CN103149319B - 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 - Google Patents

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 to stimulate the circulation of the blood and cause the muscles and joints to relax, the detection 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 pharmaceutical technology sectors.

Background technology

Drug Standard of Ministry of Public Health of the Peoples Republic of China (Traditional Chinese medicine historical preparation) the 13 WS ₃relaxing that-B-2624-97 provides is made up of 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, use only microscopical characters method, omnibearing detection cannot be realized to the quality of end product quality, lower to the requirement of product quality, adds somewhat to the possibility that the product of inferior quality puts goods on the market.Present invention employs the multinomial detection by multiple spot multiaspect of microscopical characters method, four thin-layer identification methods and content assaying method, this detection method is reliable and stable and accuracy is high, more effectively ensure that the product quality put goods on the market, facilitate the control strengthened in preparation process product quality, ensure validity and the stability of product.

Summary of the invention

The object of this invention is to provide 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, this quality determining method is reliable and stable and accuracy is high, effectively ensures the product quality put goods on the market.

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

Chinese medicine preparation of the present invention is made up of 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 any one formulation described in pharmacy, and its quality determining method comprises discrimination method and content assaying method:

Differentiate:

(1) get this product, put basis of microscopic observation: corolla fragment is yellow, has rufous or yellowish-brown secretor; Secretory cell similar round, containing faint yellow to rufous secretion, its peripheral cell makes radial arrangement; Scalariform tracheid is faint yellow to golden yellow, pit marshalling;

(2) this product being equivalent to raw medicinal herbs 13.78 ~ 20.67g is got, porphyrize, add diethyl ether 20 ~ 40ml, place 1 hour, jolting constantly, filter, filtrate volatilizes, residue adds ethyl acetate 1ml makes dissolving, 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, test according to thin-layered chromatography, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel thin-layer plate, with n-hexane-ethyl acetate-diethylamine 9 ~ 20:3 ~ 6:0 ~ 1 for developping agent, launch, take out, dry, inspect under putting ultraviolet lamp, in test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the principal spot of aobvious same color,

(3) 4-methoxysalicylaldehyde reference substance is got, add methyl alcohol and make the solution of every 1 ml containing 1mg, product solution in contrast, test according to thin-layered chromatography, draw each 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, with sherwood oil 60 ~ 90 DEG C-ethyl acetate-glacial acetic acid 15 ~ 25:1 ~ 4:0.1 ~ 0.8 for developping agent, launch, take out, dry, spray is with dinitrophenylhydrazine ethanol test solution, in test sample chromatogram, on the position corresponding to reference substance chromatogram, the spot of aobvious same color;

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

(5) this product being equivalent to raw medicinal herbs 13.78 ~ 20.67g is got, 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, leave standstill, get supernatant medicinal material solution in contrast, test according to thin-layered chromatography, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with acetate-methanol-formic acid-water 5 ~ 9:0.3 ~ 0.6:1 ~ 3:1 ~ 4 for developping agent, launch, take out, dry, in test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the principal spot of aobvious same color,

Assay:

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

The preparation of reference substance solution gets 4-methoxysalicylaldehyde reference substance in right amount, accurately weighed, adds 60% methyl alcohol and makes the solution of 1m1 containing 20 μ g, shake up, to obtain final product;

This product being 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 process 30 ~ 40 minutes, let cool, more weighed weight, the weight of less loss is supplied with 60% methyl alcohol, filter, discard just filtrate, subsequent filtrate aperture is that 0.45 μm of miillpore filter filters, and to obtain final product;

Determination method is accurate respectively draws reference substance solution and each 10 μ 1 of need testing solution, injection liquid chromatography, measures, to obtain final product;

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

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

Method one:

Differentiate:

(1) get this product, put basis of microscopic observation: corolla fragment is yellow, has rufous or yellowish-brown secretor; Secretory cell similar round, containing faint yellow to rufous secretion, its peripheral cell makes radial arrangement; Scalariform tracheid is faint yellow to golden yellow, pit marshalling;

(2) get this product being equivalent to raw medicinal herbs 17.23g, porphyrize, add diethyl ether 30ml, place 1 hour, jolting constantly, filter, filtrate volatilizes, residue adds ethyl acetate 1ml makes dissolving, 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 thin-layered chromatography test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively in same silica G F ₂₅₄on thin layer plate, with n-hexane-ethyl acetate 9:1 for developping agent, launch, take out, dry, inspect under putting ultraviolet lamp 254nm, in test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the principal spot of aobvious same color;

