CN102526561A - Quality control method for Sanjin preparation - Google Patents
Quality control method for Sanjin preparation Download PDFInfo
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- CN102526561A CN102526561A CN2011100283172A CN201110028317A CN102526561A CN 102526561 A CN102526561 A CN 102526561A CN 2011100283172 A CN2011100283172 A CN 2011100283172A CN 201110028317 A CN201110028317 A CN 201110028317A CN 102526561 A CN102526561 A CN 102526561A
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Abstract
The invention relates to a quality control method for a Sanjin preparation, which comprises the following steps of: measuring the content of Hydroxy asiaticoside and/ or asiaticoside with a high performance liquid chromatography-evaporation light-scattering detection (HPLC-ELSD) method; and measuring the content of wild rose glycoside and/or rose glycoside with the HPLC-ELSD method. With the quality control method for the Sanjin preparation, the step of measuring the content of wild rose glycoside and/or rose glycoside with the HPLC-ELSD method is added on the basis of measuring the content of Hydroxy asiaticoside and/or asiaticoside with the HPLC-ELSD method, and therefore, the quality control indexes of the Sanjin preparation are more comprehensive. The quality control method for the Sanjin preparation has the advantages of good degree of precision, good reproducibility and good stability.
Description
Technical field
The present invention relates to a kind of method of quality control of pharmaceutical preparation, particularly relate to a kind of Chinese medicine preparation, the method for quality control of SANJIN PIAN.
Background technology
Three gold medal preparations are Chinese medicine preparation of being processed into through extraction by Chinese medicine of the five flavours such as Radix Rosae Laevigatae, Rhizoma Smilacis Scobinicaulis (Rhizoma Smilacis Chinensis), Herba lygodii, YANGKAIKOU, Herba Centellaes, the existing sale on the market, and its pharmacological action is: antibiotic; Antiinflammatory, detumescence, heat clearing away; Diuresis; Analgesia, antioxidant radical, human body immunity improving power etc.Function cure mainly into: heat-clearing and toxic substances removing, dampness removing is treating stranguria, kidney tonifying.Be used for damp-heat in lower-JIAO, pyretic stranguria, oliguria with reddish urine drenches the puckery pain of drop; Acute and chronic pyelonephritis, cystitis, urinary tract infection belong to the syndrome of dampness-heat diffusing downward person that suffers from a deficiency of the kidney.The prescription of SANJIN PIAN and method of quality control have been listed version Chinese Pharmacopoeia in 2000 in.And in 2003 the revision increased the assay item, being described below SANJIN PIAN in the revised quality standard:
[prescription] Radix Rosae Laevigatae, Rhizoma Smilacis Chinensis, YANGKAIKOU, Herba lygodii, Herba Centellae
[method for making] above five tastes, the decocte with water secondary, 2 hours for the first time, 1 hour for the second time, collecting decoction filtered, and filtrating is condensed into the clear paste that relative density is 1.15～1.20 (50～60 ℃), drying; Add right amount of auxiliary materials, mixing is processed granule, is pressed into 1000 (small pieces) or 600 (sheet), and coating promptly gets.
[character] these article are coated tablet or Film coated tablets, remove coating after, show brown to pitchy; Sour in the mouth, puckery, little hardship.
15 of these article (small pieces) or 10 (sheet) are got in [discriminating] (1), remove coating, and porphyrize adds ethanol 15ml; Supersound process 20 minutes filters, the filtrating evaporate to dryness, and residue adds the sodium hydroxide solution 20ml of 0.01mol/L; Slight fever makes dissolving, extracts with ether 10ml jolting, discards ether extracted liquid; Water layer reuse ethyl acetate 10ml jolting is extracted, and ethyl acetate extraction liquid is concentrated into 1ml, as need testing solution (water layer is subsequent use).Other gets Radix Rosae Laevigatae control medicinal material 2.5g, shines medical material solution in pairs with legal system.According to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B) test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose; With chloroform-methanol (17: 3) is developing solvent; Launch, take out, dry; Spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the speckle colour developing.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show speckle identical more than.
(2) get the water layer under the item of [discriminating] (1), extract with water saturated n-butyl alcohol 15ml, obtain n-butanol layer, with the saturated water 5ml washing of n-butyl alcohol, discard water layer, n-butyl alcohol liquid is put evaporate to dryness in the water-bath, and residue adds methanol 1ml makes dissolving, as need testing solution.Other gets the asiaticoside reference substance, adds methanol and processes the solution that every 1ml contains 1mg, as reference substance solution.According to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B) test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose; With chloroform-methanol-water (7: 3: 0.5) is developing solvent; Launch, take out, dry; Spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the speckle colour developing.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color.
[inspection] should meet each item regulation (an appendix I of Chinese Pharmacopoeia version in 2000 D) relevant under the tablet item
[assay] got 50 of these article (small pieces) or 30 (sheet), removes coating, and precision takes by weighing, and porphyrize is got about 2.5g, and accurate the title decides; Put in the tool plug conical flask, the accurate methanol 100ml that adds, close plug, the accurate title, decided supersound process (power 160W, frequency 50kHz) 45 minutes; Put coldly, accurate claim surely, add methanol and supply the weight that subtracts mistake, shake up, filter, discard filtrating just; The accurate subsequent filtrate 50ml that draws, evaporate to dryness, residue add the sodium hydroxide solution 30ml of 0.05mol/L, make dissolving, with water saturated n-butanol extraction 3 times, 20ml at every turn; Discard water layer, merge n-butanol layer, with the saturated water washing twice of n-butyl alcohol, each 10ml discards water layer; N-butyl alcohol liquid reduction vaporization is to doing, and residue adds an amount of dissolve with methanol, is transferred in the 5ml measuring bottle, adds methanol to scale, as need testing solution.It is an amount of that other gets the asiaticoside reference substance, adds methanol and process the solution that every 1ml contains 1mg, as reference substance solution.(an appendix VI of Chinese Pharmacopoeia version in 2000 B) scans wavelength according to thin layer chromatography: λ S=550nm, measure test sample trap integrated value and reference substance trap integrated value, and calculate, promptly get.
Every of these article contain Herba Centellae with asiaticoside (C
48H
78O
19) meter, small pieces must not be less than 0.18mg; Sheet must not be less than 0.30mg.
[function with cure mainly] heat-clearing and toxic substances removing, dampness removing is treating stranguria, kidney tonifying.Be used for damp-heat in lower-JIAO, pyretic stranguria, oliguria with reddish urine, the puckery pain of pouring drop; Acute and chronic pyelonephritis, cystitis, urinary tract infection belongs to the syndrome of dampness-heat diffusing downward person that suffers from a deficiency of the kidney.
[usage and consumption] is oral, large stretch of one time 3,5 of small pieces one time, 3～4 times on the one.
[specification] (1) small pieces are equivalent to crude drug 2.1g (2) sheet and are equivalent to crude drug 3.5g
[storage] sealing.
Because the quality control content assaying method that above-mentioned standard adopts is traditional thin layer chromatography (TLCS method), has precision, repeatability, less stable, the deficiency that colour developing is unstable, measurement deviation is big.
Application number is that 200510200471.8 one Chinese patent application relates to a kind of three gold medal pharmaceutical preparatioies and method for preparing and method of quality control of treating diseases such as urinary system; It is that Radix Rosae Laevigatae, Rhizoma Smilacis Chinensis, YANGKAIKOU, Herba lygodii, Herba Centellae are processed acceptable dosage form on the pharmaceutics; The preparation disintegrative that obtains is good; Bioavailability is high, and particularly their supplementary product consumption is all fewer, is particularly suitable for the old people and swallow tablet or the inconvenient patient of capsule take; The present invention also provides the method for quality control of this preparation, can guarantee the quality of the pharmaceutical preparations for preparing and make things convenient for industrial production requirement; Compared with prior art, the pharmaceutical preparation that provides is used for acute and chronic pyelonephritis, and treatment of diseases such as chronic urinary tract infection have reasonable effect; Preparation technology provided by the invention is advanced, and is simple; But and the little patients life-time service of these preparation untoward reaction provided by the invention.Method of quality control related in this application comprises the thin layer chromatography differential test method of (1) Radix Rosae Laevigatae medical material, asiaticoside, diosgenin; The content test method of asiaticoside, all or part of composition of asiaticoside in (2) the three gold medal preparations.This method has still adopted thin layer chromatography (TLCS method), still has precision, repeatability, less stable, the deficiency that colour developing is unstable, measurement deviation is big.
