dose of group of 60min present composition preparation and rotundin can significantly improve pain group value behind result's (table 2) medicine, and the pain group of each medication group value all improves very significantly during 120min.Present composition preparation and rotundin do not have significant difference, and the duration is longer.

Table 2 present composition preparation is to the influence of electric shouting pain

Compare with distilled water ^*P＜0.05, ^*P＜0.01, ^{* *}P＜0.001.

2.1.3 hot plate is caused the influence of pain:

With female mice some place respectively to measure on 55 ℃ ± 0.5 ℃ the hot plate pain threshold detector lick the metapedes time in latent period, surpass 30 seconds latent period or the happiness leaper abandons it.Select 40 of qualified mouse to divide four groups at random, the lament swallow is heated up in a steamer water respectively, present composition preparation 0.48g/kg and 0.96g/kg.Rotundin 40mg/kg surveys behind the medicine 60, and the 120min mouse licks the metapedes time in latent period.Result's (table 3) present composition preparation 0.96g/kg and rotundin can obviously prolong to be licked the metapedes time in latent period.Both do not have significant difference.

Table 3 present composition preparation causes the influence of pain to the mouse hot plate

Compare with distilled water ^*P＜0.05, ^*P＜0.01, ^{* *}P＜0.001

2.2 spasmolysis

2.2.1 to the rabbit duodenal influence of exsomatizing:

5 of rabbit, 2-3kg shoots the back dead and takes out duodenum rapidly by 10 of the stripped myenteron samples of routine preparation.Respectively the intestines sample is suspended in the constant temperature Maxwell banyan groove of basket tyrode's solution 37 ± 0.5 ℃ of dark long-pending 20ml, after one section myenteron shrinkage curve of record is treated steadily earlier, carries out following experiment (experiments is all done 10 times) one by one.Influence to spontaneous activity: add present composition preparation 0.24-4.8 * 10 ^-4G/ml, myenteron is loose, and spontaneous activity disappears, and is 0.77 ± 0.49cm before the shrinkage amplitude administration, step-down behind the medicine-0.84 ± 0.59cm P<o.001.Show the spontaneous activity tool very obvious suppression effect of medicine to myenteron.

Influence to barium chloride:

With barium chloride 25 * 10 ^-4Behind the g/ml, the myenteron contraction reaches 2.79 ± 1.97cm and adds present composition preparation 4.8-7.210 immediately ^-4G/ml.Shrinkage curve is reduced to-0.28 ± 0.78cm P < 0.001.Add present composition preparation 4.8 * 10 in the ban ^-4G/ml re-uses barium chloride 25 * 10 after myenteron is suppressed ^-4G/ml, myenteron still are holddown.The proof medicine is the effect of putting forth energy altogether of antagonism barium chloride fully not only. and its effect is strong and lasting.

Influence to the acetate choline:

Use Ach2.5-5 * 10 ^-6G/ml, myenteron shrink and reach 3.75 ± 2.2cm, add present composition preparation 4.8-9.6 * 10 ^-4G/ml, curve is reduced to 0.87 ± 1.7cm, P<0.01.Show that present composition preparation also has the obvious suppression effect to Ach.

2.2.2 influence to the rabbit isolated uterine:

Get house and exempt from the uterus and prepare 8 of isolated uterine samples, the uterus sample is placed contain the perseverance of wearing krebs solution and move back Magnus' bath (volume 20mL, 3.8 ± 0.5 ℃), write down a cross-talk palace spontaneous activity treat stable after. carry out following experiment (every experiment is all done 14 times) one by one.

Influence to spontaneous activity:

Add present composition preparation 2.4-4.8 * 10 ^-4G/ml, the uterus is loose immediately, and shrinkage amplitude is reduced to-0.92 ± 1.5cm P by 1.28 ± 0.76<0.001 the prompting medicine has had strong inhibitory effects to uterine smooth muscle.

Influence to oxytocin:

During with oxytocin 0.1 μ g/ml, uterus muscle is its amplitude of spastic contraction and reaches 2.42 ± 0.69cm. adding present composition preparation 1.2-3.6 * 10 ^-4G/ml, curve progressively reduce to-0.27 ± 0.59cm, P<0.001.Use present composition preparation 2.4-4.8 * 10 in the ban ^-4Behind the g/ml, add oxytocin 0.1-0.2 μ g/ml. uterine contractile curve again and still be-0.27 ± 0.57cm.The proof medicine has complete resistant function and longer duration to oxytocin.

Turn round in body, electricity thorn and three kinds of analgesic model of hot plate, the present composition preparation of doses all shows significantly or the analgesic effect of highly significant.Electricity thorn causes in the pain present composition preparation heavy dose of (O.96g/kg) and rotundin and asks indifference in analgesia intensity and when continuing, and low dose of (0.48g/kg) plays a role later. the same very significant analgesia role that shows of 120min behind the medicine with heavy dose and rotundin.Present composition preparation needs heavy dose of significant analgesia role that just have in hot plate experiment, with also no significant difference of rotundin.Turn round that this survival dose less (0.24g/kg and 0.48g/kg) can suppress writhing response significantly in the body experiment, but effect is not as good as rotundin.

Present composition preparation causes pain at electricity thorn and hot plate and touches in the type for two kinds, no matter is in analgesia intensity and does not all have significant difference on the time with rotundin, proves that this medicine has the logical effect in town preferably really.