(3) 4-methoxysalicylaldehyde reference substance is got, add methyl alcohol and make the solution of every 1 ml containing 1mg, product solution in contrast, test according to thin-layered chromatography, draw each 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, with with sherwood oil 60 ~ 90 DEG C-ethyl acetate-glacial acetic acid 20:3:0.5 for developping agent, launch, take out, dry, spray is with dinitrophenylhydrazine ethanol test solution, in test sample chromatogram, on the position corresponding to reference substance chromatogram, the spot of aobvious same color;

(4) get this product being equivalent to raw medicinal herbs 20.67g, porphyrize, add methyl alcohol 50ml, ultrasonic process 30 minutes, filter, filtrate evaporate to dryness, the residue 20ml that adds water makes dissolving, adjusts pH value to 1 ~ 2, shake up, add ethyl acetate and extract 2 times, often with hydrochloric acid

Secondary 20ml, combined ethyl acetate liquid, evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution, separately gets protocatechualdehyde

Reference substance, adds methyl alcohol and makes 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, with methenyl choloride-acetone-formic acid 60:5:2 for developping agent, launch, take out, dry, spray, with 5% ferric trichloride ethanolic solution, is placed to spot development clear, in test sample chromatogram, on the position corresponding to reference substance chromatogram, the spot of aobvious same color;

(5) this product being equivalent to raw medicinal herbs 17.23g is got, 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, leave standstill, get supernatant medicinal material solution in contrast, test according to thin-layered chromatography, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with acetate-methanol-formic acid-water 7:0.4:2:3 for developping agent, launch, take out, dry, in test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the principal spot of aobvious same color,

Assay:

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

The preparation of reference substance solution gets 4-methoxysalicylaldehyde reference substance in right amount, accurately weighed, adds 60% methyl alcohol and makes the solution of 1m1 containing 20 μ g, shake up, to obtain final product;

This product being 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 process 30 minutes, power is 250W, and frequency is 40kHz, lets cool, weighed weight again, supplies the weight of less loss with 60% methyl alcohol, filter, discard just filtrate, subsequent filtrate aperture is that 0.45 μm of miillpore filter filters, and to obtain final product;

Determination method is accurate respectively draws reference substance solution and each 10 μ 1 of need testing solution, injection liquid chromatography, measures, to obtain final product;

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

Method two:

Differentiate:

(1) get this product, put basis of microscopic observation: corolla fragment is yellow, has rufous or yellowish-brown secretor; Secretory cell similar round, containing faint yellow to rufous secretion, its peripheral cell makes radial arrangement; Scalariform tracheid is faint yellow to golden yellow, pit marshalling;

(2) get this product being equivalent to raw medicinal herbs 13.78g, porphyrize, add diethyl ether 20ml, place 1 hour, jolting constantly, filter, filtrate volatilizes, residue adds ethyl acetate 1ml makes dissolving, 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 thin-layered chromatography test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively in same silica G F ₂₅₄on thin layer plate, with n-hexane-ethyl acetate 17:3 for developping agent, launch, take out, dry, inspect under putting ultraviolet lamp 254nm, in test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the principal spot of aobvious same color;

(3) 4-methoxysalicylaldehyde reference substance is got, add methyl alcohol and make the solution of every 1 ml containing 1mg, product solution in contrast, test according to thin-layered chromatography, draw each 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, with sherwood oil 60 ~ 90 DEG C-ethyl acetate-glacial acetic acid 15:1:0.1 for developping agent, launch, take out, dry, spray is with dinitrophenylhydrazine ethanol test solution, in test sample chromatogram, on the position corresponding to reference substance chromatogram, the spot of aobvious same color;

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

(5) this product being equivalent to raw medicinal herbs 13.78g is got, 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, leave standstill, get supernatant medicinal material solution in contrast, test according to thin-layered chromatography, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with acetate-methanol-formic acid-water 5:0.3:1:1 for developping agent, launch, take out, dry, in test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the principal spot of aobvious same color,

Assay:

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

The preparation of reference substance solution gets 4-methoxysalicylaldehyde reference substance in right amount, accurately weighed, adds 60% methyl alcohol and makes the solution of 1m1 containing 20 μ g, shake up, to obtain final product;