Because above-mentioned defective, it is 200410050066.8 one Chinese patent application that the applicant has proposed application number on July 2nd, 2004, and has authorized patent for invention.This patent discloses a kind of new method of quality control of three gold medal preparations, and this method comprises the assay item, and what assay adopted is the HPLC-ELSD method.
The inventor has carried out a large amount of research on this basis again; And find through the separate study of system; Rosamultin and multiflorin are the characteristic active component of Radix Rosae Laevigatae; And Radix Rosae Laevigatae is the necessary component of three gold medal preparations, so increase rosamultin and multiflorin are very necessary for the index components of controlling the three gold medal qualities of the pharmaceutical preparations.And existing quality standard and method of quality control all do not contain rosamultin and/or multiflorin Determination on content; It is thus clear that; Existing quality standard and method of quality control are comprehensive inadequately to the quality testing of three gold medal preparations; Quality control is still existed certain defective, certainly will have certain drug risk.Given this, the inventor provides a kind of new method of quality control.
Summary of the invention
The object of the present invention is to provide a kind of method of quality control of three gold medal preparations; On the basis of ZL200410050066.8, improved content assaying method; HPLC-ELSD method assay through effective ingredient rosamultin and/or multiflorin; Hold the quality of relevant medicine more accurately, reduce drug risk, improve the quality of products.
For realizing above-mentioned purpose, the present invention adopts following technical scheme:
A kind of method of quality control of three gold medal preparations comprises the step that adopts the HPLC-ELSD method to measure asiaticoside and/or asiaticoside content, and wherein, this method also comprises the step that adopts the HPLC-ELSD method to measure rosamultin and/or multiflorin content.
The inventor finds through the separate study of system; Rosamultin and multiflorin are the characteristic active component of Radix Rosae Laevigatae; And Radix Rosae Laevigatae is the necessary component of three gold medal preparations, so increase rosamultin and multiflorin are very necessary for the index components of controlling the three gold medal qualities of the pharmaceutical preparations.The present invention introduces the mass content of rosamultin and/or multiflorin first, thereby makes the quality control index of three gold medal preparations more comprehensive.
According to aforesaid method of quality control; Wherein, the described employing HPLC-ELSD method step of measuring rosamultin and/or multiflorin content comprises the assay of preparation, rosamultin and/or multiflorin of preparation, the need testing solution of chromatographic condition and system suitability test, reference substance solution.
According to aforesaid method of quality control, wherein, said chromatographic condition and system suitability test are to use octadecylsilane chemically bonded silica to be filler; Volume ratio is that 48: 52 methanol-water is a mobile phase; Detect with evaporative light scattering detector, number of theoretical plate calculates by the rosamultin peak should be not less than 2000.
According to aforesaid method of quality control, wherein, following method is adopted in the preparation of said reference substance solution: get rosamultin respectively and the multiflorin reference substance is an amount of, accurately claim surely, add methanol and process the mixed solution that every 1ml contains 0.5mg, promptly get.
Rosamultin according to the invention and multiflorin reference substance adopt following method self-control: with the Radix Rosae Laevigatae rhizome, pulverize, after extracting solvent extraction; With the extracting solution concentrating under reduced pressure, carry out solvent extraction or go up macroporous resin adsorption, and water, 30%, 40～90% ethanol elutions successively; Collect 40～90% ethanol elution things, reclaim solvent, again with extract or eluate process column chromatography for separation; Or with extracting solution be concentrated into do after, directly pass through column chromatography for separation; The fraction that will contain rosamultin and multiflorin separates through silica gel column chromatography once more, collects the fraction that contains rosamultin and multiflorin, must rosamultin and multiflorin coarse-grain, add again recrystallization reagent repeatedly recrystallization promptly get.
According to aforesaid method of quality control, wherein, following method is adopted in the preparation of said need testing solution: get 30 small pieces of SANJIN PIAN or 20 sheets, remove coating, the accurate title, decided porphyrize; Get about 1.5g, the accurate title, decide, and the accurate methanol 50ml that adds claims to decide weight, and supersound process is 45 minutes under power 250W, frequency 40kHz condition; Put coldly, claim again decide weight, supply the weight that subtracts mistake, shake up filtration with methanol; Precision is measured subsequent filtrate 25ml, reclaims solvent to doing, and residue adds water 20ml makes dissolving, extracts 3 times with water saturated n-butyl alcohol jolting, each 15ml; Merge n-butanol extracting liquid, with ammonia solution washing 2 times, each 15ml gets n-butyl alcohol liquid, and decompression and solvent recovery is to doing; Residue is used dissolve with methanol, is transferred in the 5ml measuring bottle, adds methanol to scale, shakes up, and promptly gets.
According to aforesaid method of quality control, wherein, in the step of the assay of said rosamultin and/or multiflorin; Assay adopts following method; Accurate respectively reference substance solution 5 μ l, 10 μ l and need testing solution 10～20 μ l of drawing inject chromatograph of liquid, measure; Calculate the content of rosamultin, multiflorin respectively with external standard two-point method logarithmic equation, promptly get; Every of these article contain Radix Rosae Laevigatae with rosamultin (C
36H
58O
10) meter, small pieces must not be less than 0.10mg; Sheet must not be less than 0.16mg; Contain Radix Rosae Laevigatae with multiflorin (C
36H
58O
10) meter, small pieces must not be less than 0.10mg; Sheet must not be less than 0.16mg.
According to aforesaid method of quality control, wherein said employing HPLC-ELSD method is measured asiaticoside and/or asiaticoside content comprises the steps:
Measure according to HPLC (an appendix VI of Chinese Pharmacopoeia version in 2010 D);
Chromatographic condition and system suitability test: use octadecylsilane chemically bonded silica to be filler; Volume ratio is that 48: 52 methanol-water is a mobile phase; Detect or use the UV-detector detection with evaporative light scattering detector, detection wavelength 210nm, number of theoretical plate calculates by the asiaticoside peak should be not less than 2000;
The preparation of reference substance solution: it is an amount of to get the asiaticoside reference substance respectively, accurate claims surely, adds methanol and processes every 1ml and contain the solution of 0.2mg and the solution that every 1ml contains 0.6mg respectively, promptly gets;
The preparation of need testing solution: get this article 30 small pieces or 20 sheets, remove coating, the accurate title, decide, and porphyrize is got about 1.5g; The accurate title, decide, and the accurate methanol 50ml that adds claims to decide weight, and supersound process is 45 minutes under the condition of power 250W, frequency 40kHz, puts cold; Claim to decide weight again, supply the weight that subtracts mistake with methanol, shake up, filter, precision is measured subsequent filtrate 25ml; Reclaim solvent to doing, residue adds water 20ml makes dissolving, extracts 3 times with water saturated n-butyl alcohol jolting, and each 15ml merges n-butanol extracting liquid; With ammonia solution washing 2 times, each 15ml gets n-butyl alcohol liquid, and decompression and solvent recovery is to doing, and residue is used dissolve with methanol; Be transferred in the 5ml measuring bottle, add methanol, shake up, promptly get to scale;
Algoscopy: accurate respectively reference substance solution each 10 μ l and need testing solution 5～10 μ l that draw above-mentioned two kinds of concentration, inject chromatograph of liquid, measure, calculate with external standard two-point method logarithmic equation, promptly get; Every of these article contain Herba Centellae with asiaticoside (C
48H
78O
20) meter, small pieces must not be less than 0.22mg; Sheet must not be less than 0.35mg.
The inventor is surprised to find that; The method for preparing of its need testing solution was just the same when the preparation of its need testing solution of process of the content of employing HPLC-ELSD method mensuration rosamultin and/or multiflorin was measured asiaticoside and/or asiaticoside content with employing HPLC-ELSD method; On the basis of adopting HPLC-ELSD method mensuration asiaticoside and/or asiaticoside content, do not need to prepare in addition need testing solution; Thereby simplified quality control process, practiced thrift cost.
Method of quality control of the present invention also further comprises the step that three gold medal preparations are differentiated.
Thin layer chromatography is adopted in said discriminating.