The myenteron experiment of exsomatizing shows, no matter present composition preparation still all is the highly significant inhibiting effect to the spastic contraction of myenteron that barium chloride and acetate choline cause to myenteron spontaneous activity.It should be noted that after present composition preparation suppresses the excitation of barium chloride.Myenteron is no longer excited when re-using barium chloride through flushing, needs repeatedly to wash and can recover, and proves that this medicine is to the strong lasting resistant function of barium chloride tool.And to the excitation of Ach, present composition preparation needs big consumption the obvious suppression effect just occur, and the duration is not long yet.Infer that thus medicine possibly be to directly act on muscle to the inhibiting effect of the myenteron that exsomatizes, rather than acting through the acceptor of myenteron.

The twin very exhibited strong inhibition effect that also shows of convulsion that present composition preparation causes the spontaneous activity and the oxytocin of rabbit isolated uterine.Except resisting the excitation of oxytocin fully; The uterus Duan Junxu of all present composition preparations effect is flushing repeatedly repeatedly, and oxytocin stimulates for several times, and muscle just can return to the excited level before the medicine; Explanation is to the strong spasmolysis of the uterus convulsion true tool of twin this medicine, and longer duration.Endometrium PGRaa increases unusually during dysmenorrhoea. and cause pain sensation fiber responsive, the contraction of the twin property of uterus convulsion, regional flow is virgin to descend, and causes the patient to have an intense pain.

More than the experiment tentative confirmation pharmacological action of present composition preparation analgesia spasmolysis.

Experimental example 2: moutan bark and the experiment of content of bornyl alcohol assay method

In order to ensure measuring real result, accurate, the present invention extracts Paeonol in the present composition sheet and borneol fully when guaranteeing to prepare need testing solution through a series of investigations.

Gas chromatography determination Paeonol and content of bornyl alcohol are adopted in this experiment, and analysis speed is fast, and is highly sensitive, and specificity is strong, applied range, and reappearance and accuracy all are superior to thin-layer chromatography-ultraviolet spectrophotometry, TLCS.

1, the selection of assay method:

The preparation of need testing solution: get the about 1g of present composition preparation, the accurate title, decide, and puts in the separating funnel, adds water 10ml; Jolting constantly makes dissolving, extracts (8,5,5,5ml) 4 times with ethyl acetate, merges extract, puts in the 25ml measuring bottle; Add ethyl acetate to scale, shake up, accurate respectively reference substance liquid 5 ml and inner mark solution 1 ml of drawing shakes up; Filter with miillpore filter (0.45 μ m), get subsequent filtrate, promptly get need testing solution.

The preparation of negative control sample solution: get the 3.36g Rhizoma Chuanxiong power and be broken into fragment, extracting in water volatile oil 2 hours, the WS after the distillation inclines and, dregs of a decoction boiling 1 hour again; Filter, filtrating merges with the above-mentioned WS, and being condensed into relative density is the clear cream of 1.20～1.25 (70 ℃); Add ethanol and make that to contain alcohol amount be 50%, stir, left standstill 24 hours; Draw supernatant, reclaim ethanol, be evaporated to thick paste (about 6.6～6.8g).Taking polyethylene glycol 6000 3.12g in addition, heating, treat whole fusions after; Add above-mentioned thick paste, rhizoma chuanxiong volatile oil and borneol, stir, from top to bottom; Splash in the whiteruss, with the dripping pill drop that is shaped to the greatest extent and the erasing liquor paraffin body, process 100 balls and lack the negative preparation of moutan bark.Get the about 1g of the negative preparation preparation of moutan bark, the accurate title, decide, and puts in the separating funnel, adds water 10ml; Jolting constantly makes dissolving, extracts (8,5,5,5ml) 4 times with ethyl acetate, merges extract, puts in the 25ml measuring bottle; Add ethyl acetate to scale, shake up, accurate respectively reference substance liquid 5 ml and inner mark solution 1 ml of drawing shakes up; Filter with miillpore filter (0.45 μ m), get subsequent filtrate, promptly get the negative control sample solution.

The preparation of reference substance solution: get borneol reference substance 70mg, Paeonol reference substance 8mg puts in the 25ml measuring bottle, adds the ethyl acetate dissolving and is diluted to scale.It is an amount of to get eugenol, adds ethyl acetate and processes every milliliter of solution that contains 20 μ g.The accurate reference substance solution 5ml that draws, the accurate eugenol inner mark solution 1ml that adds shakes up, and promptly gets borneol and Paeonol reference substance solution.

Accurate need testing solution, negative control sample solution, the reference substance solution 2 μ l of drawing, inject gas chromatograph is measured.

1.1 the selection of chromatographic column

The chromatographic column of experiment selected opposed polarity, DB1301 capillary column, 5%OV-17,10%PEG-20M, Μ lTRA-2 fused-silica capillary column.Take all factors into consideration from the chromatographic behavior of 3 kinds of materials, better with 10%PEG-20M.

1.2 the investigation of Paeonol and borneol extraction conditions in the present composition sheet

1.2.1 extract the selection of mother liquor

Borneol and Paeonol all are prone in organic solvent, dissolve.These article are to be the pill that auxiliary material is processed with the Macrogol 6000.PEG6000 is prone to dissolve in water, makes dripping pill be dissolved in water so adopt, and the method with organic solvent extraction prepares sample solution again.

1.2.2 extraction solvent is investigated

PEG6000 is prone to dissolve in water, makes dripping pill be dissolved in water so adopt, and the method with organic solvent extraction prepares sample solution again.Extract 4 times, the 5th extract sample introduction is that the result is negative.

Adopt sherwood oil (8,5,5,5 ml) to extract, extract 4 times (8,5,5,5ml) with ethyl acetate and compare, the result shows that sherwood oil can not extract fully.

