CN103149319A

CN103149319A - Quality detection method for Chinese herbal preparation with effects of stimulating circulation of blood and causing muscles and joints to relax and activating blood circulation and dissipating blood stasis

Info

Publication number: CN103149319A
Application number: CN2013100689880A
Authority: CN
Inventors: 黄小华; 付彬; 张�浩; 耿小秀; 王海亮
Original assignee: BEILIN PHARMACEUTICAL Co Ltd XI'AN
Current assignee: BEILIN PHARMACEUTICAL Co Ltd XI'AN
Priority date: 2013-03-05
Filing date: 2013-03-05
Publication date: 2013-06-12
Anticipated expiration: 2033-03-05
Also published as: CN103149319B

Abstract

The invention relates to a quality detection method for Chinese herbal preparation with effects of stimulating the circulation of the blood and causing the muscles and joints to relax and activating blood circulation and dissipating blood stasis. The Chinese herbal preparation comprises the following components of 3.05 percent of red flower, 11.41 percent of vinegar rhizoma cyperi, 15.21 percent of fried rhizoma cibotii, 7.6 percent of cortex periplocae, 11.41 percent of caulis trachelospermi, 11.41 percent of lycopodium clavatum, 11.41 percent of herba lycopi, 15.21 percent of mistletoe, 11.41 percent of caulis spatholobi and 1.9 percent of calcined natural copper which are prepared into any one dosage form in pharmaceutics. The Chinese herbal preparation has the effects of stimulating the circulation of the blood and causing the muscles and joints to relax and activating blood circulation and dissipating blood stasis and is used for arthralgia and myalgia, limb contracture, lumbar spinal cord ache and traumatic injury. The provided quality detection method is stable and reliable and is superior to the detection method provided by the thirteenth volume WS3-B-2624-97 of drug standard (traditional Chinese medicine prescription preparation) in ministry of health of China, and the product quality is effectively guaranteed.

Description

A kind ofly stimulate the circulation of the blood and cause the muscles and joints to relax, the quality determining method of the Chinese medicine preparation of promoting blood circulation to remove blood stasis

Technical field

The present invention relates to a kind ofly to stimulate the circulation of the blood and cause the muscles and joints to relax, the quality determining method of the Chinese medicine preparation of promoting blood circulation to remove blood stasis, belong to the pharmaceutical technology field.

Background technology

The 13 WS of Drug Standard of Ministry of Public Health of the Peoples Republic of China (Traditional Chinese medicine historical preparation) ₃relaxing that-B-2624-97 provides made by safflower 80g, rhizoma cyperi (system) 300g, rhizoma cibotii (system) 400g, cortex periplocae 200g, caulis trachelospermi 300g, lycopodium calvatum 300g, herba lycopi 300g, mistletoe 400g, reticulate millettia 300g, native copper (forging) 50g through blood circulation promoting slice, its quality determining method is single, only adopted micro-discrimination method, quality to end product quality can't realize omnibearing detection, requirement to product quality is lower, has increased to a certain extent the possibility that the product of inferior quality puts goods on the market.The present invention has adopted micro-discrimination method, four thin-layer identification methods and content assaying method by the multinomial detection of multiple spot multiaspect, this detection method is reliable and stable and accuracy is high, the more effective product quality put goods on the market that guaranteed, promoted to have strengthened the control to product quality in preparation process, ensured validity and the stability of product.

Summary of the invention

The purpose of this invention is to provide a kind ofly stimulate the circulation of the blood and cause the muscles and joints to relax, the quality determining method of the Chinese medicine preparation of promoting blood circulation to remove blood stasis, this quality determining method is reliable and stable and accuracy is high, effectively guarantees the product quality put goods on the market.

For achieving the above object, the present invention has adopted following technical scheme:

Chinese medicine preparation of the present invention is made by safflower 3.05%, prepared RHIZOMA CYPERI with vinegar 11.41%, boiling hot rhizoma cibotii 15.21%, cortex periplocae 7.6%, caulis trachelospermi 11.41%, lycopodium calvatum 11.41%, herba lycopi 11.41%, mistletoe 15.21%, reticulate millettia 11.41%, pyritum ignited 1.9%, this Chinese medicine preparation can be the above any formulation of pharmacy, and its quality determining method comprises discrimination method and content assaying method:

Differentiate:

(1) get this product, put micro-Microscopic observation: corolla fragment yellow has rufous or yellowish-brown secretor; The secretory cell similar round, containing faint yellow to rufous secretion, its peripheral cell is made radial arrangement; Scalariform tracheid is faint yellow to golden yellow, the pit marshalling;

(2) get this product that is equivalent to raw medicinal herbs 13.78～20.67g, porphyrize, 20～40ml adds diethyl ether, place 1 hour, jolting constantly, filter, filtrate volatilizes, residue adds ethyl acetate 1ml to be made to dissolve, as need testing solution, separately get rhizoma cyperi control medicinal material 0.5g～1g, be made in the same way of control medicinal material solution, according to thin-layered chromatography, test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel thin-layer plate, take normal hexane-ethyl acetate-diethylamine 9～20:3～6:0～1 is developping agent, launch, take out, dry, put under ultraviolet lamp and inspect, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the principal spot of aobvious same color,

(3) get the 4-methoxysalicylaldehyde reference substance, add methyl alcohol and make the solution of every 1 ml containing 1mg, product solution in contrast, according to thin-layered chromatography, test, draw each the 5 μ l of need testing solution under reference substance solution and above-mentioned (2) item, put respectively on same silica gel g thin-layer plate, take 60～90 ℃-ethyl acetate-glacial acetic acid of sherwood oil, 15～25:1～4:0.1～0.8 is developping agent, launch, take out, dry, spray is with dinitrophenylhydrazine ethanol test solution, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color;

(4) get this product that is equivalent to raw medicinal herbs 13.78～27.56g, porphyrize, add methyl alcohol 40～60ml, ultrasonic processing 30 minutes, filter, the filtrate evaporate to dryness, residue adds water 20ml to be made to dissolve, adjust pH value to 1～2 with hydrochloric acid, shake up, adding ethyl acetate extracts 2 times, each 20ml, combined ethyl acetate liquid, evaporate to dryness, residue adds methyl alcohol 1ml to be made to dissolve, as need testing solution, separately get the protocatechualdehyde reference substance, add methyl alcohol and make the solution of every 1ml containing 0.5mg, product solution in contrast, according to thin-layered chromatography, test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take methenyl choloride-ethyl acetate-acetone-formic acid 10～60:0～8:0～5:1～2 is developping agent, launch, take out, dry, spray is with 5% ferric trichloride ethanolic solution, be placed to the spot colour developing clear, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color,

(5) get this product that is equivalent to raw medicinal herbs 13.78～20.67g, porphyrize, add 80% acetone 20～30ml, flood 1 hour, jolting constantly, filter, filtrate is as need testing solution, separately get safflower control medicinal material 0.5g, add 80% acetone 10ml, jolting 15 minutes, standing, get supernatant medicinal material solution in contrast, according to thin-layered chromatography, test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take ethyl acetate-methyl alcohol-formic acid-water 5～9:0.3～0.6:1～3:1～4 is developping agent, launch, take out, dry, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the principal spot of aobvious same color,

Assay:

Chromatographic condition and system suitability be take octadecylsilane chemically bonded silica as filling agent; Methanol-water-the acetic acid (40: 60: 2) of take is mobile phase; Detect wavelength 278nm, theoretical cam curve is pressed the 4-methoxysalicylaldehyde peak and is calculated, and should be not less than 2000;

It is appropriate that the 4-methoxysalicylaldehyde reference substance is got in the preparation of reference substance solution, accurately weighed, adds 60% methyl alcohol and make the solution of 1m1 containing 20 μ g, shakes up, and obtains;

This product that is equivalent to raw medicinal herbs 13.78g is got in the preparation of need testing solution, accurately weighed, porphyrize, get about 1.5g, accurately weighed, put in tool plug conical flask, precision adds 60% methyl alcohol 25m1, weighed weight, backflow or ultrasonic processing 30～40 minutes, let cool, more weighed weight, supply the weight of less loss with 60% methyl alcohol, filter, discard just filtrate, subsequent filtrate is that 0.45 μ m miillpore filter filters with aperture, obtains;

Determination method is accurate reference substance solution and each 10 μ 1 of need testing solution of drawing respectively, and the injection liquid chromatography, measure, and obtains;

This product that is equivalent to the 1g raw medicinal herbs contains cortex periplocae with 4-methoxysalicylaldehyde (C ₈h ₈o ₃) meter, must not be less than 101.58 μ g.

