CN103983702B - A kind of quality determining method being used for the treatment of the Tibetan medicinal composition of stomach trouble - Google Patents

A kind of quality determining method being used for the treatment of the Tibetan medicinal composition of stomach trouble Download PDF

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CN103983702B
CN103983702B CN201310630096.5A CN201310630096A CN103983702B CN 103983702 B CN103983702 B CN 103983702B CN 201310630096 A CN201310630096 A CN 201310630096A CN 103983702 B CN103983702 B CN 103983702B
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CN103983702A (en
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张国霞
姬良亮
王维波
魏学明
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Gansu Qizheng Tibetan Medicine Co Ltd
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Gansu Qizheng Tibetan Medicine Co Ltd
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Abstract

The invention belongs to the field of Chinese medicines, be specifically related to a kind of quality testing being used for the treatment of the pharmaceutical composition of stomach trouble.Described quality determining method comprises and detecting the thin-layer qualitative of contained gallic acid, Tibet inula root, cloves, nutmeg and safflower, and/or the assay step to gallic acid in contained myrobalan, terminaliae billericae,fructus, emblic and safflower institute hydroxyl carthamin yellow A-containing.Described detection method is through many people repeatedly repeatable operation, and result accurately, favorable reproducibility, specificity be strong, meets accurate, easy, sensitive, principle fast, can the quality of effective testing product.

Description

A kind of quality determining method being used for the treatment of the Tibetan medicinal composition of stomach trouble
Technical field
The invention belongs to the field of Chinese medicines, be specifically related to a kind of quality testing being used for the treatment of the pharmaceutical composition of stomach trouble.
Background technology
Stomach trouble, is actually the general designation of many diseases.They have similar symptom, as upper abdomen gastral cavilty portion discomfort, pain, after meal glutted, belch, return acid, even Nausea and vomiting etc.Stomach trouble common clinically has the innocent and malignant tumour of acute gastritis, chronic gastritis, gastric ulcer, duodenal ulcer, gastroduodenal complex ulcer, polyp of stomach, gastric concretion, stomach, also has prolapse of gastric mucosa, acute gastric dilatation, pyloric stenosis etc.
The stomach trouble that it has often been said, generally refers to gastritis and duodenal ulcer disease.Often betide between 40-50 year, the male sex is more than women.The symptom of stomach trouble can very light also can be very heavy, modal have epigastric discomfort or pain, nausea,vomiting,diarrhea, poor appetite.The symptom of gastritis and duodenal ulcer is then upper abdomen burning pain, and particularly between two meals, early occur before the meal or after drinking orange juice, coffee, severe patient can have tarry stool, melena or bloody stool.
Tibetan medicine's theory is thought, there is " grand " (gas), " red bar " (fire), the large factor of " Baconic " (Tu Heshui) three in human body; Diet is precise and tiny, meat, blood, fat, bone, marrow, essence seven kinds of material bases; Stool, urine, sweat three kinds of excretas.Three large factors arrange seven kinds of material bases and three kinds of excremental operation changes.The conveying decomposition of " grand " main qi and blood, limb activity, five senses, food and Reproductive Performance etc.; " red bar " can hair tonic heat energy, mediation body wet look, pipe to hunger and thirst digestion, courage and insight wisdom etc.; " Baconic " carries liquid, reconciles the girth of a garment, is responsible for the sense of taste, sleep and personality etc.Think that the reason of ill is the imbalance of three large factors in environment, the impact of weather and daily life and body.Disease is divided into heat symptom-complex and the large class of sympotoms caused by cold factors two by it, and patient is divided into " grand " type, " red bar " type and " Baconic " type.Being called " wooden cloth is sick " of the comprehensive disease mixed when " grand ", " red bar " " Baconic " and " blood " and " yellow water " disease.It is when stomach is fallen ill, and symptom is sick just as Baconic, can cause gastral cavity pain, abdominal distension, inverse, indigestion of vomitting, and even gastroenteritic ulcer is hemorrhage waits disease.
Chinese patent CN101653580A discloses a kind of medicine for the treatment of stomach trouble, and its prescription bulk drug consists of roasted FRUCTUS CHEBULAE, Calcite, Ji Shoucao, Oletum Trogopterori paste, elecampane, seed of pomegranate, pawpaw, agalloch eaglewood, cloves, tufa, safflower, nutmeg, in one's early teens and SEMEN TSAOKO.This pharmaceutical composition to alleviation and treatment fullness and oppression of chest and abdomen and gastral cavity pain have certain curative effect, but for gastric ulcer even the symptom such as ulcer bleeding but rarely have curative effect, especially for comprehensive pain and the gastric ulcer symptom of having a stomach-ache caused by " wooden cloth is sick ", curative effect is very micro-.Further, in view of the requirement for drug quality and stability in this area, the method effectively can carrying out quality testing to this pharmaceutical composition in the document, is not provided yet.
Summary of the invention
For this reason, technical matters to be solved by this invention is to provide one effectively can treat stomach trouble, be particularly useful for have a stomach-ache pain, gastric ulcer and hemorrhage pharmaceutical composition and preparation method thereof that " wooden cloth is sick " causes, the prior quality determining method that there is provided a kind of above-mentioned composition.
For solving the problems of the technologies described above, the quality determining method being used for the treatment of the Tibetan medicinal composition of stomach trouble of the present invention, the bulk drug composition of described composition comprises: myrobalan 150-250 weight portion, terminaliae billericae,fructus 5-20 weight portion, emblic 20-80 weight portion, hooker winghead root 20-80 weight portion, Tibet inula root 20-80 weight portion, tufa 5-20 weight portion, safflower 5-20 weight portion, cloves 5-20 weight portion, cardamom 5-20 weight portion, nutmeg 5-20 weight portion, tsaoko 5-20 weight portion, agalloch eaglewood 5-20 weight portion, seed of pomegranate 5-20 weight portion, Indian Herba Swertiae bimaculatae 1-20 weight portion, pawpaw 1-20 weight portion, styrax 1-20 weight portion, cowherb reaches summer 1-20 weight portion,
Described quality determining method comprises the step of following Qualitive test and/or assay:
A) Qualitive test of gallic acid
Get described pharmaceutical composition 0.5-5 weight portion, add methyl alcohol 1-30 parts by volume, ultrasonic process 10-40 minute, filter, filtrate is as myrobalan, terminaliae billericae,fructus, emblic need testing solution;
Separately get gallic acid reference substance, add methyl alcohol and make the solution of every 1 parts by volume containing 0.0005-0.003 weight portion gallic acid, product solution in contrast;
By the bulk drug prescription preparation of described pharmaceutical composition containing the negative sample composition of described myrobalan, terminaliae billericae,fructus and emblic, and get described negative sample composition 0.5-5 weight portion, add methyl alcohol 1-30 parts by volume, ultrasonic process 10-40 minute, filter, filtrate is not as containing the negative sample solution of myrobalan, terminaliae billericae,fructus and emblic;
Test according to thin-layered chromatography, draw above-mentioned need testing solution, negative sample solution and reference substance solution each 1-4 μ l, put respectively on same silica gel g thin-layer plate, take volume ratio as the methenyl choloride-acetic ether-methanoic acid of 2-10:1-7:0.1-2.5 be developping agent, launch, take out, dry, spray take w/v as the ferric trichloride ethanolic solution of 1%-3%, inspects under daylight; In test sample chromatogram, on the position corresponding to reference substance chromatogram, the spot of aobvious same color;
B) Qualitive test of Tibet inula root
Get described pharmaceutical composition 0.5-5 weight portion, add diethyl ether 10-50 parts by volume, close plug, ultrasonic process 10-40 minute, filters, filtrate evaporate to dryness, and adding volume ratio is that the ether-acetic acid ethyl ester solution 0.5-2 parts by volume of l:1 makes it dissolve, as Tibet inula root need testing solution;
Separately get Tibet inula root control medicinal material 0.05-1 weight portion, add diethyl ether 0.5-2 parts by volume, close plug, ultrasonic process 10-40 minute, and leave standstill, supernatant is as Tibet inula root control medicinal material solution;
The negative sample composition of described Tibet inula root is not contained by the bulk drug prescription preparation of described pharmaceutical composition, get described negative sample composition 0.5-5 weight portion, add diethyl ether 10-50 parts by volume, close plug, ultrasonic process 10-40 minute, filters, filtrate evaporate to dryness, adding volume ratio is that the ether-acetic acid ethyl ester solution 0.5-2 parts by volume of l:1 makes it dissolve, as the negative sample solution not containing Tibet inula root;
Test according to thin-layered chromatography, draw need testing solution, negative sample solution and control medicinal material solution each 1-8 μ l, put respectively on same silica gel g thin-layer plate, take volume ratio as 60 DEG C of-90 DEG C of petroleum ether-ethyl acetates of 12-22:0.5-7 be developping agent, launch, take out, dry, spray with the secret potassium test solution of rare iodate (see " Chinese Pharmacopoeia " version in 2010); In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color;
C) Qualitive test of cloves
Get described pharmaceutical composition 0.5-5 weight portion, add diethyl ether 10-50 parts by volume, close plug, ultrasonic process 10-40 minute, filters, filtrate evaporate to dryness, and adding volume ratio is that the ether-acetic acid ethyl ester solution 0.5-2 parts by volume of l:1 makes it dissolve, as cloves need testing solution;
Separately get cloves control medicinal material 0.05-0.5 weight portion, add diethyl ether 0.5-5 parts by volume, close plug, ultrasonic process 10-40 minute, and leave standstill, supernatant is as cloves control medicinal material solution;
The negative sample composition of described cloves is not contained by the bulk drug prescription preparation of described pharmaceutical composition, get described negative sample composition 0.5-5 weight portion, add diethyl ether 10-50 parts by volume, close plug, ultrasonic process 10-40 minute, filters, filtrate evaporate to dryness, adding volume ratio is that the ether-acetic acid ethyl ester solution 0.5-2 parts by volume of l:1 makes it dissolve, as the negative sample solution not containing cloves;
Test according to thin-layered chromatography, draw need testing solution, negative sample solution and control medicinal material solution each 1-4 μ l, put respectively on same silica gel g thin-layer plate, take volume ratio as 60 DEG C of-90 DEG C of petroleum ether-ethyl acetates of 0.5-7.3:0.5-2.5 be developping agent, launch, take out, dry, spray with anisaldehyde test solution (see " Chinese Pharmacopoeia " version in 2010), be heated to spot development clear; In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color;
D) myristic Qualitive test
Get described pharmaceutical composition 0.5-5 weight portion, add diethyl ether 10-50 parts by volume, close plug, ultrasonic process 10-40 minute, filters, filtrate evaporate to dryness, and adding volume ratio is that the ether-acetic acid ethyl ester solution 0.5-2 parts by volume of l:1 makes it dissolve, as nutmeg need testing solution;
Separately get nutmeg control medicinal material 0.05-0.5 weight portion, add diethyl ether 1-10 parts by volume, close plug, ultrasonic process 10-40 minute, filters, filtrate evaporate to dryness, the ether-acetic acid ethyl ester solution 0.5-2 parts by volume adding volume ratio 1:1 makes it dissolve, as nutmeg control medicinal material solution;
Described myristic negative sample composition is not contained by the bulk drug prescription preparation of described pharmaceutical composition, get described negative sample composition 0.5-5 weight portion, add diethyl ether 10-50 parts by volume, close plug, ultrasonic process 10-40 minute, filters, filtrate evaporate to dryness, adding volume ratio is that the ether-acetic acid ethyl ester solution 0.5-2 parts by volume of l:1 makes it dissolve, as not containing myristic negative sample solution;
According to TLC test, draw need testing solution, negative sample solution and control medicinal material solution each 2-8 μ l, put respectively on same silica gel g thin-layer plate, take volume ratio as 60 DEG C of-90 DEG C of petroleum ether-ethyl acetates of 15-25:0.5-2.5 be developping agent, launch, take out, dry, spray is 3%-8% vanillic aldehyde concentrated sulfuric acid solution with w/v, is heated to spot development clear; In test sample chromatogram, with test sample chromatogram, with on control medicinal material chromatogram relevant position, show the spot of same color;
E) Qualitive test of safflower
Get described pharmaceutical composition 0.5-5 weight portion, add diethyl ether 10-50 parts by volume, close plug, ultrasonic process 10-40 minute, filters, add 80% acetone 1-10 parts by volume, ultrasonic process 10-40 minute after residue volatilizes, and leave standstill, supernatant is as safflower need testing solution;
Separately get safflower control medicinal material 0.05-0.5 weight portion, add 80% acetone 1-10 parts by volume, ultrasonic process 10-40 minute, leave standstill, supernatant is as safflower control medicinal material solution;
The negative sample composition of described safflower is not contained by the bulk drug prescription preparation of described pharmaceutical composition, get described negative sample composition 0.5-5 weight portion, add diethyl ether 10-50 parts by volume, close plug, ultrasonic process 10-40 minute, filters, 80% acetone 1-10 parts by volume is added after residue volatilizes, ultrasonic process 10-40 minute, leaves standstill, and supernatant is as the negative sample solution not containing safflower;
Test according to thin-layered chromatography, drawing need testing solution and negative sample solution 5-15 μ l, control medicinal material solution 2-8 μ l, put respectively on same silica gel g thin-layer plate, take volume ratio as the acetate-methanol of 2-8:0.5-4 is developping agent, exhibition is to about 3cm, take out, dry, then take volume ratio as the acetic ether-methanoic acid-water-methanol of 3-12:0.1-5:0.1-5:0.1-1 be developping agent, launch, take out, dry, inspect under daylight; In test sample chromatogram, with test sample chromatogram, with on control medicinal material chromatogram relevant position, show the spot of same color;
F) assay of gallic acid in myrobalan, terminaliae billericae,fructus, emblic
Get described pharmaceutical composition 0.05-2 weight portion, put in tool plug conical flask, add 50% methyl alcohol 25-75 parts by volume, weighed weight, ultrasonic process 10-45min under power 100W, frequency 40kHz condition, let cool, more weighed weight, the weight of less loss is supplied with 50% methyl alcohol, shake up, filter, get filtrate, as need testing solution;
Get gallic acid reference substance appropriate, accurately weighed, add 50% methyl alcohol and make the solution of every 1 parts by volume containing gallic acid 0.00001-0.0001 weight portion, product solution in contrast;
By the bulk drug prescription preparation of described pharmaceutical composition containing the negative sample composition of described myrobalan, terminaliae billericae,fructus and emblic, and get described negative sample composition 0.05-2 weight portion, put in tool plug conical flask, add 50% methyl alcohol 25-75 parts by volume, weighed weight, ultrasonic process 10-45min under power 100W, frequency 40kHz condition, let cool, weighed weight again, supply the weight of less loss with 50% methyl alcohol, shake up, filter, get filtrate, as the negative sample solution not containing myrobalan, terminaliae billericae,fructus, emblic;
According to high performance liquid chromatography test, take octadecylsilane chemically bonded silica as filling agent, with 0.1% phosphoric acid solution of acetonitrile-0.1% triethylamine of volume ratio 0.1-5:95-99.9 for mobile phase; Measure at 273 ± 2nm determined wavelength, column temperature are 25-40 DEG C; Theoretical cam curve calculates by gallic acid peak and is not less than 1000; And precision draws reference substance solution, need testing solution, negative sample solution each 5-20 μ l respectively, injection liquid chromatography, measures, and every 1 weight portion of preparation in gallic acid, must not be less than 0.017 weight portion containing myrobalan, terminaliae billericae,fructus, emblic;
G) assay of safflower institute hydroxyl carthamin yellow A-containing
Get described pharmaceutical composition 0.5-3 weight portion, accurately weighed, put in tool plug conical flask, precision adds 25% methyl alcohol 20-80 parts by volume, and weighed weight, in power 250W, ultrasonic process 20-60 minute under frequency 40kHz condition, let cool, more weighed weight, the weight of less loss is supplied with 25% methyl alcohol, shake up, filter, get subsequent filtrate, as need testing solution;
Get hydroxyl radical carthamin yellow carthamus A reference substance appropriate, accurately weighed, add the solution that 25% methyl alcohol makes every 1 parts by volume hydroxyl carthamin yellow A-containing 0.000005-0.00005 weight portion, product solution in contrast;
Do not contain the negative sample composition of described safflower by the bulk drug prescription preparation of described pharmaceutical composition, get described negative sample composition 0.5-3 weight portion, accurately weighed, put in tool plug conical flask, precision adds 25% methyl alcohol 20-80 parts by volume, weighed weight, under power 250W, frequency 40kHz condition, ultrasonic process 20-60 minute, lets cool, weighed weight again, supply the weight of less loss with 25% methyl alcohol, shake up, filter, get subsequent filtrate, as the negative sample solution not containing safflower;
According to high performance liquid chromatography test, take octadecylsilane chemically bonded silica as filling agent; With methanol-acetonitrile-0.2% phosphoric acid solution of volume ratio 15-30:0.5-5:65-85 for mobile phase; Measure at 403 ± 2nm determined wavelength, column temperature are 25-40 DEG C; Number of theoretical plate calculates should be not less than 3000 by hydroxyl radical carthamin yellow carthamus A peak; Accurate absorption reference substance solution, need testing solution, negative sample solution each 5-20 μ l respectively, injection liquid chromatography, measures, and every 1 weight portion of preparation in hydroxyl radical carthamin yellow carthamus A, must not be less than 0.00025 weight portion containing safflower;
The pass of described weight portion and parts by volume is g/ml.