This product being 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 process 40 minutes, power is 250W, and frequency is 40kHz, lets cool, weighed weight again, supplies the weight of less loss with 60% methyl alcohol, filter, discard just filtrate, subsequent filtrate aperture is that 0.45 μm of miillpore filter filters, and to obtain final product;

Determination method is accurate respectively draws reference substance solution and each 10 μ 1 of need testing solution, injection liquid chromatography, measures, to obtain final product;

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

Method three:

Differentiate:

(1) get this product, put basis of microscopic observation: corolla fragment is yellow, has rufous or yellowish-brown secretor; Secretory cell similar round, containing faint yellow to rufous secretion, its peripheral cell makes radial arrangement; Scalariform tracheid is faint yellow to golden yellow, pit marshalling;

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

(3) 4-methoxysalicylaldehyde reference substance is got, add methyl alcohol and make the solution of every 1 ml containing 1mg, product solution in contrast, test according to thin-layered chromatography, draw each 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, with sherwood oil 60 ~ 90 DEG C-ethyl acetate-glacial acetic acid 25:4:0.8 for developping agent, launch, take out, dry, spray is with dinitrophenylhydrazine ethanol test solution, in test sample chromatogram, on the position corresponding to reference substance chromatogram, the spot of aobvious same color;

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

(5) this product being equivalent to raw medicinal herbs 20.67g is got, 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, leave standstill, get supernatant medicinal material solution in contrast, test according to thin-layered chromatography, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with acetate-methanol-formic acid-water 9:0.6:3:4 for developping agent, launch, take out, dry, in test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the principal spot of aobvious same color,

Assay:

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

The preparation of reference substance solution gets 4-methoxysalicylaldehyde reference substance in right amount, accurately weighed, adds 60% methyl alcohol and makes the solution of 1m1 containing 20 μ g, shake up, to obtain final product;

This product being 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, the weight of less loss is supplied with 60% methyl alcohol, filter, discard just filtrate, subsequent filtrate aperture is that 0.45 μm of miillpore filter filters, and to obtain final product;

Determination method is accurate respectively draws reference substance solution and each 10 μ 1 of need testing solution, injection liquid chromatography, measures, to obtain final product;

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

Of the present inventionly 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, wherein this Chinese medicine preparation makes any one formulation in 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, 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 Drug Standard of Ministry of Public Health of the Peoples Republic of China (Traditional Chinese medicine historical preparation) the 13 WS ₃the detection method that-B-2624-97 provides, the more effective quality that ensure that product.

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 tastes, is first ground into fine powder by safflower, prepared RHIZOMA CYPERI with vinegar, boiling hot rhizoma cibotii, cortex periplocae, sieves.Pyritum ignited boiling 2 hours, then add caulis trachelospermi, lycopodium calvatum, herba lycopi, mistletoe, reticulate millettia decoction secondary, 3 hours first times, second time 2 hours, collecting decoction, filters.Filtrate leaves standstill 8 ~ 10 hours, and supernatant concentration becomes relative density to be 1.25 ~ 1.35(70 DEG C) thick paste, add the mixing of above-mentioned fine powder, add that starch is appropriate, maltose 39g, make particle, dry, be pressed into 1000, film coating, obtain final product.

Two, research process:

1, instrument and reagent

Instrument: Agilent 1100 type high performance liquid chromatograph, G1314A type UV-detector (Agilent Technologies of the U.S.); 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 Corporation).

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

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

2,4-methoxysalicylaldehyde reference substance solution is got in the selection measuring wavelength, with ultraviolet-visible pectrophotometer, in 190 ~ 400nm wavelength coverage, carries out sweep measuring.Result shows, 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; Determined wavelength: 278nm; Sample size: 10 μ l.

Under conditions selected, 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 4-methoxysalicylaldehyde peak.

4, the preparation of need testing solution

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

(2), the determination of extracting method gets this product, porphyrize, gets about 1.5g, two parts, accurately weighed, put in tool plug conical flask, precision adds 60% methyl alcohol 25ml, weighed weight, a ultrasonic process (power 250W, frequency 40kHz) 40 minutes, another part of refluxing extraction 40 minutes, cools, weighed weight, supply the weight of less loss with 60% methyl alcohol, shake up, filter.Subsequent filtrate miillpore filter (0.45 μm) filters, and measures, 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 selects simple and easy to do ultrasonic method as extracting method.(3), the determination of extraction time gets this product, porphyrize, gets about 1.5g, four parts, accurately weighed, put in tool plug conical flask, precision adds 60% methyl alcohol 25ml, weighed weight, respectively ultrasonic process (power 250W, frequency 40kHz) 10,20,30,40 minutes, let cool, more weighed weight, the weight of less loss is supplied with 60% methyl alcohol, shake up, filter.Subsequent filtrate miillpore filter (0.45 μm) filters, and measures, the results are shown in Table 2.