1.2.3 the investigation of extraction solvent amount

Adopting ethyl acetate to extract 4 times (8,8,5,5ml), ethyl acetate respectively extracts 4 times (5,5,5,5ml), ethyl acetate and extracts 4 (8,5,5,5ml) extractions; The result shows; Ethyl acetate extracts 4 times (5,5,5,5ml) and can not extract fully, and ethyl acetate extracts 4 (8,5,5,5ml) institutes and reaches effect and ethyl acetate extraction 4 times (8,8,5,5ml) basically identical.Therefore, select for use ethyl acetate to extract (8,8,5,5ml) 4 times.

Experimental example 3: Paeonol and content of bornyl alcohol assay method confirmatory experiment

Detecting instrument (room temperature detection): Agilent 4890 type gas chromatographs

Producer: Agilent Techologies Anjelen Sci. & Tech. Inc (China)

Stationary phase: carbowax-20M

Coating concentration: 10%

Column temperature: 170-200 ℃ of carrier: Chromaxorbw (Aw-Dmcs)

The reference substance source:

Eugenol is purchased lot number: the 0736-9913 in Nat'l Pharmaceutical & Biological Products Control Institute

Paeonol is purchased lot number: the 0736-9913 in Nat'l Pharmaceutical & Biological Products Control Institute

Borneol is purchased lot number: the 0715-9909 in Nat'l Pharmaceutical & Biological Products Control Institute

Test sample lot number: 04081201,04091302,04101403

1. content assaying method is investigated:

1.1 the mensuration of the range of linearity and correction factor:

The preparation of borneol and Paeonol reference substance solution

Get borneol reference substance 280mg, Paeonol reference substance 32mg puts in the 50ml measuring bottle, adds the ethyl acetate dissolving and is diluted to scale.

The preparation of inner mark solution

It is an amount of to get eugenol, adds ethyl acetate and processes every milliliter of solution that contains 20 μ g.

The range of linearity

The accurate reference substance solution 1,3,5,7 of drawing, 9ml places the 10ml measuring bottle respectively, is diluted to scale with ethyl acetate, shakes up, and the accurate 5ml that draws adds inner mark solution 1ml.Get solution 1 μ l inject gas chromatograph, the record peak area is a horizontal ordinate with reference substance concentration, and the ratio of the chromatographic peak area of reference substance and internal standard compound is ordinate, calculates the regression equation of borneol, Paeonol respectively.

Borneol: Y=0.112+4.3074*X, r=0.9996, the range of linearity 0.47～4.23 μ g;

Paeonol: Y=0.02366+1.1474*X, r=0.9996, the range of linearity 0.053～0.477 μ g.

1.2 precision test

Get reference substance solution, repeat sample introduction 6 times, calculate the RSD of the chromatographic peak area ratio of borneol and internal standard compound, Paeonol and internal standard compound.RSD is respectively 1.2%, 1.8% as a result.

1.3 replica test

Get lot number and be 6 parts in 04081201,04091302,04101403 sample, measure the content (n=3) of borneol and Paeonol, its relative standard deviation RSD is respectively 1.6% and 2.0%.

1.4 recovery test

Adopt the application of sample recovery method.Get the sample of known content, add borneol reference substance and Paeonol reference substance, press the operation of sample size assay method.The result sees table 4.

Table 4 recovery test result (n=5)

1.5 sample determination

Precision takes by weighing the about 1g of these article, adds water 10ml, and jolting constantly makes dissolving.With ethyl acetate extract 4 times (8,5,5,5ml).Extract merges to be put in the 25ml measuring bottle, adds ethyl acetate to scale, shakes up.Precision is measured 5ml, and the accurate inner mark solution 1ml that adds shakes up, and gets this solution 2 μ l, and the chromatogram sample introduction calculates content.The result sees table 5.

Borneol and Paeonol are measured result (n=3) in table 5 sample

Experimental example 4: moutan bark thin layer discrimination test

Mainly contain phenolic compounds such as Paeonol in the moutan bark, this constituents is that polarity is on the low side, can be dissolved in polar solvent, like sherwood oil etc.This prescription flavour of a drug are more, and complicated component utilizes thin-layer chromatography to detect phenolic compounds such as mainly containing Paeonol in the moutan bark among the present invention, identify with this and contain the Paeonol medicinal material in preparation.

1, the selection of thin layer chromatography:

Common thin-layer chromatography has silica gel thin-layer chromatography, ply of paper to analyse etc.Mainly contain phenolic compounds such as Paeonol in the moutan bark, silica gel column chromatography makes an experiment.

2, the preparation of test sample:

2.1 the extraction of phenolic compounds such as Paeonol:

This prescription taste of traditional Chinese medicine is more, and composition is complicated, need extract phenolic compounds such as Paeonols in the moutan bark in the preparation.

Adopt L ₉3 ⁽⁴⁾The orthogonal experiment design is investigated the need testing solution method for distilling, sees table 6.

Table 6

9 parts of need testing solutions have been prepared through above-mentioned experimental program.

The result shows:

Extract ratio of solvent:

Use ether to extract, impurity is more in the need testing solution extract, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on show the close overlapping spot of a lot of colors;

Use sherwood oil (60～90 ℃) to extract, impurity is less in the need testing solution extract, but extractability is not strong, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on show the very more weak spot of color;

Use sherwood oil (30～60 ℃) to extract, effect is better, and it is less to make need testing solution impurity, clear spot.

Extracting mode compares:

The cold-maceration time is longer;

It is big than extraction that ultrasonic method is extracted intensity, can extract some impurity effects more and differentiate that extracting process is more suitable.

Take all factors into consideration and confirm that the test sample preparation method is: get present composition preparation 1g, grind, add water 10ml stirring and make dissolving; With sherwood oil (30～60 ℃), extract each 10ml 2 times; Merge sherwood oil liquid, put the tepidarium Back stroke to 2ml, as need testing solution.