Described thin layer chromatography comprises the steps:
(1) get SANJIN PIAN 15 small pieces or 10 sheets, remove coating, porphyrize adds ethanol 15ml; Supersound process 20 minutes filters, the filtrating evaporate to dryness; Residue adds 0.01mol/L sodium hydroxide solution 20ml, and slight fever makes dissolving, extracts with ether 10ml jolting; Discard ether solution, water liquid reuse ethyl acetate 10ml jolting is extracted, and aqueous solution is subsequent use; Acetic acid ethyl fluid is concentrated into about 1ml, as need testing solution; Other gets Radix Rosae Laevigatae control medicinal material 2.5g, shines medical material solution in pairs with legal system; According to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2010) test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate; With chloroform-methanol (17: 3) is developing solvent; Launch, take out, dry; Spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the speckle colour developing; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the principal spot of two or more same colors;
(2) get the aqueous solution under (1) item, extract with water saturated n-butyl alcohol 15ml, obtain n-butyl alcohol liquid, with the saturated water 5ml washing of n-butyl alcohol, discard water lotion, n-butyl alcohol liquid evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution; Other gets the asiaticoside reference substance, adds methanol and processes the solution that every 1ml contains 1mg, as reference substance solution; According to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2010 B) test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate; With chloroform-methanol-water (7: 3: 0.5) is developing solvent; Launch, take out, dry; Spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the speckle colour developing; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color;
(3) get SANJIN PIAN 10 small pieces or 6 sheets, remove coating, porphyrize adds ethanol 50ml, supersound process 30 minutes; Filter, filtrating adds hydrochloric acid 5ml, and reflux 2 hours is put coldly, transfers to neutrality with 40% sodium hydroxide solution; Steam to there not being the alcohol flavor, residue adds hot water 40ml makes dissolving, with dichloromethane jolting extraction 2 times (40ml, 30ml); Combined dichloromethane extracting solution, evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution; Other gets Rhizoma Smilacis Chinensis control medicinal material 5g, shines medical material solution in pairs with legal system; Get the diosgenin reference substance again, add methanol and process the solution that every 1ml contains 0.5mg, as reference substance solution; According to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2010) test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate; With cyclohexane extraction-ethyl acetate (4: 1) is developing solvent, launches, and takes out; Dry; Spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the speckle colour developing at 105 ℃, puts under the ultra-violet lamp 365nm and inspects; In the test sample chromatograph, with control medicinal material chromatograph relevant position on, show the fluorescence speckle of two or more same colors; With reference substance chromatograph relevant position on, show the fluorescence speckle of same color;
(4) get SANJIN PIAN 15 small pieces or 10 sheets, porphyrize adds methanol 100ml, and supersound process 20 minutes filters; Filtrating is concentrated into about 10ml, is added on the neutral alumina post of 100～200 orders, 5g, internal diameter 1cm, with methanol 50ml eluting, collects effluent and eluent; Evaporate to dryness, residue add water 20ml dissolving, extract 2 times with the ethyl acetate jolting, each 15ml; Combined ethyl acetate liquid, evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution; Other gets YANGKAIKOU control medicinal material 5g, adds water 100ml, and reflux 1 hour filters, the filtrating evaporate to dryness, and residue adds methanol 20ml, shines medical material solution in pairs with legal system; According to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2010) test, draw each 5～10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate; With chloroform-acetone-water (6: 14: 1) is developing solvent, launches, and takes out; Dry, put under the ultra-violet lamp 365nm and inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence speckle of same color.
The method of the invention not only can detect SANJIN PIAN; Can also detect any other dosage form of three gold medal preparations, as: sugar coated tablet, film coated tablet, enteric coated tablet, capsule, hard capsule, soft capsule, oral liquid, suck agent, granule, electuary, pill, powder, unguentum, sublimed preparation, suspensoid, powder, solution, injection, suppository, ointment, plaster, cream, spray, drop, patch.Preparation of the present invention, peroral dosage form preferably, as: capsule, tablet, oral liquid, granule, pill, powder, sublimed preparation, unguentum etc.Can also detect with Herba Centellae, multiflorin, rosamultin is preparation, Herba Centellae medical material and extract thereof, multiflorin medical material and extract, rosamultin and the extract thereof of raw material.
Characteristics of the present invention are:
1, the present invention has increased the content of HPLC-ELSD method mensuration rosamultin and/or multiflorin on the basis of HPLC-ELSD method mensuration asiaticoside content, makes the quality control index of three gold medal preparations more comprehensive;
2, the inventive method precision, repeatability, good stability.
Description of drawings
Fig. 1 is a rosamultin reference substance equation of linear regression, and its regression equation is: Y=0.6482X-7.3328, r=0.9992;
Fig. 2 is a multiflorin reference substance equation of linear regression, and its regression equation is: Y=0.8516X-8.9913, r=0.9993;
Fig. 3 is that rosamultin mixes contrast liquid chromatogram (ELSD), wherein rosamultin Rt=42.394min with multiflorin; Multiflorin Rt=32.135min;
Fig. 4 is sample liquid chromatogram (ELSD), wherein rosamultin Rt=42.256min; Multiflorin Rt=32.086min;
Fig. 5 is 0910136 batch of a SANJIN PIAN (ELSD);
Fig. 6 is 0910144 batch of a SANJIN PIAN (ELSD);
Fig. 7 is 0910160 batch of a SANJIN PIAN (ELSD);
Fig. 8 is 0910167 batch of a SANJIN PIAN (ELSD);
Fig. 9 is 0910175 batch of a SANJIN PIAN (ELSD);
Figure 10 is 0910181 batch of a SANJIN PIAN (ELSD);
Figure 11 is 0910183 batch of a SANJIN PIAN (ELSD);
Figure 12 is 0910191 batch of a SANJIN PIAN (ELSD);
Figure 13 is 0910192 batch of a SANJIN PIAN (ELSD);
Figure 14 is 0911005 batch of a SANJIN PIAN (ELSD);
Figure 15 is 0911006 batch of a SANJIN PIAN (ELSD);
Figure 16 is 0911021 batch of a SANJIN PIAN (ELSD);
Figure 17 is that rosamultin mixes contrast liquid chromatogram (UV), wherein rosamultin Rt=42.394min with multiflorin; Multiflorin Rt=32.135min;
Figure 18 is 0910136 batch of a SANJIN PIAN (UV);
Figure 19 is 0910144 batch of a SANJIN PIAN (UV);
Figure 20 is 0910160 batch of a SANJIN PIAN (UV);
Figure 21 is 0910167 batch of a SANJIN PIAN (UV);
Figure 22 is 0910175 batch of a SANJIN PIAN (UV);
Figure 23 is 0910181 batch of a SANJIN PIAN (UV);
Figure 24 is 0910183 batch of a SANJIN PIAN (UV);
Figure 25 is 0910191 batch of a SANJIN PIAN (UV);
Figure 26 is 0910192 batch of a SANJIN PIAN (UV);
Figure 27 is 0911005 batch of a SANJIN PIAN (UV);
Figure 28 is 0911006 batch of a SANJIN PIAN (UV);
Figure 29 is 0911021 batch of a SANJIN PIAN (UV).
The specific embodiment
Below be the specific embodiment of the present invention, described embodiment is in order to further describe the present invention, rather than restriction the present invention.
[embodiment 1]
Rosamultin and multiflorin assay are newly-increased [assay] standard.
Radix Rosae Laevigatae is main flavour of a drug in the SANJIN PIAN side, and the inventor finds that through the separate study of system rosamultin and multiflorin are the characteristic active component of Radix Rosae Laevigatae, so increase rosamultin and multiflorin are very necessary for the index components of controlling this quality.This standard has increased the content of HPLC-ELSD method mensuration rosamultin and multiflorin on the basis of asiaticoside assay, make the quality control index of SANJIN PIAN more comprehensive.
1, instrument and reagent
Instrument: Waters 2695 high performance liquid chromatographs, evaporative light scattering detector (Alltech-ELSD2000), (UV-detector is Waters 996), Empower chromatographic work station.
Reagent: methanol is chromatographically pure, and water is ultra-pure water, and other reagent are analytical pure.
Reference substance: self-control; Because of " Chinese pharmacopoeia is not recorded rosamultin and multiflorin chemical reference substance; So the inventor has carried out separating preparation to rosamultin with multiflorin, and has carried out structural identification and purity test by " the new Chinese medicine quality standard research is with the requirement of reference substance investigative technique ", under selected chromatographic condition, measure; Calculating content by normalization method is 98.5%, and the result shows that prepared rosamultin and multiflorin chemical reference substance meet assay and use the reference substance requirement.
Rosamultin and multiflorin reference substance adopt following method self-control: with the Radix Rosae Laevigatae rhizome, pulverize, after extracting solvent extraction; With the extracting solution concentrating under reduced pressure, carry out solvent extraction or go up macroporous resin adsorption, and water, 30%, 40～90% ethanol elutions successively; Collect 40～90% ethanol elution things, reclaim solvent, again with extract or eluate process column chromatography for separation; Or with extracting solution be concentrated into do after, directly pass through column chromatography for separation; The fraction that will contain rosamultin and multiflorin separates through silica gel column chromatography once more, collects the fraction that contains rosamultin and multiflorin, must rosamultin and multiflorin coarse-grain, add again recrystallization reagent repeatedly recrystallization promptly get.