The comparison of table 2 extraction time

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

In sum, the preparation of need testing solution, gets this product 20, accurately weighed, porphyrize, gets about 1.5g, accurately weighed, put in tool plug conical flask, precision adds 60% methyl alcohol 25m1, weighed weight, ultrasonic process (power 250W, frequency 40kHz) 30 minutes, lets cool, weighed weight again, supplies the weight of less loss with 60% methyl alcohol, filter, discard just filtrate, subsequent filtrate miillpore filter (0.45 μm) filters, and to obtain final product.

5, interference test

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

6, the investigation of linear relationship

Accurate absorption 4-methoxysalicylaldehyde reference substance solution (0.023mg/ml )4,8,10,12,16,20 μ l, by the chromatographic condition sample introduction drafted, 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, obtains typical curve equation Y=6228.9X-18.329(r=0.9998), with peak area value to sample size (μ g) mapping, obtain a straight line.Above result shows, 4-methoxysalicylaldehyde is within the scope of 0.092 ~ 0.46 μ g, and integrating peak areas value and sample size are good linear relationship.

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

Table 4 Precision test result

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

8, stability test gets 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 result 4-methoxysalicylaldehyde peak area is 2.05%, shows that need testing solution is basicly stable in 24 hours.

9, replica test is by the content assaying method drafted, and prepares 6 parts of need testing solutions respectively 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 result 4-methoxysalicylaldehyde content is 1.65%, shows that the repeatability of this method is good.

10, recovery test gets 4-methoxysalicylaldehyde reference substance (0.2888mg/ml) 8ml, to be settled in 200 ml volumetric flasks, to shake up with 60% methanol dilution, for subsequent use; Get test sample (201101001, content) 0.292 mg/g of known content again) about 0.75g, 6 parts, accurately weighed, precision adds above-mentioned reference substance solution 25ml respectively, measures by the content assaying method drafted, with the following formulae discovery recovery, the results are shown in Table 7.

Table 7 recovery test result

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

11, sample tests gets three batches of pilot scale element sheets and Film coated tablets, and measure the content of its 4-methoxysalicylaldehyde as stated above, measurement result is in table 8.

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

According to the assay result of 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, feed intake the content (0.273% of 4-methoxysalicylaldehyde in cortex periplocae medicinal material, every sheet is 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 calculating 4-methoxysalicylaldehyde in every sheet is: 79.5%, in conjunction with the content limit (must not 0.20% be less than) of production actual conditions and " Chinese Pharmacopoeia " version in 2010 cortex periplocae medicinal material, the tentative every sheet of this product contains cortex periplocae with 4-methoxysalicylaldehyde (C ₈h ₈o ₃) meter, 70 μ g must not be less than.

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

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

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

Embodiment

Embodiment listed hereinafter 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 fine powder, sieve.Pyritum ignited 13.1g boiling 2 hours, then adds caulis trachelospermi 78.6g, lycopodium calvatum 78.6g, herba lycopi 78.6g, mistletoe 104.8g, reticulate millettia 78.6g decocts secondary, 3 hours first times, second time 2 hours, collecting decoction, filter.Filtrate leaves standstill 8 ~ 10 hours, and supernatant concentration becomes relative density to be 1.25 ~ 1.35(70 DEG C) thick paste, add the mixing of above-mentioned fine powder, add that starch is appropriate, maltose 39g, make particle, dry, be pressed into 1000, film coating, obtain final product.

This Chinese medicine preparation adopts following detection method:

Differentiate:

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

(2) get this product 25, porphyrize, add diethyl ether 30ml, places 1 hour, jolting constantly, and filter, filtrate volatilizes, and residue adds ethyl acetate 1ml makes dissolving, 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 (Chinese Pharmacopoeia version in 2010 annex VI B) test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively in same silica G F ₂₅₄on thin layer plate, with n-hexane-ethyl acetate (9:1) for developping agent, launch, take out, dry, inspect under putting ultraviolet lamp (254nm).In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the principal spot of aobvious same color.