Further, described quality determining method, comprises the step of following Qualitive test and/or assay:
A) Qualitive test of gallic acid
Get described pharmaceutical composition 1 weight portion, add methyl alcohol 5 parts by volume, ultrasonic process 20 minutes, filter, filtrate is as myrobalan, terminaliae billericae,fructus, emblic need testing solution;
Separately get gallic acid reference substance, add methyl alcohol and make the solution of every 1 parts by volume containing 0.001 weight portion gallic acid, product solution in contrast;
By the bulk drug prescription preparation of described pharmaceutical composition containing the negative sample composition of described myrobalan, terminaliae billericae,fructus and emblic, and get described negative sample composition 1 weight portion, add methyl alcohol 5 parts by volume, ultrasonic process 20 minutes, filter, filtrate is not as containing the negative sample solution of myrobalan, terminaliae billericae,fructus and emblic;
Test according to thin-layered chromatography, draw each 2 μ l of above-mentioned need testing solution, negative sample solution and reference substance solution, put respectively on same silica gel g thin-layer plate, take volume ratio as the methenyl choloride-acetic ether-methanoic acid of 6:4:1 be developping agent, launch, take out, dry, spray take w/v as the ferric trichloride ethanolic solution of 2%, inspects under daylight; In test sample chromatogram, on the position corresponding to reference substance chromatogram, the spot of aobvious same color;
B) Qualitive test of Tibet inula root
Get described pharmaceutical composition 2 weight portion, add diethyl ether 20 parts by volume, close plug, ultrasonic process 20 minutes, filters, filtrate evaporate to dryness, and adding volume ratio is that ether-acetic acid ethyl ester solution 1 parts by volume of l:1 makes it dissolve, as Tibet inula root need testing solution;
Separately get Tibet inula root control medicinal material 0.2 weight portion, add diethyl ether 1 parts by volume, close plug, ultrasonic process 20 minutes, and leave standstill, supernatant is as Tibet inula root control medicinal material solution;
The negative sample composition of described Tibet inula root is not contained by the bulk drug prescription preparation of described pharmaceutical composition, get described negative sample composition 2 weight portion, add diethyl ether 20 parts by volume, close plug, ultrasonic process 20 minutes, filters, filtrate evaporate to dryness, adding volume ratio is that ether-acetic acid ethyl ester solution 1 parts by volume of l:1 makes it dissolve, as the negative sample solution not containing Tibet inula root;
According to thin-layered chromatography test, draw each 4 μ l of need testing solution, negative sample solution and control medicinal material solution, put respectively on same silica gel g thin-layer plate, take volume ratio as 60 DEG C of-90 DEG C of petroleum ether-ethyl acetates of 17:3 be developping agent, launch, take out, dry, spray with the secret potassium test solution of rare iodate; In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color;
C) Qualitive test of cloves
Get described pharmaceutical composition 2 weight portion, add diethyl ether 20 parts by volume, close plug, ultrasonic process 20 minutes, filters, filtrate evaporate to dryness, and adding volume ratio is that ether-acetic acid ethyl ester solution 1 parts by volume of l:1 makes it dissolve, as cloves need testing solution;
Separately get cloves control medicinal material 0.2 weight portion, add diethyl ether 3 parts by volume, close plug, ultrasonic process 20 minutes, and leave standstill, supernatant is as cloves control medicinal material solution;
The negative sample composition of described cloves is not contained by the bulk drug prescription preparation of described pharmaceutical composition, get described negative sample composition 2 weight portion, add diethyl ether 20 parts by volume, close plug, ultrasonic process 20 minutes, filters, filtrate evaporate to dryness, adding volume ratio is that ether-acetic acid ethyl ester solution 1 parts by volume of l:1 makes it dissolve, as the negative sample solution not containing cloves;
Test according to thin-layered chromatography, draw each 2 μ l of need testing solution, negative sample solution and control medicinal material solution, put respectively on same silica gel g thin-layer plate, take volume ratio as 60 DEG C of-90 DEG C of petroleum ether-ethyl acetates of 3:1 be developping agent, launch, take out, dry, spray, with anisaldehyde test solution, is heated to spot development clear; In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color;
D) myristic Qualitive test
Get described pharmaceutical composition 2 weight portion, add diethyl ether 20 parts by volume, close plug, ultrasonic process 20 minutes, filters, filtrate evaporate to dryness, and adding volume ratio is that ether-acetic acid ethyl ester solution 1 parts by volume of l:1 makes it dissolve, as nutmeg need testing solution;
Separately get nutmeg control medicinal material 0.2 weight portion, add diethyl ether 5 parts by volume, close plug, ultrasonic process 20 minutes, and filter, filtrate evaporate to dryness, ether-acetic acid ethyl ester solution 1 parts by volume adding volume ratio 1:1 makes it dissolve, as nutmeg control medicinal material solution;
Described myristic negative sample composition is not contained by the bulk drug prescription preparation of described pharmaceutical composition, get described negative sample composition 2 weight portion, add diethyl ether 20 parts by volume, close plug, ultrasonic process 20 minutes, filters, filtrate evaporate to dryness, adding volume ratio is that ether-acetic acid ethyl ester solution 1 parts by volume of l:1 makes it dissolve, as not containing myristic negative sample solution;
According to TLC test, draw each 5 μ l of need testing solution, negative sample solution and control medicinal material solution, put respectively on same silica gel g thin-layer plate, take volume ratio as 60 DEG C of-90 DEG C of petroleum ether-ethyl acetates of 20:1 be developping agent, launch, take out, dry, spray is 5% vanillic aldehyde concentrated sulfuric acid solution with w/v, is heated to spot development clear; In test sample chromatogram, with test sample chromatogram, with on control medicinal material chromatogram relevant position, show the spot of same color;
E) Qualitive test of safflower
Get described pharmaceutical composition 2 weight portion, add diethyl ether 20 parts by volume, close plug, ultrasonic process 20 minutes, filters, add 80% acetone 5 parts by volume, ultrasonic process 20 minutes after residue volatilizes, and leave standstill, supernatant is as safflower need testing solution;
Separately get safflower control medicinal material 0.2 weight portion, add 80% acetone 5 parts by volume, ultrasonic process 20 minutes, leave standstill, supernatant is as safflower control medicinal material solution;
The negative sample composition of described safflower is not contained by the bulk drug prescription preparation of described pharmaceutical composition, get described negative sample composition 2 weight portion, add diethyl ether 20 parts by volume, close plug, ultrasonic process 20 minutes, filters, 80% acetone 5 parts by volume is added after residue volatilizes, ultrasonic process 20 minutes, leaves standstill, and supernatant is as the negative sample solution not containing safflower;
Test according to thin-layered chromatography, drawing need testing solution and negative sample solution 10 μ l, control medicinal material solution 5 μ l, put respectively on same silica gel g thin-layer plate, take volume ratio as the acetate-methanol of 5:2 is developping agent, exhibition is to about 3cm, take out, dry, then take volume ratio as the acetic ether-methanoic acid-water-methanol of 7:2:3:0.4 be developping agent, launch, take out, dry, inspect under daylight; In test sample chromatogram, with test sample chromatogram, with on control medicinal material chromatogram relevant position, show the spot of same color;
F) assay of gallic acid in myrobalan, terminaliae billericae,fructus, emblic
Get described pharmaceutical composition 0.1 weight portion, put in tool plug conical flask, add 50% methyl alcohol 50 parts by volume, weighed weight, ultrasonic process 30min under power 100W, frequency 40kHz condition, let cool, more weighed weight, the weight of less loss is supplied with 50% methyl alcohol, shake up, filter, get filtrate, as need testing solution;
Get gallic acid reference substance appropriate, accurately weighed, add 50% methyl alcohol and make the solution of every 1 parts by volume containing gallic acid 0.00004 weight portion, product solution in contrast;
By the bulk drug prescription preparation of described pharmaceutical composition containing the negative sample composition of described myrobalan, terminaliae billericae,fructus and emblic, and get described negative sample composition 0.1 weight portion, put in tool plug conical flask, add 50% methyl alcohol 50 parts by volume, weighed weight, under power 100W, frequency 40kHz condition, ultrasonic process 30min, lets cool, weighed weight again, supply the weight of less loss with 50% methyl alcohol, shake up, filter, get filtrate, as the negative sample solution not containing myrobalan, terminaliae billericae,fructus, emblic;
According to high performance liquid chromatography test, take octadecylsilane chemically bonded silica as filling agent, with 0.1% phosphoric acid solution of acetonitrile-0.1% triethylamine of volume ratio 1:99 for mobile phase; Measure at 273nm determined wavelength, column temperature are 30 DEG C; Theoretical cam curve calculates by gallic acid peak and is not less than 1000; And precision draws reference substance solution, need testing solution, each 10 μ l of negative sample solution respectively, injection liquid chromatography, measures, and every 1 weight portion of preparation in gallic acid, must not be less than 0.017 weight portion containing myrobalan, terminaliae billericae,fructus, emblic;
G) assay of safflower institute hydroxyl carthamin yellow A-containing
Get described pharmaceutical composition 1.5 weight portion, accurately weighed, put in tool plug conical flask, precision adds 25% methyl alcohol 50 parts by volume, and weighed weight, in power 250W, ultrasonic process 40 minutes under frequency 40kHz condition, let cool, more weighed weight, the weight of less loss is supplied with 25% methyl alcohol, shake up, filter, get subsequent filtrate, as need testing solution;
Get hydroxyl radical carthamin yellow carthamus A reference substance appropriate, accurately weighed, add the solution that 25% methyl alcohol makes every 1 parts by volume hydroxyl carthamin yellow A-containing 0.00002 weight portion, product solution in contrast;
Do not contain the negative sample composition of described safflower by the bulk drug prescription preparation of described pharmaceutical composition, get described negative sample composition 1.5 weight portion, accurately weighed, put in tool plug conical flask, precision adds 25% methyl alcohol 50 parts by volume, weighed weight, under power 250W, frequency 40kHz condition, ultrasonic process 40 minutes, lets cool, weighed weight again, supply the weight of less loss with 25% methyl alcohol, shake up, filter, get subsequent filtrate, as the negative sample solution not containing safflower;
According to high performance liquid chromatography test, take octadecylsilane chemically bonded silica as filling agent; With methanol-acetonitrile-0.2% phosphoric acid solution of volume ratio 22:2:76 for mobile phase; Measure at 403nm determined wavelength, column temperature are 30 DEG C; Number of theoretical plate calculates should be not less than 3000 by hydroxyl radical carthamin yellow carthamus A peak; Accurate absorption reference substance solution, need testing solution, each 10 μ l of negative sample solution respectively, injection liquid chromatography, measures, and every 1 weight portion of preparation in hydroxyl radical carthamin yellow carthamus A, must not be less than 0.00025 weight portion containing safflower.
The bulk drug of described composition consists of:
Myrobalan 180-220 weight portion, terminaliae billericae,fructus 8-15 weight portion, emblic 30-70 weight portion, hooker winghead root 30-70 weight portion, Tibet inula root 30-70 weight portion, tufa 8-15 weight portion, safflower 8-15 weight portion, cloves 8-15 weight portion, cardamom 8-15 weight portion, nutmeg 8-15 weight portion, tsaoko 8-15 weight portion, agalloch eaglewood 8-15 weight portion, seed of pomegranate 8-15 weight portion, Indian Herba Swertiae bimaculatae 5-15 weight portion, pawpaw 5-15 weight portion, styrax 5-15 weight portion, cowherb reaches summer 5-15 weight portion.
The bulk drug of described composition consists of:
Myrobalan 201.7 weight portion, terminaliae billericae,fructus 12.6 weight portion, emblic 50.4 weight portion, hooker winghead root 50.4 weight portion, Tibet inula root 50.4 weight portion, tufa 11.8 weight portion, safflower 11.8 weight portion, cloves 11.8 weight portion, cardamom 11.8 weight portion, nutmeg 11.8 weight portion, tsaoko 11.8 weight portion, agalloch eaglewood 11.8 weight portion, seed of pomegranate 11.8 weight portion, Indian Herba Swertiae bimaculatae 10.1 weight portion, pawpaw 10.1 weight portion, styrax 10.1 weight portion, cowherb reach summer 10.1 weight portion.
Described composition makes clinical or pharmaceutically acceptable tablet, capsule, granule, powder, pill, sustained release agent or oral liquid.
Described pharmaceutical composition prepares by the following method:
Above-mentioned ten seven flavor medicine materials, wherein, Tibet inula root, cloves, cardamom, nutmeg, tsaoko, agalloch eaglewood, styrax, safflower and tufa are pulverized, and sieve; Myrobalan, terminaliae billericae,fructus, emblic, cowherb reach summer boiling twice, collecting decoction, and filter, filtrate is condensed into cream clearly, for subsequent use; Hooker winghead root, seed of pomegranate, India's camphor tree bud dish and pawpaw, add alcohol reflux and extract secondary, merging filtrate, filtrate recycling ethanol, to appropriate, adds above-mentioned clear cream, be condensed into cream clearly, drying, adds fine medicinal material powder, mixing, by pharmacy conventional method, add customary adjuvant and make the tablet, capsule, granule, powder, pill, sustained release agent or the oral liquid that accept clinically.
Further, described pharmaceutical composition prepares by the following method: according to the powder that selected weight portion gets Tibet inula root, tufa, safflower, cloves, cardamom, nutmeg, tsaoko, agalloch eaglewood, styrax are crushed to 0.01-200 micron; Separately get the myrobalan of selected weight portion, terminaliae billericae,fructus, emblic and cowherb to reach the summer and add the water accounting for drug weight 5-20 times of weight, extract 1-5 time, obtain Aqueous extracts; Get surplus stock hooker winghead root, seed of pomegranate, Indian Herba Swertiae bimaculatae and pawpaw and add the ethanol that the concentration accounting for drug weight 5-20 times of weight is greater than 20%, extract 1-5 time, obtain alcohol extract; Respectively described Aqueous extracts, alcohol extract are concentrated into the medicinal extract that relative density is 0.5-1.35, mixes with aforementioned powder pulverized powder after dry, add customary adjuvant conveniently technique make clinical acceptable tablet, capsule, granule, powder, pill, sustained release agent or oral liquid.
Quality determining method of the present invention establishes the described thin-layer identification method being used for the treatment of contained gallic acid, Tibet inula root, cloves, nutmeg, safflower in the pharmaceutical composition of stomach trouble, and specificity is strong, and reappearance is better; And establish method for measuring is carried out to the content of gallic acid, safflower institute hydroxyl carthamin yellow A-containing contained by myrobalan, terminaliae billericae,fructus, emblic in prescription preparation, this detection method not only specificity is strong, and negative noiseless.Described detection method is through many people repeatedly repeatable operation, and result accurately, favorable reproducibility, specificity be strong, meets accurate, easy, sensitive, principle fast, can the quality of effective testing product.
Accompanying drawing explanation
In order to make content of the present invention be more likely to be clearly understood, below according to a particular embodiment of the invention and by reference to the accompanying drawings, the present invention is further detailed explanation, wherein
Fig. 1 is gallic acid of the present invention qualification thin-layer chromatogram; Wherein, label 1-3 is test sample, and label 4-5 is gallic acid reference substance, and label 6 is not containing the negative sample of myrobalan, terminaliae billericae,fructus, emblic;
Fig. 2 is Tibet inula root of the present invention qualification thin-layer chromatogram; Wherein, label 1-3 is test sample, and label 4-5 is Tibet inula root control medicinal material, and label 6 is not containing the negative sample of Tibet inula root;
Fig. 3 is cloves of the present invention qualification thin-layer chromatogram; Wherein, label 1-3 is test sample, and label 4-5 is cloves control medicinal material, and label 6 is not containing the negative sample of cloves;
Fig. 4 is nutmeg of the present invention qualification thin-layer chromatogram; Wherein, label 1-3 is test sample, and label 4-5 is nutmeg control medicinal material, and label 6 is not containing myristic negative sample;
Fig. 5 is safflower of the present invention qualification thin-layer chromatogram; Wherein, label 1-3 is test sample, and label 4-5 is safflower control medicinal material, and label 6 is not containing the negative sample of safflower;
Fig. 6 is gallic acid chromatogram contained by myrobalan of the present invention, terminaliae billericae,fructus, emblic; Wherein, label A-C be respectively hydroxyl radical carthamin yellow carthamus A reference substance, test sample, not containing myrobalan, terminaliae billericae,fructus, emblic negative sample;
Fig. 7 is the peak area of gallic acid and the linear relationship chart of reference substance concentration;
Fig. 8 is safflower institute of the present invention hydroxyl carthamin yellow A-containing chromatogram; Wherein, label A-C be respectively hydroxyl radical carthamin yellow carthamus A reference substance, safflower test sample, not containing the negative sample of safflower;
Fig. 9 is the peak area of hydroxyl radical carthamin yellow carthamus A and the linear relationship chart of reference substance concentration.