Specifically adopt the self-control of following method: get fresh Radix Rosae Laevigatae rhizome 10Kg, pulverize,, each 2 hours, filter concentrated extractum with 60% alcohol reflux 2 times.Extractum adding distil water 1000mL makes homodisperse, adds same volumes of acetic acid ethyl ester extraction three times, and combined ethyl acetate concentrates and reclaims solvent, gets ethyl acetate extractum.Ethyl acetate extractum is used the silica gel column chromatography crude separation, and with chloroform/methanol (10: 0～0: 10) gradient elution, each gradient elution 3000mL, TLC detect and merge same section, obtain 9 major parts.Wherein be rich in the fraction of rosamultin and multiflorin; With chloroform/methanol (10: 1) is that eluant carries out silica gel column chromatography, collects the fraction be rich in rosamultin, is recrystallization in 10: 1 the chloroform/methanol mixed solution in volume ratio; Get white indefinite form solid, i.e. rosamultin; The fraction of multiflorin is rich in collection, is to press reversed phase column chromatography during eluant carries out repeatedly with 55% methanol, is recrystallization in 10: 1 the chloroform/methanol mixed solution in volume ratio, white indefinite form solid, i.e. multiflorin.
2, the selection of chromatographic condition
Chromatographic column: Nova-pak C18 (3.9 * 150mm, 4um);
Mobile phase: methanol-water (48: 52); Flow velocity: 0.8ml/min;
Drift tube temperature: 90 ℃; Gas flow rate: 2.4L/min;
In mobile phase is selected, tested the separating effect of the methanol-water system of different proportion, the result is the best with methanol-water (48: 52), and retention time is suitable, and separating effect can meet the demands.
Under selected condition, the separating degree of the rosamultin of three batches of test samples and multiflorin chromatographic peak, the result of number of theoretical plate see table 1.
The result of table 1, rosamultin and multiflorin number of theoretical plate and separating degree
Rosamultin and multiflorin peak, left side separating degree: R
1=2 (t
R-t
R1)/(W+W
1)
t
R-be the retention time at one peak, back in adjacent two peaks
t
R1-be the retention time at last peak in adjacent two peaks
W, W
1The peak width at these adjacent two peaks
According to result of the test, consider the influence of the system factors such as preparation and temperature of instrument, chromatographic column, mobile phase, the regulation number of theoretical plate press the calculating of rosamultin peak, should be not less than 2000.
3, the investigation of the range of linearity
The preparation of reference substance solution: the rosamultin and the multiflorin reference substance that are dried to constant weight of learning from else's experience 105 ℃ is an amount of; The accurate title, decide; Add methanol process the mixed solution that every 1ml contains 1mg (the real 54.25mg that gets of rosamultin, multiflorin is real gets 51.20mg, is settled to 50ml; Promptly be respectively 1.085mg/ml, 1.024mg/ml), promptly get.
Measure: accurate respectively absorption mixing reference substance solution solution 1 μ l, 2 μ l, 4 μ l, 6 μ l, 8 μ l, 10 μ l inject chromatograph of liquid; Measuring peak area, is abscissa with the natural logrithm of peak area, is vertical coordinate with the natural logrithm of sample size; Regression Calculation; Get linear equation, measure the result and see table 2, table 3.
Table 2, the linear result that investigates of rosamultin reference substance
Above result shows that in sample size 1.085～10.85 μ g scopes, the natural logrithm of rosamultin peak area and the natural logrithm of sample size have good linear relationship.
Table 3, the linear result that investigates of multiflorin reference substance
Above result shows that in sample size 1.024～10.24 μ g scopes, the natural logrithm at multiflorin peak and the natural logrithm of sample size have good linear relationship.
4, the research of need testing solution method for preparing
(1) extracts choice of Solvent
Investigate 50% methanol respectively, 80% methanol, methanol, ethanol is as extracting reagent, and the contrast different solvents is to the influence of extraction effect.Concrete grammar is:
60 of these article of getting (lot number 0910021) are removed coating, and accurate the title decides, and porphyrize is got about 1.5g respectively, and accurate title is fixed; The accurate difference that adds is extracted solvent 50ml, claims to decide weight, and supersound process (power 250W, frequency 40kHz) 45 minutes is put cold; Claim to decide weight again, supply the weight that subtracts mistake with the corresponding solvent that extracts, shake up, filter, precision is measured subsequent filtrate 25ml; Reclaim solvent to doing, residue adds water 20ml makes dissolving, extracts 3 times with water saturated n-butyl alcohol jolting, and each 15ml merges n-butanol extracting liquid; With ammonia solution washing 2 times, each 15ml gets n-butyl alcohol liquid, and decompression and solvent recovery is to doing, and residue is used dissolve with methanol; Be transferred in the 5ml measuring bottle, add methanol, shake up to scale, sample introduction 10 μ l, the result sees table 4.
Table 4, extraction choice of Solvent
Experimental result shows that 80% methanol is suitable basically with the methanol extraction effect, but during 80% methanol extraction, impurity is more, the easy emulsifying of post-processed.So select methanol as extracting solvent.
(2) investigation of method for distilling and extraction time.
Supersound extraction is investigated in test, and Suo Shi extracts, three kinds of method for distilling of heating and refluxing extraction, and different extraction times reach equilibrated influence to extraction.
Supersound extraction is specially: get 60 of these article (lot number 0910021), remove coating, the accurate title, decide, and porphyrize is got about 1.5g respectively; The accurate title, decide, and the accurate methanol solvate 50ml that adds claims to decide weight, supersound process (power 250W, frequency 40kHz) different time; Put coldly, claim again decide weight, supply the weight that subtracts mistake, shake up filtration with methanol; Precision is measured subsequent filtrate 25ml, reclaims solvent to doing, and residue adds water 20ml makes dissolving, extracts 3 times with water saturated n-butyl alcohol jolting, each 15ml; Merge n-butanol extracting liquid, with ammonia solution washing 2 times, each 15ml gets n-butyl alcohol liquid, and decompression and solvent recovery is to doing; Residue is used dissolve with methanol, is transferred in the 5ml measuring bottle, adds methanol to scale, shakes up.
Suo Shi extracts and is specially: get 60 of these article (lot number 0910021), remove coating, the accurate title, decide, and porphyrize is got about 1.5g respectively, and precision is claimed fixed, puts in the apparatus,Soxhlet's, and accurate adding methanol 50ml extracted respectively 4 hours, and 8 hours, 12 hours.Extracting solution reclaims solvent to doing, and operates with method from " residue adds water 20ml " and supersound process, processes need testing solution.
Heating and refluxing extraction is specially: get 60 of these article (lot number 0910021), remove coating, the accurate title, decide, and porphyrize is got about 1.5g respectively, and accurate title is fixed, and in the flask, accurate adding methanol 50ml claims to decide weight, difference reflux 1 hour, 2 hours, 4 hours at the bottom of the horizontalization.Rise and supersound process is operated with method from " put cold, as to claim decide weight again ", process need testing solution.
Above-mentioned need testing solution sample introduction 10 μ l measure the result and see table 5.
The investigation of table 5, Different Extraction Method and extraction time
| Different Extraction Method and time | The content of rosamultin (μ g/ sheet) | The content of multiflorin (μ g/ sheet) |
| Supersound extraction 30 minutes | 302.4 | 323.7 |
| Supersound extraction 45 minutes | 345.3 | 378.8 |
| Supersound extraction 60 minutes | 348.1 | 379.4 |
| Suo Shi extracted 4 hours | 292.5 | 313.6 |
| Suo Shi extracted 8 hours | 326.9 | 355.8 |
| Suo Shi extracted 12 hours | 345.4 | 374.2 |
| Reflux 1 hour | 316.8 | 339.5 |
| |
344.2 | 376.1 |
| |
346.3 | 377.8 |
Experimental result shows that supersound process 45min has reached poised state, and extracts 12 hours with Suo Shi, and 2 hours effects of heating and refluxing extraction are suitable basically.So method for distilling is decided to be: supersound process 45min.
(3) investigation of purification process
Traditional saponin purification process is adopted in test.That is: the methanol extract liquid of sample, the residue behind the evaporate to dryness dissolves with aqueous alkali, extracts with the water-saturated n-butanol jolting, continues with the method for water washing n-butanol extracting liquid.