(3) get 4-methoxysalicylaldehyde reference substance, add methyl alcohol and make the solution of every 1 ml containing 1mg, product solution in contrast.Test according to thin-layered chromatography (Chinese Pharmacopoeia version in 2010 annex VI B), draw each 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, with sherwood oil (60 ~ 90 DEG C)-ethyl acetate-glacial acetic acid (20:3:0.5) for developping agent, launch, take out, dry, spray with dinitrophenylhydrazine ethanol test solution.In test sample chromatogram, on the position corresponding to reference substance chromatogram, the spot of aobvious same color.

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

(5) get this product 25, 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, leave standstill, get supernatant medicinal material solution in contrast.According to thin-layered chromatography (Chinese Pharmacopoeia version in 2010 annex VI B) test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with acetate-methanol-formic acid-water (7:0.4:2:3) for developping agent, launch, take out, dry.In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the principal spot of aobvious same color.

Assay:

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

The preparation of reference substance solution gets 4-methoxysalicylaldehyde reference substance in right amount, accurately weighed, adds 60% methyl alcohol and makes the solution of 1m1 containing 20 μ g, shake up, to obtain final product.

This product 20 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 process (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, subsequent filtrate miillpore filter (0.45 μm) filters, and to obtain final product.

Determination method is accurate respectively draws reference substance solution and each 10 μ 1 of need testing solution, injection liquid chromatography, measures, to obtain final product.

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

Embodiment 2

Safflower 21g, prepared RHIZOMA CYPERI with vinegar 78.6g, boiling hot rhizoma cibotii 104.8g, cortex periplocae 52.4g are ground into fine powder, sieve.Pyritum ignited 13.1g boiling 2 hours, then adds caulis trachelospermi 78.6g, lycopodium calvatum 78.6g, herba lycopi 78.6g, mistletoe 104.8g, reticulate millettia 78.6g decocts secondary, 3 hours first times, second time 2 hours, collecting decoction, filter.Filtrate leaves standstill 8 ~ 10 hours, and supernatant concentration becomes relative density to be 1.25 ~ 1.35(70 DEG C) thick paste, add the mixing of above-mentioned fine powder, add that starch is appropriate, maltose 39g, make particle, dry, be pressed into 1000, film coating, obtain final product.

This Chinese medicine preparation adopts following detection method:

Differentiate:

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

(2) get this product 20, porphyrize, add diethyl ether 20ml, places 1 hour, jolting constantly, and filter, filtrate volatilizes, and residue adds ethyl acetate 1ml makes dissolving, 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 (Chinese Pharmacopoeia version in 2010 annex VI B) test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively in same silica G F ₂₅₄on thin layer plate, with n-hexane-ethyl acetate (17:3) for developping agent, launch, take out, dry, inspect under putting ultraviolet lamp (254nm).In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the principal spot of aobvious same color.

(3) get 4-methoxysalicylaldehyde reference substance, add methyl alcohol and make the solution of every 1 ml containing 1mg, product solution in contrast.Test according to thin-layered chromatography (Chinese Pharmacopoeia version in 2010 annex VI B), draw each 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, with sherwood oil (60 ~ 90 DEG C)-ethyl acetate-glacial acetic acid (15:1:0.1) for developping agent, launch, take out, dry, spray with dinitrophenylhydrazine ethanol test solution.In test sample chromatogram, on the position corresponding to reference substance chromatogram, the spot of aobvious same color.

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

(5) get this product 20, 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, leave standstill, get supernatant medicinal material solution in contrast.According to thin-layered chromatography (Chinese Pharmacopoeia version in 2010 annex VI B) test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with acetate-methanol-formic acid-water (5:0.3:1:1) for developping agent, launch, take out, dry.In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the principal spot of aobvious same color.

Assay:

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

The preparation of reference substance solution gets 4-methoxysalicylaldehyde reference substance in right amount, accurately weighed, adds 60% methyl alcohol and makes the solution of 1m1 containing 20 μ g, shake up, to obtain final product.

This product 20 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 process (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, subsequent filtrate miillpore filter (0.45 μm) filters, and to obtain final product.

Determination method is accurate respectively draws reference substance solution and each 10 μ 1 of need testing solution, injection liquid chromatography, measures, to obtain final product.