The quality determining method of this Chinese medicine preparation can be:

Method one:

Differentiate:

(2) get this product that is equivalent to raw medicinal herbs 17.23g, porphyrize, 30ml adds diethyl ether, place 1 hour, jolting constantly, filter, filtrate volatilizes, residue adds ethyl acetate 1ml to be made to dissolve, and as need testing solution, separately gets rhizoma cyperi control medicinal material 0.5g, be made in the same way of 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 thin layer plate, the normal hexane-ethyl acetate 9:1 of take is developping agent, launches, and takes out, and dries, put under ultraviolet lamp 254nm and inspect, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the principal spot of aobvious same color;

(3) get the 4-methoxysalicylaldehyde reference substance, add methyl alcohol and make the solution of every 1 ml containing 1mg, product solution in contrast, according to thin-layered chromatography, test, draw each the 5 μ l of need testing solution under reference substance solution and above-mentioned (2) item, put respectively on same silica gel g thin-layer plate, take 60～90 ℃-ethyl acetate of sherwood oil-glacial acetic acid 20:3:0.5 as developping agent, launch, take out, dry, spray is with dinitrophenylhydrazine ethanol test solution, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color;

(4) get this product that is equivalent to raw medicinal herbs 20.67g, porphyrize, add methyl alcohol 50ml, and ultrasonic processing 30 minutes filters, the filtrate evaporate to dryness, and residue adds water 20ml to be made to dissolve, and with hydrochloric acid, adjusts pH value to 1～2, shakes up, and adds ethyl acetate and extracts 2 times, every

Inferior 20ml, combined ethyl acetate liquid, evaporate to dryness, residue adds methyl alcohol 1ml to be made to dissolve, and as need testing solution, separately gets protocatechualdehyde

Reference substance, add methyl alcohol and make the solution of every 1ml containing 0.5mg, product solution in contrast, according to the thin-layered chromatography test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, methenyl choloride-acetone-formic acid the 60:5:2 of take is developping agent, launches, and takes out, dry, spray, with 5% ferric trichloride ethanolic solution, is placed to the spot colour developing clear, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color;

(5) get this product that is equivalent to raw medicinal herbs 17.23g, porphyrize, add 80% acetone 30ml, flood 1 hour, jolting constantly, filter, filtrate is as need testing solution, separately get safflower control medicinal material 0.5g, add 80% acetone 10ml, jolting 15 minutes, standing, get supernatant medicinal material solution in contrast, according to thin-layered chromatography, test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, ethyl acetate-methyl alcohol-formic acid-water the 7:0.4:2:3 of take is developping agent, launch, take out, dry, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the principal spot of aobvious same color,

Assay:

This product that is equivalent to raw medicinal herbs 13.78g is got in the preparation of need testing solution, accurately weighed, porphyrize, get about 1.5g, accurately weighed, put in tool plug conical flask, precision adds 60% methyl alcohol 25m1, weighed weight, ultrasonic processing 30 minutes, power is 250W, and frequency is 40kHz, lets cool, weighed weight again, supply the weight of less loss with 60% methyl alcohol, filter, discard just filtrate, subsequent filtrate is that 0.45 μ m miillpore filter filters with aperture, obtains;

Method two:

Differentiate:

(2) get this product that is equivalent to raw medicinal herbs 13.78g, porphyrize, 20ml adds diethyl ether, place 1 hour, jolting constantly, filter, filtrate volatilizes, residue adds ethyl acetate 1ml to be made to dissolve, and as need testing solution, separately gets rhizoma cyperi control medicinal material 1g, be made in the same way of 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 thin layer plate, the normal hexane-ethyl acetate 17:3 of take is developping agent, launches, and takes out, and dries, put under ultraviolet lamp 254nm and inspect, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the principal spot of aobvious same color;

(3) get the 4-methoxysalicylaldehyde reference substance, add methyl alcohol and make the solution of every 1 ml containing 1mg, product solution in contrast, according to thin-layered chromatography, test, draw each the 5 μ l of need testing solution under reference substance solution and above-mentioned (2) item, put respectively on same silica gel g thin-layer plate, 60～90 ℃-ethyl acetate of the sherwood oil-glacial acetic acid 15:1:0.1 of take is developping agent, launch, take out, dry, spray is with dinitrophenylhydrazine ethanol test solution, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color;

(4) get this product that is equivalent to raw medicinal herbs 13.78g, porphyrize, add methyl alcohol 40ml, ultrasonic processing 30 minutes, filter, the filtrate evaporate to dryness, residue adds water 20ml to be made to dissolve, adjust pH value to 1～2 with hydrochloric acid, shake up, adding ethyl acetate extracts 2 times, each 20ml, combined ethyl acetate liquid, evaporate to dryness, residue adds methyl alcohol 1ml to be made to dissolve, as need testing solution, separately get the protocatechualdehyde reference substance, add methyl alcohol and make the solution of every 1ml containing 0.5mg, product solution in contrast, according to thin-layered chromatography, test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, methenyl choloride-ethyl acetate-formic acid the 10:8:1 of take is developping agent, launch, take out, dry, spray is with 5% ferric trichloride ethanolic solution, be placed to the spot colour developing clear, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color,

(5) get this product that is equivalent to raw medicinal herbs 13.78g, porphyrize, add 80% acetone 20ml, flood 1 hour, jolting constantly, filter, filtrate is as need testing solution, separately get safflower control medicinal material 0.5g, add 80% acetone 10ml, jolting 15 minutes, standing, get supernatant medicinal material solution in contrast, according to thin-layered chromatography, test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, ethyl acetate-methyl alcohol-formic acid-water the 5:0.3:1:1 of take is developping agent, launch, take out, dry, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the principal spot of aobvious same color,

Assay:

This product that is equivalent to raw medicinal herbs 13.78g is got in the preparation of need testing solution, accurately weighed, porphyrize, get about 1.5g, accurately weighed, put in tool plug conical flask, precision adds 60% methyl alcohol 25m1, weighed weight, ultrasonic processing 40 minutes, power is 250W, and frequency is 40kHz, lets cool, weighed weight again, supply the weight of less loss with 60% methyl alcohol, filter, discard just filtrate, subsequent filtrate is that 0.45 μ m miillpore filter filters with aperture, obtains;

Method three:

Differentiate:

(2) get this product that is equivalent to raw medicinal herbs 20.67g, porphyrize, 40ml adds diethyl ether, place 1 hour, jolting constantly, filter, filtrate volatilizes, residue adds ethyl acetate 1ml to be made to dissolve, as need testing solution, separately get rhizoma cyperi control medicinal material 1g, be made in the same way of control medicinal material solution, according to thin-layered chromatography, test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, normal hexane-ethyl acetate-diethylamine the 20:6:1 of take is developping agent, launch, take out, dry, put under ultraviolet lamp 365nm and inspect, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the principal spot of aobvious same color,

(3) get the 4-methoxysalicylaldehyde reference substance, add methyl alcohol and make the solution of every 1 ml containing 1mg, product solution in contrast, according to thin-layered chromatography, test, draw each the 5 μ l of need testing solution under reference substance solution and above-mentioned (2) item, put respectively on same silica gel g thin-layer plate, 60～90 ℃-ethyl acetate of the sherwood oil-glacial acetic acid 25:4:0.8 of take is developping agent, launch, take out, dry, spray is with dinitrophenylhydrazine ethanol test solution, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color;

(4) get this product that is equivalent to raw medicinal herbs 27.56g, porphyrize, add methyl alcohol 60ml, ultrasonic processing 30 minutes, filter, the filtrate evaporate to dryness, residue adds water 20ml to be made to dissolve, adjust pH value to 1～2 with hydrochloric acid, shake up, adding ethyl acetate extracts 2 times, each 20ml, combined ethyl acetate liquid, evaporate to dryness, residue adds methyl alcohol 1ml to be made to dissolve, as need testing solution, separately get the protocatechualdehyde reference substance, add methyl alcohol and make the solution of every 1ml containing 0.5mg, product solution in contrast, according to thin-layered chromatography, test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, methenyl choloride-acetone-formic acid the 60:5:2 of take is developping agent, launch, take out, dry, spray is with 5% ferric trichloride ethanolic solution, be placed to the spot colour developing clear, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color,

(5) get this product that is equivalent to raw medicinal herbs 20.67g, porphyrize, add 80% acetone 30ml, flood 1 hour, jolting constantly, filter, filtrate is as need testing solution, separately get safflower control medicinal material 0.5g, add 80% acetone 10ml, jolting 15 minutes, standing, get supernatant medicinal material solution in contrast, according to thin-layered chromatography, test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, ethyl acetate-methyl alcohol-formic acid-water the 9:0.6:3:4 of take is developping agent, launch, take out, dry, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the principal spot of aobvious same color,

Assay:

This product that is equivalent to raw medicinal herbs 13.78g is got in the preparation of need testing solution, accurately weighed, porphyrize, get about 1.5g, accurately weighed, put in tool plug conical flask, precision adds 60% methyl alcohol 25m1, weighed weight, refluxing extraction 40 minutes, let cool, more weighed weight, supply the weight of less loss with 60% methyl alcohol, filter, discard just filtrate, subsequent filtrate is that 0.45 μ m miillpore filter filters with aperture, obtains;

Of the present inventionly a kind ofly stimulate the circulation of the blood and cause the muscles and joints to relax, the quality determining method of the Chinese medicine preparation of promoting blood circulation to remove blood stasis, wherein this Chinese medicine preparation is made any formulation on pharmacy by safflower 3.05%, prepared RHIZOMA CYPERI with vinegar 11.41%, boiling hot rhizoma cibotii 15.21%, cortex periplocae 7.6%, caulis trachelospermi 11.41%, lycopodium calvatum 11.41%, herba lycopi 11.41%, mistletoe 15.21%, reticulate millettia 11.41%, pyritum ignited 1.9%, by regulating channels and activating collateral, promoting blood circulation to remove blood stasis, for arthralgia and myalgia, the limbs contraction, aching pain in waist and back, traumatic injury.The quality determining method of the said preparation provided is stable, reliable, is better than the 13 WS of Drug Standard of Ministry of Public Health of the Peoples Republic of China (Traditional Chinese medicine historical preparation) ₃the detection method that-B-2624-97 provides, the more effective quality that guarantees product.

The inventor has done following research to the content of 4-methoxysalicylaldehyde in this Chinese medicine preparation:

One, experimental drug

1. the prescription of experimental drug:

Safflower 21g prepared RHIZOMA CYPERI with vinegar 78.6g scalds rhizoma cibotii 104.8g

Cortex periplocae 52.4g caulis trachelospermi 78.6g lycopodium calvatum 78.6g

Herba lycopi 78.6g mistletoe 104.8g reticulate millettia 78.6g

Pyritum ignited 13.1g

2. the preparation of experimental drug: above ten flavors, first safflower, prepared RHIZOMA CYPERI with vinegar, boiling hot rhizoma cibotii, cortex periplocae are ground into to fine powder, sieve.Pyritum ignited boiling 2 hours, then add caulis trachelospermi, lycopodium calvatum, herba lycopi, mistletoe, reticulate millettia to decoct secondary, and 3 hours for the first time, 2 hours for the second time, collecting decoction, filtered.Standing 8～10 hours of filtrate, it is 1.25～1.35(70 ℃ that supernatant concentration becomes relative density) thick paste, add above-mentioned fine powder to mix, add that starch is appropriate, maltose 39g, granulation, drying, be pressed into 1000, film coating, obtain.

Two, research process:

1, instrument and reagent

Instrument: Agilent 1100 type high performance liquid chromatographs, G1314A type UV-detector (U.S. Agilent Technologies); BP211D type electronic analytical balance (German Sai Duolisi group company); KQ250B type ultrasonic cleaner (power 250W, frequency 40kHz), (Kunshan Ultrasonic Instruments Co., Ltd.); UV-1800 type ultraviolet-visible pectrophotometer, (Japanese Shimadzu company).

Reagent: methyl alcohol is chromatographically pure (U.S. Honeywell company); Water is redistilled water; All the other reagent are analyzes pure (Xi'an chemical reagent factory).

Reference substance: the 4-methoxysalicylaldehyde reference substance is purchased from Nat'l Pharmaceutical & Biological Products Control Institute (lot number 0790-200202 assay is used).

2, the 4-methoxysalicylaldehyde reference substance solution is got in the selection of measuring wavelength, uses ultraviolet-visible pectrophotometer, carries out sweep measuring in 190～400nm wavelength coverage.Result shows, the 4-methoxysalicylaldehyde maximum absorption wavelength is 278nm.

3, chromatographic condition

Chromatographic column: Agilent TC-C ₁₈(250mm * 4.6mm 5 μ) chromatographic column; Mobile phase: methanol-water-acetic acid (40:60:2); Flow velocity: 1ml/min; Column temperature: room temperature; Detect wavelength: 278nm; Sample size: 10 μ l.

Under selected condition, in 4-methoxysalicylaldehyde and preparation, other component chromatographic peak can reach baseline separation, and the degree of separation of 4-methoxysalicylaldehyde and adjacent peak is 3.26,5.81.Number of theoretical plate counts 15806 with the 4-methoxysalicylaldehyde peak.

4, the preparation of need testing solution

(1), extract definite list of references of solvent and " assay of 4-methoxysalicylaldehyde under cortex periplocae medicinal material item of Chinese pharmacopoeia version in 2010 determines that 60% methyl alcohol is for extracting solvent.

(2), definite this product of getting of extracting method, porphyrize, get about 1.5g, two parts, accurately weighed, put in tool plug conical flask, precision adds 60% methyl alcohol 25ml, weighed weight, a ultrasonic processing (power 250W, frequency 40kHz) 40 minutes, another part of refluxing extraction 40 minutes, cooled, weighed weight, supply the weight of less loss with 60% methyl alcohol, shake up, filter.Miillpore filter for subsequent filtrate (0.45 μ m) filters, and measures, and the results are shown in Table 1.