Test is selected else with ethyl acetate as extracting solution, as comparing.The result sees table 6.
Table 6, purification process are investigated
| Purification solvent | N-butyl alcohol | Ethyl acetate |
| The content of rosamultin (μ g/ sheet) | 344.6 | 345.2 |
| The content of multiflorin (μ g/ sheet) | 378.5 | 376.7 |
The result shows, n-butyl alcohol or ethyl acetate extraction effect basically identical, but in the test operation process, find; During ethyl acetate extraction, solution emulsifying more easily, the processing time is longer; And from extracting solution colour and test sample chromatogram, the impurity of ethyl acetate extraction is more.Therefore, select the extraction solvent of n-butyl alcohol for use as purification.
(4) selection of n-butanol extraction number of times
For investigating the n-butanol extraction number of times, after extracting 3 times, continue with water saturated n-butanol extraction the 4th, the 5th; Reduction vaporization is to doing respectively; Residue dissolves with methanol 1ml, sample introduction, and the result shows in the 4th water-saturated n-butanol jolting extracting solution has not had rosamultin and multiflorin; So jolting is extracted 3 times, rosamultin and multiflorin can extract fully.So purification process adopts water-saturated n-butanol to extract 3 times.
5, precision test
20 of these article of getting (lot number 0910021) are equipped with need testing solution by text [assay] below legal system, and sample introduction 20 μ l repeat sample introduction 6 times, measure rosamultin and multiflorin area value, and calculate content respectively, and the result sees table 7.
Table 7, Precision test result
Above result shows that the precision of used chromatograph of liquid is good.
6, stability test
Get need testing solution (lot number 0910021), at room temperature preserve, respectively at 0,1,2,4,6,8,24 hour interval; The accurate need testing solution 20 μ l that draw inject chromatograph of liquid, measure rosamultin and multiflorin peak area value; And calculating content respectively, the result sees table 8.
Table 8, stability test result
Result of the test shows that need testing solution was measured in 24 hours, its relative deviation is respectively 2.78%, 2.83%, explains that need testing solution measured in 24 hours, and it is stable that the result keeps.
7, repeatability test
Get 6 parts of the SANJIN PIANs of same lot number (0910021 batch), prepare need testing solution respectively in accordance with the law, record peak area value respectively, and calculate rosamultin and multiflorin content respectively, RSD is respectively: 3.38%, 3.73%, and the result sees table 9.Result of the test shows that the method repeatability is good.
Table 9, reproducible test results
8, application of sample recovery test
Get with repeatability test same lot number SANJIN PIAN (the assay result is: rosamultin 335.3 μ g/ sheets, multiflorin 379.6 μ g/ sheets, average sheet is heavy: 0.2790g) about 0.75g; Parallel 6 parts, the accurate title, decide, and precision adds rosamultin and mixes reference substance solution (concentration is respectively: 1.085 μ g/ml, 1.024 μ g/ml) 1ml with multiflorin respectively; Prepare application of sample in accordance with the law and reclaim need testing solution, measure the result, with the formula calculate recovery rate; The result sees table 10, table 11.
Table 10, rosamultin application of sample recovery test result
Table 11, multiflorin application of sample recovery test result
Result of the test shows: the response rate is between 95%～100%, and application of sample reclaims good.
9, sample determination
(1) measure the content of rosamultin and multiflorin in 12 batches of SANJIN PIANs (film-coat large stretch of) according to rosamultin and multiflorin [assay] method, the mensuration result sees table 12.
Table 12,12 crowdes of assay results of SANJIN PIAN (sheet)
According to above mensuration result, the content fluctuation range of rosamultin is 89.3～354.0 μ g/ sheets in totally 12 batches of SANJIN PIANs (sheet) of being surveyed, and the content fluctuation range of multiflorin is 85.4～376.3 μ g/ sheets.It is thus clear that the content fluctuation is bigger, average content is respectively 204.7 μ g/ sheets, 202.6 μ g/ sheets; Because the content that factors such as medical material content difference, operation cause fluctuation is formulated limit with 80% of average content, so content limit is tentative be: every of these article contain Radix Rosae Laevigatae with rosamultin (C in order to avoid
36H
58O
10) meter, sheet must not be less than 0.16mg; Contain Radix Rosae Laevigatae with multiflorin (C
36H
58O
10) meter, sheet must not be less than 0.16mg.
Cause does not have sample cuttings to supply to measure temporarily, converts so press recipe quantity, small pieces content is fixed tentatively be: every of these article contain Radix Rosae Laevigatae with rosamultin (C
36H
58O
10) meter, small pieces must not be less than 0.10mg; Contain Radix Rosae Laevigatae with multiflorin (C
36H
58O
10) meter, small pieces must not be less than 0.10mg.Unified mutually with the content limit of sheet.
Though the composition of being surveyed does not have tangible uv absorption, under the separation condition of this experiment, adopt UV-detector, detect under the wavelength at 210nm, also can realize effective mensuration, adopt UV-detector to detect simultaneously and calculate 12 lot sample article content results and see table 13.
Table 13,12 crowdes of assay results of SANJIN PIAN (sheet) (ultraviolet detection method)
Visible according to above mensuration result, ultraviolet detection method result and evaporative light scattering detector detection method be basically identical as a result, and therefore, above-mentioned two kinds of detectors all can be used as the assay detector of this kind.(the ultraviolet detection method spectrogram sees Figure 17 to Figure 29 for details)
The effective ingredient rosamultin in the content that the method for quality control of the SANJIN PIAN that this embodiment provided comprises the effective ingredient asiaticoside that adopts in the high effective liquid chromatography for measuring SANJIN PIAN and asiaticoside and the employing high effective liquid chromatography for measuring SANJIN PIAN and the content of multiflorin.Concrete grammar is following:
SANJIN PIAN, the method that provides according to pharmacopeia preparation, or buy in Guangxi or any one tame pharmacy of China is produced and is provided by Guilin three gold medal pharmaceutical Co. Ltds.
Assay:
(1) effective ingredient asiaticoside and the asiaticoside in the employing high effective liquid chromatography for measuring SANJIN PIAN, concrete operation method is seen an appendix VI of Chinese Pharmacopoeia version in 2010 D.
Chromatographic condition and system suitability test: use octadecylsilane chemically bonded silica to be filler; Volume ratio is that 48: 52 methanol-water is a mobile phase; Detect or use the UV-detector detection with evaporative light scattering detector, detection wavelength 210nm, number of theoretical plate calculates by the asiaticoside peak should be not less than 2000;
The preparation of reference substance solution: it is an amount of to get the asiaticoside reference substance respectively, accurate claims surely, adds methanol and processes every 1ml and contain the solution of 0.2mg and the solution that every 1ml contains 0.6mg respectively, promptly gets;
The preparation of need testing solution: get this article 20 sheets, remove coating, the accurate title, decide, and porphyrize is got about 1.5g; The accurate title, decide, and the accurate methanol 50ml that adds claims to decide weight, and supersound process is 45 minutes under the condition of power 250W, frequency 40kHz, puts cold; Claim to decide weight again, supply the weight that subtracts mistake with methanol, shake up, filter, precision is measured subsequent filtrate 25ml; Reclaim solvent to doing, residue adds water 20ml makes dissolving, extracts 3 times with water saturated n-butyl alcohol jolting, and each 15ml merges n-butanol extracting liquid; With ammonia solution washing 2 times, each 15ml gets n-butyl alcohol liquid, and decompression and solvent recovery is to doing, and residue is used dissolve with methanol; Be transferred in the 5ml measuring bottle, add methanol, shake up, promptly get to scale;
Algoscopy: accurate respectively reference substance solution each 10 μ l and need testing solution 5～10 μ l that draw above-mentioned two kinds of concentration, inject chromatograph of liquid, measure, calculate with external standard two-point method logarithmic equation, promptly get; Every of these article contain Herba Centellae with asiaticoside (C
48H
78O
20) meter, sheet must not be less than 0.35mg.
(2) effective ingredient rosamultin and the multiflorin in the employing high effective liquid chromatography for measuring SANJIN PIAN, concrete operation method is seen an appendix VI of Chinese Pharmacopoeia version in 2010 D.
Chromatographic condition and system suitability test: use octadecylsilane chemically bonded silica to be filler; Methanol-water (48: 52) is a mobile phase; Detect with evaporative light scattering detector.Number of theoretical plate calculates by the rosamultin peak should be not less than 2000.
The preparation of reference substance solution: it is an amount of to get rosamultin and multiflorin reference substance (adopting the method self-control described in the embodiment 1) respectively, and accurate title is fixed, adds methanol and processes the mixed solution that every 1ml contains 0.5mg, promptly gets.