Embodiment
The quality testing of embodiment 1 hard capsule
[prescription] myrobalan 201.7g, terminaliae billericae,fructus 12.6g, emblic 50.4g, hooker winghead root 50.4g, Tibet inula root 50.4g, tufa 11.8g, safflower 11.8g, cloves 11.8g, cardamom 11.8g, nutmeg 11.8g, tsaoko 11.8g, agalloch eaglewood 11.8g, seed of pomegranate 11.8g, Indian Herba Swertiae bimaculatae 10.1g, pawpaw 10.1g, styrax 10.1g, cowherb reach summer 10.1g.
[method] is got Tibet inula root, cloves, cardamom, nutmeg, tsaoko, agalloch eaglewood, styrax, safflower and tufa and is ground into 40-50 micron fine powder; Get myrobalan, terminaliae billericae,fructus, emblic, cowherb reach summer boiling twice, first time adds the water accounting for drug weight 12 times of weight, second time adds the water accounting for drug weight 10 times of weight, each 1.5 hours, collecting decoction, filter, filtrate is concentrated into relative density and is about 1.05-1.10(70 DEG C) clear cream, for subsequent use; Hooker winghead root, seed of pomegranate, India's river deer bud dish and pawpaw, add 70% alcohol reflux and extract secondary, first time adds 70% ethanol accounting for drug weight 10 times of weight, add for the second time and account for 70% of drug weight 9 times of weight, each 2 hours, filter, merging filtrate, filtrate recycling ethanol, to appropriate, adds above-mentioned clear cream, being concentrated into relative density is 1.10-1.15(70 DEG C) clear cream, vacuum or spraying dry, add fine medicinal material powder, mixing, add customary adjuvant, make clinical acceptable hard shell capsules.
[quality determining method]
The TLC distinguish of A, gallic acid
Get above-mentioned hard shell capsules 1g, add methyl alcohol 5ml, ultrasonic process 20 minutes, filter, filtrate is as myrobalan, terminaliae billericae,fructus, emblic need testing solution.
Separately get gallic acid reference substance, add methyl alcohol and make the solution of every 1ml containing 1mg, product solution in contrast.
In prescription ratio and the preparation technology of above-mentioned capsule, configuration not containing the negative sample of myrobalan, terminaliae billericae,fructus, emblic, and is made not containing the negative sample solution of myrobalan, terminaliae billericae,fructus, emblic by the compound method of above-mentioned need testing solution.
Test according to thin-layered chromatography " Chinese Pharmacopoeia " version in 2010 annex VIB, draw each 2 μ l of need testing solution, negative sample solution and reference substance solution respectively, put respectively on same silica gel g thin-layer plate, with methenyl choloride-acetic ether-methanoic acid (6:4:1) for developping agent, launch, take out, dry, spray, with 2% ferric trichloride ethanolic solution, is inspected under daylight; In test sample chromatogram, on the position corresponding to reference substance chromatogram, the spot of aobvious same color.Thin-layer chromatogram is shown in Fig. 1.
The TLC distinguish of B, Tibet inula root
Get above-mentioned hard shell capsules 2g, add diethyl ether 20ml, close plug, ultrasonic process 20 minutes, and filter, filtrate evaporate to dryness ,-ethyl acetate (l:1) the solution 1ml that adds diethyl ether makes dissolving, as Tibet inula root need testing solution.
Separately get Tibet inula root control medicinal material 0.2g, add diethyl ether 1ml, close plug, ultrasonic process 20 minutes, and leave standstill, supernatant is as Tibet inula root control medicinal material solution.
In above-mentioned capsule prescription ratio and preparation technology, configuration not containing the negative sample of Tibet inula root, and is made not containing the negative sample solution of Tibet inula root by the compound method of above-mentioned need testing solution.
Test according to thin-layered chromatography " Chinese Pharmacopoeia " version in 2010 annex VIB, draw each 4 μ l of need testing solution, negative sample solution and control medicinal material solution, put respectively on same silica gel g thin-layer plate, with sherwood oil (60 DEG C-90 DEG C)-ethyl acetate (17:3) for developping agent, launch, take out, dry, spray with the secret potassium test solution of rare iodate; In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color.Thin layer detects spectrogram and sees Fig. 2.
The TLC distinguish of C, cloves
Get above-mentioned hard shell capsules 2g, add diethyl ether 20ml, close plug, ultrasonic process 20 minutes, and filter, filtrate evaporate to dryness ,-ethyl acetate (l:1) the solution 1ml that adds diethyl ether makes dissolving, as cloves need testing solution.
Separately get cloves control medicinal material 0.2g, add diethyl ether 3ml, close plug, ultrasonic process 20 minutes, and leave standstill, supernatant is as cloves control medicinal material solution.
In above-mentioned capsule prescription ratio and preparation technology, configuration not containing the negative sample of cloves, and is made not containing the negative sample solution of cloves by the compound method of above-mentioned need testing solution.
Test according to thin-layered chromatography " Chinese Pharmacopoeia " version in 2010 annex VIB, draw each 2 μ l of need testing solution, negative sample solution and control medicinal material solution, put respectively on same silica gel g thin-layer plate, with sherwood oil (60 DEG C-90 DEG C)-ethyl acetate (3:1) for developping agent, launch, take out, dry, spray, with anisaldehyde test solution, is heated to spot development clear; In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color.Thin layer detects spectrogram and sees place shown in Fig. 3 circle.
D, myristic TLC distinguish
Get above-mentioned hard shell capsules 2g, add diethyl ether 20ml, close plug, ultrasonic process 20 minutes, and filter, filtrate evaporate to dryness ,-ethyl acetate (l:1) the solution 1ml that adds diethyl ether makes dissolving, as nutmeg need testing solution.
Separately get nutmeg control medicinal material 0.2g, add diethyl ether 5ml, close plug, ultrasonic process 20 minutes, filters, filtrate evaporate to dryness, and residue-ethyl acetate (l:1) the solution 1ml that adds diethyl ether makes dissolving, as nutmeg control medicinal material solution.
In prescription ratio and the preparation technology of above-mentioned capsule, configuration not containing myristic negative sample, and is made not containing myristic negative sample solution by the compound method of above-mentioned need testing solution.
Test according to thin-layered chromatography " Chinese Pharmacopoeia " version in 2010 annex VIB, draw each 5 μ l of need testing solution, negative sample solution and control medicinal material solution, put respectively on same silica gel g thin-layer plate, with sherwood oil (60 DEG C-90 DEG C)-ethyl acetate (20:1) for developping agent, launch, take out, dry, spray, with 5% vanillic aldehyde concentrated sulfuric acid solution, is heated to spot development clear; In test sample chromatogram, with test sample chromatogram, with on control medicinal material chromatogram relevant position, show the spot of same color.Thin layer detects spectrogram and sees Fig. 4.
The TLC distinguish of E, safflower
Get above-mentioned hard shell capsules 2g, add diethyl ether 20ml, close plug, ultrasonic process 20 minutes, filters, add 80% acetone 5ml, ultrasonic process 20 minutes after residue volatilizes, and leave standstill, supernatant is as safflower need testing solution.
Separately get safflower control medicinal material 0.2g, add 80% acetone 5ml, ultrasonic process 20 minutes, leave standstill, supernatant is as safflower control medicinal material solution.
In above-mentioned capsule prescription ratio and preparation technology, configuration not containing the negative sample of safflower, and is made not containing the negative sample solution of safflower by the compound method of above-mentioned need testing solution.
Test according to thin-layered chromatography " Chinese Pharmacopoeia " version in 2010 annex VIB, draw need testing solution and negative sample solution 10 μ l, control medicinal material solution 5 μ l, put respectively on same silica gel g thin-layer plate, with acetate-methanol (5:2) for developping agent, exhibition is to about 3cm, take out, dry, then with acetic ether-methanoic acid-water-methanol (7:2:3:0.4) for developping agent, launch, take out, dry, inspect under daylight; In test sample chromatogram, with test sample chromatogram, with on control medicinal material chromatogram relevant position, show the spot of same color.Thin layer detects spectrogram and sees Fig. 5.
Gallic acid content contained by F, myrobalan, terminaliae billericae,fructus, emblic measures
(1) instrument and reagent
Aglient1100 high performance liquid chromatograph (Agilent company of the U.S.): G1314A UV-vis detector, G1314A binary pump, Agilent1100 chromatographic work station.
SK8200HP ultrasonic cleaner (power 250W, frequency 40kHz) (Shanghai High Kudos Science Instrument Co., Ltd.).
Acetonitrile (chromatographically pure); Methyl alcohol (analyzing pure, chromatographically pure).
Gallic acid reference substance (lot number: 110831-200803), purchased from Chinese pharmaceutical biological product qualification institute.
(2) chromatographic condition
Octadecyl silane is filling agent; Chromatographic column: ZORBAXEclipsepluslC18(4.6 × 250mm, 5.0 μm, Dalian is according to Lyntech Corporation (US) 10177 South 77th East Avenue Tulsa, Oklahoma 74133 U.S.);
Mobile phase: acetonitrile-containing 0.1% phosphoric acid solution (l:99) of 0.1% triethylamine is mobile phase;
Flow velocity: 1.0ml/min;
Determined wavelength: 273nm;
Column temperature: 30 DEG C.
(3) chromatographic system employment and suitability test (E & ST)
Under above chromatographic condition, better, theoretical cam curve calculates should be not less than 1000 by gallic acid for gallic acid and all the other impurity peaks separating effects.
(4) preparation of reference substance solution and need testing solution
The preparation of reference substance solution: get gallic acid reference substance appropriate, accurately weighed, add 50% methyl alcohol and make the solution of every 1ml containing gallic acid 44.72 μ g, shake up, to obtain final product.
The preparation of need testing solution: get described capsule preparations, porphyrize, gets about O.lg, accurately weighed, put in tool plug conical flask, precision adds 50% methyl alcohol 5Oml, weighed weight, ultrasonic process (power 100W, frequency 40kHz) 30min, let cool, more weighed weight, the weight of less loss is supplied with 50% methyl alcohol, shake up, filter, get subsequent filtrate, to obtain final product.
The preparation of negative sample solution: get not containing the negative sample 0.1g of myrobalan, terminaliae billericae,fructus, emblic medicinal material, be made in the same way of negative sample solution, obtain final product.
(5) negative interference test
In each 10 μ l injection liquid chromatographies of accurate absorption need testing solution, negative sample solution and reference substance solution, from chromatogram, on the position corresponding to reference substance solution chromatographic peak retention time, the chromatographic peak that need testing solution has identical retention time occurs, and negative sample solution at this wavelength without absorption, noiseless to the assay of gallic acid in this product.Chromatogram A, B, C as shown in Figure 6.
(6) investigation of linear relationship
Accurate absorption reference substance solution (44.72 μ g/ml) 2,4,6,8,10 μ l respectively, injection liquid chromatography, measure gallic acid peak area, gallic acid between 0.08944 μ g ~ 0.4472 μ g in the concentration of peak area and reference substance be good linear relationship, its regression equation is Y=3007157.871X-1221.2(r=0.9998).Result is as shown in table 1 and Fig. 7.
The typical curve of table 1 gallic acid
(7) precision test
The accurate need testing solution 10 μ l drawing same concentration, continuous sample introduction 5 times, measures the peak area of gallic acid, the results are shown in Table 2.
Table 2 Precision test result
Test shows, precision is good, and RSD is respectively 0.57%.
(8) stability test
The accurate need testing solution 10 μ l drawing same concentration, respectively at 0,1,2,6,24h sample introduction, measure the peak area of chromatographic peak, the results are shown in Table 3.
Table 3 stability test result
Test shows, result is good at 0 ~ 24h internal stability, and RSD is respectively 0.53%.
(9) reappearance test
(lot number: 01) 5 parts, carry out sample preparation, sample introduction according to sample preparation methods calculates content and the RSD of gallic acid, the results are shown in Table 4 to get test sample.
Table 4 reproducible test results
Test shows, same batch sample samples 5 parts of mensuration, and result reappearance is better, and RSD is 0.90%.
(10) recovery test
Get test sample powder (lot number: 01, content is 2.565%) the about 50mg of known content, accurately weighed, accurately add a certain amount of gallic acid reference substance, measure, according to peak area value in accordance with the law, try to achieve the content of gallic acid respectively, calculate the recovery, measurement result is in table 5.
The average recovery test of table 5 gallic acid
Result shows, this law recovery is good, and RSD is 4.40%.
(11) sample tests
Get capsule sample respectively and be about 0.1g, accurately weighed, put in tool plug conical flask, precision adds 50% methyl alcohol 50ml, weighed weight, ultrasonic process (power 250W, frequency 40kHz) 30 minutes, let cool, more weighed weight, the weight of less loss is supplied with 50% methyl alcohol, shake up, filter, get subsequent filtrate, obtain need testing solution (lot number is labeled as 01,02,03,071101,071102,071103 successively).The each lot number need testing solution 10 μ l of accurate absorption, measures in accordance with the law, calculates the content of gallic acid, the results are shown in Table 6.
The assay result of gallic acid in table 6 sample
According to the content assaying method of capsule pharmaceutical standard, 6 batch samples are measured, float downward 20% as the lower limit of this product content using minimum: i.e. 21.79 × 80%=17.43 (mg/g), therefore order the every 1g of this product temporarily containing gallic acid (C 7h 60 5), must not 17mg be less than.
G, safflower institute hydroxyl carthamin yellow A-containing assay
(1) instrument and reagent
Aglient1100 high performance liquid chromatograph (Agilent company of the U.S.): G1314A UV-vis detector, G1314A binary pump, Agilent1100 chromatographic work station.
SK8200HP ultrasonic cleaner (power 250W, frequency 40kHz) (Shanghai High Kudos Science Instrument Co., Ltd.).
Acetonitrile (chromatographically pure); Methyl alcohol (analyzing pure, chromatographically pure).
Hydroxyl radical carthamin yellow carthamus A reference substance (lot number: 111637-200905), purchased from Chinese pharmaceutical biological product qualification institute.
(2) chromatographic condition
Octadecyl silane is filling agent; Chromatographic column: KromasilE56526-C18(4.6 × 250mm, 5.0 μm, Dalian is according to Lyntech Corporation (US) 10177 South 77th East Avenue Tulsa, Oklahoma 74133 U.S.);
Mobile phase: methanol-acetonitrile-0.2% phosphoric acid solution (22:2:76);
Flow velocity: 1.0ml/min;
Determined wavelength: 403nm;
Column temperature: 30 DEG C.
(3) chromatographic system employment and suitability test (E & ST)
Under above chromatographic condition, better, theoretical cam curve calculates should be not less than 3000 by hydroxyl radical carthamin yellow carthamus A for hydroxyl radical carthamin yellow carthamus A and all the other impurity peaks separating effects.
(4) preparation of reference substance solution and need testing solution
The preparation of reference substance solution: take hydroxyl radical carthamin yellow carthamus A reference substance appropriate, accurately weighed, add the solution that 25% methyl alcohol makes every 1ml hydroxyl carthamin yellow A-containing 18.688 μ g, shake up, to obtain final product.
The preparation of need testing solution: get described capsule preparations, porphyrize, gets about 1.5g, accurately weighed, put in tool plug conical flask, precision adds 25% methyl alcohol 50ml, weighed weight, ultrasonic process (power 250W, frequency 40kHz) 40 minutes, let cool, more weighed weight, the weight of less loss is supplied with 25% methyl alcohol, shake up, filter, get subsequent filtrate, to obtain final product.
The preparation of negative sample solution: get not containing the negative sample 1.5g of flos carthami, be made in the same way of negative sample solution, obtain final product.
(5) negative interference test
In each 10 μ l injection liquid chromatographies of accurate absorption need testing solution, negative sample solution and reference substance solution, from chromatogram, on the position corresponding to reference substance solution chromatographic peak retention time, the chromatographic peak that need testing solution has identical retention time occurs, and negative sample solution at this wavelength without absorption, noiseless to the assay of hydroxyl radical carthamin yellow carthamus A in this product.Chromatogram A, B, C as shown in Figure 8.
(6) investigation of linear relationship
Accurate absorption reference substance solution (18.688 μ g/ml) 2,4,6,8,10,12 μ l respectively, injection liquid chromatography, measure hydroxyl radical carthamin yellow carthamus A peak area, hydroxyl radical carthamin yellow carthamus A between 0.037376 μ g ~ 0.224256 μ g in the concentration of peak area and reference substance be good linear relationship, its regression equation is Y=3546697.654X-9332.134(r=0.9998).Result is as shown in table 7 and Fig. 9.
The typical curve of table 7 hydroxyl radical carthamin yellow carthamus A
(7) precision test
The accurate need testing solution 10 μ l drawing same concentration, continuous sample introduction 5 times, measures the peak area of chromatographic peak, the results are shown in Table 8.
Table 8 Precision test result
Test shows, precision is good, and RSD is respectively 0.77%.
(8) stability test
The accurate need testing solution 10 μ l drawing same concentration, respectively at 0,1,2,6,24h sample introduction, measure the peak area of chromatographic peak, the results are shown in Table 9.
