CN102526561B - Detection method for Sanjin preparation - Google Patents

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

A kind of detection method of three gold medal preparations

Technical field

The present invention relates to a kind of detection method of pharmaceutical preparation, particularly relate to a kind of Chinese medicine preparation, the detection method of SANJIN PIAN.

Background technology

Three gold medal preparations are through extracting the Chinese medicine preparation of being processed into 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 market, its pharmacological action is: antibiotic, antiinflammatory, detumescence, heat clearing away, diuresis, analgesia, antioxidant radical improves immunity of organisms 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 formula of SANJIN PIAN and method of quality control have been listed version Chinese Pharmacopoeia in 2000 in.And increased the assay item, being described below SANJIN PIAN in revised quality standard in revision in 2003:

[prescription] Radix Rosae Laevigatae, Rhizoma Smilacis Chinensis, YANGKAIKOU, Herba lygodii, Herba Centellae

[method for making] above five tastes decoct with water secondary, and 2 hours for the first time, 1 hour for the second time, collecting decoction filtered, and it is 1.15～1.20(50～60 ℃ that filtrate is condensed into relative density) clear paste, drying; Add right amount of auxiliary materials, mixing, granulation is pressed into 1000 (small pieces) or 600 (sheet), coating, and get final product.

[character] this product is coated tablet or Film coated tablets, and is after removing coating, aobvious brown to pitchy; Sour in the mouth, puckery, little hardship.

15 of this product (small pieces) or 10 (sheet) are got in [discriminating] (1), remove coating, porphyrize, add ethanol 15ml, supersound process 20 minutes filters, the filtrate evaporate to dryness, residue adds the sodium hydroxide solution 20ml of 0.01mol/L, and slight fever makes dissolving, extracts with ether 10ml jolting, discard ether extracted liquid, water layer extracts with ethyl acetate 10ml jolting again, and ethyl acetate extraction liquid is concentrated into 1ml, as need testing solution (water layer is standby).Separately get Radix Rosae Laevigatae control medicinal material 2.5g, be made in the same way of control medicinal material solution.Test according to thin layer chromatography (appendix VI B of Chinese Pharmacopoeia version in 2000), draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate take sodium carboxymethyl cellulose as adhesive, take chloroform-methanol (17:3) as developing solvent, launch, take out, dry, spray is heated to the speckle colour developing clear with 10% ethanol solution of sulfuric acid.In the test sample chromatograph, with control medicinal material chromatograph corresponding position on, aobvious speckle identical more than.

(2) get the water layer under the item of [discriminating] (1), extract with water saturated n-butyl alcohol 15ml, divide and get 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 water-bath, and residue adds methanol 1ml makes dissolving, as need testing solution.Separately get the asiaticoside reference substance, add methanol and make the solution that every 1ml contains 1mg, in contrast product solution.Test according to thin layer chromatography (appendix VI B of Chinese Pharmacopoeia version in 2000), draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate take sodium carboxymethyl cellulose as adhesive, take chloroform-methanol-water (7:3:0.5) as developing solvent, launch, take out, dry, spray is heated to the speckle colour developing clear with 10% ethanol solution of sulfuric acid.In the test sample chromatograph, with reference substance chromatograph corresponding position on, the speckle of aobvious same color.

[inspection] should meet every regulation relevant under the tablet item (appendix I D of Chinese Pharmacopoeia version in 2000)

[assay] gets 50 of this product (small pieces) or 30 (sheet), remove coating, precision takes, porphyrize, get approximately 2.5g, accurately weighed, put in tool plug conical flask, precision adds methanol 100ml, close plug, accurately weighed, supersound process (power 160W, frequency 50kHz) 45 minutes, let cool, accurately weighed, add methanol and supply the weight of less loss, shake up, filter, discard just filtrate, the accurate subsequent filtrate 50ml that draws, evaporate to dryness, residue adds the sodium hydroxide solution 30ml of 0.05mol/L, make dissolving, with water saturated n-butanol extraction 3 times, each 20ml, discard water layer, merge n-butanol layer, with twice of the saturated water washing of n-butyl alcohol, each 10ml, discard water layer, n-butyl alcohol liquid reduction vaporization is to doing, residue adds appropriate dissolve with methanol, be transferred in the 5ml measuring bottle, add methanol to scale, as need testing solution.Separately get the asiaticoside reference substance appropriate, add methanol and make the solution that every 1ml contains 1mg, in contrast product solution.(appendix VI B of Chinese Pharmacopoeia version in 2000) scans wavelength according to thin layer chromatography: λ S=550nm, measure test sample trap integrated value and reference substance trap integrated value, and calculate, and get final product.

Every of this product contains 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 is equivalent to crude drug 3.5g

[storage] sealing.

The quality control content assaying method that adopts due to above-mentioned standard is traditional thin layer chromatography (TLCS method), has precision, repeatability, less stable, and the deficiency unstable, that measurement deviation is large develops the color.

application number is that 200510200471.8 Chinese patent application relates to a kind of three gold medal pharmaceutical preparatioies and preparation method and method of quality control for the treatment of the diseases such as urinary system, it is with Radix Rosae Laevigatae, Rhizoma Smilacis Chinensis, YANGKAIKOU, Herba lygodii, Herba Centellae is made acceptable dosage form on pharmaceutics, the preparation disintegrative that obtains is good, bioavailability is high, particularly their supplementary product consumption is all fewer, be 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 facilitate the requirement of suitability for industrialized production, compared with prior art, the pharmaceutical preparation that provides is used for Acute and chronic pyelonephritis, and the treatment of the diseases such as Chronic Urinary Tract Infection has reasonable effect, preparation technology provided by the invention is advanced, and is simple, and these preparation untoward reaction provided by the invention are little, can supply patient's life-time service.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) three gold medal preparations.The method has still adopted thin layer chromatography (TLCS method), still has precision, repeatability, less stable, and the deficiency unstable, that measurement deviation is large develops the color.