The preparation of need testing solution: get SANJIN PIAN 20 sheets, remove coating, the accurate title, decide, and porphyrize is got about 1.5g, and accurate title is fixed; The accurate methanol 50ml that adds claims decide weight, and supersound process (power 250W, frequency 40kHz) 45 minutes is put coldly, and weight decided in title again; Supply the weight that subtracts mistake with methanol, shake up, filter, precision is measured subsequent filtrate 25ml, reclaims solvent to doing, and residue adds water 20ml makes dissolving; Extract 3 times with water saturated n-butyl alcohol jolting, each 15ml merges n-butanol extracting liquid, and with ammonia solution washing 2 times, each 15ml gets n-butyl alcohol liquid; Decompression and solvent recovery is to doing, and residue is used dissolve with methanol, is transferred in the 5ml measuring bottle, adds methanol to scale, shakes up, and promptly gets.
Algoscopy: respectively accurate reference substance solution 5 μ l, the 10 μ l and need testing solution 10～20 μ l of drawing, inject chromatograph of liquid, mensuration is calculated the content of rosamultin, multiflorin respectively with external standard two-point method logarithmic equation, promptly gets.
Every of these article contain Radix Rosae Laevigatae with rosamultin (C
36H
58O
10) meter, sheet must not be less than 0.16mg; Contain Radix Rosae Laevigatae with multiflorin (C
36H
58O
10) meter, sheet must not be less than 0.16mg.
The effective ingredient rosamultin in the content that the method for quality control of the SANJIN PIAN that this embodiment provided comprises the effective ingredient asiaticoside that adopts in the high effective liquid chromatography for measuring SANJIN PIAN and asiaticoside and the employing high effective liquid chromatography for measuring SANJIN PIAN and the content of multiflorin.Concrete grammar is following:
SANJIN PIAN, the method that provides according to pharmacopeia preparation, or buy in Guangxi or any one tame pharmacy of China is produced and is provided by Guilin three gold medal pharmaceutical Co. Ltds.
Assay:
(1) effective ingredient asiaticoside and the asiaticoside in the employing high effective liquid chromatography for measuring SANJIN PIAN, concrete operation method is seen an appendix VI of Chinese Pharmacopoeia version in 2010 D.
Chromatographic condition and system suitability test: use octadecylsilane chemically bonded silica to be filler; Volume ratio is that 48: 52 methanol-water is a mobile phase; Detect or use the UV-detector detection with evaporative light scattering detector, detection wavelength 210nm, number of theoretical plate calculates by the asiaticoside peak should be not less than 2000;
The preparation of reference substance solution: it is an amount of to get the asiaticoside reference substance respectively, accurate claims surely, adds methanol and processes every 1ml and contain the solution of 0.2mg and the solution that every 1ml contains 0.6mg respectively, promptly gets;
The preparation of need testing solution: get SANJIN PIAN 30 small pieces, remove coating, the accurate title, decide, and porphyrize is got about 1.5g; The accurate title, decide, and the accurate methanol 50ml that adds claims to decide weight, and supersound process is 45 minutes under the condition of power 250W, frequency 40kHz, puts cold; Claim to decide weight again, supply the weight that subtracts mistake with methanol, shake up, filter, precision is measured subsequent filtrate 25ml; Reclaim solvent to doing, residue adds water 20ml makes dissolving, extracts 3 times with water saturated n-butyl alcohol jolting, and each 15ml merges n-butanol extracting liquid; With ammonia solution washing 2 times, each 15ml gets n-butyl alcohol liquid, and decompression and solvent recovery is to doing, and residue is used dissolve with methanol; Be transferred in the 5ml measuring bottle, add methanol, shake up, promptly get to scale;
Algoscopy: accurate respectively reference substance solution each 10 μ l and need testing solution 5～10 μ l that draw above-mentioned two kinds of concentration, inject chromatograph of liquid, measure, calculate with external standard two-point method logarithmic equation, promptly get; Every of these article contain Herba Centellae with asiaticoside (C
48H
78O
20) meter, small pieces must not be less than 0.22mg.
(2) effective ingredient rosamultin and the multiflorin in the employing high effective liquid chromatography for measuring SANJIN PIAN, concrete operation method is seen an appendix VI of Chinese Pharmacopoeia version in 2010 D.
Chromatographic condition and system suitability test: use octadecylsilane chemically bonded silica to be filler; Methanol-water (48: 52) is a mobile phase; Detect with evaporative light scattering detector.Number of theoretical plate calculates by the rosamultin peak should be not less than 2000.
The preparation of reference substance solution: it is an amount of to get rosamultin and multiflorin reference substance (adopting the method self-control described in the embodiment 1) respectively, and accurate title is fixed, adds methanol and processes the mixed solution that every 1ml contains 0.5mg, promptly gets.
The preparation of need testing solution: get SANJIN PIAN 30 small pieces, remove coating, the accurate title, decide, and porphyrize is got about 1.5g, and accurate title is fixed; The accurate methanol 50ml that adds claims decide weight, and supersound process (power 250W, frequency 40kHz) 45 minutes is put coldly, and weight decided in title again; Supply the weight that subtracts mistake with methanol, shake up, filter, precision is measured subsequent filtrate 25ml, reclaims solvent to doing, and residue adds water 20ml makes dissolving; Extract 3 times with water saturated n-butyl alcohol jolting, each 15ml merges n-butanol extracting liquid, and with ammonia solution washing 2 times, each 15ml gets n-butyl alcohol liquid; Decompression and solvent recovery is to doing, and residue is used dissolve with methanol, is transferred in the 5ml measuring bottle, adds methanol to scale, shakes up, and promptly gets.
Algoscopy: respectively accurate reference substance solution 5 μ l, the 10 μ l and need testing solution 10～20 μ l of drawing, inject chromatograph of liquid, mensuration is calculated the content of rosamultin, multiflorin respectively with external standard two-point method logarithmic equation, promptly gets.
Every of these article contain Radix Rosae Laevigatae with rosamultin (C
36H
58O
10) meter, small pieces must not be less than 0.10mg; Contain Radix Rosae Laevigatae with multiflorin (C
36H
58O
10) meter, small pieces must not be less than 0.10mg.
The method of quality control of the SANJIN PIAN that this embodiment provided comprises the effective ingredient asiaticoside that adopts in the high effective liquid chromatography for measuring SANJIN PIAN and the content of asiaticoside, effective ingredient rosamultin and the content of multiflorin and the step that SANJIN PIAN is differentiated in the employing high effective liquid chromatography for measuring SANJIN PIAN; Wherein the assay of the assay of asiaticoside and asiaticoside, rosamultin and multiflorin is with embodiment 2; The step of differentiating is to adopt thin layer chromatography to differentiate that concrete steps are following:
(1) get SANJIN PIAN 10 sheets, remove coating, porphyrize adds ethanol 15ml; Supersound process 20 minutes filters, the filtrating evaporate to dryness; Residue adds 0.01mol/L sodium hydroxide solution 20ml, and slight fever makes dissolving, extracts with ether 10ml jolting; Discard ether solution, water liquid reuse ethyl acetate 10ml jolting is extracted, and aqueous solution is subsequent use; Acetic acid ethyl fluid is concentrated into about 1ml, as need testing solution; Other gets Radix Rosae Laevigatae control medicinal material 2.5g, shines medical material solution in pairs with legal system; According to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2010) test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate; With chloroform-methanol (17: 3) is developing solvent; Launch, take out, dry; Spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the speckle colour developing; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the principal spot of two or more same colors;
(2) get the aqueous solution under (1) item, extract with water saturated n-butyl alcohol 15ml, obtain n-butyl alcohol liquid, with the saturated water 5ml washing of n-butyl alcohol, discard water lotion, n-butyl alcohol liquid evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution; Other gets the asiaticoside reference substance, adds methanol and processes the solution that every 1ml contains 1mg, as reference substance solution; According to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2010 B) test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate; With chloroform-methanol-water (7: 3: 0.5) is developing solvent; Launch, take out, dry; Spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the speckle colour developing; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color;
(3) get SANJIN PIAN 6 sheets, remove coating, porphyrize adds ethanol 50ml, supersound process 30 minutes; Filter, filtrating adds hydrochloric acid 5ml, and reflux 2 hours is put coldly, transfers to neutrality with 40% sodium hydroxide solution; Steam to there not being the alcohol flavor, residue adds hot water 40ml makes dissolving, with dichloromethane jolting extraction 2 times (40ml, 30ml); Combined dichloromethane extracting solution, evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution; Other gets Rhizoma Smilacis Chinensis control medicinal material 5g, shines medical material solution in pairs with legal system; Get the diosgenin reference substance again, add methanol and process the solution that every 1ml contains 0.5mg, as reference substance solution; According to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2010) test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate; With cyclohexane extraction-ethyl acetate (4: 1) is developing solvent, launches, and takes out; Dry; Spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the speckle colour developing at 105 ℃, puts under the ultra-violet lamp 365nm and inspects; In the test sample chromatograph, with control medicinal material chromatograph relevant position on, show the fluorescence speckle of two or more same colors; With reference substance chromatograph relevant position on, show the fluorescence speckle of same color;
(4) get SANJIN PIAN 10 sheets, porphyrize adds methanol 100ml, and supersound process 20 minutes filters; Filtrating is concentrated into about 10ml, is added on the neutral alumina post of 100～200 orders, 5g, internal diameter 1cm, with methanol 50ml eluting, collects effluent and eluent; Evaporate to dryness, residue add water 20ml dissolving, extract 2 times with the ethyl acetate jolting, each 15ml; Combined ethyl acetate liquid, evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution; Other gets YANGKAIKOU control medicinal material 5g, adds water 100ml, and reflux 1 hour filters, the filtrating evaporate to dryness, and residue adds methanol 20ml, shines medical material solution in pairs with legal system; According to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2010) test, draw each 5～10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate; With chloroform-acetone-water (6: 14: 1) is developing solvent, launches, and takes out; Dry, put under the ultra-violet lamp 365nm and inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence speckle of same color.