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

Embodiment 3

Safflower 21g, prepared RHIZOMA CYPERI with vinegar 78.6g, boiling hot rhizoma cibotii 104.8g, cortex periplocae 52.4g are ground into fine powder, sieve.Pyritum ignited 13.1g boiling 2 hours, then adds caulis trachelospermi 78.6g, lycopodium calvatum 78.6g, herba lycopi 78.6g, mistletoe 104.8g, reticulate millettia 78.6g decocts secondary, 3 hours first times, second time 2 hours, collecting decoction, filter.Filtrate leaves standstill 8 ~ 10 hours, and supernatant concentration becomes relative density to be 1.25 ~ 1.35(70 DEG C) thick paste, add the mixing of above-mentioned fine powder, add that starch is appropriate, maltose 39g, make particle, dry, be pressed into 1000, film coating, obtain final product.

This Chinese medicine preparation adopts following detection method:

Differentiate:

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

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

(3) get 4-methoxysalicylaldehyde reference substance, add methyl alcohol and make the solution of every 1 ml containing 1mg, product solution in contrast.Test according to thin-layered chromatography (Chinese Pharmacopoeia version in 2010 annex VI B), draw each 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, with sherwood oil (60 ~ 90 DEG C)-ethyl acetate-glacial acetic acid (25:4:0.8) for developping agent, launch, take out, dry, spray with dinitrophenylhydrazine ethanol test solution.In test sample chromatogram, on the position corresponding to reference substance chromatogram, the spot of aobvious same color.

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

(5) get this product 30, 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, leave standstill, get supernatant medicinal material solution in contrast.According to thin-layered chromatography (Chinese Pharmacopoeia version in 2010 annex VI B) test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with acetate-methanol-formic acid-water (9:0.6:3:4) for developping agent, launch, take out, dry.In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the principal spot of aobvious same color.

Assay:

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

The preparation of reference substance solution gets 4-methoxysalicylaldehyde reference substance in right amount, accurately weighed, adds 60% methyl alcohol and makes the solution of 1m1 containing 20 μ g, shake up, to obtain final product.

This product 20 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, the weight of less loss is supplied with 60% methyl alcohol, filter, discard just filtrate, subsequent filtrate miillpore filter (0.45 μm) filters, and to obtain final product.

Determination method is accurate respectively draws reference substance solution and each 10 μ 1 of need testing solution, injection liquid chromatography, measures, to obtain final product.

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

Claims (1)

1. one kind stimulate the circulation of the blood and cause the muscles and joints to relax, the detection method of the Chinese medicine preparation of promoting blood circulation to remove blood stasis, this Chinese medicine preparation is made up of 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 detection method of this Chinese medicine preparation comprises discrimination method and content assaying method:

Differentiate:

(1) get this product, put basis of microscopic observation: corolla fragment is yellow, has rufous or yellowish-brown secretor; Secretory cell similar round, containing faint yellow to rufous secretion, its peripheral cell makes radial arrangement; Scalariform tracheid is faint yellow to golden yellow, pit marshalling;

(2) this product being equivalent to raw medicinal herbs 13.78 ~ 20.67g is got, porphyrize, add diethyl ether 20 ~ 40ml, place 1 hour, jolting constantly, filter, filtrate volatilizes, residue adds ethyl acetate 1ml makes dissolving, 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, test according to thin-layered chromatography, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel thin-layer plate, with n-hexane-ethyl acetate 9:1 or n-hexane-ethyl acetate 17:3 or n-hexane-ethyl acetate-diethylamine 20:6:1 for developping agent, launch, take out, dry, inspect under putting ultraviolet lamp, in test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the principal spot of aobvious same color,

(3) 4-methoxysalicylaldehyde reference substance is got, add methyl alcohol and make the solution of every 1 ml containing 1mg, product solution in contrast, test according to thin-layered chromatography, draw each 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, with sherwood oil 60 ~ 90 DEG C-ethyl acetate-glacial acetic acid 15 ~ 25:1 ~ 4:0.1 ~ 0.8 for developping agent, launch, take out, dry, spray is with dinitrophenylhydrazine ethanol test solution, in test sample chromatogram, on the position corresponding to reference substance chromatogram, the spot of aobvious same color;

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

(5) this product being equivalent to raw medicinal herbs 13.78 ~ 20.67g is got, 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, leave standstill, get supernatant medicinal material solution in contrast, test according to thin-layered chromatography, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with acetate-methanol-formic acid-water 5 ~ 9:0.3 ~ 0.6:1 ~ 3:1 ~ 4 for developping agent, launch, take out, dry, in test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the principal spot of aobvious same color,