Due to defects, it is 200410050066.8 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 the method comprises the assay item, and what assay adopted is the HPLC-ELSD method.

The inventor has carried out again a large amount of research on this basis, and find through the Separation Research 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, therefore 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 the mensuration of rosamultin and/or multiflorin content, as seen, existing quality standard and method of quality control are comprehensive not 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 detection method.

Summary of the invention

The object of the present invention is to provide a kind of detection method of three gold medal preparations, improved content assaying method on the basis of ZL200410050066.8, HPLC-ELSD method assay by effective ingredient rosamultin and/or multiflorin, hold more accurately the quality of relevant medicine, reduce drug risk, improve the quality of products.

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

A kind of detection method of three gold medal preparations comprises the step that adopts the HPLC-ELSD method to measure asiaticoside and/or asiaticoside content, and wherein, the method also comprises the step that adopts the HPLC-ELSD method to measure rosamultin and/or multiflorin content.

The inventor finds through the Separation Research 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, therefore 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 detection method, 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 detection method, wherein, described chromatographic condition and system suitability test are to be filler with octadecylsilane chemically bonded silica; Volume ratio is that the methanol-water of 48:52 is mobile phase; Detect with evaporative light scattering detector, number of theoretical plate should be not less than 2000 by rosamultin peak calculating.

According to aforesaid detection method, wherein, following methods is adopted in the preparation of described reference substance solution: get respectively rosamultin and the multiflorin reference substance is appropriate, and accurately weighed, add methanol and make the mixed solution that every 1ml contains 0.5mg, and get final product.

Rosamultin of the present invention and multiflorin reference substance adopt following method self-control: with the rhizome of Radix Rosae Laevigatae, pulverize, after extracting solvent extraction, with the extracting solution concentrating under reduced pressure, carry out solvent extraction or upper macroporous resin adsorption, and water, 30%, 40～90% ethanol elutions successively, collect 40～90% ethanol elution things, reclaim solvent, then 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 again, and collection contains the fraction of rosamultin and multiflorin, gets rosamultin and multiflorin coarse-grain, then adds recrystallization reagent recrystallization and get final product repeatedly.

according to aforesaid detection method, wherein, following methods is adopted in the preparation of described need testing solution: get 30 small pieces of SANJIN PIAN or 20 sheets, remove coating, accurately weighed, porphyrize, get approximately 1.5g, accurately weighed, precision adds methanol 50ml, weighed weight, at power 250W, under frequency 40kHz condition, supersound process is 45 minutes, let cool, weighed weight again, supply the weight of less loss with methanol, shake up, filter, precision measures subsequent filtrate 25ml, reclaim solvent to doing, residue adds water 20ml makes dissolving, extract 3 times with water saturated n-butyl alcohol jolting, each 15ml, merge n-butanol extracting liquid, with ammonia solution washing 2 times, each 15ml, get n-butyl alcohol liquid, decompression and solvent recovery is to doing, the residue dissolve with methanol, be transferred in the 5ml measuring bottle, add methanol to scale, shake up, and get final product.

According to aforesaid detection method, wherein, in the step of the assay of described rosamultin and/or multiflorin, assay adopts following methods, precision is drawn reference substance solution 5 μ l, 10 μ l and need testing solution 10～20 μ l respectively, and the injection liquid chromatography is measured, calculate respectively the content of rosamultin, multiflorin with external standard two-point method logarithmic equation, and get final product; Every of this product contains 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 detection method, wherein said employing HPLC-ELSD method is measured asiaticoside and/or asiaticoside content comprises the steps:

Measure according to high performance liquid chromatography (appendix VI D of Chinese Pharmacopoeia version in 2010);

Chromatographic condition and system suitability: be filler with octadecylsilane chemically bonded silica; Volume ratio is that the methanol-water of 48:52 is mobile phase; Detect or use UV-detector to detect with evaporative light scattering detector, detecting wavelength 210nm, number of theoretical plate should be not less than 2000 by asiaticoside peak calculating;

The preparation of reference substance solution: get respectively the asiaticoside reference substance appropriate, accurately weighed, add methanol and make solution and the every 1ml that every 1ml contains respectively 0.2mg and contain the solution of 0.6mg, and get final product;

the preparation of need testing solution: get this product 30 small pieces or 20 sheets, remove coating, accurately weighed, porphyrize, get approximately 1.5g, accurately weighed, precision adds methanol 50ml, weighed weight, at power 250W, under the condition of frequency 40kHz, supersound process is 45 minutes, let cool, weighed weight again, supply the weight of less loss with methanol, shake up, filter, precision measures subsequent filtrate 25ml, reclaim solvent to doing, residue adds water 20ml makes dissolving, extract 3 times with water saturated n-butyl alcohol jolting, each 15ml, merge n-butanol extracting liquid, with ammonia solution washing 2 times, each 15ml, get n-butyl alcohol liquid, decompression and solvent recovery is to doing, the residue dissolve with methanol, be transferred in the 5ml measuring bottle, add methanol to scale, shake up, and get final product,

Algoscopy: precision is drawn reference substance solution each 10 μ l and need testing solution 5～10 μ l of above-mentioned two kinds of concentration respectively, and the injection liquid chromatography is measured, and with the calculating of external standard two-point method logarithmic equation, and get final product; Every of this product contains 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, when the preparation of its need testing solution of process of the content of employing HPLC-ELSD method mensuration rosamultin and/or multiflorin is measured asiaticoside and/or asiaticoside content with employing HPLC-ELSD method, the preparation method of its need testing solution is just the same, do not need to prepare in addition need testing solution on the basis of adopting HPLC-ELSD method mensuration asiaticoside and/or asiaticoside content, thereby simplified quality control process, saved cost.