The method of quality control of the SANJIN PIAN that this embodiment provided comprises the effective ingredient asiaticoside that adopts in the high effective liquid chromatography for measuring SANJIN PIAN and the content of asiaticoside, effective ingredient rosamultin and the content of multiflorin and the step that SANJIN PIAN is differentiated in the employing high effective liquid chromatography for measuring SANJIN PIAN; Wherein the assay of the assay of asiaticoside and asiaticoside, rosamultin and multiflorin is with embodiment 3; The step of differentiating is to adopt thin layer chromatography to differentiate that concrete steps are following:
(1) get SANJIN PIAN 15 small pieces, remove coating, porphyrize adds ethanol 15ml; Supersound process 20 minutes filters, the filtrating evaporate to dryness; Residue adds 0.01mol/L sodium hydroxide solution 20ml, and slight fever makes dissolving, extracts with ether 10ml jolting; Discard ether solution, water liquid reuse ethyl acetate 10ml jolting is extracted, and aqueous solution is subsequent use; Acetic acid ethyl fluid is concentrated into about 1ml, as need testing solution; Other gets Radix Rosae Laevigatae control medicinal material 2.5g, shines medical material solution in pairs with legal system; According to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2010) test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate; With chloroform-methanol (17: 3) is developing solvent; Launch, take out, dry; Spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the speckle colour developing; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the principal spot of two or more same colors;
(2) get the aqueous solution under (1) item, extract with water saturated n-butyl alcohol 15ml, obtain n-butyl alcohol liquid, with the saturated water 5ml washing of n-butyl alcohol, discard water lotion, n-butyl alcohol liquid evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution; Other gets the asiaticoside reference substance, adds methanol and processes the solution that every 1ml contains 1mg, as reference substance solution; According to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2010 B) test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate; With chloroform-methanol-water (7: 3: 0.5) is developing solvent; Launch, take out, dry; Spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the speckle colour developing; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color;
(3) get SANJIN PIAN 10 small pieces, remove coating, porphyrize adds ethanol 50ml, supersound process 30 minutes; Filter, filtrating adds hydrochloric acid 5ml, and reflux 2 hours is put coldly, transfers to neutrality with 40% sodium hydroxide solution; Steam to there not being the alcohol flavor, residue adds hot water 40ml makes dissolving, with dichloromethane jolting extraction 2 times (40ml, 30ml); Combined dichloromethane extracting solution, evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution; Other gets Rhizoma Smilacis Chinensis control medicinal material 5g, shines medical material solution in pairs with legal system; Get the diosgenin reference substance again, add methanol and process the solution that every 1ml contains 0.5mg, as reference substance solution; According to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2010) test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate; With cyclohexane extraction-ethyl acetate (4: 1) is developing solvent, launches, and takes out; Dry; Spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the speckle colour developing at 105 ℃, puts under the ultra-violet lamp 365nm and inspects; In the test sample chromatograph, with control medicinal material chromatograph relevant position on, show the fluorescence speckle of two or more same colors; With reference substance chromatograph relevant position on, show the fluorescence speckle of same color;
(4) get SANJIN PIAN 15 small pieces, porphyrize adds methanol 100ml, and supersound process 20 minutes filters; Filtrating is concentrated into about 10ml, is added on the neutral alumina post of 100～200 orders, 5g, internal diameter 1cm, with methanol 50ml eluting, collects effluent and eluent; Evaporate to dryness, residue add water 20ml dissolving, extract 2 times with the ethyl acetate jolting, each 15ml; Combined ethyl acetate liquid, evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution; Other gets YANGKAIKOU control medicinal material 5g, adds water 100ml, and reflux 1 hour filters, the filtrating evaporate to dryness, and residue adds methanol 20ml, shines medical material solution in pairs with legal system; According to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2010) test, draw each 5～10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate; With chloroform-acetone-water (6: 14: 1) is developing solvent, launches, and takes out; Dry, put under the ultra-violet lamp 365nm and inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence speckle of same color.
Test Example 1
This Test Example is identified adopting the homemade rosamultin of method described in the embodiment of the invention 1 and the chemical constitution of multiflorin reference substance.
Gjy-4:2 α, 3 β, 19 α-trihydroxy Usu-12 alkene-28 acid, 28-O-β-D-glucoside; Rosamultin; Rosamultin;
1H-NMR(CD
3OD)δ：0.77(3H，s)，0.81(3H，s)，0.93(3H，d，J＝6.5Hz，H-30)，1.01(6H，s)，1.20(3H，s)，1.33(3H，s)，2.51(1H，s，H-18)，2.91(1H，d，J＝9.6Hz，H-3)，5.31(1H，brs，H-12)，5.32(1H，d，J＝8.0Hz，H-1′)；
13C-NMR(CD
3OD)δ：48.2(t，C-1)，69.5(d，C-2)，84.5(d，C-3)，40.5(s，C-4)，56.7(d，C-5)，19.7(t，C-6)，34.1(t，C-7)，41.3(s，C-8)，48.6(d，C-9)，39.2(s，C-10)，24.8(t，C-11)，129.5(d，C-12)，139.7(s，C-13)，42.7(s，C-14)，29.6(t，C-15)，26.5(t，C-16)，49.5(s，C-17)，54.9(d，C-18)，73.7(s，C-19)，42.9(d，C-20)，27.2(t，C-21)，38.3(t，C-22)，29.4(q，C-23)，16.7(q，C-24)，17.2(q，C-25)，17.7(q，C-26)，24.7(q，C-27)，178.5(s，C-28)，27.1(q，C-29)，17.5(q，C-30)，95.8(d，C-1′)，73.8(d，C-2′)，78.5(d，C-3′)，71.1(d，C-4′)，78.3(d，C-5′)，62.4(t，C-6′)。
Gjy-6:2 α, 3 α, 19 α-trihydroxy Usu-12-alkene-28 acid, 28-O-β-D-glucoside; Multiflorin; Euscaphicoside; The Fructus Rosae Normalis glycosides; Kajiichigoside F1;
1H-NMR(CD3OD)δ：0.76(3H，s)，0.88(3H，s)，0.92(3H，d，J＝6.5Hz，H-30)，0.97(3H，s)，0.98(3H，s)，1.25(3H，s)，1.32(3H，s)，2.50(1H，s，H-18)，3.32(1H，brs，H-3)，3.92(1H，m，H-2)，5.30(1H，brs，H-12)，5.32(1H，d，J＝8.0Hz，H-1′)；
13C-NMR(CD3OD)δ：42.8(t，C-1)，67.2(d，C-2)，80.1(d，C-3)，39.5(s，C-4)，49.3(d，C-5)，19.3(t，C-6)，34.0(t，C-7)，41.4(s，C-8)，48.2(d，C-9)，39.4(s，C-10)，24.7(t，C-11)，129.6(d，C-12)，139.7(s，C-13)，42.7(s，C-14)，29.6(t，C-15)，26.6(t，C-16)，49.6(s，C-17)，55.0(d，C-18)，73.6(s，C-19)，43.0(d，C-20)，27.3(t，C-21)，39.0(t，C-22)，29.3(q，C-23)，22.4(q，C-24)，16.9(q，C-25)，17.6(q，C-26)，24.8(q，C-27)，178.5(s，C-28)，27.0(q，C-29)，16.6(q，C-30)，95.8(d，C-1′)，73.8(d，C-2′)，78.6(d，C-3′)，71.1(d，C-4′)，78.3(d，C-5′)，62.4(t，C-6′)。
Claims (8)
1. the method for quality control of a gold medal preparation; Comprise the step that adopts the HPLC-ELSD method to measure asiaticoside and/or asiaticoside content; It is characterized in that this method also comprises the step that adopts the HPLC-ELSD method to measure rosamultin and/or multiflorin content.