Assay:

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

The preparation of reference substance solution gets 4-methoxysalicylaldehyde reference substance in right amount, accurately weighed, adds 60% methyl alcohol and makes the solution of 1m1 containing 20 μ g, shake up, to obtain final product;

This product being 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 process 30 ~ 40 minutes, let cool, more weighed weight, the weight of less loss is supplied with 60% methyl alcohol, filter, discard just filtrate, subsequent filtrate aperture is that 0.45 μm of miillpore filter filters, and to obtain final product;

Determination method is accurate respectively draws reference substance solution and each 10 μ 1 of need testing solution, injection liquid chromatography, measures, to obtain final product.

2. according to claim 1ly a kind ofly to stimulate the circulation of the blood and cause the muscles and joints to relax, the detection method of the Chinese medicine preparation of promoting blood circulation to remove blood stasis, this Chinese medicine preparation is made up of 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 detection method of this Chinese medicine preparation comprises discrimination method and content assaying method:

Differentiate:

(1) get this product, put basis of microscopic observation: corolla fragment is yellow, has rufous or yellowish-brown secretor; Secretory cell similar round, containing faint yellow to rufous secretion, its peripheral cell makes radial arrangement; Scalariform tracheid is faint yellow to golden yellow, pit marshalling;

(2) get this product being equivalent to raw medicinal herbs 17.23g, porphyrize, add diethyl ether 30ml, place 1 hour, jolting constantly, filter, filtrate volatilizes, residue adds ethyl acetate 1ml makes dissolving, 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 thin-layered chromatography test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively in same silica G F ₂₅₄on thin layer plate, with n-hexane-ethyl acetate 9:1 for developping agent, launch, take out, dry, inspect under putting ultraviolet lamp 254nm, in test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the principal spot of aobvious same color;

(3) 4-methoxysalicylaldehyde reference substance is got, add methyl alcohol and make the solution of every 1 ml containing 1mg, product solution in contrast, test according to thin-layered chromatography, draw each 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, with sherwood oil 60 ~ 90 DEG C-ethyl acetate-glacial acetic acid 20:3:0.5 for developping agent, launch, take out, dry, spray is with dinitrophenylhydrazine ethanol test solution, in test sample chromatogram, on the position corresponding to reference substance chromatogram, the spot of aobvious same color;

(4) get this product being equivalent to raw medicinal herbs 20.67g, porphyrize, add methyl alcohol 50ml, ultrasonic process 30 minutes, filter, filtrate evaporate to dryness, the residue 20ml that adds water makes dissolving, with hydrochloric acid adjust pH to 1 ~ 2, shakes up, adds ethyl acetate and extract 2 times, often

Secondary 20ml, combined ethyl acetate liquid, evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution, separately gets protocatechualdehyde

Reference substance, adds methyl alcohol and makes 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, with methenyl choloride-acetone-formic acid 60:5:2 for developping agent, launch, take out, dry, spray, with 5% ferric trichloride ethanolic solution, is placed to spot development clear, in test sample chromatogram, on the position corresponding to reference substance chromatogram, the spot of aobvious same color;

(5) this product being equivalent to raw medicinal herbs 17.23g is got, 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, leave standstill, get supernatant medicinal material solution in contrast, test according to thin-layered chromatography, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with acetate-methanol-formic acid-water 7:0.4:2:3 for developping agent, launch, take out, dry, in test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the principal spot of aobvious same color,

Assay:

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

The preparation of reference substance solution gets 4-methoxysalicylaldehyde reference substance in right amount, accurately weighed, adds 60% methyl alcohol and makes the solution of 1m1 containing 20 μ g, shake up, to obtain final product;

This product being 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 process 30 minutes, power is 250W, and frequency is 40kHz, lets cool, weighed weight again, supplies the weight of less loss with 60% methyl alcohol, filter, discard just filtrate, subsequent filtrate aperture is that 0.45 μm of miillpore filter filters, and to obtain final product;

Determination method is accurate respectively draws reference substance solution and each 10 μ 1 of need testing solution, injection liquid chromatography, measures, to obtain final product.