Detection method of the present invention also further comprises the step that three gold medal preparations are differentiated.

Thin layer chromatography is adopted in described discriminating.

Described thin layer chromatography comprises the steps:

(1) get SANJIN PIAN 15 small pieces or 10 sheets, remove coating, porphyrize, add ethanol 15ml, supersound process 20 minutes filters the filtrate evaporate to dryness, residue adds 0.01mol/L sodium hydroxide solution 20ml, slight fever makes dissolving, extracts with ether 10ml jolting, discards ether solution, water liquid extracts with ethyl acetate 1Oml jolting again, and aqueous solution is standby; Acetic acid ethyl fluid is concentrated into approximately 1ml, as need testing solution; Separately get Radix Rosae Laevigatae control medicinal material 2.5g, be made in the same way of control medicinal material solution; Test according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2010), draw each 1O μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take San Lv Jia Wan – methanol (17:3) as developing solvent, launch, take out, dry, spray is heated to the speckle colour developing clear with 10% ethanol solution of sulfuric acid; In the test sample chromatograph, with control medicinal material chromatograph corresponding position on, the principal spot of aobvious two or more same colors;

(2) get the aqueous solution under (1) item, extract with water saturated n-butyl alcohol 15ml, divide and get 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; Separately get the asiaticoside reference substance, add methanol and make the solution that every 1ml contains 1mg, in contrast product solution; Test according to thin layer chromatography (appendix VI B of Chinese Pharmacopoeia version in 2010), draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take chloroform-methanol-water (7:3:0.5) as developing solvent, launch, take out, dry, spray is heated to the speckle colour developing clear with 10% ethanol solution of sulfuric acid; In the test sample chromatograph, with reference substance chromatograph corresponding position on, the speckle of aobvious same color;

(3) get SANJIN PIAN 10 small pieces or 6 sheets, remove coating, porphyrize, add ethanol 50ml, supersound process 30 minutes filters, filtrate adds hydrochloric acid 5ml, and reflux 2 hours lets cool, transfer to neutrality with 40% sodium hydroxide solution, steam to distinguishing the flavor of without alcohol, residue heating water 40ml makes dissolving, extract 2 times (40ml, 30ml), combined dichloromethane extracting solution with the dichloromethane jolting, evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution; Separately get Rhizoma Smilacis Chinensis control medicinal material 5g, be made in the same way of control medicinal material solution; Get again the diosgenin reference substance, add methanol and make the solution that every 1ml contains 0.5mg, in contrast product solution; Test according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2010), draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take Huan Ji Wan – ethyl acetate (4:1) as developing solvent, launch, take out, dry, spray is heated to the speckle colour developing at 105 ℃ clear with 10% ethanol solution of sulfuric acid, puts under ultra-violet lamp 365nm and inspects; In the test sample chromatograph, with control medicinal material chromatograph relevant position on, the fluorescence speckle of aobvious two or more same colors; With reference substance chromatograph relevant position on, the fluorescence speckle of aobvious same color;

(4) get SANJIN PIAN 15 small pieces or 10 sheets, porphyrize adds methanol 100ml, supersound process 20 minutes filters, and filtrate is concentrated into approximately 10ml, be added on the neutral alumina column of 100～200 orders, 5g, internal diameter 1cm, with methanol 50ml eluting, collect 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 adds methanol 1ml makes dissolving, as need testing solution; Separately get YANGKAIKOU control medicinal material 5g, add water 100ml, reflux 1 hour filters, and filtrate evaporate to dryness, residue add methanol 20ml, are made in the same way of control medicinal material solution; Test according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2010), draw each 5～10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take San Lv Jia Wan – Bing Tong – water (6:14:1) as developing solvent, launch, take out, dry, put under ultra-violet lamp 365nm and inspect; In the test sample chromatograph, with control medicinal material chromatograph corresponding position on, the fluorescence speckle of aobvious 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 preparation, Herba Centellae medical material and extract thereof, multiflorin medical material and extract, rosamultin and extract thereof take Herba Centellae, multiflorin, rosamultin as 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 rosamultin reference substance equation of linear regression, and its regression equation is: Y=0.6482X-7.3328, r=0.9992;

Fig. 2 is 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 SANJIN PIAN (ELSD);

Fig. 6 is 0910144 batch of SANJIN PIAN (ELSD);

Fig. 7 is 0910160 batch of SANJIN PIAN (ELSD);

Fig. 8 is 0910167 batch of SANJIN PIAN (ELSD);

Fig. 9 is 0910175 batch of SANJIN PIAN (ELSD);

Figure 10 is 0910181 batch of SANJIN PIAN (ELSD);

Figure 11 is 0910183 batch of SANJIN PIAN (ELSD);

Figure 12 is 0910191 batch of SANJIN PIAN (ELSD);

Figure 13 is 0910192 batch of SANJIN PIAN (ELSD);

Figure 14 is 0911005 batch of SANJIN PIAN (ELSD);

Figure 15 is 0911006 batch of SANJIN PIAN (ELSD);

Figure 16 is 0911021 batch of 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 SANJIN PIAN (UV);

Figure 19 is 0910144 batch of SANJIN PIAN (UV);

Figure 20 is 0910160 batch of SANJIN PIAN (UV);

Figure 21 is 0910167 batch of SANJIN PIAN (UV);

Figure 22 is 0910175 batch of SANJIN PIAN (UV);

Figure 23 is 0910181 batch of SANJIN PIAN (UV);

Figure 24 is 0910183 batch of SANJIN PIAN (UV);

Figure 25 is 0910191 batch of SANJIN PIAN (UV);

Figure 26 is 0910192 batch of SANJIN PIAN (UV);

Figure 27 is 0911005 batch of SANJIN PIAN (UV);

Figure 28 is 0911006 batch of SANJIN PIAN (UV);

Figure 29 is 0911021 batch of SANJIN PIAN (UV).