The comparison of table 1 extracting method

Result shows, circumfluence method and ultrasonic method measurement result no significant difference, therefore select simple and easy to do ultrasonic method as extracting method.(3), definite this product of getting of extraction time, porphyrize, get about 1.5g, four parts, accurately weighed, put in tool plug conical flask, precision adds 60% methyl alcohol 25ml, weighed weight, ultrasonic processing (power 250W respectively, frequency 40kHz) 10,20,30,40 minutes, let cool, more weighed weight, supply the weight of less loss with 60% methyl alcohol, shake up, filter.Miillpore filter for subsequent filtrate (0.45 μ m) filters, and measures, and the results are shown in Table 2.

The comparison of table 2 extraction time

Result shows, extracts 30 minutes and the content of 40 minutes 4-methoxysalicylaldehydes approaches, therefore select ultrasonic 30 minutes as extraction time.

In sum, the preparation of need testing solution, get 20 of this product, accurately weighed, porphyrize, get about 1.5g, accurately weighed, put in tool plug conical flask, precision adds 60% methyl alcohol 25m1, weighed weight, ultrasonic processing (power 250W, frequency 40kHz) 30 minutes, let cool, weighed weight again, supply the weight of less loss with 60% methyl alcohol, filter, discard just filtrate, miillpore filter for subsequent filtrate (0.45 μ m) filters, and obtains.

5, interference test

Get not the preparation containing the cortex periplocae medicinal material, according to the preparation method of need testing solution, be made in the same way of blank solution, by the chromatographic condition of drafting sample introduction respectively, result shows, with 4-methoxysalicylaldehyde chromatographic peak relevant position, without chromatographic peak, is occurring, negative noiseless.

6, the investigation of linear relationship

The accurate 4-methoxysalicylaldehyde reference substance solution (0.023mg/ml that draws )4,8,10,12,16,20 μ l, by the chromatographic condition sample introduction of drafting, measure peak area, the results are shown in Table 3.

Table 3 4-methoxysalicylaldehyde linear relationship measurement result

With peak area, sample size is returned, is obtained typical curve equation Y=6228.9X-18.329(r=0.9998), to sample size (μ g) mapping, obtain a straight line with peak area value.Above result shows, 4-methoxysalicylaldehyde is in 0.092～0.46 μ g scope, and peak area integrated value and sample size are good linear relationship.

7, precision test is got the need testing solution prepared, and by the chromatographic condition of drafting, measures, and repeats sample introduction 6 times, the results are shown in Table 4.

Table 4 Precision test result

The RSD of 4-methoxysalicylaldehyde peak area is 0.97% as a result, shows that the precision of this method is good.

8, stability test is got test liquid respectively at 0 hour, 2 hours, 4 hours, 6 hours, 8 hours, 24 hours sample introductions, the results are shown in Table 5.

Table 5 stability test result

The RSD of 4-methoxysalicylaldehyde peak area is 2.05% as a result, shows that need testing solution is basicly stable in 24 hours.

9, replica test, by the content assaying method of drafting, prepares respectively 6 parts of need testing solutions to same batch sample, measures the content of 4-methoxysalicylaldehyde, the results are shown in Table 6.

Table 6 reproducible test results

The RSD of 4-methoxysalicylaldehyde content is 1.65% as a result, shows that the repeatability of this method is good.

10, recovery test is got 4-methoxysalicylaldehyde reference substance (0.2888mg/ml) 8ml, with 60% methyl alcohol, dilutes and is settled in 200 ml volumetric flasks, shakes up, standby; Get again test sample (201101001, content) 0.292 mg/g of known content) about 0.75g, 6 parts, accurately weighed, precision adds above-mentioned reference substance solution 25ml respectively, by the content assaying method of drafting, measures, with following formula calculate recovery rate, the results are shown in Table 7.

Table 7 recovery test result

Result shows, this method 4-methoxysalicylaldehyde average recovery is good.

11, the mensuration of sample is got three batches of pilot scale element sheets and Film coated tablets, measures as stated above the content of its 4-methoxysalicylaldehyde, and measurement result is in Table 8.

4-methoxysalicylaldehyde assay result in three batches of plain sheets of table 8 and Film coated tablets

Assay result according to 4-methoxysalicylaldehyde in 3 batches of pilot scale element sheets and Film coated tablets, the content no significant difference of element sheet and Film coated tablets 4-methoxysalicylaldehyde, the content (0.273% of 4-methoxysalicylaldehyde in the cortex periplocae medicinal material feeds intake, every containing cortex periplocae medicinal material 0.052g), the average content of 3 batches of pilot scale Film coated tablets 4-methoxysalicylaldehydes is 112.9 μ g/ sheets, the rate of transform of calculating every middle 4-methoxysalicylaldehyde is: 79.5%, in conjunction with production actual conditions and the " content limit (must not be less than 0.20%) of a cortex periplocae medicinal material of Chinese pharmacopoeia version in 2010, every of tentative this product contains cortex periplocae with 4-methoxysalicylaldehyde (C ₈h ₈o ₃) meter, must not be less than 70 μ g.

12, the assay of cortex periplocae medicinal material is got the about 0.3g of cortex periplocae medicinal powder (crossing sieve No. three) of different batches, accurately weighed, puts in tool plug triangular flask, precision adds 60% methyl alcohol 25ml, weighs, and ultrasonic extraction (power 250W, frequency 40kHz) 30 minutes, let cool, supply the weight of less loss with 60% methyl alcohol, shake up, filter, miillpore filter for subsequent filtrate (0.45 μ m) filters, according to the chromatographic condition of drafting, measure the content of 4-methoxysalicylaldehyde, measurement result is in Table 9.

The assay result of three batches of cortex periplocae medicinal materials of table 9

In the three batches of cortex periplocae medicinal materials, the measurement result of 4-methoxysalicylaldehyde all meets " the regulation under cortex periplocae medicinal material item of Chinese pharmacopoeia version in 2010.

Embodiment

Hereinafter listed embodiment should not be construed as limiting the invention.

Embodiment 1

Safflower 21g, prepared RHIZOMA CYPERI with vinegar 78.6g, boiling hot rhizoma cibotii 104.8g, cortex periplocae 52.4g are ground into to fine powder, sieve.Pyritum ignited 13.1g boiling 2 hours, then add caulis trachelospermi 78.6g, lycopodium calvatum 78.6g, herba lycopi 78.6g, mistletoe 104.8g, reticulate millettia 78.6g to decoct secondary, and 3 hours for the first time, 2 hours for the second time, collecting decoction, filtered.Standing 8～10 hours of filtrate, it is 1.25～1.35(70 ℃ that supernatant concentration becomes relative density) thick paste, add above-mentioned fine powder to mix, add that starch is appropriate, maltose 39g, granulation, drying, be pressed into 1000, film coating, obtain.

This Chinese medicine preparation adopts following detection method:

Differentiate:

(1) get this product, put micro-Microscopic observation: corolla fragment yellow has rufous or yellowish-brown secretor (safflower).The secretory cell similar round, containing faint yellow to rufous secretion, its peripheral cell is made radial arrangement (rhizoma cyperi).Scalariform tracheid is faint yellow to golden yellow, pit marshalling (rhizoma cibotii).