2.2 the investigation of need testing solution and control medicinal material solution point sample amount:

Get present composition preparation 1g, grind, add water 10ml stirring and make dissolving, with sherwood oil (30～60 ℃), extract 2 times, each 10ml merges sherwood oil liquid, puts the tepidarium Back stroke to 2ml, as need testing solution.

Get the Paeonol reference substance, add ethanol and process the solution that per 1 ml contains 1mg, as reference substance solution.

Drawing above-mentioned two kinds of solution, 2 μ l, 5 μ l, 10 μ l respectively, put respectively on same silica gel g thin-layer plate, is developping agent with cyclohexane-ethyl acetate (3:1), launches, and takes out, and dries, and spray is with 5% ferric trichloride ethanolic solution, and it is clear that hot blast blows to the spot colour developing.

Experimental result is following:

When Paeonol reference substance solution point sample amount was 2 μ l, spot intensity was lower, should not observe; When the point sample amount was 5 μ l, clear spot on the Paeonol reference substance solution thin layer was of moderate size; When the point sample amount was 10 μ l, spot was too big, hangover.

Therefore, the best point sample amount of confirming the Paeonol reference substance solution is 4 μ l.

When need testing solution point sample amount was 2 μ l, spot intensity was lower on the corresponding position of Paeonol reference substance, should not observe; When the point sample amount was 5 μ l, spot was obvious on the corresponding position of Paeonol reference substance, and spot size is moderate, and front and back are noiseless; When the point sample amount was 10 μ l, spot was bigger on the corresponding position of Paeonol reference substance, and obviously hangover links to each other with front and back fluorescence spot.

Dimension confirms that the best point sample amount of test sample is 5 μ l.

2.3 the selection of developping agent:

Prepare need testing solution and control medicinal material solution as stated above.Draw each 5 μ l of above-mentioned reference substance solution and need testing solution, launch with following developping agent respectively, take out, dry, spray is with 5% ferric trichloride ethanolic solution, and it is clear that hot blast blows to the spot colour developing.Test findings is following: be developping agent, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on show the spot of same color.

Method 1: developping agent is cyclohexane-ethyl acetate (1:8), and developping agent polarity is bigger, and test sample, Paeonol spot and R f value are near 0.85, and the aurantiamarin spot does not separate with the front and back spot in the test sample chromatogram;

Method 2: developping agent is cyclohexane-ethyl acetate (3:1), and polarity is suitable, and the Paeonol spot and R f value is near 0.45, and the Paeonol spot separates well in the test sample chromatogram, and front and back are noiseless;

Method 3: developping agent is cyclohexane-ethyl acetate (8:1), and polarity is on the low side, and the Paeonol spot does not separate with the front and back spot in the test sample chromatogram.

Therefore, confirm that the optimum thin-layer developing agent of moutan bark composition is cyclohexane-ethyl acetate (3:1) in the separating traditional Chinese medicine composite preparation.

2.4 the selection of color condition:

Do not have color under the liposoluble ingredient daylight in the moutan bark, but behind the spray developer, show color, therefore, consider to observe and the exclusive developer of phenols behind the universal chromogenic reagent of straight use.Universal developer commonly used is 10% ethanol solution of sulfuric acid.

Prepare need testing solution, control medicinal material solution as stated above.Get two silica gel thin-layers respectively point sample, launch, dry.

Method one: the ethanol solution of sulfuric acid that thin layer 1 sprays with 10%;

Method two: thin layer 2 sprays 5% ferric trichloride ethanolic solution;

Experimental result shows:

The method skim is observed under daylight, and need testing solution and control medicinal material solution chromatogram have the spot of observing same color, but spot is unintelligible;

The method two thin layer the spray 5% ferric trichloride ethanolic solution, with the corresponding position of Paeonol on, need testing solution has the fluorescence spot of obvious same color, clear spot, front and back are noiseless.

Therefore the best color condition of confirming liposoluble ingredient in the separating traditional Chinese medicine composite preparation is spray 5% ferric trichloride ethanolic solution.

Experimental example 5: Ligusticum wallichii thin layer discrimination test

Mainly contain volatilization compounds such as ligustrazine etc. in the Ligusticum wallichii.This compounds has the preparation characteristic of himself.

1, test sample preparation method's selection

1.1 extract the selection of solvent and method for distilling

Get present composition preparation 2g, porphyrize adds sherwood oil 10ml, filters, and filtrating is as need testing solution 1.

Get present composition preparation 2g, porphyrize, the 10ml that adds diethyl ether filters, and filtrating is as need testing solution 2.

Get present composition preparation 2g, porphyrize, the 10ml that adds diethyl ether after ultrasonic 30 minutes, filters, and filtrating is as need testing solution 3.

Get Ligusticum wallichii control medicinal material 1g, add ethanol 5ml, sonicated 5 minutes filters, and filtrating is as control medicinal material solution.

According to the thin-layered chromatography test, draw each 10 μ l of above-mentioned two kinds of solution, put in same silica G F respectively ₂₅₄On the thin layer plate, be developping agent, launch, take out, dry with cyclohexane-ethyl acetate (3:1).Put under the ultraviolet lamp (365nm) and inspect.

The result shows:

Need testing solution 1 adopts sherwood oil to make the extraction solvent, be difficult to the volatile ingredient in the Ligusticum wallichii is all extracted, in the test sample chromatogram, with the corresponding position of Ligusticum wallichii control medicinal material chromatogram on, the fluorescence spot that apparent color is more weak;

Need testing solution 2,3 adopts ether to make the extraction solvent, can preferably the volatile ingredient in the Ligusticum wallichii all be extracted, and does not need ultrasonic; Soak and get final product; In the test sample chromatogram, with the corresponding position of Ligusticum wallichii control medicinal material chromatogram on, show the fluorescence spot of same color.And clear spot, rounding.