3. according to claim 1ly a kind ofly to stimulate the circulation of the blood and cause the muscles and joints to relax, the detection method of the Chinese medicine preparation of promoting blood circulation to remove blood stasis, this Chinese medicine preparation is made up of 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 detection method of this Chinese medicine preparation comprises discrimination method and content assaying method:

Differentiate:

(1) get this product, put basis of microscopic observation: corolla fragment is yellow, has rufous or yellowish-brown secretor; Secretory cell similar round, containing faint yellow to rufous secretion, its peripheral cell makes radial arrangement; Scalariform tracheid is faint yellow to golden yellow, pit marshalling;

(2) get this product being equivalent to raw medicinal herbs 13.78g, porphyrize, add diethyl ether 20ml, place 1 hour, jolting constantly, filter, filtrate volatilizes, residue adds ethyl acetate 1ml makes dissolving, 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 thin-layered chromatography test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively in same silica G F ₂₅₄on thin layer plate, with n-hexane-ethyl acetate 17:3 for developping agent, launch, take out, dry, inspect under putting ultraviolet lamp 254nm, in test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the principal spot of aobvious same color;

(3) 4-methoxysalicylaldehyde reference substance is got, add methyl alcohol and make the solution of every 1 ml containing 1mg, product solution in contrast, test according to thin-layered chromatography, draw each 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, with sherwood oil 60 ~ 90 DEG C-ethyl acetate-glacial acetic acid 15:1:0.1 for developping agent, launch, take out, dry, spray is with dinitrophenylhydrazine ethanol test solution, in test sample chromatogram, on the position corresponding to reference substance chromatogram, the spot of aobvious same color;

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

(5) this product being equivalent to raw medicinal herbs 13.78g is got, 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, leave standstill, get supernatant medicinal material solution in contrast, test according to thin-layered chromatography, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with acetate-methanol-formic acid-water 5:0.3:1:1 for developping agent, launch, take out, dry, in test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the principal spot of aobvious same color,

Assay:

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

The preparation of reference substance solution gets 4-methoxysalicylaldehyde reference substance in right amount, accurately weighed, adds 60% methyl alcohol and makes the solution of 1m1 containing 20 μ g, shake up, to obtain final product;

This product being 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 process 40 minutes, power is 250W, and frequency is 40kHz, lets cool, weighed weight again, supplies the weight of less loss with 60% methyl alcohol, filter, discard just filtrate, subsequent filtrate aperture is that 0.45 μm of miillpore filter filters, and to obtain final product;

Determination method is accurate respectively draws reference substance solution and each 10 μ 1 of need testing solution, injection liquid chromatography, measures, to obtain final product.

4. according to claim 1ly a kind ofly to stimulate the circulation of the blood and cause the muscles and joints to relax, the detection method of the Chinese medicine preparation of promoting blood circulation to remove blood stasis, this Chinese medicine preparation is made up of 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 detection method of this Chinese medicine preparation comprises discrimination method and content assaying method:

Differentiate:

(1) get this product, put basis of microscopic observation: corolla fragment is yellow, has rufous or yellowish-brown secretor; Secretory cell similar round, containing faint yellow to rufous secretion, its peripheral cell makes radial arrangement; Scalariform tracheid is faint yellow to golden yellow, pit marshalling;

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

(3) 4-methoxysalicylaldehyde reference substance is got, add methyl alcohol and make the solution of every 1 ml containing 1mg, product solution in contrast, test according to thin-layered chromatography, draw each 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, with sherwood oil 60 ~ 90 DEG C-ethyl acetate-glacial acetic acid 25:4:0.8 for developping agent, launch, take out, dry, spray is with dinitrophenylhydrazine ethanol test solution, in test sample chromatogram, on the position corresponding to reference substance chromatogram, the spot of aobvious same color;

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

(5) this product being equivalent to raw medicinal herbs 20.67g is got, 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, leave standstill, get supernatant medicinal material solution in contrast, test according to thin-layered chromatography, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with acetate-methanol-formic acid-water 9:0.6:3:4 for developping agent, launch, take out, dry, in test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the principal spot of aobvious same color,

Assay:

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

The preparation of reference substance solution gets 4-methoxysalicylaldehyde reference substance in right amount, accurately weighed, adds 60% methyl alcohol and makes the solution of 1m1 containing 20 μ g, shake up, to obtain final product;

This product being 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, the weight of less loss is supplied with 60% methyl alcohol, filter, discard just filtrate, subsequent filtrate aperture is that 0.45 μm of miillpore filter filters, and to obtain final product;

Determination method is accurate respectively draws reference substance solution and each 10 μ 1 of need testing solution, injection liquid chromatography, measures, to obtain final product.