(2) get 25 of this product, porphyrize, the 30ml that adds diethyl ether, place 1 hour, and jolting constantly filters, and filtrate volatilizes, and residue adds ethyl acetate 1ml to be made to dissolve, as need testing solution.Separately get rhizoma cyperi control medicinal material 0.5g, be made in the same way of control medicinal material solution.According to thin-layered chromatography (appendix VI B of Chinese Pharmacopoeia version in 2010) test, draw each 10 μ l of above-mentioned two kinds of solution, put in same silica G F respectively ₂₅₄on thin layer plate, the normal hexane-ethyl acetate (9:1) of take is developping agent, launches, and takes out, and dries, and puts under ultraviolet lamp (254nm) and inspects.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the principal spot of aobvious same color.

(3) get the 4-methoxysalicylaldehyde reference substance, add methyl alcohol and make the solution of every 1 ml containing 1mg, product solution in contrast.According to thin-layered chromatography (appendix VI B of Chinese Pharmacopoeia version in 2010), test, draw each the 5 μ l of need testing solution under reference substance solution and [discriminating] (2) item, put respectively on same silica gel g thin-layer plate, sherwood oil (60～90 ℃)-ethyl acetate-glacial acetic acid (20:3:0.5) of take is developping agent, launch, take out, dry, spray is with dinitrophenylhydrazine ethanol test solution.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color.

(4) get 30 of this product, porphyrize, add methyl alcohol 50ml, ultrasonic processing 30 minutes, filter the filtrate evaporate to dryness, residue adds water 20ml to be made to dissolve, and with hydrochloric acid, adjusts pH value to 1～2, shakes up, adding ethyl acetate extracts 2 times, each 20ml, combined ethyl acetate liquid, evaporate to dryness, residue adds methyl alcohol 1ml to be made to dissolve, as need testing solution.Separately get the protocatechualdehyde reference substance, add methyl alcohol and make the solution of every 1ml containing 0.5mg, product solution in contrast.According to thin-layered chromatography (appendix VI B of Chinese Pharmacopoeia version in 2010), test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, methenyl choloride-acetone-the formic acid (60:5:2) of take is developping agent, launch, take out, dry, spray, with 5% ferric trichloride ethanolic solution, is placed to the spot colour developing clear.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color.

(5) get 25 of this product, porphyrize, add 80% acetone 30ml, floods 1 hour, and jolting constantly, filter, and filtrate is as need testing solution.Separately get safflower control medicinal material 0.5g, add 80% acetone 10ml, jolting 15 minutes, standing, get supernatant medicinal material solution in contrast.According to thin-layered chromatography (appendix VI B of Chinese Pharmacopoeia version in 2010) test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, ethyl acetate-methyl alcohol-formic acid-the water (7:0.4:2:3) of take is developping agent, launch, take out, dry.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the principal spot of aobvious same color.

Assay:

Chromatographic condition and system suitability be take octadecylsilane chemically bonded silica as filling agent; Methanol-water-the acetic acid (40: 60: 2) of take is mobile phase; Detect wavelength 278nm.Theoretical cam curve is pressed the 4-methoxysalicylaldehyde peak and is calculated, and should be not less than 2000.

It is appropriate that the 4-methoxysalicylaldehyde reference substance is got in the preparation of reference substance solution, accurately weighed, adds 60% methyl alcohol and make the solution of 1m1 containing 20 μ g, shakes up, and obtains.

20 of this product are got in the preparation of need testing solution, accurately weighed, porphyrize, get about 1.5g, accurately weighed, put in tool plug conical flask, precision adds 60% methyl alcohol 25m1, weighed weight, ultrasonic processing (power 250W, frequency 40kHz) 30 minutes, let cool, more weighed weight, supply the weight of less loss with 60% methyl alcohol, filter, discard just filtrate, miillpore filter for subsequent filtrate (0.45 μ m) filters, and obtains.

Determination method is accurate reference substance solution and each 10 μ 1 of need testing solution of drawing respectively, and the injection liquid chromatography, measure, and obtains.

Every of this product contains cortex periplocae with 4-methoxysalicylaldehyde (C ₈h ₈o ₃) meter, must not be less than 70 μ g.

Embodiment 2

This Chinese medicine preparation adopts following detection method:

Differentiate:

(2) get 20 of this product, porphyrize, the 20ml that adds diethyl ether, place 1 hour, and jolting constantly filters, and filtrate volatilizes, and residue adds ethyl acetate 1ml to be made to dissolve, as need testing solution.Separately get rhizoma cyperi control medicinal material 1g, be made in the same way of control medicinal material solution.According to thin-layered chromatography (appendix VI B of Chinese Pharmacopoeia version in 2010) test, draw each 10 μ l of above-mentioned two kinds of solution, put in same silica G F respectively ₂₅₄on thin layer plate, the normal hexane-ethyl acetate (17:3) of take is developping agent, launches, and takes out, and dries, and puts under ultraviolet lamp (254nm) and inspects.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the principal spot of aobvious same color.

(3) get the 4-methoxysalicylaldehyde reference substance, add methyl alcohol and make the solution of every 1 ml containing 1mg, product solution in contrast.According to thin-layered chromatography (appendix VI B of Chinese Pharmacopoeia version in 2010), test, draw each the 5 μ l of need testing solution under reference substance solution and [discriminating] (2) item, put respectively on same silica gel g thin-layer plate, sherwood oil (60～90 ℃)-ethyl acetate-glacial acetic acid (15:1:0.1) of take is developping agent, launch, take out, dry, spray is with dinitrophenylhydrazine ethanol test solution.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color.

(4) get 20 of this product, porphyrize, add methyl alcohol 40ml, ultrasonic processing 30 minutes, filter the filtrate evaporate to dryness, residue adds water 20ml to be made to dissolve, and with hydrochloric acid, adjusts pH value to 1～2, shakes up, adding ethyl acetate extracts 2 times, each 20ml, combined ethyl acetate liquid, evaporate to dryness, residue adds methyl alcohol 1ml to be made to dissolve, as need testing solution.Separately get the protocatechualdehyde reference substance, add methyl alcohol and make the solution of every 1ml containing 0.5mg, product solution in contrast.According to thin-layered chromatography (appendix VI B of Chinese Pharmacopoeia version in 2010), test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, chloromethanes-ethyl acetate-the formic acid (10:8:1) of take is developping agent, launch, take out, dry, spray, with 5% ferric trichloride ethanolic solution, is placed to the spot colour developing clear.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color.

(5) get 20 of this product, porphyrize, add 80% acetone 20ml, floods 1 hour, and jolting constantly, filter, and filtrate is as need testing solution.Separately get safflower control medicinal material 0.5g, add 80% acetone 10ml, jolting 15 minutes, standing, get supernatant medicinal material solution in contrast.According to thin-layered chromatography (appendix VI B of Chinese Pharmacopoeia version in 2010) test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, ethyl acetate-methyl alcohol-formic acid-the water (5:0.3:1:1) of take is developping agent, launch, take out, dry.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the principal spot of aobvious same color.

Assay:

20 of this product are got in the preparation of need testing solution, accurately weighed, porphyrize, get about 1.5g, accurately weighed, put in tool plug conical flask, precision adds 60% methyl alcohol 25m1, weighed weight, ultrasonic processing (power 250W, frequency 40kHz) 40 minutes, let cool, more weighed weight, supply the weight of less loss with 60% methyl alcohol, filter, discard just filtrate, miillpore filter for subsequent filtrate (0.45 μ m) filters, and obtains.