Table 9 stability test result
Test shows, result is good at 0 ~ 24h internal stability, and RSD is respectively 0.95%.
(9) reappearance test
(lot number: 01) 5 parts, carry out sample preparation, sample introduction according to sample preparation methods calculates content and the RSD of hydroxyl radical carthamin yellow carthamus A, the results are shown in Table 10 to get test sample.
Table 10 reproducible test results
Test shows, same batch sample samples 5 parts of mensuration, and result reappearance is better, and RSD is 1.05%.
(10) recovery test
The test sample powder getting known content is about 0.75g, accurately weighed, accurately adds a certain amount of hydroxyl radical carthamin yellow carthamus A, measures in accordance with the law, according to peak area value, tries to achieve the content of hydroxyl radical carthamin yellow carthamus A respectively, and calculate the recovery, measurement result is in table 11.
The average recovery test of table 11 hydroxyl radical carthamin yellow carthamus A
Result shows, this law recovery is good, and RSD is 1.34%.
(11) sample tests
Get capsule sample respectively and be about 4g, accurately weighed, put in tool plug conical flask, precision adds 25% methyl alcohol 50ml, weighed weight, ultrasonic process (power 250W, frequency 40kHz) 40 minutes, let cool, more weighed weight, the weight of less loss is supplied with methyl alcohol, shake up, filter, get subsequent filtrate, obtain need testing solution (lot number is labeled as 01,02,03,071101,071102,071103 successively).Accurate absorption need testing solution 10 μ l, measures in accordance with the law, calculates the content of hydroxyl radical carthamin yellow carthamus A, the results are shown in Table 12.
The assay result of hydroxyl radical carthamin yellow carthamus A in table 12 sample
According to the content assaying method of capsule pharmaceutical standard, 6 batch samples are measured, float downward 20% as the lower limit of this product content using minimum: i.e. 313.21 × 80%=250.57 (μ g/g), therefore order the every 1g of this product temporarily containing safflower with hydroxyl radical carthamin yellow carthamus A (C 27h 30o l5) meter, 250 μ g must not be less than.
The quality testing of embodiment 2 powder
[prescription] myrobalan 150.5g, terminaliae billericae,fructus 5.8g, emblic 20.6g, hooker winghead root 20.6g, Tibet inula root 20.6g, tufa 5.3g, safflower 5.3g, cloves 5.3g, cardamom 5.3g, nutmeg 5.3g, tsaoko 5.3g, agalloch eaglewood 5.3g, seed of pomegranate 5.3g, Indian Herba Swertiae bimaculatae 1.1g, pawpaw 1.1g, styrax 1.1g, cowherb reach summer 1.1g.
More than [technique], 17 tastes, are ground into meal, sieve, and by pharmacy conventional method, add customary adjuvant, make the powder accepted clinically.
[quality determining method]
The TLC distinguish of A, gallic acid
Get above-mentioned powder 0.6g, add methyl alcohol 2ml, ultrasonic process 10 minutes, filter, filtrate is as myrobalan, terminaliae billericae,fructus, emblic need testing solution.
Separately get gallic acid reference substance, add methyl alcohol and make the solution of every 1ml containing 0.6mg, product solution in contrast.
In prescription ratio and preparation technology, configuration not containing the negative sample of myrobalan, terminaliae billericae,fructus, emblic, and is made not containing the negative sample solution of myrobalan, terminaliae billericae,fructus, emblic by the compound method of above-mentioned need testing solution.
Test according to thin-layered chromatography " Chinese Pharmacopoeia " version in 2010 annex VIB, draw each 1 μ l of need testing solution, negative sample solution and reference substance solution, put respectively on same silica gel g thin-layer plate, with methenyl choloride-acetic ether-methanoic acid (7:5:1.5) for developping agent, launch, take out, dry, spray, with 2% ferric trichloride ethanolic solution, is inspected under daylight; In test sample chromatogram, on the position corresponding to reference substance chromatogram, the spot of aobvious same color.
B. the TLC distinguish of Tibet inula root
Get above-mentioned powder 0.6g, add diethyl ether 10ml, close plug, ultrasonic process 20 minutes, and filter, filtrate evaporate to dryness ,-ethyl acetate (l:1) the solution 0.5ml that adds diethyl ether makes dissolving, as Tibet inula root need testing solution.
Separately get Tibet inula root control medicinal material 0.1g, add diethyl ether 0.5ml, close plug, ultrasonic process 20 minutes, and leave standstill, supernatant is as Tibet inula root control medicinal material solution.
In prescription ratio and preparation technology, configuration not containing the negative sample of Tibet inula root, and is made not containing the negative sample solution of Tibet inula root by the compound method of above-mentioned need testing solution.
Test according to thin-layered chromatography " Chinese Pharmacopoeia " version in 2010 annex VIB, draw each 2 μ l of need testing solution, negative sample solution and control medicinal material solution, put respectively on same silica gel g thin-layer plate, with sherwood oil (60 DEG C-90 DEG C)-ethyl acetate (15:2) for developping agent, launch, take out, dry, spray with the secret potassium test solution of rare iodate; In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color.
The TLC distinguish of C, cloves
Get above-mentioned powder 0.6g, add diethyl ether 10ml, close plug, ultrasonic process 20 minutes, and filter, filtrate evaporate to dryness ,-ethyl acetate (l:1) the solution 0.5ml that adds diethyl ether makes dissolving, as cloves need testing solution.
Separately get cloves control medicinal material 0.1g, add diethyl ether 1ml, close plug, ultrasonic process 20 minutes, and leave standstill, supernatant is as cloves control medicinal material solution.
In prescription ratio and preparation technology, configuration not containing the negative sample of cloves, and is made not containing the negative sample solution of cloves by the compound method of above-mentioned need testing solution.
Test according to thin-layered chromatography " Chinese Pharmacopoeia " version in 2010 annex VIB, draw each 1 μ l of need testing solution, negative sample solution and control medicinal material solution, put respectively on same silica gel g thin-layer plate, with sherwood oil (60 DEG C-90 DEG C)-ethyl acetate (1:0.8) for developping agent, launch, take out, dry, spray, with anisaldehyde test solution, is heated to spot development clear; In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color
D, myristic TLC distinguish
Get above-mentioned powder 0.6g, add diethyl ether 10ml, close plug, ultrasonic process 20 minutes, and filter, filtrate evaporate to dryness ,-ethyl acetate (l:1) the solution 0.5ml that adds diethyl ether makes dissolving, as nutmeg need testing solution.
Separately get nutmeg control medicinal material 0.1g, add diethyl ether 2ml, close plug, ultrasonic process 20 minutes, and filter, filtrate evaporate to dryness ,-ethyl acetate (l:1) the solution 0.5ml that adds diethyl ether makes dissolving, as nutmeg control medicinal material solution.
In prescription ratio and preparation technology, configuration not containing myristic negative sample, and is made not containing myristic negative sample solution by the compound method of above-mentioned need testing solution.
Test according to thin-layered chromatography " Chinese Pharmacopoeia " version in 2010 annex VIB, draw each 2 μ l of need testing solution, negative sample solution and control medicinal material solution, put respectively on same silica gel g thin-layer plate, with sherwood oil (60 DEG C-90 DEG C)-ethyl acetate (17:0.8) for developping agent, launch, take out, dry, spray, with 5% vanillic aldehyde concentrated sulfuric acid solution, is heated to spot development clear; In test sample chromatogram, with test sample chromatogram, with on control medicinal material chromatogram relevant position, show the spot of same color.
The TLC distinguish of E, safflower
Get above-mentioned powder 0.6g, add diethyl ether 10ml, close plug, ultrasonic process 20 minutes, filters, add 80% acetone 1.5ml, ultrasonic process 20 minutes after residue volatilizes, and leave standstill, supernatant is as safflower need testing solution.
Separately get safflower control medicinal material 0.1g, add 80% acetone 2ml, ultrasonic process 20 minutes, leave standstill, supernatant is as safflower control medicinal material solution.
In prescription ratio and preparation technology, configuration not containing the negative sample of safflower, and is made not containing the negative sample solution of safflower by the compound method of above-mentioned need testing solution.
Test according to thin-layered chromatography " Chinese Pharmacopoeia " version in 2010 annex VIB, draw need testing solution and negative sample solution 5 μ l, control medicinal material solution 2 μ l, put respectively on same silica gel g thin-layer plate, with acetate-methanol (4:1) for developping agent, exhibition is to about 3cm, take out, dry, then with acetic ether-methanoic acid-water-methanol (9:2.5:1.8:0.5) for developping agent, launch, take out, dry, inspect under daylight; In test sample chromatogram, with test sample chromatogram, with on control medicinal material chromatogram relevant position, show the spot of same color.
Gallic acid content contained by F, myrobalan, terminaliae billericae,fructus, emblic measures
The preparation of reference substance solution: get gallic acid reference substance appropriate, accurately weighed, add 50% methyl alcohol and make the solution of every 1ml containing gallic acid 20 μ g, to obtain final product.
The preparation of need testing solution: get above-mentioned powder 0.06g, accurately weighed, put in tool plug conical flask, precision adds 50% methyl alcohol 25ml, weighed weight, ultrasonic process (power 100W, frequency 40kHz) 10min, let cool, more weighed weight, the weight of less loss is supplied with 50% methyl alcohol, shake up, filter, get subsequent filtrate, as need testing solution.
According to high performance liquid chromatography (" Chinese Pharmacopoeia " 2010 editions one annex VI D), take octadecylsilane chemically bonded silica as filling agent; With acetonitrile-containing 0.1% phosphoric acid solution (2:98) of 0.1% triethylamine for mobile phase; Determined wavelength is 273nm; Column temperature is measure at 25 DEG C; Number of theoretical plate calculates should be not less than 1000 by gallic acid peak.
Accurate absorption reference substance solution and each 5 μ l of need testing solution respectively, injection liquid chromatography, measures, to obtain final product.
The every 1g of described powder in gallic acid, must not be less than 17mg containing myrobalan, terminaliae billericae,fructus, emblic.
G, safflower institute hydroxyl carthamin yellow A-containing assay
The preparation of reference substance solution: take hydroxyl radical carthamin yellow carthamus A reference substance appropriate, accurately weighed, add the solution that 25% methyl alcohol makes every 1ml hydroxyl carthamin yellow A-containing 18.688 μ g, shake up, to obtain final product.
The preparation of need testing solution: get Powder preparation, porphyrize, gets about 0.6g, accurately weighed, put in tool plug conical flask, precision adds 25% methyl alcohol 25ml, weighed weight, ultrasonic process (power 250W, frequency 40kHz) 25 minutes, let cool, more weighed weight, the weight of less loss is supplied with 25% methyl alcohol, shake up, filter, get subsequent filtrate, to obtain final product.
Testing according to high performance liquid chromatography " Chinese Pharmacopoeia " 2010 editions one annex VI D, take octadecylsilane chemically bonded silica as filling agent; With methanol-acetonitrile-0.2% phosphoric acid solution (20:2:78) for mobile phase; Determined wavelength is 403nm; Column temperature is 25 DEG C; Number of theoretical plate calculates should be not less than 3000 by hydroxyl radical carthamin yellow carthamus A peak.
Accurate absorption reference substance solution and each 5 μ l of need testing solution respectively, injection liquid chromatography, measures, to obtain final product.
The every 1g of described powder in hydroxyl radical carthamin yellow carthamus A, must not be less than 250 μ g containing safflower.
The discriminating of embodiment 3 pill
[prescription] myrobalan 249.7g, terminaliae billericae,fructus 19.8g, emblic 79.1g, hooker winghead root 79.1g, Tibet inula root 79.1g, tufa 19.2g, safflower 19.2g, cloves 19.2g, cardamom 19.2g, nutmeg 19.2g, tsaoko 19.2g, agalloch eaglewood 19.2g, seed of pomegranate 19.2g, Indian Herba Swertiae bimaculatae 18.9g, pawpaw 18.9g, styrax 18.9g, cowherb reach summer 18.9g.
More than [technique], 17 tastes, are ground into meal, sieve, and by pharmacy conventional method, add customary adjuvant, make the pill accepted clinically.
[Quality evaluation]
A. the TLC distinguish of gallic acid
Get above-mentioned pill 3g, add methyl alcohol 20ml, ultrasonic process 30 minutes, filter, filtrate is as myrobalan, terminaliae billericae,fructus, emblic need testing solution.
Separately get gallic acid reference substance, add methyl alcohol and make the solution of every 1ml containing 2mg, product solution in contrast.
In prescription ratio and preparation technology, configuration not containing the negative sample of myrobalan, terminaliae billericae,fructus, emblic, and is made not containing the negative sample solution of myrobalan, terminaliae billericae,fructus, emblic by the compound method of above-mentioned need testing solution.
Test according to thin-layered chromatography " Chinese Pharmacopoeia " version in 2010 annex VIB, draw each 3 μ l of need testing solution, negative sample solution and reference substance solution, put respectively on same silica gel g thin-layer plate, with methenyl choloride-acetic ether-methanoic acid (3:1.5:0.5) for developping agent, launch, take out, dry, spray, with 2% ferric trichloride ethanolic solution, is inspected under daylight; In test sample chromatogram, on the position corresponding to reference substance chromatogram, the spot of aobvious same color.
The TLC distinguish of B, Tibet inula root
Get above-mentioned pill 2.5g, add diethyl ether 25ml, close plug, ultrasonic process 10 minutes, and filter, filtrate evaporate to dryness ,-ethyl acetate (l:1) the solution 1.5ml that adds diethyl ether makes dissolving, as Tibet inula root need testing solution.
Separately get Tibet inula root control medicinal material 0.8g, add diethyl ether 1.5ml, close plug, ultrasonic process 10 minutes, and leave standstill, supernatant is as Tibet inula root control medicinal material solution.
In prescription ratio and preparation technology, configuration not containing the negative sample of Tibet inula root, and is made not containing the negative sample solution of Tibet inula root by the compound method of above-mentioned need testing solution.
Test according to thin-layered chromatography " Chinese Pharmacopoeia " version in 2010 annex VIB, draw each 5 μ l of need testing solution, negative sample solution and control medicinal material solution, put respectively on same silica gel g thin-layer plate, with sherwood oil (60 DEG C-90 DEG C)-ethyl acetate (12:1.5) for developping agent, launch, take out, dry, spray with the secret potassium test solution of rare iodate; In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color.
The TLC distinguish of C, cloves
Get above-mentioned pill 2.5g, add diethyl ether 25ml, close plug, ultrasonic process 10 minutes, and filter, filtrate evaporate to dryness ,-ethyl acetate (l:1) the solution 1.5ml that adds diethyl ether makes dissolving, as cloves need testing solution.
Separately get cloves control medicinal material 0.4g, add diethyl ether 4ml, close plug, ultrasonic process 10 minutes, and leave standstill, supernatant is as cloves control medicinal material solution.
In prescription ratio and preparation technology, configuration not containing the negative sample of cloves, and is made not containing the negative sample solution of cloves by the compound method of above-mentioned need testing solution.
Test according to thin-layered chromatography " Chinese Pharmacopoeia " version in 2010 annex VIB, draw each 3 μ l of need testing solution, negative sample solution and control medicinal material solution, put respectively on same silica gel g thin-layer plate, with sherwood oil (60 DEG C-90 DEG C)-ethyl acetate (2.5:1.5) for developping agent, launch, take out, dry, spray, with anisaldehyde test solution, is heated to spot development clear; In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color.
D, myristic TLC distinguish
Get above-mentioned pill 2.5g, add diethyl ether 25ml, close plug, ultrasonic process 10 minutes, and filter, filtrate evaporate to dryness ,-ethyl acetate (l:1) the solution 1.5ml that adds diethyl ether makes dissolving, as nutmeg need testing solution.
Separately get nutmeg control medicinal material 0.4g, add diethyl ether 8ml, close plug, ultrasonic process 10 minutes, and filter, filtrate evaporate to dryness ,-ethyl acetate (l:1) the solution 1.5ml that adds diethyl ether makes dissolving, as nutmeg control medicinal material solution.
In prescription ratio and preparation technology, configuration not containing myristic negative sample, and is made not containing myristic negative sample solution by the compound method of above-mentioned need testing solution.
Test according to thin-layered chromatography " Chinese Pharmacopoeia " version in 2010 annex VIB, draw each 6 μ l of need testing solution, negative sample solution and control medicinal material solution, put respectively on same silica gel g thin-layer plate, with sherwood oil (60 DEG C-90 DEG C)-ethyl acetate (16:0.7) for developping agent, launch, take out, dry, spray, with 5% vanillic aldehyde concentrated sulfuric acid solution, is heated to spot development clear; In test sample chromatogram, with test sample chromatogram, with on control medicinal material chromatogram relevant position, show the spot of same color.
The TLC distinguish of E, safflower
Get above-mentioned pill 2.5g, add diethyl ether 25ml, close plug, ultrasonic process 10 minutes, filters, add 80% acetone 8ml, ultrasonic process 10 minutes after residue volatilizes, and leave standstill, supernatant is as safflower need testing solution.