The specific embodiment

Be below 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 SANJIN PIAN side, and the inventor is through the Separation Research discovery of system, and rosamultin and multiflorin are the characteristic active component of Radix Rosae Laevigatae, therefore increase rosamultin and multiflorin are very necessary for the index components of controlling this product 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: Waters2695 high performance liquid chromatograph, evaporative light scattering detector (Alltech-ELSD2000), (UV-detector is Waters996), 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 " Chinese pharmacopoeia is not recorded rosamultin and multiflorin chemical reference substance, therefore the inventor has carried out separating preparation to rosamultin with multiflorin, and structural identification and purity test have been carried out by " the research of the chemical standard product specification requirement of new Chinese medicine quality standard research ", measure under selected chromatographic condition, calculating content by normalization method is 98.5%, and result shows that prepared rosamultin and multiflorin chemical reference substance meet assay reference substance requirement.

Rosamultin and multiflorin reference substance adopt following method self-control: with the rhizome of Radix Rosae Laevigatae, pulverize, after extracting solvent extraction, with the extracting solution concentrating under reduced pressure, carry out solvent extraction or upper macroporous resin adsorption, and water, 30%, 40～90% ethanol elutions successively, collect 40～90% ethanol elution things, reclaim solvent, then 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 again, and collection contains the fraction of rosamultin and multiflorin, gets rosamultin and multiflorin coarse-grain, then adds recrystallization reagent recrystallization and get final product repeatedly.

Specifically adopt the self-control of following method: get fresh Radix Rosae Laevigatae rhizome 10Kg, pulverize, with 60% alcohol reflux 2 times, each 2 hours, filter, concentrate and to get extractum.Extractum adding distil water 1000mL makes Uniform Dispersion, adds same volumes of acetic acid ethyl ester extraction three times, combined ethyl acetate, and the concentration and recovery solvent gets ethyl acetate extract.Ethyl acetate extract silica gel column chromatography crude separation, (TLC combining data detection same section obtains 9 major parts for the gradient elution of 10:0～0:10), each gradient elution 3000mL with chloroform/methanol.Wherein be rich in the fraction of rosamultin and multiflorin, carrying out silica gel column chromatography take chloroform/methanol (10:1) as eluant, collect the fraction be rich in rosamultin, is recrystallization in the chloroform/methanol mixed solution of 10:1 in volume ratio, get white indefinite form solid, i.e. rosamultin; The fraction of multiflorin is rich in collection, repeatedly carries out middle pressure reversed phase column chromatography take 55% methanol as eluant, is recrystallization in the chloroform/methanol mixed solution of 10:1 in volume ratio, gets 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, for the best, retention time is suitable take methanol-water (48:52) for result, 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, number of theoretical plate the results are shown in 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 a rear peak 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 impact of the system factors such as the preparation of instrument, chromatographic column, mobile phase and temperature, the regulation number of theoretical plate press rosamultin peak calculating, 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 appropriate, accurately weighed, add methanol and make mixed solution (the real 54.25mg that gets of rosamultin that every 1ml contains 1mg, the real 51.20mg that gets of multiflorin, be settled to 50ml, namely be respectively 1.085mg/ml, 1.024mg/ml), and get final product.

Measure: the accurate absorption mixed reference substance solution solution 1 μ l, 2 μ l, 4 μ l, 6 μ l, 8 μ l, 10 μ l injection liquid chromatographies respectively, measure peak area, take the natural logrithm of peak area as abscissa, take the natural logrithm of sample size as vertical coordinate, regression Calculation, get linear equation, measurement result sees Table 2, table 3.

Table 2, the linear result of investigating of rosamultin reference substance

Above result shows, 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 of investigating of multiflorin reference substance

Above result shows, 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 preparation method

(1) extract the selection of solvent

Investigate respectively 50% methanol, 80% methanol, methanol, ethanol is as extracting reagent, the impact of contrast different solvents on extraction effect.Concrete grammar is:

get 60 of this product (lot number 0910021), remove coating, accurately weighed, porphyrize, get respectively approximately 1.5g, accurately weighed, precision adds different solvents 50ml, weighed weight, supersound process (power 250W, frequency 40kHz) 45 minutes, let cool, weighed weight again, supply the weight of less loss with corresponding extraction solvent, shake up, filter, precision measures subsequent filtrate 25ml, reclaim solvent to doing, residue adds water 20ml makes dissolving, extract 3 times with water saturated n-butyl alcohol jolting, each 15ml, merge n-butanol extracting liquid, with ammonia solution washing 2 times, each 15ml, get n-butyl alcohol liquid, decompression and solvent recovery is to doing, the residue dissolve with methanol, be transferred in the 5ml measuring bottle, add methanol to scale, shake up, sample introduction 10 μ l, the results are shown in Table 4.

Table 4, the selection of extracting solvent

Experimental result shows that 80% methanol is substantially suitable with the methanol extraction effect, but during 80% methanol extraction, impurity is more, the easy emulsifying of post-processed.Therefore select methanol as extracting solvent.

(2) investigation of extracting method and extraction time.