Therefore, get present composition preparation 2g, porphyrize, the 10ml that adds diethyl ether filters, and filtrating is as need testing solution 2.

2, the investigation of need testing solution point sample amount:

Draw need testing solution 5,10,20 μ l points respectively on same silica G plate, launch, examine and know.Test findings is following:

When need testing solution point sample amount was 5 μ l, spot intensity was lower, should not observe;

When need testing solution point sample amount was 10 μ l, the fluorescence clear spot was of moderate size on the thin layer, did not have hangover, and front and back are noiseless;

When need testing solution point sample amount was 20 μ l, the fluorescence spot was bigger on the need testing solution thin layer, linked to each other with front and back fluorescence spot.

Therefore, the best point sample amount of confirming need testing solution is 10 μ l.

Experimental example 6: borneol thin layer discrimination test

1, test sample preparation method's investigation:

Borneol all has volatility and similar polarity is arranged with moutan bark.Therefore, the Paeonol preparation method is closely similar in the test sample preparation method of borneol and the moutan bark, and the present invention differentiates that to the borneol thin layer test sample preparation method carries out preferably through verification experimental verification.

Get present composition preparation content 1g, grind, add water 10ml stirring and make dissolving, with sherwood oil (30～60 ℃), extract 2 times, each 10ml merges sherwood oil liquid, puts tepidarium Back stroke to 2 ml, as need testing solution.

Get the borneol reference substance, add ethanol and process the solution that per 1 ml contains 1mg, as reference substance solution.

Drawing each 5 μ l of reference substance solution and need testing solution, put respectively on same silica gel g thin-layer plate, is developping agent with sherwood oil (60～90 ℃) ethyl acetates (17:3); Launch, take out, dry; Spray is with 5% vanillic aldehyde sulfuric acid solution, about 5 minutes of 105 ℃ of bakings.

The result shows, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color.Can prepare the borneol need testing solution with the test sample preparation method in the moutan bark identification check.

2. the investigation of test sample, reference substance solution point sample amount:

The present invention investigates need testing solution, reference substance solution point sample amount.

Draw need testing solution, reference substance solution 2,5,8 μ l points respectively on same silica G plate, launch, examine and know.Test findings is following:

When the point sample amount was 2 μ l, spot intensity was lower, should not observe;

When the point sample amount was 5 μ l, the fluorescence clear spot was of moderate size on the thin layer, did not have hangover, and front and back are noiseless;

When the point sample amount was 8 μ l, the fluorescence spot was bigger on the need testing solution thin layer, linked to each other with front and back fluorescence spot.

Therefore, confirm that best point sample amount is 5 μ l.

3, the selection of developping agent:

Draw each 5 μ l of reference substance solution and need testing solution; Put respectively on same silica gel g thin-layer plate, with sherwood oil (60～90 ℃)-ethyl acetate (17:3), sherwood oil (30～60 ℃)-ethyl acetate (17:3), ether-ethyl acetate (17:3), be developping agent, launch; Take out; Dry, spray is with 5% vanillic aldehyde sulfuric acid solution, about 5 minutes of 105 ℃ of bakings.The result shows, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color.

Method 1: developping agent is sherwood oil (60～90 ℃)-ethyl acetate (17:3), and polarity is suitable, and spot and R f value is near 0.45, and spot separates good, and front and back are noiseless;

Method 2: developping agent is sherwood oil (30～60 ℃)-ethyl acetate (17:3), and polarity and sherwood oil (60～90 ℃)-(17:3) is similar for ethyl acetate, is nothing like sherwood oil (60～90 ℃) but launch effect, and spot has hangover, diffusion phenomena,

Method 3: developping agent is ether-ethyl acetate (17:3), and polarity is bigger than normal, and spot does not separate with the front and back spot in the test sample chromatogram.

Therefore, confirm that the optimum thin-layer developing agent of borneol composition is sherwood oil (60～90 ℃)-ethyl acetate (17:3) in the separating traditional Chinese medicine composite preparation.

4. the investigation of color condition

Adopt spray with 5% vanillic aldehyde sulfuric acid solution respectively, about 5 minutes of 105 ℃ of bakings; 5% vanillic aldehyde sulfuric acid solution compares in the method that is heated to the spot colour developing,

The result shows:

Spray is with 5% vanillic aldehyde sulfuric acid solution, and about 5 minutes of 105 ℃ of bakings, temperature was higher, and color speed is fast, and the time is controlled, the colour developing clear spot.

Spray is being heated to the spot colour developing with 5% vanillic aldehyde sulfuric acid solution, and temperature is wayward, and developing time can not be empty, influences the spot colour developing.

Therefore, select spray, developed the color in about 5 minutes 105 ℃ of bakings with 5% vanillic aldehyde sulfuric acid solution.

Following embodiment all can realize the effect of above-mentioned experimental example.

Embodiment

Embodiment 1

Moutan bark 20.1g Ligusticum wallichii 33.6g borneol 2.18g

More than three flavors, moutan bark is ground into meal, according to the following percolation of liquid extract and extract item (" an appendix I of Chinese pharmacopoeia version in 2000 O), makes solvent with 90% ethanol, cold soaking 24 hours, diacolation slowly, collection percolate, recovery ethanol.Rhizoma Chuanxiong power is broken into fragment, extracting in water volatile oil 2 hours, and the WS after the distillation inclines and; Dregs of a decoction boiling 1 hour again filters, and filtrating merges with the above-mentioned WS; Being condensed into relative density is the clear cream of 1.20～1.25 (70 ℃), adds ethanol and makes that to contain alcohol amount be 50%, stirs; Left standstill 24 hours, and drew supernatant, reclaim ethanol.Merge moutan bark and Ligusticum wallichii soup, continue to be evaporated to thick paste (about 6.6～6.8g).Taking polyethylene glycol 6000 31.2g in addition, heating, treat whole fusions after, add above-mentioned thick paste, rhizoma chuanxiong volatile oil and borneol, stir, from top to bottom, splash in the whiteruss, with the dripping pill drop that is shaped to the greatest extent and the erasing liquor paraffin body, process 1000 balls, promptly get.