Separately get safflower control medicinal material 0.4g, add 80% acetone 8ml, ultrasonic process 10 minutes, leave standstill, supernatant is as safflower control medicinal material solution.
In prescription ratio and preparation technology, configuration not containing the negative sample of safflower, and is made not containing the negative sample solution of safflower by the compound method of above-mentioned need testing solution.
Test according to thin-layered chromatography " Chinese Pharmacopoeia " version in 2010 annex VIB, draw need testing solution and negative sample solution 12 μ l, control medicinal material solution 6 μ l, put respectively on same silica gel g thin-layer plate, with acetate-methanol (3:0.8) for developping agent, exhibition is to about 3cm, take out, dry, then with acetic ether-methanoic acid-water-methanol (11:4.5:4.5:0.2) for developping agent, launch, take out, dry, inspect under daylight; In test sample chromatogram, with test sample chromatogram, with on control medicinal material chromatogram relevant position, show the spot of same color.
Gallic acid content contained by F, myrobalan, terminaliae billericae,fructus, emblic measures
The preparation of reference substance solution: get gallic acid reference substance appropriate, accurately weighed, add 50% methyl alcohol and make the solution of every 1ml containing gallic acid 50 μ g, to obtain final product.
The preparation of need testing solution: get above-mentioned pill 1g, accurately weighed, put in tool plug conical flask, precision adds 50% methyl alcohol 6Oml, weighed weight, ultrasonic process (power 100W, frequency 40kHz) 40min, let cool, more weighed weight, the weight of less loss is supplied with 50% methyl alcohol, shake up, filter, get subsequent filtrate, as need testing solution.
According to high performance liquid chromatography (" Chinese Pharmacopoeia " 2010 editions one annex VI D), take octadecylsilane chemically bonded silica as filling agent; With acetonitrile-containing 0.1% phosphoric acid solution (4:96) of 0.1% triethylamine for mobile phase; Determined wavelength is 273nm; Column temperature is measure at 35 DEG C; Number of theoretical plate calculates should be not less than 1000 by gallic acid peak.
Accurate absorption reference substance solution and each 15 μ l of need testing solution respectively, injection liquid chromatography, measures, to obtain final product.
The every 1g of pill in gallic acid, must not be less than 17mg containing myrobalan, terminaliae billericae,fructus, emblic.
G, safflower institute hydroxyl carthamin yellow A-containing assay
The preparation of reference substance solution: take hydroxyl radical carthamin yellow carthamus A reference substance appropriate, accurately weighed, add the solution that 25% methyl alcohol makes every 1ml hydroxyl carthamin yellow A-containing 40 μ g, shake up, to obtain final product.
The preparation of need testing solution: get described pill, porphyrize, gets about 2g, accurately weighed, put in tool plug conical flask, precision adds 25% methyl alcohol 50ml, weighed weight, ultrasonic process (power 250W, frequency 40kHz) 45 minutes, let cool, more weighed weight, the weight of less loss is supplied with 25% methyl alcohol, shake up, filter, get subsequent filtrate, to obtain final product.
Testing according to high performance liquid chromatography " Chinese Pharmacopoeia " 2010 editions one annex VI D, take octadecylsilane chemically bonded silica as filling agent; With methanol-acetonitrile-0.2% phosphoric acid solution (24:2:74) for mobile phase; Determined wavelength is 403nm; Column temperature is 35 DEG C; Number of theoretical plate calculates should be not less than 3000 by hydroxyl radical carthamin yellow carthamus A peak.
Accurate absorption reference substance solution and each 15 μ l of need testing solution respectively, injection liquid chromatography, measures, to obtain final product.
The every 1g of described pill in hydroxyl radical carthamin yellow carthamus A, must not be less than 250 μ g containing safflower.
The discriminating of embodiment 4 tablet
[prescription] myrobalan 201.7g, terminaliae billericae,fructus 12.6g, emblic 50.4g, hooker winghead root 50.4g, Tibet inula root 50.4g, tufa 11.8g, safflower 11.8g, cloves 11.8g, cardamom 11.8g, nutmeg 11.8g, tsaoko 11.8g, agalloch eaglewood 11.8g, seed of pomegranate 11.8g, Indian Herba Swertiae bimaculatae 10.1g, pawpaw 10.1g, styrax 10.1g, cowherb reach summer 10.1g.
[technique] is got Tibet inula root, cloves, cardamom, nutmeg, tsaoko, agalloch eaglewood, styrax, safflower and tufa and is pulverized, and crosses 200 mesh sieves; Myrobalan, terminaliae billericae,fructus, emblic, cowherb reach summer boiling twice, and first time adds water 10 times of weight, and second time adds water 10 times of weight, each 2 hours, collecting decoction, and filter, filtrate is concentrated into relative density and is about 1.05-1.10(70 DEG C) clear cream, for subsequent use; Hooker winghead root, seed of pomegranate, India's river deer bud dish and pawpaw, add 70% alcohol reflux and extract secondary, first time adds 70% ethanol 8 times of weight, second time adds 70% ethanol 7 times of weight, each 2 hours, filters, merging filtrate, filtrate recycling ethanol, to appropriate, adds above-mentioned clear cream, being concentrated into relative density is 1.10-1.15(70 DEG C) clear cream, vacuum or spraying dry, add fine medicinal material powder, mixing, dry granulation, it is appropriate to add lubricant, compressing tablet, film coating and get final product.
[Quality evaluation]
The TLC distinguish of A, gallic acid
Get above-mentioned tablet 1g, add methyl alcohol 5ml, ultrasonic process 20 minutes, filter, filtrate is as myrobalan, terminaliae billericae,fructus, emblic need testing solution.
Separately get gallic acid reference substance, add methyl alcohol and make the solution of every 1ml containing 1mg, product solution in contrast.
In prescription ratio and preparation technology, configuration not containing the negative sample of myrobalan, terminaliae billericae,fructus, emblic, and is made not containing the negative sample solution of myrobalan, terminaliae billericae,fructus, emblic by the compound method of above-mentioned need testing solution.
Test according to thin-layered chromatography " Chinese Pharmacopoeia " version in 2010 annex VIB, draw each 2 μ l of need testing solution, negative sample solution and reference substance solution, put respectively on same silica gel g thin-layer plate, with methenyl choloride-acetic ether-methanoic acid (6:4:1) for developping agent, launch, take out, dry, spray, with 2% ferric trichloride ethanolic solution, is inspected under daylight; In test sample chromatogram, on the position corresponding to reference substance chromatogram, the spot of aobvious same color.
The TLC distinguish of B, Tibet inula root
Get above-mentioned tablet 2g, add diethyl ether 20ml, close plug, ultrasonic process 20 minutes, and filter, filtrate evaporate to dryness ,-ethyl acetate (l:1) the solution 1ml that adds diethyl ether makes dissolving, as Tibet inula root need testing solution.
Separately get Tibet inula root control medicinal material 0.2g, add diethyl ether 1ml, close plug, ultrasonic process 20 minutes, and leave standstill, supernatant is as Tibet inula root control medicinal material solution.
In prescription ratio and preparation technology, configuration not containing the negative sample of Tibet inula root, and is made not containing the negative sample solution of Tibet inula root by the compound method of above-mentioned need testing solution.
Test according to thin-layered chromatography " Chinese Pharmacopoeia " version in 2010 annex VIB, draw each 4 μ l of need testing solution, negative sample solution and control medicinal material solution, put respectively on same silica gel g thin-layer plate, with sherwood oil (60 DEG C-90 DEG C)-ethyl acetate (17:3) for developping agent, launch, take out, dry, spray with the secret potassium test solution of rare iodate; In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color.
The TLC distinguish of C, cloves
Get above-mentioned tablet 2g, add diethyl ether 20ml, close plug, ultrasonic process 20 minutes, and filter, filtrate evaporate to dryness ,-ethyl acetate (l:1) the solution 1ml that adds diethyl ether makes dissolving, as cloves need testing solution.
Separately get cloves control medicinal material 0.2g, add diethyl ether 3ml, close plug, ultrasonic process 20 minutes, and leave standstill, supernatant is as cloves control medicinal material solution.
In prescription ratio and preparation technology, configuration not containing the negative sample of cloves, and is made not containing the negative sample solution of cloves by the compound method of above-mentioned need testing solution.
Test according to thin-layered chromatography " Chinese Pharmacopoeia " version in 2010 annex VIB, draw each 2 μ l of need testing solution, negative sample solution and control medicinal material solution, put respectively on same silica gel g thin-layer plate, with sherwood oil (60 DEG C-90 DEG C)-ethyl acetate (3:1) for developping agent, launch, take out, dry, spray, with anisaldehyde test solution, is heated to spot development clear; In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color.
D, myristic TLC distinguish
Get above-mentioned tablet 2g, add diethyl ether 20ml, close plug, ultrasonic process 20 minutes, and filter, filtrate evaporate to dryness ,-ethyl acetate (l:1) the solution 1ml that adds diethyl ether makes dissolving, as nutmeg need testing solution.
Separately get nutmeg control medicinal material 0.2g, add diethyl ether 5ml, close plug, ultrasonic process 20 minutes, and filter, filtrate evaporate to dryness ,-ethyl acetate (l:1) the solution 1ml that adds diethyl ether makes dissolving, as nutmeg control medicinal material solution.
In prescription ratio and preparation technology, configuration not containing myristic negative sample, and is made not containing myristic negative sample solution by the compound method of above-mentioned need testing solution.
Test according to thin-layered chromatography " Chinese Pharmacopoeia " version in 2010 annex VIB, draw each 5 μ l of need testing solution, negative sample solution and control medicinal material solution, put respectively on same silica gel g thin-layer plate, with sherwood oil (60 DEG C-90 DEG C)-ethyl acetate (20:1) for developping agent, launch, take out, dry, spray, with 5% vanillic aldehyde concentrated sulfuric acid solution, is heated to spot development clear; In test sample chromatogram, with test sample chromatogram, with on control medicinal material chromatogram relevant position, show the spot of same color.
The TLC distinguish of E, safflower
Get above-mentioned tablet 2g, add diethyl ether 20ml, close plug, ultrasonic process 20 minutes, filters, add 80% acetone 5ml, ultrasonic process 20 minutes after residue volatilizes, and leave standstill, supernatant is as safflower need testing solution.
Separately get safflower control medicinal material 0.2g, add 80% acetone 5ml, ultrasonic process 20 minutes, leave standstill, supernatant is as safflower control medicinal material solution.
In prescription ratio and preparation technology, configuration not containing the negative sample of safflower, and is made not containing the negative sample solution of safflower by the compound method of above-mentioned need testing solution.
Test according to thin-layered chromatography " Chinese Pharmacopoeia " version in 2010 annex VIB, draw need testing solution and negative sample solution 10 μ l, control medicinal material solution 5 μ l, put respectively on same silica gel g thin-layer plate, with acetate-methanol (5:2) for developping agent, exhibition is to about 3cm, take out, dry, then with acetic ether-methanoic acid-water-methanol (7:2:3:0.4) for developping agent, launch, take out, dry, inspect under daylight; In test sample chromatogram, with test sample chromatogram, with on control medicinal material chromatogram relevant position, show the spot of same color.
Gallic acid content contained by F, myrobalan, terminaliae billericae,fructus, emblic measures
The preparation of reference substance solution: get gallic acid reference substance appropriate, accurately weighed, add 50% methyl alcohol and make the solution of every 1ml containing gallic acid 40 μ g, to obtain final product.
The preparation of need testing solution: get above-mentioned tablet 0.1g, accurately weighed, put in tool plug conical flask, precision adds 50% methyl alcohol 5Oml, weighed weight, ultrasonic process (power 100W, frequency 40kHz) 30min, let cool, more weighed weight, the weight of less loss is supplied with 50% methyl alcohol, shake up, filter, get subsequent filtrate, as need testing solution.
According to high performance liquid chromatography (" Chinese Pharmacopoeia " 2010 editions one annex VI D), take octadecylsilane chemically bonded silica as filling agent; With acetonitrile-containing 0.1% phosphoric acid solution (1:99) of 0.1% triethylamine for mobile phase; Determined wavelength is 273nm; Column temperature is measure at 30 DEG C; Number of theoretical plate calculates should be not less than 1000 by gallic acid peak.
Accurate absorption reference substance solution and each 10 μ l of need testing solution respectively, injection liquid chromatography, measures, to obtain final product.
The every 1g of described tablet in gallic acid, must not be less than 17mg containing myrobalan, terminaliae billericae,fructus, emblic.
G, safflower institute hydroxyl carthamin yellow A-containing assay
The preparation of reference substance solution: take hydroxyl radical carthamin yellow carthamus A reference substance appropriate, accurately weighed, add the solution that 25% methyl alcohol makes every 1ml hydroxyl carthamin yellow A-containing 18.688 μ g, shake up, to obtain final product.
The preparation of need testing solution: get tablet, porphyrize, gets about 1.5g, accurately weighed, put in tool plug conical flask, precision adds 25% methyl alcohol 50ml, weighed weight, ultrasonic process (power 250W, frequency 40kHz) 40 minutes, let cool, more weighed weight, the weight of less loss is supplied with 25% methyl alcohol, shake up, filter, get subsequent filtrate, to obtain final product.
Testing according to high performance liquid chromatography " Chinese Pharmacopoeia " 2010 editions one annex VI D, take octadecylsilane chemically bonded silica as filling agent; With methanol-acetonitrile-0.2% phosphoric acid solution (22:2:76) for mobile phase; Determined wavelength is 403nm; Column temperature is 30 DEG C; Number of theoretical plate calculates should be not less than 3000 by hydroxyl radical carthamin yellow carthamus A peak.
Accurate absorption reference substance solution and each 10 μ l of need testing solution respectively, injection liquid chromatography, measures, to obtain final product.
The every 1g of described tablet in hydroxyl radical carthamin yellow carthamus A, must not be less than 250 μ g containing safflower.
The discriminating of embodiment 5 granule
[prescription] myrobalan 180.2g, terminaliae billericae,fructus 8.3g, emblic 30.4g, hooker winghead root 30.4g, Tibet inula root 30.4g, tufa 8.1g, safflower 8.1g, cloves 8.1g, cardamom 8.1g, nutmeg 8.1g, tsaoko 8.1g, agalloch eaglewood 8.1g, seed of pomegranate 8.1g, Indian Herba Swertiae bimaculatae 5.6g, pawpaw 5.6g, styrax 5.6g, cowherb reach summer 5.6g.
More than [technique], 17 tastes, are ground into meal, sieve, and by pharmacy conventional method, add customary adjuvant, make the granule accepted clinically.
[Quality evaluation]
The TLC distinguish of A, gallic acid
Get above-mentioned granule 4.5g, add methyl alcohol 30ml, ultrasonic process 40 minutes, filter, filtrate is as myrobalan, terminaliae billericae,fructus, emblic need testing solution.
Separately get gallic acid reference substance, add methyl alcohol and make the solution of every 1ml containing 2.5mg, product solution in contrast.
In prescription ratio and preparation technology, configuration not containing the negative sample of myrobalan, terminaliae billericae,fructus, emblic, and is made not containing the negative sample solution of myrobalan, terminaliae billericae,fructus, emblic by the compound method of above-mentioned need testing solution.
Test according to thin-layered chromatography " Chinese Pharmacopoeia " version in 2010 annex VIB, draw each 4 μ l of need testing solution, negative sample solution and reference substance solution, put respectively on same silica gel g thin-layer plate, with methenyl choloride-acetic ether-methanoic acid (9:6:2) for developping agent, launch, take out, dry, spray, with 2% ferric trichloride ethanolic solution, is inspected under daylight; In test sample chromatogram, on the position corresponding to reference substance chromatogram, the spot of aobvious same color.
The TLC distinguish of B, Tibet inula root
Get above-mentioned granule 4.5g, add diethyl ether 45ml, close plug, ultrasonic process 40 minutes, and filter, filtrate evaporate to dryness ,-ethyl acetate (l:1) the solution 2ml that adds diethyl ether makes dissolving, as Tibet inula root need testing solution.
Separately get Tibet inula root control medicinal material 1g, add diethyl ether 2ml, close plug, ultrasonic process 40 minutes, and leave standstill, supernatant is as Tibet inula root control medicinal material solution.
In prescription ratio and preparation technology, configuration not containing the negative sample of Tibet inula root, and is made not containing the negative sample solution of Tibet inula root by the compound method of above-mentioned need testing solution.
Test according to thin-layered chromatography " Chinese Pharmacopoeia " version in 2010 annex VIB, draw each 8 μ l of need testing solution, negative sample solution and control medicinal material solution, put respectively on same silica gel g thin-layer plate, with sherwood oil (60 DEG C-90 DEG C)-ethyl acetate (21:5) for developping agent, launch, take out, dry, spray with the secret potassium test solution of rare iodate; In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color.
The TLC distinguish of C, cloves
Get above-mentioned granule 4.5g, add diethyl ether 45ml, close plug, ultrasonic process 40 minutes, and filter, filtrate evaporate to dryness ,-ethyl acetate (l:1) the solution 2ml that adds diethyl ether makes dissolving, as cloves need testing solution.