Supersound extraction is investigated in test, and Soxhlet is extracted, three kinds of extracting method of heating and refluxing extraction, and different extraction times reach the impact of balance on extraction.

supersound extraction is specially: get 60 of this product (lot number 0910021), remove coating, accurately weighed, porphyrize, get respectively approximately 1.5g, accurately weighed, precision adds methanol solvate 50ml, weighed weight, supersound process (power 250W, frequency 40kHz) different time, let cool, weighed weight again, supply the weight of less loss with methanol, shake up, filter, precision measures subsequent filtrate 25ml, reclaim solvent to doing, residue adds water 20ml makes dissolving, extract 3 times with water saturated n-butyl alcohol jolting, each 15ml, merge n-butanol extracting liquid, with ammonia solution washing 2 times, each 15ml, get n-butyl alcohol liquid, decompression and solvent recovery is to doing, the residue dissolve with methanol, be transferred in the 5ml measuring bottle, add methanol to scale, shake up.

Soxhlet is extracted and is specially: get 60 of this product (lot number 0910021), remove coating, accurately weighed, porphyrize is got respectively approximately 1.5g, and is accurately weighed, puts in apparatus,Soxhlet's, and precision adds methanol 50ml, extracted respectively 4 hours, and 8 hours, 12 hours.Extracting solution reclaims solvent to doing, and operates with method with supersound process from " residue adds water 20ml ", makes need testing solution.

Heating and refluxing extraction is specially: gets 60 of this product (lot number 0910021), removes coating, and accurately weighed, porphyrize, get respectively approximately 1.5g, accurately weighed, at the bottom of horizontalization, in flask, precision adds methanol 50ml, weighed weight, reflux is 1 hour respectively, and 2 hours, 4 hours.Play with supersound process operating with method from " let cool, more weighed weight ", make need testing solution.

Above-mentioned need testing solution sample introduction 10 μ l, measurement result sees 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
Soxhlet was extracted 4 hours	292.5	313.6
			Soxhlet was extracted 8 hours	326.9	355.8
Soxhlet was extracted 12 hours	345.4	374.2
			Reflux 1 hour	316.8	339.5

Reflux 2 hours	344.2	376.1
			Reflux 4 hours	346.3	377.8

Experimental result shows, supersound process 45min has reached poised state, and extracts 12 hours with Soxhlet, and 2 hours effects of heating and refluxing extraction are substantially suitable.Therefore extracting method 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 after 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.

The test alternative uses ethyl acetate as extracting solution, as a comparison.The results are shown in 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

Result shows, n-butyl alcohol or ethyl acetate extraction effect are basically identical, but finds in the test operation process, during ethyl acetate extraction, the easier emulsifying of solution, the processing time is longer, and from extracting solution colour and test sample chromatogram, the impurity of ethyl acetate extraction is more.Therefore, select n-butyl alcohol as the extraction solvent of 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 result shows in the 4th water-saturated n-butanol jolting extracting solution without rosamultin and multiflorin, therefore jolting is extracted 3 times, rosamultin and multiflorin can extract fully.Therefore purification process adopts water-saturated n-butanol to extract 3 times.

5, precision test

Get 20 of this product (lot number 0910021), press text [assay] below legal system available test sample solution, sample introduction 20 μ l repeat sample introduction 6 times, measure rosamultin and multiflorin area value, and calculate respectively content, the results are shown in Table 7.

Table 7, Precision test result

Above result shows, the precision of chromatograph of liquid used is good.

6, stability test

Get need testing solution (lot number 0910021), at room temperature preserve, respectively at the interval of 0,1,2,4,6,8,24 hour, the accurate need testing solution 20 μ l that draw, the injection liquid chromatography is measured rosamultin and multiflorin peak area value, and calculate respectively content, the results are shown in Table 8.

Table 8, stability test result

Result of the test shows, need testing solution was measured in 24 hours, and its relative deviation is respectively 2.78%, 2.83%, illustrates that need testing solution measured in 24 hours, and it is stable that result keeps.

7, repeatability test

Get 6 parts of the SANJIN PIAN of same lot number (0910021 batch), prepare respectively need testing solution in accordance with the law, record respectively peak area value, and calculate respectively rosamultin and multiflorin content, RSD is respectively: 3.38%, 3.73%, the results are shown in Table 9.Result of the test shows, the method repeatability is good.

Table 9, reproducible test results

8, application of sample recovery test

(the assay result is: rosamultin 335.3 μ g/ sheets to get SANJIN PIAN with the same lot number of repeatability test, multiflorin 379.6 μ g/ sheets, average sheet is heavy: 0.2790g) about 0.75g, and parallel 6 parts, accurately weighed, precision adds rosamultin to mix 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, measurement result is with following formula calculate recovery rate, the results are shown in 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 the middle rosamultin of 12 batches of SANJIN PIAN (film-coat is large stretch of) and multiflorin according to rosamultin and multiflorin [assay] method, measurement result sees Table 12.

Table 12,12 batches of assay results of SANJIN PIAN (sheet)

According to above measurement result, in totally 12 batches of SANJIN PIAN (sheet) of surveying, the content fluctuation range of rosamultin is 89.3～354.0 μ g/ sheets, and the content fluctuation range of multiflorin is 85.4～376.3 μ g/ sheets.As seen the content fluctuation is larger, and average content is respectively 204.7 μ g/ sheets, 202.6 μ g/ sheets; For the content fluctuation of avoiding causing due to factors such as medical material content difference, operations, formulate limit with 80% of average content, therefore content limit is tentative be: every of this product contains 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.

Cause does not have sample cuttings temporarily for measuring, and converts therefore press recipe quantity, small pieces content is fixed tentatively be: every of this product contains 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.

Although the composition of surveying without obvious uv absorption, under the separation condition of this experiment, adopts UV-detector, detects under wavelength at 210nm, also can realize effective mensuration, adopts simultaneously UV-detector to detect and calculate 12 batch sample content results to see Table 13.