Embodiment 3

This Chinese medicine preparation adopts following detection method:

Differentiate:

(2) get 30 of this product, porphyrize, the 40ml that adds diethyl ether, place 1 hour, and jolting constantly filters, and filtrate volatilizes, and residue adds ethyl acetate 1ml to be made to dissolve, as need testing solution.Separately get rhizoma cyperi control medicinal material 1g, be made in the same way of control medicinal material solution.According to thin-layered chromatography (appendix VI B of Chinese Pharmacopoeia version in 2010), test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, normal hexane-ethyl acetate-the diethylamine (20:6:1) of take is developping agent, launch, take out, dry, put under ultraviolet lamp (365nm) and inspect.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the principal spot of aobvious same color.

(3) get the 4-methoxysalicylaldehyde reference substance, add methyl alcohol and make the solution of every 1 ml containing 1mg, product solution in contrast.According to thin-layered chromatography (appendix VI B of Chinese Pharmacopoeia version in 2010), test, draw each the 5 μ l of need testing solution under reference substance solution and [discriminating] (2) item, put respectively on same silica gel g thin-layer plate, sherwood oil (60～90 ℃)-ethyl acetate-glacial acetic acid (25:4:0.8) of take is developping agent, launch, take out, dry, spray is with dinitrophenylhydrazine ethanol test solution.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color.

(4) get 40 of this product, porphyrize, add methyl alcohol 60ml, ultrasonic processing 30 minutes, filter the filtrate evaporate to dryness, residue adds water 20ml to be made to dissolve, and with hydrochloric acid, adjusts pH value to 1～2, shakes up, adding ethyl acetate extracts 2 times, each 20ml, combined ethyl acetate liquid, evaporate to dryness, residue adds methyl alcohol 1ml to be made to dissolve, as need testing solution.Separately get the protocatechualdehyde reference substance, add methyl alcohol and make the solution of every 1ml containing 0.5mg, product solution in contrast.According to thin-layered chromatography (appendix VI B of Chinese Pharmacopoeia version in 2010), test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, methenyl choloride-acetone-the formic acid (60:5:2) of take is developping agent, launch, take out, dry, spray, with 5% ferric trichloride ethanolic solution, is placed to the spot colour developing clear.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color.

(5) get 30 of this product, porphyrize, add 80% acetone 30ml, floods 1 hour, and jolting constantly, filter, and filtrate is as need testing solution.Separately get safflower control medicinal material 0.5g, add 80% acetone 10ml, jolting 15 minutes, standing, get supernatant medicinal material solution in contrast.According to thin-layered chromatography (appendix VI B of Chinese Pharmacopoeia version in 2010) test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, ethyl acetate-methyl alcohol-formic acid-the water (9:0.6:3:4) of take is developping agent, launch, take out, dry.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the principal spot of aobvious same color.

Assay:

20 of this product are got in the preparation of need testing solution, accurately weighed, porphyrize, get about 1.5g, accurately weighed, put in tool plug conical flask, precision adds 60% methyl alcohol 25m1, weighed weight, refluxing extraction 40 minutes, let cool, more weighed weight, supply the weight of less loss with 60% methyl alcohol, filter, discard just filtrate, miillpore filter for subsequent filtrate (0.45 μ m) filters, and obtains.

Claims

One kind stimulate the circulation of the blood and cause the muscles and joints to relax, the quality determining method of the Chinese medicine preparation of promoting blood circulation to remove blood stasis, this Chinese medicine preparation is made by safflower 3.05%, prepared RHIZOMA CYPERI with vinegar 11.41%, boiling hot rhizoma cibotii 15.21%, cortex periplocae 7.6%, caulis trachelospermi 11.41%, lycopodium calvatum 11.41%, herba lycopi 11.41%, mistletoe 15.21%, reticulate millettia 11.41%, pyritum ignited 1.9%, it is characterized in that the quality determining method of this Chinese medicine preparation comprises discrimination method and content assaying method:

Differentiate:

(1) get this product, put micro-Microscopic observation: corolla fragment yellow has rufous or yellowish-brown secretor; The secretory cell similar round, containing faint yellow to rufous secretion, its peripheral cell is made radial arrangement; Scalariform tracheid is faint yellow to golden yellow, the pit marshalling;

(2) get this product that is equivalent to raw medicinal herbs 13.78～20.67g, porphyrize, 20～40ml adds diethyl ether, place 1 hour, jolting constantly, filter, filtrate volatilizes, residue adds ethyl acetate 1ml to be made to dissolve, as need testing solution, separately get rhizoma cyperi control medicinal material 0.5g～1g, be made in the same way of control medicinal material solution, according to thin-layered chromatography, test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel thin-layer plate, take normal hexane-ethyl acetate-diethylamine 9～20:3～6:0～1 is developping agent, launch, take out, dry, put under ultraviolet lamp and inspect, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the principal spot of aobvious same color,

(3) get the 4-methoxysalicylaldehyde reference substance, add methyl alcohol and make the solution of every 1 ml containing 1mg, product solution in contrast, according to thin-layered chromatography, test, draw each the 5 μ l of need testing solution under reference substance solution and above-mentioned (2) item, put respectively on same silica gel g thin-layer plate, take 60～90 ℃-ethyl acetate-glacial acetic acid of sherwood oil, 15～25:1～4:0.1～0.8 is developping agent, launch, take out, dry, spray is with dinitrophenylhydrazine ethanol test solution, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color;

(4) get this product that is equivalent to raw medicinal herbs 13.78～27.56g, porphyrize, add methyl alcohol 40～60ml, ultrasonic processing 30 minutes, filter, the filtrate evaporate to dryness, residue adds water 20ml to be made to dissolve, adjust pH value to 1～2 with hydrochloric acid, shake up, adding ethyl acetate extracts 2 times, each 20ml, combined ethyl acetate liquid, evaporate to dryness, residue adds methyl alcohol 1ml to be made to dissolve, as need testing solution, separately get the protocatechualdehyde reference substance, add methyl alcohol and make the solution of every 1ml containing 0.5mg, product solution in contrast, according to thin-layered chromatography, test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take methenyl choloride-ethyl acetate-acetone-formic acid 10～60:0～8:0～5:1～2 is developping agent, launch, take out, dry, spray is with 5% ferric trichloride ethanolic solution, be placed to the spot colour developing clear, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color,

(5) get this product that is equivalent to raw medicinal herbs 13.78～20.67g, porphyrize, add 80% acetone 20～30ml, flood 1 hour, jolting constantly, filter, filtrate is as need testing solution, separately get safflower control medicinal material 0.5g, add 80% acetone 10ml, jolting 15 minutes, standing, get supernatant medicinal material solution in contrast, according to thin-layered chromatography, test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take ethyl acetate-methyl alcohol-formic acid-water 5～9:0.3～0.6:1～3:1～4 is developping agent, launch, take out, dry, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the principal spot of aobvious same color,

Assay:

Chromatographic condition and system suitability be take octadecylsilane chemically bonded silica as filling agent; Methanol-water-the acetic acid of take is mobile phase at 40: 60: 2; Detect wavelength 278nm, theoretical cam curve is pressed the 4-methoxysalicylaldehyde peak and is calculated, and should be not less than 2000;

It is appropriate that the 4-methoxysalicylaldehyde reference substance is got in the preparation of reference substance solution, accurately weighed, adds 60% methyl alcohol and make the solution of 1m1 containing 20 μ g, shakes up, and obtains;