Separately get cloves control medicinal material 0.5g, add diethyl ether 5ml, close plug, ultrasonic process 40 minutes, and leave standstill, supernatant is as cloves control medicinal material solution.
In prescription ratio and preparation technology, configuration not containing the negative sample of cloves, and is made not containing the negative sample solution of cloves by the compound method of above-mentioned need testing solution.
Test according to thin-layered chromatography " Chinese Pharmacopoeia " version in 2010 annex VIB, draw each 4 μ l of need testing solution, negative sample solution and control medicinal material solution, put respectively on same silica gel g thin-layer plate, with sherwood oil (60 DEG C-90 DEG C)-ethyl acetate (7:2.4) for developping agent, launch, take out, dry, spray, with anisaldehyde test solution, is heated to spot development clear; In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color.
D, myristic TLC distinguish
Get above-mentioned granule 4.5g, add diethyl ether 45ml, close plug, ultrasonic process 40 minutes, and filter, filtrate evaporate to dryness ,-ethyl acetate (l:1) the solution 2ml that adds diethyl ether makes dissolving, as nutmeg need testing solution.
Separately get nutmeg control medicinal material 0.5g, add diethyl ether 10ml, close plug, ultrasonic process 40 minutes, and filter, filtrate evaporate to dryness ,-ethyl acetate (l:1) the solution 2ml that adds diethyl ether makes dissolving, as nutmeg control medicinal material solution.
In prescription ratio and preparation technology, configuration not containing myristic negative sample, and is made not containing myristic negative sample solution by the compound method of above-mentioned need testing solution.
Test according to thin-layered chromatography " Chinese Pharmacopoeia " version in 2010 annex VIB, draw each 8 μ l of need testing solution, negative sample solution and control medicinal material solution, put respectively on same silica gel g thin-layer plate, with sherwood oil (60 DEG C-90 DEG C)-ethyl acetate (24:2.2) for developping agent, launch, take out, dry, spray, with 5% vanillic aldehyde concentrated sulfuric acid solution, is heated to spot development clear; In test sample chromatogram, with test sample chromatogram, with on control medicinal material chromatogram relevant position, show the spot of same color.
The TLC distinguish of E, safflower
Get above-mentioned granule 4.5g, add diethyl ether 45ml, close plug, ultrasonic process 40 minutes, filters, add 80% acetone 10ml, ultrasonic process 40 minutes after residue volatilizes, and leave standstill, supernatant is as safflower need testing solution.
Separately get safflower control medicinal material 0.5g, add 80% acetone 10ml, ultrasonic process 40 minutes, leave standstill, supernatant is as safflower control medicinal material solution.
In prescription ratio and preparation technology, configuration not containing the negative sample of safflower, and is made not containing the negative sample solution of safflower by the compound method of above-mentioned need testing solution.
Test according to thin-layered chromatography " Chinese Pharmacopoeia " version in 2010 annex VIB, draw need testing solution and negative sample solution 15 μ l, control medicinal material solution 8 μ l, put respectively on same silica gel g thin-layer plate, with acetate-methanol (6:2.5) for developping agent, exhibition is to about 3cm, take out, dry, then with acetic ether-methanoic acid-water-methanol (10:3:3:0.5) for developping agent, launch, take out, dry, inspect under daylight; In test sample chromatogram, with on control medicinal material chromatogram relevant position, show the spot of same color.
Gallic acid content contained by F, myrobalan, terminaliae billericae,fructus, emblic measures
The preparation of reference substance solution: get gallic acid reference substance appropriate, accurately weighed, add 50% methyl alcohol and make the solution of every 1ml containing gallic acid 80 μ g, to obtain final product.
The preparation of need testing solution: get above-mentioned granule 1.8g, accurately weighed, put in tool plug conical flask, precision adds 50% methyl alcohol 75ml, weighed weight, ultrasonic process (power 100W, frequency 40kHz) 45min, let cool, more weighed weight, the weight of less loss is supplied with 50% methyl alcohol, shake up, filter, get subsequent filtrate, as need testing solution.
According to high performance liquid chromatography (" Chinese Pharmacopoeia " 2010 editions one annex VI D), take octadecylsilane chemically bonded silica as filling agent; With acetonitrile-containing 0.1% phosphoric acid solution (5:95) of 0.1% triethylamine for mobile phase; Determined wavelength is 273nm; Column temperature is measure at 40 DEG C; Number of theoretical plate calculates should be not less than 1000 by gallic acid peak.
Accurate absorption reference substance solution and each 20 μ l of need testing solution respectively, injection liquid chromatography, measures, to obtain final product.
The every 1g of described granule in gallic acid, must not be less than 17mg containing myrobalan, terminaliae billericae,fructus, emblic.
G, safflower institute hydroxyl carthamin yellow A-containing assay
The preparation of reference substance solution: take hydroxyl radical carthamin yellow carthamus A reference substance appropriate, accurately weighed, add the solution that 25% methyl alcohol makes every 1ml hydroxyl carthamin yellow A-containing 50 μ g, shake up, to obtain final product.
The preparation of need testing solution: get granule, porphyrize, gets about 2.5g, accurately weighed, put in tool plug conical flask, precision adds 25% methyl alcohol 75ml, weighed weight, ultrasonic process (power 250W, frequency 40kHz) 60 minutes, let cool, more weighed weight, the weight of less loss is supplied with 25% methyl alcohol, shake up, filter, get subsequent filtrate, to obtain final product.
Testing according to high performance liquid chromatography " Chinese Pharmacopoeia " 2010 editions one annex VI D, take octadecylsilane chemically bonded silica as filling agent; With methanol-acetonitrile-0.2% phosphoric acid solution (26:2:72) for mobile phase; Determined wavelength is 403nm; Column temperature is 40 DEG C; Number of theoretical plate calculates should be not less than 3000 by hydroxyl radical carthamin yellow carthamus A peak.
Accurate absorption reference substance solution and each 20 μ l of need testing solution respectively, injection liquid chromatography, measures, to obtain final product.
The every 1g of described granule in hydroxyl radical carthamin yellow carthamus A, must not be less than 250 μ g containing safflower.
The discriminating of embodiment 6 soft capsule
[prescription] myrobalan 219.8g, terminaliae billericae,fructus 14.7g, emblic 69.6g, hooker winghead root 69.6g, Tibet inula root 69.6g, tufa 14.5g, safflower 14.5g, cloves 14.5g, cardamom 14.5g, nutmeg 14.5g, tsaoko 14.5g, agalloch eaglewood 14.5g, seed of pomegranate 14.5g, Indian Herba Swertiae bimaculatae 14.3g, pawpaw 14.3g, styrax 14.3g, cowherb reach summer 14.3g.
More than [technique], 17 tastes, are ground into meal, sieve, and by pharmacy conventional method, add customary adjuvant, make the soft capsule accepted clinically.
[quality determining method]
The TLC distinguish of A, gallic acid
Get above-mentioned soft capsule 1.5g, add methyl alcohol 10ml, ultrasonic process 20 minutes, filter, filtrate is as myrobalan, terminaliae billericae,fructus, emblic need testing solution.
Separately get gallic acid reference substance, add methyl alcohol and make the solution of every 1ml containing 1.5mg, product solution in contrast.
In prescription ratio and preparation technology, configuration not containing the negative sample of myrobalan, terminaliae billericae,fructus, emblic, and is made not containing the negative sample solution of myrobalan, terminaliae billericae,fructus, emblic by the compound method of above-mentioned need testing solution.
Test according to thin-layered chromatography " Chinese Pharmacopoeia " version in 2010 annex VIB, draw each 2 μ l of need testing solution, negative sample solution and reference substance solution, put respectively on same silica gel g thin-layer plate, with methenyl choloride-acetic ether-methanoic acid (5:3:1.8) for developping agent, launch, take out, dry, spray, with 2% ferric trichloride ethanolic solution, is inspected under daylight; In test sample chromatogram, on the position corresponding to reference substance chromatogram, the spot of aobvious same color.
B. the TLC distinguish of Tibet inula root
Get above-mentioned soft capsule 1g, add diethyl ether 15ml, close plug, ultrasonic process 30 minutes, and filter, filtrate evaporate to dryness ,-ethyl acetate (l:1) the solution 1ml that adds diethyl ether makes dissolving, as Tibet inula root need testing solution.
Separately get Tibet inula root control medicinal material 0.5g, add diethyl ether 0.8ml, close plug, ultrasonic process 30 minutes, and leave standstill, supernatant is as Tibet inula root control medicinal material solution.
In prescription ratio and preparation technology, configuration not containing the negative sample of Tibet inula root, and is made not containing the negative sample solution of Tibet inula root by the compound method of above-mentioned need testing solution.
Test according to thin-layered chromatography " Chinese Pharmacopoeia " version in 2010 annex VIB, draw each 3 μ l of need testing solution, negative sample solution and control medicinal material solution, put respectively on same silica gel g thin-layer plate, with sherwood oil (60 DEG C-90 DEG C)-ethyl acetate (19:7) for developping agent, launch, take out, dry, spray with the secret potassium test solution of rare iodate; In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color.
The TLC distinguish of C, cloves
Get above-mentioned soft capsule 1g, add diethyl ether 15ml, close plug, ultrasonic process 30 minutes, and filter, filtrate evaporate to dryness ,-ethyl acetate (l:1) the solution 1ml that adds diethyl ether makes dissolving, as cloves need testing solution.
Separately get cloves control medicinal material 0.3g, add diethyl ether 2ml, close plug, ultrasonic process 30 minutes, and leave standstill, supernatant is as cloves control medicinal material solution.
In prescription ratio and preparation technology, configuration not containing the negative sample of cloves, and is made not containing the negative sample solution of cloves by the compound method of above-mentioned need testing solution.
Test according to thin-layered chromatography " Chinese Pharmacopoeia " version in 2010 annex VIB, draw each 2 μ l of need testing solution, negative sample solution and control medicinal material solution, put respectively on same silica gel g thin-layer plate, with sherwood oil (60 DEG C-90 DEG C)-ethyl acetate (4.5:1.2) for developping agent, launch, take out, dry, spray, with anisaldehyde test solution, is heated to spot development clear; In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color.
D, myristic TLC distinguish
Get above-mentioned soft capsule 1g, add diethyl ether 15ml, close plug, ultrasonic process 30 minutes, and filter, filtrate evaporate to dryness ,-ethyl acetate (l:1) the solution 1ml that adds diethyl ether makes dissolving, as nutmeg need testing solution.
Separately get nutmeg control medicinal material 0.3g, add diethyl ether 4ml, close plug, ultrasonic process 30 minutes, and filter, filtrate evaporate to dryness ,-ethyl acetate (l:1) the solution 1ml that adds diethyl ether makes dissolving, as nutmeg control medicinal material solution.
In prescription ratio and preparation technology, configuration not containing myristic negative sample, and is made not containing myristic negative sample solution by the compound method of above-mentioned need testing solution.
Test according to thin-layered chromatography " Chinese Pharmacopoeia " version in 2010 annex VIB, draw each 4 μ l of need testing solution, negative sample solution and control medicinal material solution, put respectively on same silica gel g thin-layer plate, with sherwood oil (60 DEG C-90 DEG C)-ethyl acetate (17:0.8) for developping agent, launch, take out, dry, spray, with 5% vanillic aldehyde concentrated sulfuric acid solution, is heated to spot development clear; In test sample chromatogram, with on control medicinal material chromatogram relevant position, show the spot of same color.
The TLC distinguish of E, safflower
Get above-mentioned soft capsule 1g, add diethyl ether 15ml, close plug, ultrasonic process 30 minutes, filters, add 80% acetone 3ml, ultrasonic process 30 minutes after residue volatilizes, and leave standstill, supernatant is as safflower need testing solution.
Separately get safflower control medicinal material 0.3g, add 80% acetone 4ml, ultrasonic process 30 minutes, leave standstill, supernatant is as safflower control medicinal material solution.
In prescription ratio and preparation technology, configuration not containing the negative sample of safflower, and is made not containing the negative sample solution of safflower by the compound method of above-mentioned need testing solution.
Test according to thin-layered chromatography " Chinese Pharmacopoeia " version in 2010 annex VIB, draw need testing solution and negative sample solution 8 μ l, control medicinal material solution 4 μ l, put respectively on same silica gel g thin-layer plate, with acetate-methanol (7:3.5) for developping agent, exhibition is to about 3cm, take out, dry, then with acetic ether-methanoic acid-water-methanol (5:1.5:3.2:0.8) for developping agent, launch, take out, dry, inspect under daylight; In test sample chromatogram, with on control medicinal material chromatogram relevant position, show the spot of same color.
Gallic acid content contained by F, myrobalan, terminaliae billericae,fructus, emblic measures
The preparation of reference substance solution: get gallic acid reference substance appropriate, accurately weighed, add 50% methyl alcohol and make the solution of every 1ml containing gallic acid 30 μ g, to obtain final product.
The preparation of need testing solution: get above-mentioned soft capsule 0.5g, accurately weighed, put in tool plug conical flask, precision adds 50% methyl alcohol 3Oml, weighed weight, ultrasonic process (power 100W, frequency 40kHz) 20min, let cool, more weighed weight, the weight of less loss is supplied with 50% methyl alcohol, shake up, filter, get subsequent filtrate, as need testing solution;
According to high performance liquid chromatography (" Chinese Pharmacopoeia " 2010 editions one annex VI D), take octadecylsilane chemically bonded silica as filling agent; With acetonitrile-containing 0.1% phosphoric acid solution (3:97) of 0.1% triethylamine for mobile phase; Determined wavelength is 273nm; Column temperature is measure at 30 DEG C; Number of theoretical plate calculates should be not less than 1000 by gallic acid peak.
Accurate absorption reference substance solution and each 10 μ l of need testing solution respectively, injection liquid chromatography, measures, to obtain final product.
The every 1g of described soft capsule in gallic acid, must not be less than 17mg containing myrobalan, terminaliae billericae,fructus, emblic.
G, safflower institute hydroxyl carthamin yellow A-containing assay
The preparation of reference substance solution: take hydroxyl radical carthamin yellow carthamus A reference substance appropriate, accurately weighed, add the solution that 25% methyl alcohol makes every 1ml hydroxyl carthamin yellow A-containing 30 μ g, shake up, to obtain final product.
The preparation of need testing solution: get soft capsule, porphyrize, gets about 1g, accurately weighed, put in tool plug conical flask, precision adds 25% methyl alcohol 40ml, weighed weight, ultrasonic process (power 250W, frequency 40kHz) 30 minutes, let cool, more weighed weight, the weight of less loss is supplied with 25% methyl alcohol, shake up, filter, get subsequent filtrate, to obtain final product.
Testing according to high performance liquid chromatography " Chinese Pharmacopoeia " 2010 editions one annex VI D, take octadecylsilane chemically bonded silica as filling agent; With methanol-acetonitrile-0.2% phosphoric acid solution (18:2:80) for mobile phase; Determined wavelength is 403nm; Column temperature is 30 DEG C; Number of theoretical plate calculates should be not less than 3000 by hydroxyl radical carthamin yellow carthamus A peak.
Accurate absorption reference substance solution and each 10 μ l of need testing solution respectively, injection liquid chromatography, measures, to obtain final product.
The every 1g of described soft capsule in hydroxyl radical carthamin yellow carthamus A, must not be less than 250 μ g containing safflower.
Contrast experiment's example
experimental example 1: the TLC distinguish of gallic acid
(1) instrument
Tool plug conical flask, electronic scales, transfer pipet, ultrasound wave extraction apparatus, funnel, filter paper, sample applicator, silica G plate, chromatography cylinder.
(2) reference substance and test sample
Gallic acid reference substance (lot number: 110831-200803), purchased from Chinese pharmaceutical biological product qualification institute;
Test sample, embodiment 1 gained capsule.
(3) reagent
Methyl alcohol, absolute ethyl alcohol, 70% ethanol, methenyl choloride, ethyl acetate, formic acid, acetone, 2% acidic alcohol, water.
(4) method of inspection:
Extraction solvent: methyl alcohol, absolute ethyl alcohol, 70% ethanol, 2% acidic alcohol.
Extracting method: jolting and ultrasonic.
Developping agent: acetic ether-methanoic acid-water (12:2.5:2), methenyl choloride-acetic ether-methanoic acid (6:4:1), methenyl choloride-acetone-formic acid (7:3:1).
Select above Extraction solvent, extracting method and developping agent to test respectively, result is as follows:
Result shows, and is that Extraction solvent carries out ultrasonic extraction, and with methenyl choloride-acetic ether-methanoic acid (6:4:1) for developping agent, is conducive to the TLC distinguish of gallic acid most with methyl alcohol.
experimental example 2: the TLC distinguish of Tibet inula root
(1) instrument
Tool plug conical flask, electronic scales, transfer pipet, ultrasound wave extraction apparatus, funnel, filter paper, evaporating dish, water-bath, sample applicator, silica G plate, chromatography cylinder.
(2) control medicinal material and test sample
Tibet inula root control medicinal material (lot number: 121090-201002), purchased from Chinese pharmaceutical biological product qualification institute;
Test sample, embodiment 1 gained capsule.