2. method of quality control according to claim 1; It is characterized in that the step that described employing HPLC-ELSD method is measured rosamultin and/or multiflorin content comprises the assay of preparation, rosamultin and/or multiflorin of preparation, the need testing solution of chromatographic condition and system suitability test, reference substance solution.
3. method of quality control according to claim 2 is characterized in that, said chromatographic condition and system suitability test are to use octadecylsilane chemically bonded silica to be filler; Volume ratio is that 48: 52 methanol-water is a mobile phase; Detect with evaporative light scattering detector, number of theoretical plate calculates by the rosamultin peak should be not less than 2000.
4. method of quality control according to claim 2; It is characterized in that following method is adopted in the preparation of said reference substance solution: get rosamultin respectively and the multiflorin reference substance is an amount of, accurate claim fixed; Add methanol and process the mixed solution that every 1ml contains 0.5mg, promptly get.
5. method of quality control according to claim 4 is characterized in that, said rosamultin and multiflorin reference substance adopt following method self-control: with the Radix Rosae Laevigatae rhizome; Pulverize, after extracting solvent extraction, the extracting solution concentrating under reduced pressure; Carry out solvent extraction or go up macroporous resin adsorption, and water, 30%, 40～90% ethanol elutions successively, 40～90% ethanol elution things collected; Reclaim solvent; Again with extract or eluate through column chromatography for separation, or with extracting solution be concentrated into do after, directly pass through column chromatography for separation; The fraction that will contain rosamultin and multiflorin separates through silica gel column chromatography once more, collects the fraction that contains rosamultin and multiflorin, must rosamultin and multiflorin coarse-grain, add again recrystallization reagent repeatedly recrystallization promptly get.
6. method of quality control according to claim 2 is characterized in that, following method is adopted in the preparation of said need testing solution: get 30 small pieces of these article or 20 sheets, remove coating, the accurate title, decided porphyrize; Get about 1.5g, the accurate title, decide, and the accurate methanol 50ml that adds claims to decide weight, and ultrasonic place is 45 minutes under power 250W, frequency 40kHz condition; Put coldly, claim again decide weight, supply the weight that subtracts mistake, shake up filtration with methanol; Precision is measured subsequent filtrate 25ml, reclaims solvent to doing, and residue adds water 20ml makes dissolving, extracts 3 times with water saturated n-butyl alcohol jolting, each 15ml; Merge n-butanol extracting liquid, with ammonia solution washing 2 times, each 15ml gets n-butyl alcohol liquid, and decompression and solvent recovery is to doing; Residue is used dissolve with methanol, is transferred in the 5ml measuring bottle, adds methanol to scale, shakes up, and promptly gets.
7. method of quality control according to claim 2; It is characterized in that in the step of the assay of said rosamultin and/or multiflorin, assay adopts following method: accurate respectively reference substance solution 5 μ l, 10 μ l and need testing solution 10～20 μ l of drawing; Inject chromatograph of liquid; Measure, calculate the content of rosamultin, multiflorin respectively, promptly get with external standard two-point method logarithmic equation; Every of these article contain Radix Rosae Laevigatae with rosamultin (C
36H
58O
10) meter, small pieces must not be less than 0.10mg; Sheet must not be less than 0.16mg; Contain Radix Rosae Laevigatae with multiflorin (C
36H
58O
10) meter, small pieces must not be less than 0.10mg; Sheet must not be less than 0.16mg.
8. method of quality control according to claim 1 is characterized in that, said employing HPLC-ELSD method is measured asiaticoside and/or asiaticoside content comprises the steps:
Measure according to HPLC (an appendix VI of Chinese Pharmacopoeia version in 2010 D);
Chromatographic condition and system suitability test: use octadecylsilane chemically bonded silica to be filler; Volume ratio is that 48: 52 methanol-water is a mobile phase; Detect or use the UV-detector detection with evaporative light scattering detector, detection wavelength 210nm, number of theoretical plate calculates by the asiaticoside peak should be not less than 2000;
The preparation of reference substance solution: it is an amount of to get the asiaticoside reference substance respectively, accurate claims surely, adds methanol and processes every 1ml and contain the solution of 0.2mg and the solution that every 1ml contains 0.6mg respectively, promptly gets;
The preparation of need testing solution: get this article 30 small pieces or 20 sheets, remove coating, the accurate title, decide, and porphyrize is got about 1.5g; The accurate title, decide, and the accurate methanol 50ml that adds claims to decide weight, and supersound process is 45 minutes under the condition of power 250W, frequency 40kHz, puts cold; Claim to decide weight again, supply the weight that subtracts mistake with methanol, shake up, filter, precision is measured subsequent filtrate 25ml; Reclaim solvent to doing, residue adds water 20ml makes dissolving, extracts 3 times with water saturated n-butyl alcohol jolting, and each 15ml merges n-butanol extracting liquid; With ammonia solution washing 2 times, each 15ml gets n-butyl alcohol liquid, and decompression and solvent recovery is to doing, and residue is used dissolve with methanol; Be transferred in the 5ml measuring bottle, add methanol, shake up, promptly get to scale;
Algoscopy: accurate respectively reference substance solution each 10 μ l and need testing solution 5～10 μ l that draw above-mentioned two kinds of concentration, inject chromatograph of liquid, measure, calculate with external standard two-point method logarithmic equation, promptly get; Every of these article contain Herba Centellae with asiaticoside (C
48H
78O
20) meter, small pieces must not be less than 0.22mg; Sheet must not be less than 0.35mg.
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| CN109001307A (en) * | 2018-06-08 | 2018-12-14 | 桂林三金药业股份有限公司 | The HPLC characteristic spectrum and its construction method of three gold preparations |
| CN111289680A (en) * | 2018-12-08 | 2020-06-16 | 九芝堂股份有限公司 | Method for identifying cherokee rose fruit in Yilingjing |
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| CN1595147A (en) * | 2004-07-02 | 2005-03-16 | 桂林三金药业股份有限公司 | Quality control method for Sanjin preparation |
| CN101172131A (en) * | 2006-11-01 | 2008-05-07 | 中国医药研究开发中心有限公司 | Method for preparing potentilla plants total-triterpene extract |
| CN101503341A (en) * | 2009-03-10 | 2009-08-12 | 沈阳药科大学 | Novel technique for preparing schizandrol A and schizandrol B |
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| CN1595147A (en) * | 2004-07-02 | 2005-03-16 | 桂林三金药业股份有限公司 | Quality control method for Sanjin preparation |
| CN101172131A (en) * | 2006-11-01 | 2008-05-07 | 中国医药研究开发中心有限公司 | Method for preparing potentilla plants total-triterpene extract |
| CN101503341A (en) * | 2009-03-10 | 2009-08-12 | 沈阳药科大学 | Novel technique for preparing schizandrol A and schizandrol B |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109001307A (en) * | 2018-06-08 | 2018-12-14 | 桂林三金药业股份有限公司 | The HPLC characteristic spectrum and its construction method of three gold preparations |
| CN109001307B (en) * | 2018-06-08 | 2021-05-04 | 桂林三金药业股份有限公司 | HPLC (high Performance liquid chromatography) characteristic spectrum of Sanjin preparation and construction method thereof |
| CN111289680A (en) * | 2018-12-08 | 2020-06-16 | 九芝堂股份有限公司 | Method for identifying cherokee rose fruit in Yilingjing |
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Address after: 541004 No.9, South Renmin Road, Lingui District, Guilin City, Guangxi Zhuang Autonomous Region Patentee after: Sanjin Pharmaceutical Co., Ltd., Guilin Address before: 541004 No. 1 Jinxing Road, Guilin, the Guangxi Zhuang Autonomous Region Patentee before: Sanjin Pharmaceutical Co., Ltd., Guilin |