This product that is equivalent to raw medicinal herbs 13.78g is got in the preparation of need testing solution, accurately weighed, porphyrize, get about 1.5g, accurately weighed, put in tool plug conical flask, precision adds 60% methyl alcohol 25m1, weighed weight, backflow or ultrasonic processing 30～40 minutes, let cool, more weighed weight, supply the weight of less loss with 60% methyl alcohol, filter, discard just filtrate, subsequent filtrate is that 0.45 μ m miillpore filter filters with aperture, obtains;

Determination method is accurate reference substance solution and each 10 μ 1 of need testing solution of drawing respectively, and the injection liquid chromatography, measure, and obtains.
2. according to claim 1ly a kind ofly stimulate the circulation of the blood and cause the muscles and joints to relax, the quality determining method of the Chinese medicine preparation of promoting blood circulation to remove blood stasis, this Chinese medicine preparation is made by safflower 3.05%, prepared RHIZOMA CYPERI with vinegar 11.41%, boiling hot rhizoma cibotii 15.21%, cortex periplocae 7.6%, caulis trachelospermi 11.41%, lycopodium calvatum 11.41%, herba lycopi 11.41%, mistletoe 15.21%, reticulate millettia 11.41%, pyritum ignited 1.9%, it is characterized in that the quality determining method of this Chinese medicine preparation comprises discrimination method and content assaying method:

Differentiate:

(1) get this product, put micro-Microscopic observation: corolla fragment yellow has rufous or yellowish-brown secretor; The secretory cell similar round, containing faint yellow to rufous secretion, its peripheral cell is made radial arrangement; Scalariform tracheid is faint yellow to golden yellow, the pit marshalling;

(2) get this product that is equivalent to raw medicinal herbs 17.23g, porphyrize, 30ml adds diethyl ether, place 1 hour, jolting constantly, filter, filtrate volatilizes, residue adds ethyl acetate 1ml to be made to dissolve, and as need testing solution, separately gets rhizoma cyperi control medicinal material 0.5g, be made in the same way of 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 thin layer plate, the normal hexane-ethyl acetate 9:1 of take is developping agent, launches, and takes out, and dries, put under ultraviolet lamp 254nm and inspect, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the principal spot of aobvious same color;

(3) get the 4-methoxysalicylaldehyde reference substance, add methyl alcohol and make the solution of every 1 ml containing 1mg, product solution in contrast, according to thin-layered chromatography, test, draw each the 5 μ l of need testing solution under reference substance solution and above-mentioned (2) item, put respectively on same silica gel g thin-layer plate, take 60～90 ℃-ethyl acetate of sherwood oil-glacial acetic acid 20:3:0.5 as developping agent, launch, take out, dry, spray is with dinitrophenylhydrazine ethanol test solution, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color;

(4) get this product that is equivalent to raw medicinal herbs 20.67g, porphyrize, add methyl alcohol 50ml, and ultrasonic processing 30 minutes filters, the filtrate evaporate to dryness, and residue adds water 20ml to be made to dissolve, and with hydrochloric acid, adjusts pH value to 1～2, shakes up, and adds ethyl acetate and extracts 2 times, every

Inferior 20ml, combined ethyl acetate liquid, evaporate to dryness, residue adds methyl alcohol 1ml to be made to dissolve, and as need testing solution, separately gets protocatechualdehyde

Reference substance, add methyl alcohol and make the solution of every 1ml containing 0.5mg, product solution in contrast, according to the thin-layered chromatography test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, methenyl choloride-acetone-formic acid the 60:5:2 of take is developping agent, launches, and takes out, dry, spray, with 5% ferric trichloride ethanolic solution, is placed to the spot colour developing clear, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color;

(5) get this product that is equivalent to raw medicinal herbs 17.23g, porphyrize, add 80% acetone 30ml, flood 1 hour, jolting constantly, filter, filtrate is as need testing solution, separately get safflower control medicinal material 0.5g, add 80% acetone 10ml, jolting 15 minutes, standing, get supernatant medicinal material solution in contrast, according to thin-layered chromatography, test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, ethyl acetate-methyl alcohol-formic acid-water the 7:0.4:2:3 of take is developping agent, launch, take out, dry, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the principal spot of aobvious same color,

Assay:

Chromatographic condition and system suitability be take octadecylsilane chemically bonded silica as filling agent; Methanol-water-the acetic acid of take is mobile phase at 40: 60: 2; Detect wavelength 278nm, theoretical cam curve is pressed the 4-methoxysalicylaldehyde peak and is calculated, and should be not less than 2000;

It is appropriate that the 4-methoxysalicylaldehyde reference substance is got in the preparation of reference substance solution, accurately weighed, adds 60% methyl alcohol and make the solution of 1m1 containing 20 μ g, shakes up, and obtains;

This product that is equivalent to raw medicinal herbs 13.78g is got in the preparation of need testing solution, accurately weighed, porphyrize, get about 1.5g, accurately weighed, put in tool plug conical flask, precision adds 60% methyl alcohol 25m1, weighed weight, ultrasonic processing 30 minutes, power is 250W, and frequency is 40kHz, lets cool, weighed weight again, supply the weight of less loss with 60% methyl alcohol, filter, discard just filtrate, subsequent filtrate is that 0.45 μ m miillpore filter filters with aperture, obtains;

Determination method is accurate reference substance solution and each 10 μ 1 of need testing solution of drawing respectively, and the injection liquid chromatography, measure, and obtains.
3. according to claim 1ly a kind ofly stimulate the circulation of the blood and cause the muscles and joints to relax, the quality determining method of the Chinese medicine preparation of promoting blood circulation to remove blood stasis, this Chinese medicine preparation is made by safflower 3.05%, prepared RHIZOMA CYPERI with vinegar 11.41%, boiling hot rhizoma cibotii 15.21%, cortex periplocae 7.6%, caulis trachelospermi 11.41%, lycopodium calvatum 11.41%, herba lycopi 11.41%, mistletoe 15.21%, reticulate millettia 11.41%, pyritum ignited 1.9%, it is characterized in that the quality determining method of this Chinese medicine preparation comprises discrimination method and content assaying method:

Differentiate:

(1) get this product, put micro-Microscopic observation: corolla fragment yellow has rufous or yellowish-brown secretor; The secretory cell similar round, containing faint yellow to rufous secretion, its peripheral cell is made radial arrangement; Scalariform tracheid is faint yellow to golden yellow, the pit marshalling;

(2) get this product that is equivalent to raw medicinal herbs 13.78g, porphyrize, 20ml adds diethyl ether, place 1 hour, jolting constantly, filter, filtrate volatilizes, residue adds ethyl acetate 1ml to be made to dissolve, and as need testing solution, separately gets rhizoma cyperi control medicinal material 1g, be made in the same way of 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 thin layer plate, the normal hexane-ethyl acetate 17:3 of take is developping agent, launches, and takes out, and dries, put under ultraviolet lamp 254nm and inspect, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the principal spot of aobvious same color;

(3) get the 4-methoxysalicylaldehyde reference substance, add methyl alcohol and make the solution of every 1 ml containing 1mg, product solution in contrast, according to thin-layered chromatography, test, draw each the 5 μ l of need testing solution under reference substance solution and above-mentioned (2) item, put respectively on same silica gel g thin-layer plate, 60～90 ℃-ethyl acetate of the sherwood oil-glacial acetic acid 15:1:0.1 of take is developping agent, launch, take out, dry, spray is with dinitrophenylhydrazine ethanol test solution, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color;