(3) reagent
Ether, ethyl acetate, sherwood oil, acetone, methyl alcohol, cyclohexane, methylene chloride, the secret potassium test solution of rare iodate.
(4) method of inspection:
Extraction solvent: ether, acetone, methyl alcohol.
Extracting method: ultrasonic and jolting.
Developping agent: sherwood oil (60 DEG C-90 DEG C)-ethyl acetate (17:3), cyclohexane-dichloromethane-ethyl acetate (15:5:1).Select above Extraction solvent, extracting method and developping agent to test respectively, result is as follows:
Result shows, and is that Extraction solvent carries out ultrasonic extraction, and with sherwood oil (60 DEG C-90 DEG C)-ethyl acetate (17:3) for developping agent, is conducive to the TLC distinguish of Tibet inula root most with ether.
experimental example 3: the TLC distinguish of cloves
(1) instrument
Tool plug conical flask, electronic scales, transfer pipet, ultrasound wave extraction apparatus, funnel, filter paper, evaporating dish, water-bath, sample applicator, silica G plate, chromatography cylinder.
(2) control medicinal material agent test sample
Cloves control medicinal material (lot number: 121039-201004), purchased from Chinese pharmaceutical biological product qualification institute;
Test sample, embodiment 1 gained capsule.
(3) reagent
Ether, ethyl acetate, sherwood oil, toluene, anisaldehyde test solution.
(4) method of inspection:
Extraction solvent: ether, ethyl acetate.
Extracting method: ultrasonic and immersion.
Developping agent: sherwood oil (60 DEG C-90 DEG C)-ethyl acetate (3:1), toluene-ethyl acetate (19:1).
Select above Extraction solvent, extracting method and developping agent to test respectively, result is as follows:
Result shows, and is that Extraction solvent carries out ultrasonic extraction, and with sherwood oil (60 DEG C-90 DEG C)-ethyl acetate (3:1) for developping agent, is conducive to the TLC distinguish of cloves most with ether.
experimental example 4: myristic TLC distinguish
(1) instrument
Tool plug conical flask, electronic scales, transfer pipet, ultrasound wave extraction apparatus, funnel, filter paper, evaporating dish, water-bath, sample applicator, silica G plate, chromatography cylinder.
(2) control medicinal material and test sample
Nutmeg control medicinal material (lot number: 120926-200906), purchased from Chinese pharmaceutical biological product qualification institute;
Test sample, embodiment 1 gained capsule.
(3) reagent
Ether, ethyl acetate, sherwood oil, methylene chloride, acetone, 5% vanillic aldehyde concentrated sulfuric acid solution.
(4) method of inspection:
Extraction solvent: ether, sherwood oil, methylene chloride.
Extracting method: ultrasonic and jolting.
Developping agent: sherwood oil-acetone (8:2), sherwood oil (60 DEG C-90 DEG C)-ethyl acetate (20:1).Select above Extraction solvent, extracting method and developping agent to test respectively, result is as follows:
Result shows, and is that Extraction solvent carries out ultrasonic extraction, and with sherwood oil (60 DEG C-90 DEG C)-ethyl acetate (20:1) for developping agent, is conducive to the TLC distinguish of gallic acid most with ether.
experimental example 5: the TLC distinguish of safflower
(1) instrument
Tool plug conical flask, electronic scales, transfer pipet, ultrasound wave extraction apparatus, funnel, filter paper, evaporating dish, water-bath, sample applicator, silica G plate, chromatography cylinder.
(2) control medicinal material and test sample
Safflower control medicinal material (lot number: 120907-201010), purchased from Chinese pharmaceutical biological product qualification institute;
Test sample, embodiment gained capsule.
(3) reagent
Ether, 80% acetone, ethyl acetate, methyl alcohol, formic acid.
(4) method of inspection:
Extraction solvent: ether, 80% acetone.
Extracting method: ultrasonic and jolting.
Developping agent: acetate-methanol (5:2), acetic ether-methanoic acid-water-methanol (7:2:3:0.4), with chloroform-ether-ethyl acetate (3: 2: 1).
Select above Extraction solvent, extracting method and developping agent to test respectively, result is as follows:
Result shows, and is that Extraction solvent carries out ultrasonic extraction with ether, and with acetate-methanol (5:2) and acetic ether-methanoic acid-water-methanol (7:2:3:0.4) for developping agent, is conducive to the TLC distinguish of safflower most.
experimental example 6: gallic acid content contained by myrobalan, terminaliae billericae,fructus, emblic measures
(1) instrument
Conical flask, electronic balance, 50ml transfer pipet, ultrasound wave extraction apparatus, funnel, filter paper, syringe, filter.
(2) reference substance and test sample
Gallic acid reference substance (lot number: 110831-200803), purchased from Chinese pharmaceutical biological product qualification institute;
Test sample, embodiment 1 gained capsule.
(3) reagent
Methyl alcohol, acetonitrile, triethylamine, phosphoric acid.
(4) method of inspection:
Extraction solvent: methyl alcohol, 50% methyl alcohol, ethanol.
Extraction time: ultrasonic 30 minutes, 40 minutes, 50 minutes.
Mobile phase: acetonitrile-0.1% triethylamine solution (2:98), acetonitrile-0.1 phosphoric acid solution (2:98), acetonitrile-0.1% triethylamine 0.1% phosphoric acid solution (1:99), acetonitrile-0.4% phosphoric acid solution (5:95), methyl alcohol-0.2% phosphoric acid solution (90:10).
Select above Extraction solvent, extraction time and mobile phase to test respectively, result is as follows:
Result shows, and with 50% methyl alcohol for Extraction solvent carries out ultrasonic extraction 30min, and with acetonitrile-0.1% triethylamine 0.1 phosphoric acid solution (1:99) for mobile phase, is conducive to the mensuration of gallic acid content contained by myrobalan, terminaliae billericae,fructus, emblic most.
experimental example 7: safflower institute hydroxyl carthamin yellow A-containing assay
(1) instrument
Conical flask, electronic balance, 50ml transfer pipet, ultrasound wave extraction apparatus, funnel, filter paper, syringe, filter.
(2) reference substance and test sample
Hydroxyl radical carthamin yellow carthamus A reference substance (lot number: 111637-200905), purchased from Chinese pharmaceutical biological product qualification institute;
Test sample, embodiment 1 gained capsule.
(3) reagent
Methyl alcohol, acetonitrile, phosphoric acid, ethanol.
(4) method of inspection:
Extraction solvent: methyl alcohol, 25% methyl alcohol, ethanol.
Extraction time: ultrasonic 30 minutes, 40 minutes, 50 minutes.
Mobile phase: methanol-water (28:72), methyl alcohol-0.2% phosphoric acid solution (26:74), methanol-acetonitrile-0.2% phosphoric acid solution (22:2:76), acetonitrile-0.2% phosphoric acid solution (22:78).
Select above Extraction solvent, extraction time and mobile phase to test respectively, result is as follows:
Result shows, and with 25% methyl alcohol for Extraction solvent carries out ultrasonic extraction 40min, and with methanol-acetonitrile-0.2% phosphoric acid solution (22:2:76) for mobile phase, is conducive to the mensuration of safflower institute hydroxyl carthamin yellow A-containing content most.
Obviously, above-described embodiment is only for clearly example being described, and the restriction not to embodiment.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here exhaustive without the need to also giving all embodiments.And thus the apparent change of extending out or variation be still among the protection domain of the invention.

Claims (7)

1. one kind is used for the treatment of the quality determining method of the Tibetan medicinal composition of stomach trouble, it is characterized in that, the bulk drug composition of described composition comprises: myrobalan 150-250 weight portion, terminaliae billericae,fructus 5-20 weight portion, emblic 20-80 weight portion, hooker winghead root 20-80 weight portion, Tibet inula root 20-80 weight portion, tufa 5-20 weight portion, safflower 5-20 weight portion, cloves 5-20 weight portion, cardamom 5-20 weight portion, nutmeg 5-20 weight portion, tsaoko 5-20 weight portion, agalloch eaglewood 5-20 weight portion, seed of pomegranate 5-20 weight portion, Indian Herba Swertiae bimaculatae 1-20 weight portion, pawpaw 1-20 weight portion, styrax 1-20 weight portion, cowherb reaches summer 1-20 weight portion,
Described quality determining method comprises the step of following Qualitive test and/or assay:
A) Qualitive test of gallic acid
Get described Tibetan medicinal composition 0.5-5 weight portion, add methyl alcohol 1-30 parts by volume, ultrasonic process 10-40 minute, filter, filtrate is as myrobalan, terminaliae billericae,fructus, emblic need testing solution;
Separately get gallic acid reference substance, add methyl alcohol and make the solution of every 1 parts by volume containing 0.0005-0.003 weight portion gallic acid, product solution in contrast;
By the bulk drug prescription preparation of described Tibetan medicinal composition containing the negative sample composition of described myrobalan, terminaliae billericae,fructus and emblic, and get described negative sample composition 0.5-5 weight portion, add methyl alcohol 1-30 parts by volume, ultrasonic process 10-40 minute, filter, filtrate is not as containing the negative sample solution of myrobalan, terminaliae billericae,fructus and emblic;
Test according to thin-layered chromatography, draw above-mentioned need testing solution, negative sample solution and reference substance solution each 1-4 μ l, put respectively on same silica gel g thin-layer plate, take volume ratio as the methenyl choloride-acetic ether-methanoic acid of 2-10:1-7:0.1-2.5 be developping agent, launch, take out, dry, spray take w/v as the ferric trichloride ethanolic solution of 1%-3%, inspects under daylight; In test sample chromatogram, on the position corresponding to reference substance chromatogram, the spot of aobvious same color;
B) Qualitive test of Tibet inula root
Get described Tibetan medicinal composition 0.5-5 weight portion, add diethyl ether 10-50 parts by volume, close plug, ultrasonic process 10-40 minute, filters, filtrate evaporate to dryness, and adding volume ratio is that the ether-acetic acid ethyl ester solution 0.5-2 parts by volume of l:1 makes it dissolve, as Tibet inula root need testing solution;
Separately get Tibet inula root control medicinal material 0.05-1 weight portion, add diethyl ether 0.5-2 parts by volume, close plug, ultrasonic process 10-40 minute, and leave standstill, supernatant is as Tibet inula root control medicinal material solution;
The negative sample composition of described Tibet inula root is not contained by the bulk drug prescription preparation of described Tibetan medicinal composition, get described negative sample composition 0.5-5 weight portion, add diethyl ether 10-50 parts by volume, close plug, ultrasonic process 10-40 minute, filters, filtrate evaporate to dryness, adding volume ratio is that the ether-acetic acid ethyl ester solution 0.5-2 parts by volume of l:1 makes it dissolve, as the negative sample solution not containing Tibet inula root;
Test according to thin-layered chromatography, draw need testing solution, negative sample solution and control medicinal material solution each 1-8 μ l, put respectively on same silica gel g thin-layer plate, take volume ratio as 60 DEG C of-90 DEG C of petroleum ether-ethyl acetates of 12-22:0.5-7 be developping agent, launch, take out, dry, spray with the secret potassium test solution of rare iodate; In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color;
C) Qualitive test of cloves
Get described Tibetan medicinal composition 0.5-5 weight portion, add diethyl ether 10-50 parts by volume, close plug, ultrasonic process 10-40 minute, filters, filtrate evaporate to dryness, and adding volume ratio is that the ether-acetic acid ethyl ester solution 0.5-2 parts by volume of l:1 makes it dissolve, as cloves need testing solution;
Separately get cloves control medicinal material 0.05-0.5 weight portion, add diethyl ether 0.5-5 parts by volume, close plug, ultrasonic process 10-40 minute, and leave standstill, supernatant is as cloves control medicinal material solution;
The negative sample composition of described cloves is not contained by the bulk drug prescription preparation of described Tibetan medicinal composition, get described negative sample composition 0.5-5 weight portion, add diethyl ether 10-50 parts by volume, close plug, ultrasonic process 10-40 minute, filters, filtrate evaporate to dryness, adding volume ratio is that the ether-acetic acid ethyl ester solution 0.5-2 parts by volume of l:1 makes it dissolve, as the negative sample solution not containing cloves;
Test according to thin-layered chromatography, draw need testing solution, negative sample solution and control medicinal material solution each 1-4 μ l, put respectively on same silica gel g thin-layer plate, take volume ratio as 60 DEG C of-90 DEG C of petroleum ether-ethyl acetates of 0.5-7.3:0.5-2.5 be developping agent, launch, take out, dry, spray, with anisaldehyde test solution, is heated to spot development clear; In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color;
D) myristic Qualitive test
Get described Tibetan medicinal composition 0.5-5 weight portion, add diethyl ether 10-50 parts by volume, close plug, ultrasonic process 10-40 minute, filters, filtrate evaporate to dryness, and adding volume ratio is that the ether-acetic acid ethyl ester solution 0.5-2 parts by volume of l:1 makes it dissolve, as nutmeg need testing solution;
Separately get nutmeg control medicinal material 0.05-0.5 weight portion, add diethyl ether 1-10 parts by volume, close plug, ultrasonic process 10-40 minute, filters, filtrate evaporate to dryness, the ether-acetic acid ethyl ester solution 0.5-2 parts by volume adding volume ratio 1:1 makes it dissolve, as nutmeg control medicinal material solution;
Described myristic negative sample composition is not contained by the bulk drug prescription preparation of described Tibetan medicinal composition, get described negative sample composition 0.5-5 weight portion, add diethyl ether 10-50 parts by volume, close plug, ultrasonic process 10-40 minute, filters, filtrate evaporate to dryness, adding volume ratio is that the ether-acetic acid ethyl ester solution 0.5-2 parts by volume of l:1 makes it dissolve, as not containing myristic negative sample solution;
According to TLC test, draw need testing solution, negative sample solution and control medicinal material solution each 2-8 μ l, put respectively on same silica gel g thin-layer plate, take volume ratio as 60 DEG C of-90 DEG C of petroleum ether-ethyl acetates of 15-25:0.5-2.5 be developping agent, launch, take out, dry, spray is 3%-8% vanillic aldehyde concentrated sulfuric acid solution with w/v, is heated to spot development clear; In test sample chromatogram, with on control medicinal material chromatogram relevant position, show the spot of same color;
E) Qualitive test of safflower
Get described Tibetan medicinal composition 0.5-5 weight portion, add diethyl ether 10-50 parts by volume, close plug, ultrasonic process 10-40 minute, filters, add 80% acetone 1-10 parts by volume, ultrasonic process 10-40 minute after residue volatilizes, and leave standstill, supernatant is as safflower need testing solution;
Separately get safflower control medicinal material 0.05-0.5 weight portion, add 80% acetone 1-10 parts by volume, ultrasonic process 10-40 minute, leave standstill, supernatant is as safflower control medicinal material solution;
The negative sample composition of described safflower is not contained by the bulk drug prescription preparation of described Tibetan medicinal composition, get described negative sample composition 0.5-5 weight portion, add diethyl ether 10-50 parts by volume, close plug, ultrasonic process 10-40 minute, filters, 80% acetone 1-10 parts by volume is added after residue volatilizes, ultrasonic process 10-40 minute, leaves standstill, and supernatant is as the negative sample solution not containing safflower;
Test according to thin-layered chromatography, drawing need testing solution and negative sample solution 5-15 μ l, control medicinal material solution 2-8 μ l, put respectively on same silica gel g thin-layer plate, take volume ratio as the acetate-methanol of 2-8:0.5-4 is developping agent, exhibition is to about 3cm, take out, dry, then take volume ratio as the acetic ether-methanoic acid-water-methanol of 3-12:0.1-5:0.1-5:0.1-1 be developping agent, launch, take out, dry, inspect under daylight; In test sample chromatogram, with test sample chromatogram, with on control medicinal material chromatogram relevant position, show the spot of same color;
F) assay of gallic acid in myrobalan, terminaliae billericae,fructus, emblic
Get described Tibetan medicinal composition 0.05-2 weight portion, put in tool plug conical flask, add 50% methyl alcohol 25-75 parts by volume, weighed weight, ultrasonic process 10-45min under power 100W, frequency 40kHz condition, let cool, more weighed weight, the weight of less loss is supplied with 50% methyl alcohol, shake up, filter, get filtrate, as need testing solution;
Get gallic acid reference substance appropriate, accurately weighed, add 50% methyl alcohol and make the solution of every 1 parts by volume containing gallic acid 0.00001-0.0001 weight portion, product solution in contrast;
By the bulk drug prescription preparation of described Tibetan medicinal composition containing the negative sample composition of described myrobalan, terminaliae billericae,fructus and emblic, and get described negative sample composition 0.05-2 weight portion, put in tool plug conical flask, add 50% methyl alcohol 25-75 parts by volume, weighed weight, ultrasonic process 10-45min under power 100W, frequency 40kHz condition, let cool, weighed weight again, supply the weight of less loss with 50% methyl alcohol, shake up, filter, get filtrate, as the negative sample solution not containing myrobalan, terminaliae billericae,fructus, emblic;
According to high performance liquid chromatography test, take octadecylsilane chemically bonded silica as filling agent, with 0.1% phosphoric acid solution of acetonitrile-0.1% triethylamine of volume ratio 0.1-5:95-99.9 for mobile phase; Measure at 273 ± 2nm determined wavelength, column temperature are 25-40 DEG C; Theoretical cam curve calculates by gallic acid peak and is not less than 1000; And precision draws reference substance solution, need testing solution, negative sample solution each 5-20 μ l respectively, injection liquid chromatography, measures, and every 1 weight portion of preparation in gallic acid, must not be less than 0.017 weight portion containing myrobalan, terminaliae billericae,fructus, emblic;
G) assay of safflower institute hydroxyl carthamin yellow A-containing
Get described Tibetan medicinal composition 0.5-3 weight portion, accurately weighed, put in tool plug conical flask, precision adds 25% methyl alcohol 20-80 parts by volume, and weighed weight, in power 250W, ultrasonic process 20-60 minute under frequency 40kHz condition, let cool, more weighed weight, the weight of less loss is supplied with 25% methyl alcohol, shake up, filter, get subsequent filtrate, as need testing solution;
Get hydroxyl radical carthamin yellow carthamus A reference substance appropriate, accurately weighed, add the solution that 25% methyl alcohol makes every 1 parts by volume hydroxyl carthamin yellow A-containing 0.000005-0.00005 weight portion, product solution in contrast;
Do not contain the negative sample composition of described safflower by the bulk drug prescription preparation of described Tibetan medicinal composition, get described negative sample composition 0.5-3 weight portion, accurately weighed, put in tool plug conical flask, precision adds 25% methyl alcohol 20-80 parts by volume, weighed weight, under power 250W, frequency 40kHz condition, ultrasonic process 20-60 minute, lets cool, weighed weight again, supply the weight of less loss with 25% methyl alcohol, shake up, filter, get subsequent filtrate, as the negative sample solution not containing safflower;
According to high performance liquid chromatography test, take octadecylsilane chemically bonded silica as filling agent; With methanol-acetonitrile-0.2% phosphoric acid solution of volume ratio 15-30:0.5-5:65-85 for mobile phase; Measure at 403 ± 2nm determined wavelength, column temperature are 25-40 DEG C; Number of theoretical plate calculates should be not less than 3000 by hydroxyl radical carthamin yellow carthamus A peak; Accurate absorption reference substance solution, need testing solution, negative sample solution each 5-20 μ l respectively, injection liquid chromatography, measures, and every 1 weight portion of preparation in hydroxyl radical carthamin yellow carthamus A, must not be less than 0.00025 weight portion containing safflower;
The pass of described weight portion and parts by volume is g/ml.