Table 13,12 batches of assay results of SANJIN PIAN (sheet) (ultraviolet detection method)

According to above measurement result as seen, ultraviolet detection method result and evaporative light scattering detector detection method result are basically identical, 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)

Embodiment 2

The detection method of the SANJIN PIAN that this embodiment provides comprises the content of the effective ingredient asiaticoside that adopts in the high effective liquid chromatography for measuring SANJIN PIAN and asiaticoside and adopts effective ingredient rosamultin in the high effective liquid chromatography for measuring SANJIN PIAN and the content of multiflorin.Concrete grammar is as follows:

SANJIN PIAN, the method that provides according to pharmacopeia preparation, or buy in Guangxi or any pharmacy of China, three gold medal pharmaceutical Co. Ltds produce and provide by Guilin.

Assay:

(1) effective ingredient asiaticoside and the asiaticoside in employing high effective liquid chromatography for measuring SANJIN PIAN, concrete operation method is seen appendix VI D of Chinese Pharmacopoeia version in 2010.

Chromatographic condition and system suitability: be filler with octadecylsilane chemically bonded silica; Volume ratio is that the methanol-water of 48:52 is mobile phase; Detect or use UV-detector to detect with evaporative light scattering detector, detecting wavelength 210nm, number of theoretical plate should be not less than 2000 by asiaticoside peak calculating;

The preparation of reference substance solution: get respectively the asiaticoside reference substance appropriate, accurately weighed, add methanol and make solution and the every 1ml that every 1ml contains respectively 0.2mg and contain the solution of 0.6mg, and get final product;

the preparation of need testing solution: get this product 20 sheets, remove coating, accurately weighed, porphyrize, get approximately 1.5g, accurately weighed, precision adds methanol 50ml, weighed weight, at power 250W, under the condition of frequency 40kHz, supersound process is 45 minutes, let cool, weighed weight again, supply the weight of less loss with methanol, shake up, filter, precision measures subsequent filtrate 25ml, reclaim solvent to doing, residue adds water 20ml makes dissolving, extract 3 times with water saturated n-butyl alcohol jolting, each 15ml, merge n-butanol extracting liquid, with ammonia solution washing 2 times, each 15ml, get n-butyl alcohol liquid, decompression and solvent recovery is to doing, the residue dissolve with methanol, be transferred in the 5ml measuring bottle, add methanol to scale, shake up, and get final product,

Algoscopy: precision is drawn reference substance solution each 10 μ l and need testing solution 5～10 μ l of above-mentioned two kinds of concentration respectively, and the injection liquid chromatography is measured, and with the calculating of external standard two-point method logarithmic equation, and get final product; Every of this product contains 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 employing high effective liquid chromatography for measuring SANJIN PIAN, concrete operation method is seen appendix VI D of Chinese Pharmacopoeia version in 2010.

Chromatographic condition and system suitability: be filler with octadecylsilane chemically bonded silica; Methanol-water (48:52) is mobile phase; Detect with evaporative light scattering detector.Number of theoretical plate should be not less than 2000 by rosamultin peak calculating.

The preparation of reference substance solution: get respectively rosamultin and multiflorin reference substance (adopting the method self-control described in embodiment 1) appropriate, accurately weighed, add methanol and make the mixed solution that every 1ml contains 0.5mg, and get final product.

the preparation of need testing solution: get SANJIN PIAN 20 sheets, remove coating, accurately weighed, porphyrize, get approximately 1.5g, accurately weighed, precision adds methanol 50ml, weighed weight, supersound process (power 250W, frequency 40kHz) 45 minutes, let cool, weighed weight again, supply the weight of less loss with methanol, shake up, filter, precision measures subsequent filtrate 25ml, reclaim solvent to doing, residue adds water 20ml makes dissolving, extract 3 times with water saturated n-butyl alcohol jolting, each 15ml, merge n-butanol extracting liquid, with ammonia solution washing 2 times, each 15ml, get n-butyl alcohol liquid, decompression and solvent recovery is to doing, the residue dissolve with methanol, be transferred in the 5ml measuring bottle, add methanol to scale, shake up, and get final product.

Algoscopy: precision is drawn reference substance solution 5 μ l, 10 μ l and need testing solution 10～20 μ l respectively, and the injection liquid chromatography is measured, and calculates respectively the content of rosamultin, multiflorin with external standard two-point method logarithmic equation, and get final product.

Every of this product contains 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.

Embodiment 3

The detection method of the SANJIN PIAN that this embodiment provides comprises the content of the effective ingredient asiaticoside that adopts in the high effective liquid chromatography for measuring SANJIN PIAN and asiaticoside and adopts effective ingredient rosamultin in the high effective liquid chromatography for measuring SANJIN PIAN and the content of multiflorin.Concrete grammar is as follows:

SANJIN PIAN, the method that provides according to pharmacopeia preparation, or buy in Guangxi or any pharmacy of China, three gold medal pharmaceutical Co. Ltds produce and provide by Guilin.

Assay:

(1) effective ingredient asiaticoside and the asiaticoside in employing high effective liquid chromatography for measuring SANJIN PIAN, concrete operation method is seen appendix VI D of Chinese Pharmacopoeia version in 2010.