(4) get this product that is equivalent to raw medicinal herbs 13.78g, porphyrize, add methyl alcohol 40ml, ultrasonic processing 30 minutes, filter, the filtrate evaporate to dryness, residue adds water 20ml to be made to dissolve, adjust pH value to 1～2 with hydrochloric acid, shake up, adding ethyl acetate extracts 2 times, each 20ml, combined ethyl acetate liquid, evaporate to dryness, residue adds methyl alcohol 1ml to be made to dissolve, as need testing solution, separately get the protocatechualdehyde reference substance, add methyl alcohol and make the solution of every 1ml containing 0.5mg, product solution in contrast, according to thin-layered chromatography, test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, methenyl choloride-ethyl acetate-formic acid the 10:8:1 of take is developping agent, launch, take out, dry, spray is with 5% ferric trichloride ethanolic solution, be placed to the spot colour developing clear, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color,

(5) get this product that is equivalent to raw medicinal herbs 13.78g, porphyrize, add 80% acetone 20ml, flood 1 hour, jolting constantly, filter, filtrate is as need testing solution, separately get safflower control medicinal material 0.5g, add 80% acetone 10ml, jolting 15 minutes, standing, get supernatant medicinal material solution in contrast, according to thin-layered chromatography, test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, ethyl acetate-methyl alcohol-formic acid-water the 5:0.3:1:1 of take is developping agent, launch, take out, dry, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the principal spot of aobvious same color,

Assay:

Chromatographic condition and system suitability be take octadecylsilane chemically bonded silica as filling agent; Methanol-water-the acetic acid of take is mobile phase at 40: 60: 2; Detect wavelength 278nm, theoretical cam curve is pressed the 4-methoxysalicylaldehyde peak and is calculated, and should be not less than 2000;

It is appropriate that the 4-methoxysalicylaldehyde reference substance is got in the preparation of reference substance solution, accurately weighed, adds 60% methyl alcohol and make the solution of 1m1 containing 20 μ g, shakes up, and obtains;

This product that is equivalent to raw medicinal herbs 13.78g is got in the preparation of need testing solution, accurately weighed, porphyrize, get about 1.5g, accurately weighed, put in tool plug conical flask, precision adds 60% methyl alcohol 25m1, weighed weight, ultrasonic processing 40 minutes, power is 250W, and frequency is 40kHz, lets cool, weighed weight again, supply the weight of less loss with 60% methyl alcohol, filter, discard just filtrate, subsequent filtrate is that 0.45 μ m miillpore filter filters with aperture, obtains;

Determination method is accurate reference substance solution and each 10 μ 1 of need testing solution of drawing respectively, and the injection liquid chromatography, measure, and obtains.
4. according to claim 1ly a kind ofly stimulate the circulation of the blood and cause the muscles and joints to relax, the quality determining method of the Chinese medicine preparation of promoting blood circulation to remove blood stasis, this Chinese medicine preparation is made by safflower 3.05%, prepared RHIZOMA CYPERI with vinegar 11.41%, boiling hot rhizoma cibotii 15.21%, cortex periplocae 7.6%, caulis trachelospermi 11.41%, lycopodium calvatum 11.41%, herba lycopi 11.41%, mistletoe 15.21%, reticulate millettia 11.41%, pyritum ignited 1.9%, it is characterized in that the quality determining method of this Chinese medicine preparation comprises discrimination method and content assaying method:

Differentiate:

(1) get this product, put micro-Microscopic observation: corolla fragment yellow has rufous or yellowish-brown secretor; The secretory cell similar round, containing faint yellow to rufous secretion, its peripheral cell is made radial arrangement; Scalariform tracheid is faint yellow to golden yellow, the pit marshalling;

(2) get this product that is equivalent to raw medicinal herbs 20.67g, porphyrize, 40ml adds diethyl ether, place 1 hour, jolting constantly, filter, filtrate volatilizes, residue adds ethyl acetate 1ml to be made to dissolve, as need testing solution, separately get rhizoma cyperi control medicinal material 1g, be made in the same way of control medicinal material solution, according to thin-layered chromatography, test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, normal hexane-ethyl acetate-diethylamine the 20:6:1 of take is developping agent, launch, take out, dry, put under ultraviolet lamp 365nm and inspect, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the principal spot of aobvious same color,

(3) get the 4-methoxysalicylaldehyde reference substance, add methyl alcohol and make the solution of every 1 ml containing 1mg, product solution in contrast, according to thin-layered chromatography, test, draw each the 5 μ l of need testing solution under reference substance solution and above-mentioned (2) item, put respectively on same silica gel g thin-layer plate, 60～90 ℃-ethyl acetate of the sherwood oil-glacial acetic acid 25:4:0.8 of take is developping agent, launch, take out, dry, spray is with dinitrophenylhydrazine ethanol test solution, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color;

(4) get this product that is equivalent to raw medicinal herbs 27.56g, porphyrize, add methyl alcohol 60ml, ultrasonic processing 30 minutes, filter, the filtrate evaporate to dryness, residue adds water 20ml to be made to dissolve, adjust pH value to 1～2 with hydrochloric acid, shake up, adding ethyl acetate extracts 2 times, each 20ml, combined ethyl acetate liquid, evaporate to dryness, residue adds methyl alcohol 1ml to be made to dissolve, as need testing solution, separately get the protocatechualdehyde reference substance, add methyl alcohol and make the solution of every 1ml containing 0.5mg, product solution in contrast, according to thin-layered chromatography, test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, methenyl choloride-acetone-formic acid the 60:5:2 of take is developping agent, launch, take out, dry, spray is with 5% ferric trichloride ethanolic solution, be placed to the spot colour developing clear, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color,

(5) get this product that is equivalent to raw medicinal herbs 20.67g, porphyrize, add 80% acetone 30ml, flood 1 hour, jolting constantly, filter, filtrate is as need testing solution, separately get safflower control medicinal material 0.5g, add 80% acetone 10ml, jolting 15 minutes, standing, get supernatant medicinal material solution in contrast, according to thin-layered chromatography, test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, ethyl acetate-methyl alcohol-formic acid-water the 9:0.6:3:4 of take is developping agent, launch, take out, dry, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the principal spot of aobvious same color,

Assay:

Chromatographic condition and system suitability be take octadecylsilane chemically bonded silica as filling agent; Methanol-water-the acetic acid of take is mobile phase at 40: 60: 2; Detect wavelength 278nm, theoretical cam curve is pressed the 4-methoxysalicylaldehyde peak and is calculated, and should be not less than 2000;

It is appropriate that the 4-methoxysalicylaldehyde reference substance is got in the preparation of reference substance solution, accurately weighed, adds 60% methyl alcohol and make the solution of 1m1 containing 20 μ g, shakes up, and obtains;

This product that is equivalent to raw medicinal herbs 13.78g is got in the preparation of need testing solution, accurately weighed, porphyrize, get about 1.5g, accurately weighed, put in tool plug conical flask, precision adds 60% methyl alcohol 25m1, weighed weight, refluxing extraction 40 minutes, let cool, more weighed weight, supply the weight of less loss with 60% methyl alcohol, filter, discard just filtrate, subsequent filtrate is that 0.45 μ m miillpore filter filters with aperture, obtains;

Determination method is accurate reference substance solution and each 10 μ 1 of need testing solution of drawing respectively, and the injection liquid chromatography, measure, and obtains.
According to claim 1 or 2,3,4 describedly a kind ofly stimulate the circulation of the blood and cause the muscles and joints to relax, the quality determining method of the Chinese medicine preparation of promoting blood circulation to remove blood stasis, this product that it is characterized in that being equivalent to the 1g raw medicinal herbs containing cortex periplocae with 4-methoxysalicylaldehyde C ₈h ₈o ₃meter, must not be less than 101.58 μ g.