[discriminating]

(1) get these article 1g, grind, add water 10ml stirring and make dissolving, with sherwood oil (30～60 ℃), extract 2 times, each 10ml merges sherwood oil liquid, puts tepidarium Back stroke to 2 ml, as reference substance solution.Other gets the Paeonol reference substance, adds ethanol and processes the solution that per 1 ml contains 1mg, as contrast solution.According to thin-layered chromatography (" an appendix VI of Chinese pharmacopoeia version in 2000 B) test, draw above-mentioned two kinds of solution, 5 μ l, put respectively on same silica gel g thin-layer plate; With cyclohexane-ethyl acetate (3:1) is developping agent; Launch, take out, dry; Spray is with 5% ferric trichloride ethanolic solution, and it is clear that hot blast blows to the spot colour developing.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on show the spot of same color.

(2) get this article 50 balls, porphyrize, the 10ml that adds diethyl ether filters, and filtrating is as need testing solution.Other gets Ligusticum wallichii control medicinal material 1g, adds ethanol 5ml, and sonicated 5 minutes filters, and filtrating is as control medicinal material solution.According to thin-layered chromatography (appendix VIB) test, draw above-mentioned two kinds of each 10ul of solution, put respectively on same silica GF254 thin layer plate, be developping agent with cyclohexane-ethyl acetate (3:1), launch, take out, dry.Put under the ultraviolet lamp (365nm) and inspect.In the test sample chromatogram, with the corresponding position of Ligusticum wallichii control medicinal material chromatogram on, show the spot of same color.Ethyl acetate (17:3) is a developping agent, launches, and takes out, and dries, and spray is with 5% vanillic aldehyde sulfuric acid solution, about 5 minutes of 105 ℃ of bakings.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color.

(3) get the borneol reference substance, add ethanol and process the solution that per 1 ml contains 1mg, as reference substance solution.According to thin-layered chromatography (" an appendix VI of Chinese pharmacopoeia version in 2000 B) test, draw the need testing solution 5 μ l respectively under reference substance solution and [discriminatings] (1), put respectively on same silica gel g thin-layer plate; With sherwood oil (60～90 ℃) ethyl acetates (17:3) is developping agent; Launch, take out, dry; Spray is with 5% vanillic aldehyde sulfuric acid solution, about 5 minutes of 105 ℃ of bakings.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color.

[assay] moutan bark, borneol are according to gas chromatography determination.

Chromatographic condition and system suitability test are stationary phase with the carbowax-20M, coating concentration 10%, carrier Chromaxorbw (Aw-Dmcs); Column temperature 170-200 ℃.Number of theoretical plate is pressed the eugenol peak and is calculated, and should be not less than 5000.The degree of separation at Paeonol, borneol peak and eugenol peak should be greater than 2.

It is an amount of that correction factor mensuration is got eugenol, adds ethyl acetate and process the solution that per 1 ml contains 20 μ g, shakes up, as inner mark solution; Other gets about 70 mg of borneol reference substance, about 8 mg of Paeonol reference substance, and accurate the title, decide, and puts in the 25ml measuring bottle; Add the ethyl acetate dissolving and be diluted to scale, shake up, the accurate 5ml that draws, the accurate inner mark solution 1ml that adds; Shake up, draw 2～4 μ l, inject gas chromatograph, the calculation correction factor.

Determination method: get these article under the weight differential inspection item, mixing is got about 1g, and accurate the title decides, and puts in the separating funnel; Add water 10ml, jolting constantly makes dissolving, extracts (8,5,5,5ml) 4 times with ethyl acetate, merges extract, puts in the 25ml measuring bottle; Add ethyl acetate to scale, shake up, the accurate 5ml that draws, the accurate inner mark solution 1ml that adds shakes up; Draw 2～4 μ l, inject gas chromatograph, measure, promptly get.

These article contain moutan bark with Paeonol (C ₉H ₁₀O ₃) meter, must not be less than 0.26%; Contain borneol (C ₁₀H ₁₈O) must not be less than 4.8%.

Function cures mainly: clearing heat and cooling blood, promoting blood circulation and stopping pain.Be used for light, the moderate chest impediment and cardialgia of partial heat type, the pain dysphoria with smothery sensation of holding concurrently, the tongue fur look yellow.

Usage and dosage: sublingual administration, one time 3～9,3 times on the one, during acute attack 12～18.

Specification: 40 milligrams on every ball

Embodiment 2

Moutan bark 30.1g Ligusticum wallichii 23.6g borneol 6.18g

[discriminating]

[assay] moutan bark, borneol shine gas chromatography determination:

Embodiment 3

Moutan bark 20.1g Ligusticum wallichii 33.6g borneol 2.18g

Moutan bark is ground into meal, according to the percolation under liquid extract and the extract item, makes solvent with 90% ethanol, cold soaking 24 hours, and slowly diacolation is collected percolate, reclaims ethanol.Rhizoma Chuanxiong power is broken into fragment, extracting in water volatile oil 2 hours, and the WS after the distillation inclines and; Dregs of a decoction boiling 1 hour again filters, and filtrating merges with the above-mentioned WS; Being condensed into relative density is the clear cream of 1.20～1.25 (70 ℃), adds ethanol and makes that to contain alcohol amount be 50%, stirs; Left standstill 24 hours, and drew supernatant, reclaim ethanol.Merge moutan bark and Ligusticum wallichii soup, continue to be evaporated to thick paste (about 6.6～6.8g).Add Icing Sugar and dextrin, mixing, drying is granulated, and promptly gets granule.