Chromatographic condition and system suitability: be filler with octadecylsilane chemically bonded silica; Volume ratio is that the methanol-water of 48:52 is mobile phase; Detect or use UV-detector to detect with evaporative light scattering detector, detecting wavelength 210nm, number of theoretical plate should be not less than 2000 by asiaticoside peak calculating;

The preparation of reference substance solution: get respectively the asiaticoside reference substance appropriate, accurately weighed, add methanol and make solution and the every 1ml that every 1ml contains respectively 0.2mg and contain the solution of 0.6mg, and get final product;

the preparation of need testing solution: get SANJIN PIAN 30 small pieces, remove coating, accurately weighed, porphyrize, get approximately 1.5g, accurately weighed, precision adds methanol 50ml, weighed weight, at power 250W, under the condition of frequency 40kHz, supersound process is 45 minutes, let cool, weighed weight again, supply the weight of less loss with methanol, shake up, filter, precision measures subsequent filtrate 25ml, reclaim solvent to doing, residue adds water 20ml makes dissolving, extract 3 times with water saturated n-butyl alcohol jolting, each 15ml, merge n-butanol extracting liquid, with ammonia solution washing 2 times, each 15ml, get n-butyl alcohol liquid, decompression and solvent recovery is to doing, the residue dissolve with methanol, be transferred in the 5ml measuring bottle, add methanol to scale, shake up, and get final product,

Algoscopy: precision is drawn reference substance solution each 10 μ l and need testing solution 5～10 μ l of above-mentioned two kinds of concentration respectively, and the injection liquid chromatography is measured, and with the calculating of external standard two-point method logarithmic equation, and get final product; Every of this product contains 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 employing high effective liquid chromatography for measuring SANJIN PIAN, concrete operation method is seen appendix VI D of Chinese Pharmacopoeia version in 2010.

Chromatographic condition and system suitability: be filler with octadecylsilane chemically bonded silica; Methanol-water (48:52) is mobile phase; Detect with evaporative light scattering detector.Number of theoretical plate should be not less than 2000 by rosamultin peak calculating.

The preparation of reference substance solution: get respectively rosamultin and multiflorin reference substance (adopting the method self-control described in embodiment 1) appropriate, accurately weighed, add methanol and make the mixed solution that every 1ml contains 0.5mg, and get final product.

the preparation of need testing solution: get SANJIN PIAN 30 small pieces, remove coating, accurately weighed, porphyrize, get approximately 1.5g, accurately weighed, precision adds methanol 50ml, weighed weight, supersound process (power 250W, frequency 40kHz) 45 minutes, let cool, weighed weight again, supply the weight of less loss with methanol, shake up, filter, precision measures subsequent filtrate 25ml, reclaim solvent to doing, residue adds water 20ml makes dissolving, extract 3 times with water saturated n-butyl alcohol jolting, each 15ml, merge n-butanol extracting liquid, with ammonia solution washing 2 times, each 15ml, get n-butyl alcohol liquid, decompression and solvent recovery is to doing, the residue dissolve with methanol, be transferred in the 5ml measuring bottle, add methanol to scale, shake up, and get final product.

Algoscopy: precision is drawn reference substance solution 5 μ l, 10 μ l and need testing solution 10～20 μ l respectively, and the injection liquid chromatography is measured, and calculates respectively the content of rosamultin, multiflorin with external standard two-point method logarithmic equation, and get final product.

Every of this product contains 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.

Embodiment 4

The detection method of the SANJIN PIAN that this embodiment provides comprise the effective ingredient asiaticoside that adopts in the high effective liquid chromatography for measuring SANJIN PIAN and asiaticoside content, adopt effective ingredient rosamultin and the content of multiflorin and the step that SANJIN PIAN is differentiated in the 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, concrete steps are as follows:

(1) get SANJIN PIAN 10 sheets, remove coating, porphyrize, add ethanol 15ml, supersound process 20 minutes filters the filtrate evaporate to dryness, residue adds 0.01mol/L sodium hydroxide solution 20ml, slight fever makes dissolving, extracts with ether 10ml jolting, discards ether solution, water liquid extracts with ethyl acetate 1Oml jolting again, and aqueous solution is standby; Acetic acid ethyl fluid is concentrated into approximately 1ml, as need testing solution; Separately get Radix Rosae Laevigatae control medicinal material 2.5g, be made in the same way of control medicinal material solution; Test according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2010), draw each 1O μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take San Lv Jia Wan – methanol (17:3) as developing solvent, launch, take out, dry, spray is heated to the speckle colour developing clear with 10% ethanol solution of sulfuric acid; In the test sample chromatograph, with control medicinal material chromatograph corresponding position on, the principal spot of aobvious two or more same colors;

(2) get the aqueous solution under (1) item, extract with water saturated n-butyl alcohol 15ml, divide and get 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; Separately get the asiaticoside reference substance, add methanol and make the solution that every 1ml contains 1mg, in contrast product solution; Test according to thin layer chromatography (appendix VI B of Chinese Pharmacopoeia version in 2010), draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take chloroform-methanol-water (7:3:0.5) as developing solvent, launch, take out, dry, spray is heated to the speckle colour developing clear with 10% ethanol solution of sulfuric acid; In the test sample chromatograph, with reference substance chromatograph corresponding position on, the speckle of aobvious same color;

(3) get SANJIN PIAN 6 sheets, remove coating, porphyrize, add ethanol 50ml, supersound process 30 minutes filters, filtrate adds hydrochloric acid 5ml, and reflux 2 hours lets cool, transfer to neutrality with 40% sodium hydroxide solution, steam to distinguishing the flavor of without alcohol, residue heating water 40ml makes dissolving, extract 2 times (40ml, 30ml), combined dichloromethane extracting solution with the dichloromethane jolting, evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution; Separately get Rhizoma Smilacis Chinensis control medicinal material 5g, be made in the same way of control medicinal material solution; Get again the diosgenin reference substance, add methanol and make the solution that every 1ml contains 0.5mg, in contrast product solution; Test according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2010), draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take Huan Ji Wan – ethyl acetate (4:1) as developing solvent, launch, take out, dry, spray is heated to the speckle colour developing at 105 ℃ clear with 10% ethanol solution of sulfuric acid, puts under ultra-violet lamp 365nm and inspects; In the test sample chromatograph, with control medicinal material chromatograph relevant position on, the fluorescence speckle of aobvious two or more same colors; With reference substance chromatograph relevant position on, the fluorescence speckle of aobvious same color;