2. quality determining method according to claim 1, is characterized in that, comprises the step of following Qualitive test and/or assay:
A) Qualitive test of gallic acid
Get described Tibetan medicinal composition 1 weight portion, add methyl alcohol 5 parts by volume, ultrasonic process 20 minutes, filter, filtrate is as myrobalan, terminaliae billericae,fructus, emblic need testing solution;
Separately get gallic acid reference substance, add methyl alcohol and make the solution of every 1 parts by volume containing 0.001 weight portion gallic acid, product solution in contrast;
By the bulk drug prescription preparation of described Tibetan medicinal composition containing the negative sample composition of described myrobalan, terminaliae billericae,fructus and emblic, and get described negative sample composition 1 weight portion, add methyl alcohol 5 parts by volume, ultrasonic process 20 minutes, filter, filtrate is not as containing the negative sample solution of myrobalan, terminaliae billericae,fructus and emblic;
Test according to thin-layered chromatography, draw each 2 μ l of above-mentioned need testing solution, negative sample solution and reference substance solution, put respectively on same silica gel g thin-layer plate, take volume ratio as the methenyl choloride-acetic ether-methanoic acid of 6:4:1 be developping agent, launch, take out, dry, spray take w/v as the ferric trichloride ethanolic solution of 2%, inspects under daylight; In test sample chromatogram, on the position corresponding to reference substance chromatogram, the spot of aobvious same color;
B) Qualitive test of Tibet inula root
Get described Tibetan medicinal composition 2 weight portion, add diethyl ether 20 parts by volume, close plug, ultrasonic process 20 minutes, filters, filtrate evaporate to dryness, and adding volume ratio is that ether-acetic acid ethyl ester solution 1 parts by volume of l:1 makes it dissolve, as Tibet inula root need testing solution;
Separately get Tibet inula root control medicinal material 0.2 weight portion, add diethyl ether 1 parts by volume, close plug, ultrasonic process 20 minutes, and leave standstill, supernatant is as Tibet inula root control medicinal material solution;
The negative sample composition of described Tibet inula root is not contained by the bulk drug prescription preparation of described Tibetan medicinal composition, get described negative sample composition 2 weight portion, add diethyl ether 20 parts by volume, close plug, ultrasonic process 20 minutes, filters, filtrate evaporate to dryness, adding volume ratio is that ether-acetic acid ethyl ester solution 1 parts by volume of l:1 makes it dissolve, as the negative sample solution not containing Tibet inula root;
According to thin-layered chromatography test, draw each 4 μ l of need testing solution, negative sample solution and control medicinal material solution, put respectively on same silica gel g thin-layer plate, take volume ratio as 60 DEG C of-90 DEG C of petroleum ether-ethyl acetates of 17:3 be developping agent, launch, take out, dry, spray with the secret potassium test solution of rare iodate; In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color;
C) Qualitive test of cloves
Get described Tibetan medicinal composition 2 weight portion, add diethyl ether 20 parts by volume, close plug, ultrasonic process 20 minutes, filters, filtrate evaporate to dryness, and adding volume ratio is that ether-acetic acid ethyl ester solution 1 parts by volume of l:1 makes it dissolve, as cloves need testing solution;
Separately get cloves control medicinal material 0.2 weight portion, add diethyl ether 3 parts by volume, close plug, ultrasonic process 20 minutes, and leave standstill, supernatant is as cloves control medicinal material solution;
The negative sample composition of described cloves is not contained by the bulk drug prescription preparation of described Tibetan medicinal composition, get described negative sample composition 2 weight portion, add diethyl ether 20 parts by volume, close plug, ultrasonic process 20 minutes, filters, filtrate evaporate to dryness, adding volume ratio is that ether-acetic acid ethyl ester solution 1 parts by volume of l:1 makes it dissolve, as the negative sample solution not containing cloves;
Test according to thin-layered chromatography, draw each 2 μ l of need testing solution, negative sample solution and control medicinal material solution, put respectively on same silica gel g thin-layer plate, take volume ratio as 60 DEG C of-90 DEG C of petroleum ether-ethyl acetates of 3:1 be developping agent, launch, take out, dry, spray, with anisaldehyde test solution, is heated to spot development clear; In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color;
D) myristic Qualitive test
Get described Tibetan medicinal composition 2 weight portion, add diethyl ether 20 parts by volume, close plug, ultrasonic process 20 minutes, filters, filtrate evaporate to dryness, and adding volume ratio is that ether-acetic acid ethyl ester solution 1 parts by volume of l:1 makes it dissolve, as nutmeg need testing solution;
Separately get nutmeg control medicinal material 0.2 weight portion, add diethyl ether 5 parts by volume, close plug, ultrasonic process 20 minutes, and filter, filtrate evaporate to dryness, ether-acetic acid ethyl ester solution 1 parts by volume adding volume ratio 1:1 makes it dissolve, as nutmeg control medicinal material solution;
Described myristic negative sample composition is not contained by the bulk drug prescription preparation of described Tibetan medicinal composition, get described negative sample composition 2 weight portion, add diethyl ether 20 parts by volume, close plug, ultrasonic process 20 minutes, filters, filtrate evaporate to dryness, adding volume ratio is that ether-acetic acid ethyl ester solution 1 parts by volume of l:1 makes it dissolve, as not containing myristic negative sample solution;
According to TLC test, draw each 5 μ l of need testing solution, negative sample solution and control medicinal material solution, put respectively on same silica gel g thin-layer plate, take volume ratio as 60 DEG C of-90 DEG C of petroleum ether-ethyl acetates of 20:1 be developping agent, launch, take out, dry, spray is 5% vanillic aldehyde concentrated sulfuric acid solution with w/v, is heated to spot development clear; In test sample chromatogram, with test sample chromatogram, with on control medicinal material chromatogram relevant position, show the spot of same color;
E) Qualitive test of safflower
Get described Tibetan medicinal composition 2 weight portion, add diethyl ether 20 parts by volume, close plug, ultrasonic process 20 minutes, filters, add 80% acetone 5 parts by volume, ultrasonic process 20 minutes after residue volatilizes, and leave standstill, supernatant is as safflower need testing solution;
Separately get safflower control medicinal material 0.2 weight portion, add 80% acetone 5 parts by volume, ultrasonic process 20 minutes, leave standstill, supernatant is as safflower control medicinal material solution;
The negative sample composition of described safflower is not contained by the bulk drug prescription preparation of described Tibetan medicinal composition, get described negative sample composition 2 weight portion, add diethyl ether 20 parts by volume, close plug, ultrasonic process 20 minutes, filters, 80% acetone 5 parts by volume is added after residue volatilizes, ultrasonic process 20 minutes, leaves standstill, and supernatant is as the negative sample solution not containing safflower;
Test according to thin-layered chromatography, drawing need testing solution and negative sample solution 10 μ l, control medicinal material solution 5 μ l, put respectively on same silica gel g thin-layer plate, take volume ratio as the acetate-methanol of 5:2 is developping agent, exhibition is to about 3cm, take out, dry, then take volume ratio as the acetic ether-methanoic acid-water-methanol of 7:2:3:0.4 be developping agent, launch, take out, dry, inspect under daylight; In test sample chromatogram, with test sample chromatogram, with on control medicinal material chromatogram relevant position, show the spot of same color;
F) assay of gallic acid in myrobalan, terminaliae billericae,fructus, emblic
Get described Tibetan medicinal composition 0.1 weight portion, put in tool plug conical flask, add 50% methyl alcohol 50 parts by volume, weighed weight, ultrasonic process 30min under power 100W, frequency 40kHz condition, let cool, more weighed weight, the weight of less loss is supplied with 50% methyl alcohol, shake up, filter, get filtrate, as need testing solution;
Get gallic acid reference substance appropriate, accurately weighed, add 50% methyl alcohol and make the solution of every 1 parts by volume containing gallic acid 0.00004 weight portion, product solution in contrast;
By the bulk drug prescription preparation of described Tibetan medicinal composition containing the negative sample composition of described myrobalan, terminaliae billericae,fructus and emblic, and get described negative sample composition 0.1 weight portion, put in tool plug conical flask, add 50% methyl alcohol 50 parts by volume, weighed weight, under power 100W, frequency 40kHz condition, ultrasonic process 30min, lets cool, weighed weight again, supply the weight of less loss with 50% methyl alcohol, shake up, filter, get filtrate, as the negative sample solution not containing myrobalan, terminaliae billericae,fructus, emblic;
According to high performance liquid chromatography test, take octadecylsilane chemically bonded silica as filling agent, with 0.1% phosphoric acid solution of acetonitrile-0.1% triethylamine of volume ratio 1:99 for mobile phase; Measure at 273nm determined wavelength, column temperature are 30 DEG C; Theoretical cam curve calculates by gallic acid peak and is not less than 1000; And precision draws reference substance solution, need testing solution, each 10 μ l of negative sample solution respectively, injection liquid chromatography, measures, and every 1 weight portion of preparation in gallic acid, must not be less than 0.017 weight portion containing myrobalan, terminaliae billericae,fructus, emblic;
G) assay of safflower institute hydroxyl carthamin yellow A-containing
Get described Tibetan medicinal composition 1.5 weight portion, accurately weighed, put in tool plug conical flask, precision adds 25% methyl alcohol 50 parts by volume, and weighed weight, in power 250W, ultrasonic process 40 minutes under frequency 40kHz condition, let cool, more weighed weight, the weight of less loss is supplied with 25% methyl alcohol, shake up, filter, get subsequent filtrate, as need testing solution;
Get hydroxyl radical carthamin yellow carthamus A reference substance appropriate, accurately weighed, add the solution that 25% methyl alcohol makes every 1 parts by volume hydroxyl carthamin yellow A-containing 0.00002 weight portion, product solution in contrast;
Do not contain the negative sample composition of described safflower by the bulk drug prescription preparation of described Tibetan medicinal composition, get described negative sample composition 1.5 weight portion, accurately weighed, put in tool plug conical flask, precision adds 25% methyl alcohol 50 parts by volume, weighed weight, under power 250W, frequency 40kHz condition, ultrasonic process 40 minutes, lets cool, weighed weight again, supply the weight of less loss with 25% methyl alcohol, shake up, filter, get subsequent filtrate, as the negative sample solution not containing safflower;
According to high performance liquid chromatography test, take octadecylsilane chemically bonded silica as filling agent; With methanol-acetonitrile-0.2% phosphoric acid solution of volume ratio 22:2:76 for mobile phase; Measure at 403nm determined wavelength, column temperature are 30 DEG C; Number of theoretical plate calculates should be not less than 3000 by hydroxyl radical carthamin yellow carthamus A peak; Accurate absorption reference substance solution, need testing solution, each 10 μ l of negative sample solution respectively, injection liquid chromatography, measures, and every 1 weight portion of preparation in hydroxyl radical carthamin yellow carthamus A, must not be less than 0.00025 weight portion containing safflower.
3. quality determining method according to claim 1 and 2, is characterized in that, the bulk drug of described composition consists of:
Myrobalan 180-220 weight portion, terminaliae billericae,fructus 8-15 weight portion, emblic 30-70 weight portion, hooker winghead root 30-70 weight portion, Tibet inula root 30-70 weight portion, tufa 8-15 weight portion, safflower 8-15 weight portion, cloves 8-15 weight portion, cardamom 8-15 weight portion, nutmeg 8-15 weight portion, tsaoko 8-15 weight portion, agalloch eaglewood 8-15 weight portion, seed of pomegranate 8-15 weight portion, Indian Herba Swertiae bimaculatae 5-15 weight portion, pawpaw 5-15 weight portion, styrax 5-15 weight portion, cowherb reaches summer 5-15 weight portion.
4. quality determining method according to claim 3, is characterized in that, the bulk drug of described composition consists of:
Myrobalan 201.7 weight portion, terminaliae billericae,fructus 12.6 weight portion, emblic 50.4 weight portion, hooker winghead root 50.4 weight portion, Tibet inula root 50.4 weight portion, tufa 11.8 weight portion, safflower 11.8 weight portion, cloves 11.8 weight portion, cardamom 11.8 weight portion, nutmeg 11.8 weight portion, tsaoko 11.8 weight portion, agalloch eaglewood 11.8 weight portion, seed of pomegranate 11.8 weight portion, Indian Herba Swertiae bimaculatae 10.1 weight portion, pawpaw 10.1 weight portion, styrax 10.1 weight portion, cowherb reach summer 10.1 weight portion.
5. quality determining method according to claim 4, is characterized in that, described composition makes clinical or pharmaceutically acceptable tablet, capsule, granule, powder, pill, sustained release agent or oral liquid.
6. quality determining method according to claim 5, is characterized in that, described Tibetan medicinal composition prepares by the following method:
Above-mentioned ten seven flavor medicine materials, wherein, Tibet inula root, cloves, cardamom, nutmeg, tsaoko, agalloch eaglewood, styrax, safflower and tufa are crushed to the powder of 0.01-200 micron, sieve; Myrobalan, terminaliae billericae,fructus, emblic, cowherb reach summer boiling twice, collecting decoction, and filter, filtrate is condensed into cream clearly, for subsequent use; Hooker winghead root, seed of pomegranate, India's camphor tree bud dish and pawpaw, add alcohol reflux and extract secondary, merging filtrate, filtrate recycling ethanol, to appropriate, adds above-mentioned clear cream, be condensed into medicinal extract, drying, adds fine medicinal material powder, mixing, by pharmacy conventional method, add customary adjuvant and make the tablet, capsule, granule, powder, pill, sustained release agent or the oral liquid that accept clinically.
7. the quality determining method according to claim 1,2,4,5 or 6, is characterized in that, described Tibetan medicinal composition prepares by the following method:
According to the powder that selected weight portion gets Tibet inula root, tufa, safflower, cloves, cardamom, nutmeg, tsaoko, agalloch eaglewood, styrax are crushed to 0.01-200 micron; Separately get the myrobalan of selected weight portion, terminaliae billericae,fructus, emblic and cowherb to reach the summer and add the water accounting for drug weight 5-20 times of weight, extract 1-5 time, obtain Aqueous extracts; Get surplus stock hooker winghead root, seed of pomegranate, Indian Herba Swertiae bimaculatae and pawpaw and add the ethanol that the concentration accounting for drug weight 5-20 times of weight is greater than 20%, extract 1-5 time, obtain alcohol extract; Respectively described Aqueous extracts, alcohol extract are concentrated into the medicinal extract that relative density is 0.5-1.35, mixes with aforementioned powder pulverized powder after dry, add customary adjuvant conveniently technique make clinical acceptable tablet, capsule, granule, powder, pill, sustained release agent or oral liquid.
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