Embodiment 4

Moutan bark 20.1g Ligusticum wallichii 33.6g borneol 2.18g

Moutan bark is ground into meal, according to the percolation under liquid extract and the extract item, makes solvent with 90% ethanol, cold soaking 24 hours, and slowly diacolation is collected percolate, reclaims ethanol.Rhizoma Chuanxiong power is broken into fragment, extracting in water volatile oil 2 hours, and the WS after the distillation inclines and; Dregs of a decoction boiling 1 hour again filters, and filtrating merges with the above-mentioned WS; Being condensed into relative density is the clear cream of 1.20～1.25 (70 ℃), adds ethanol and makes that to contain alcohol amount be 50%, stirs; Left standstill 24 hours, and drew supernatant, reclaim ethanol.Merge moutan bark and Ligusticum wallichii soup, continue to be evaporated to thick paste (about 6.6～6.8g).Add Icing Sugar and dextrin, mixing, drying is granulated, and is encapsulated, promptly gets.

Claims

1. clearing heat and cooling blood, the detection method of the Chinese medicine composition pill of promoting blood circulation and stopping pain is characterized in that this method comprises the steps:

The bulk drug of said pill consists of:

Moutan bark 14.08-25.12 weight portion, Ligusticum wallichii 22.4-44.8 weight portion, borneol 1.6-2.7 weight portion;

The preparation method of said pill is:

Moutan bark is ground into meal, and percolation extracts, and makes solvent with 60-100% ethanol, and cold soaking 12-48 hour, slowly diacolation was collected percolate, reclaims ethanol; Rhizoma Chuanxiong power is broken into fragment, extracting in water volatile oil 1-3 hour, and the WS after the distillation inclines and; Dregs of a decoction boiling 0.5-3 hour again filter, and filtrating merges with the above-mentioned WS; Be condensed into the clear cream that 70 ℃ of relative densities are 1.1-1.4, add ethanol and make the alcohol amount of containing be 30-70%, stir; Left standstill 12-48 hour, and drew supernatant, reclaim ethanol; Merge moutan bark and Ligusticum wallichii soup, continue to be evaporated to thick paste; According to common process, process pill;

Detection method comprises the steps:

A, assay: chromatographic condition and system suitability test are stationary phase with the carbowax-20M, coating concentration 10%, carrier Chromaxorbw Aw-Dmcs; Column temperature 170-200 ℃;

Correction factor is measured, and it is an amount of to get eugenol, adds ethyl acetate and processes the solution that every 1ml contains 10-40 μ g, shakes up, as inner mark solution; Other gets borneol reference substance 50-100mg, Paeonol reference substance 2-12mg, and accurate the title, decide, and puts in the 10-50ml measuring bottle; Add the ethyl acetate dissolving and be diluted to scale, shake up, the accurate 2-10ml that draws, the accurate inner mark solution 0.5-1.5ml that adds; Shake up, draw 2～10 μ l, inject gas chromatograph, the calculation correction factor; Determination method: get said Chinese medicine composition pill, mixing is got about 1g, and accurate the title decides, and puts in the separating funnel; Add water 5-20ml, jolting constantly makes dissolving, extracts 3-5 time with ethyl acetate, merges extract, puts in the 10-50ml measuring bottle; Add ethyl acetate to scale, shake up, the accurate 2-10ml that draws, the accurate inner mark solution 0.2-1.2ml that adds shakes up; Draw 2～8 μ l, inject gas chromatograph is measured, and promptly gets; Contain moutan bark in Paeonol, must not be less than 0.26%; Contain borneol and must not be less than 4.8%;

B, discriminating: get said Chinese medicine composition pill 0.5-2g, grind, add water 10ml and stir and make dissolving, with 30～60 ℃ of Petroleum ether extraction 2-3 time, 5-20ml at every turn, merging sherwood oil liquid is put the tepidarium Back stroke to 1-3ml, as need testing solution; Other gets the Paeonol reference substance, adds ethanol and processes the solution that per 1 ml contains 0.5-3mg, as contrast solution; Drawing above-mentioned two kinds of solution 1-10 μ l, put respectively on same silica gel g thin-layer plate, is developping agent with the cyclohexane-ethyl acetate mixed solvent of 2-4:1 ratio, launches, and takes out, and dries, and spray is with 5% ferric trichloride ethanolic solution, and it is clear that hot blast blows to the spot colour developing; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on show the spot of same color;

C, discriminating: get said Chinese medicine composition pill 0.5-5g, porphyrize, the 5-20ml that adds diethyl ether filters, and filtrating is as need testing solution; Other gets Ligusticum wallichii control medicinal material 0.5-5g, adds ethanol 1-10ml, and sonicated 1-15 minute, filter, filtrating is as control medicinal material solution; According to the thin-layered chromatography test, draw above-mentioned two kinds of each 2-20ul of solution, put in same silica G F respectively ₂₅₄On the thin layer plate, be developping agent, launch, take out, dry with the cyclohexane-ethyl acetate mixed solvent of 2-4:1 ratio; Put under the 365nm ultraviolet lamp and inspect; In the test sample chromatogram, with the corresponding position of Ligusticum wallichii control medicinal material chromatogram on, show the spot of same color;

D, discriminating: get the borneol reference substance, add ethanol and process the solution that per 1 ml contains 1mg, as reference substance solution; Get said Chinese medicine composition pill 0.5-2g, grind, add water 10ml and stir and make dissolving, with 30～60 ℃ of Petroleum ether extraction 2-3 time, 5-20ml at every turn, merging sherwood oil liquid is put the tepidarium Back stroke to 1-3ml, as need testing solution; Drawing reference substance solution and each 1-20 μ l of need testing solution, put respectively on same silica gel g thin-layer plate, is developping agent with 60～90 ℃ of sherwood oils and the ethyl acetate mixed solvent of 13-25:2-4 ratio; Launch, take out, dry; Spray is with 5% vanillic aldehyde sulfuric acid solution, about 5 minutes of 105 ℃ of bakings; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color.

2. the method for claim 1 is characterized in that the bulk drug of said pill consists of:

3. according to claim 1 or claim 2 method is characterized in that the preparation method of said pill is:

Moutan bark is ground into meal, and percolation extracts, and makes solvent with 90% ethanol, cold soaking 24 hours, and slowly diacolation is collected percolate, reclaims ethanol; Rhizoma Chuanxiong power is broken into fragment, extracting in water volatile oil 2 hours, and the WS after the distillation inclines and; Dregs of a decoction boiling 1 hour again filters, and filtrating merges with the above-mentioned WS; Be condensed into 70 ℃ of relative densities and be 1.20～1.25 clear cream, add ethanol and make that to contain the alcohol amount be 50%, stir; Left standstill 24 hours, and drew supernatant, reclaim ethanol; Merge moutan bark and Ligusticum wallichii soup, continue to be evaporated to about 6.6～6.8 weight portions of thick paste; Taking polyethylene glycol 6,000 31.2 weight portions in addition, heating, treat whole fusions after, add above-mentioned thick paste, rhizoma chuanxiong volatile oil and borneol, stir, from top to bottom, splash in the whiteruss, with the dripping pill drop that is shaped to the greatest extent and the erasing liquor paraffin body, promptly get.

4. the method for claim 1 is characterized in that this method comprises the steps:

Assay:

Chromatographic condition and system suitability test: with the carbowax-20M is stationary phase, coating concentration 10%, carrier Chromaxorbw Aw-Dmcs; Column temperature 170-200 ℃; Number of theoretical plate is pressed the eugenol peak and is calculated, and should be not less than 5000; The degree of separation at Paeonol, borneol peak and eugenol peak should be greater than 2; Correction factor is measured: it is an amount of to get eugenol, adds ethyl acetate and processes the solution that every 1ml contains 20 μ g, shakes up, as inner mark solution; Other gets the about 70mg of borneol reference substance, the about 8mg of Paeonol reference substance, and accurate the title, decide, and puts in the 25ml measuring bottle; Add the ethyl acetate dissolving and be diluted to scale, shake up, the accurate 5ml that draws, the accurate inner mark solution 1ml that adds; Shake up, draw 2～4 μ l, inject gas chromatograph, the calculation correction factor; Determination method is got said Chinese medicine composition pill, and mixing is got about 1g, and accurate the title decides, and puts in the separating funnel; 8,5,5,5ml add water 10ml, jolting constantly makes dissolving, extracts 4 times with ethyl acetate, and the ethyl acetate consumption is respectively:, merge extract; Put in the 25ml measuring bottle, add ethyl acetate, shake up, the accurate 5ml that draws, the accurate inner mark solution 1ml that adds to scale; Shake up, draw 2～4 μ l, inject gas chromatograph is measured, and promptly gets;

Differentiate:

The moutan bark thin layer is differentiated: get said Chinese medicine composition pill 1g, grind, add water 10ml stirring and make dissolving, with 30～60 ℃ of sherwood oils, extract 2 times, each 10ml merges sherwood oil liquid, puts tepidarium Back stroke to 2 ml, as need testing solution; Other gets the Paeonol reference substance, adds ethanol and processes the solution that per 1 ml contains 1mg, as contrast solution; Drawing above-mentioned two kinds of solution, 5 μ l, put respectively on same silica gel g thin-layer plate, is developping agent with 3:1 cyclohexane-ethyl acetate, launches, and takes out, and dries, and spray is with 5% ferric trichloride ethanolic solution, and it is clear that hot blast blows to the spot colour developing; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on show the spot of same color;

The Ligusticum wallichii thin layer is differentiated: get said Chinese medicine composition pill 2g, and porphyrize, the 10ml that adds diethyl ether filters, and filtrating is as need testing solution; Other gets Ligusticum wallichii control medicinal material 1g, adds ethanol 5ml, and sonicated 5 minutes filters, and filtrating is as control medicinal material solution; Draw above-mentioned two kinds of each 10ul of solution, put in same silica G F respectively ₂₅₄On the thin layer plate, be developping agent, launch, take out, dry with 3:1 cyclohexane-ethyl acetate; Put under the 365nm ultraviolet lamp and inspect; In the test sample chromatogram, with the corresponding position of Ligusticum wallichii control medicinal material chromatogram on, show the spot of same color;

The borneol thin layer is differentiated: get said Chinese medicine composition pill 1g, grind, add water 10ml stirring and make dissolving, with 30～60 ℃ sherwood oil, extract 2 times, each 10ml merges sherwood oil liquid, puts tepidarium Back stroke to 2 ml, as need testing solution; Get the borneol reference substance, add ethanol and process the solution that per 1 ml contains 1mg, as reference substance solution; Drawing reference substance solution and moutan bark and differentiate need testing solution 5 μ l respectively down, put respectively on same silica gel g thin-layer plate, is developping agent with sherwood oil-ethyl acetate of 60～90 ℃ of 17:3; Launch, take out, dry; Spray is with 5% vanillic aldehyde sulfuric acid solution, about 5 minutes of 105 ℃ of bakings; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color.