(4) get SANJIN PIAN 10 sheets, porphyrize adds methanol 100ml, supersound process 20 minutes filters, and filtrate is concentrated into approximately 10ml, be added on the neutral alumina column of 100～200 orders, 5g, internal diameter 1cm, with methanol 50ml eluting, collect 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 adds methanol 1ml makes dissolving, as need testing solution; Separately get YANGKAIKOU control medicinal material 5g, add water 100ml, reflux 1 hour filters, and filtrate evaporate to dryness, residue add methanol 20ml, are made in the same way of control medicinal material solution; Test according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2010), draw each 5～10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take San Lv Jia Wan – Bing Tong – water (6:14:1) as developing solvent, launch, take out, dry, put under ultra-violet lamp 365nm and inspect; In the test sample chromatograph, with control medicinal material chromatograph corresponding position on, the fluorescence speckle of aobvious same color.

Embodiment 5

The detection method of the SANJIN PIAN that this embodiment provides comprise the effective ingredient asiaticoside that adopts in the high effective liquid chromatography for measuring SANJIN PIAN and asiaticoside content, adopt effective ingredient rosamultin and the content of multiflorin and the step that SANJIN PIAN is differentiated in the 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, concrete steps are as follows:

(1) get SANJIN PIAN 15 small pieces, remove coating, porphyrize, add ethanol 15ml, supersound process 20 minutes filters the filtrate evaporate to dryness, residue adds 0.01mol/L sodium hydroxide solution 20ml, slight fever makes dissolving, extracts with ether 10ml jolting, discards ether solution, water liquid extracts with ethyl acetate 1Oml jolting again, and aqueous solution is standby; Acetic acid ethyl fluid is concentrated into approximately 1ml, as need testing solution; Separately get Radix Rosae Laevigatae control medicinal material 2.5g, be made in the same way of control medicinal material solution; Test according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2010), draw each 1O μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take San Lv Jia Wan – methanol (17:3) as developing solvent, launch, take out, dry, spray is heated to the speckle colour developing clear with 10% ethanol solution of sulfuric acid; In the test sample chromatograph, with control medicinal material chromatograph corresponding position on, the principal spot of aobvious two or more same colors;

(2) get the aqueous solution under (1) item, extract with water saturated n-butyl alcohol 15ml, divide and get 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; Separately get the asiaticoside reference substance, add methanol and make the solution that every 1ml contains 1mg, in contrast product solution; Test according to thin layer chromatography (appendix VI B of Chinese Pharmacopoeia version in 2010), draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take chloroform-methanol-water (7:3:0.5) as developing solvent, launch, take out, dry, spray is heated to the speckle colour developing clear with 10% ethanol solution of sulfuric acid; In the test sample chromatograph, with reference substance chromatograph corresponding position on, the speckle of aobvious same color;

(3) get SANJIN PIAN 10 small pieces, remove coating, porphyrize, add ethanol 50ml, supersound process 30 minutes filters, filtrate adds hydrochloric acid 5ml, and reflux 2 hours lets cool, transfer to neutrality with 40% sodium hydroxide solution, steam to distinguishing the flavor of without alcohol, residue heating water 40ml makes dissolving, extract 2 times (40ml, 30ml), combined dichloromethane extracting solution with the dichloromethane jolting, evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution; Separately get Rhizoma Smilacis Chinensis control medicinal material 5g, be made in the same way of control medicinal material solution; Get again the diosgenin reference substance, add methanol and make the solution that every 1ml contains 0.5mg, in contrast product solution; Test according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2010), draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take Huan Ji Wan – ethyl acetate (4:1) as developing solvent, launch, take out, dry, spray is heated to the speckle colour developing at 105 ℃ clear with 10% ethanol solution of sulfuric acid, puts under ultra-violet lamp 365nm and inspects; In the test sample chromatograph, with control medicinal material chromatograph relevant position on, the fluorescence speckle of aobvious two or more same colors; With reference substance chromatograph relevant position on, the fluorescence speckle of aobvious same color;

(4) get SANJIN PIAN 15 small pieces, porphyrize adds methanol 100ml, supersound process 20 minutes filters, and filtrate is concentrated into approximately 10ml, be added on the neutral alumina column of 100～200 orders, 5g, internal diameter 1cm, with methanol 50ml eluting, collect 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 adds methanol 1ml makes dissolving, as need testing solution; Separately get YANGKAIKOU control medicinal material 5g, add water 100ml, reflux 1 hour filters, and filtrate evaporate to dryness, residue add methanol 20ml, are made in the same way of control medicinal material solution; Test according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2010), draw each 5～10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take San Lv Jia Wan – Bing Tong – water (6:14:1) as developing solvent, launch, take out, dry, put under ultra-violet lamp 365nm and inspect; In the test sample chromatograph, with control medicinal material chromatograph corresponding position on, the fluorescence speckle of aobvious same color.

Test example 1

This test example is to adopting the homemade rosamultin of method described in the embodiment of the present invention 1 and the chemical constitution of multiflorin reference substance to identify.

Gjy-4:2 α, 3 β, 19 α-trihydroxy Usu-12 alkene-28 acid, 28-O-β-D-Glucose glycosides; 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-Glucose glycosides; Multiflorin; Euscaphicoside; Euscaphicoside; 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 (6)

1. the detection method 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, the method also comprises the step that adopts the HPLC-ELSD method to measure rosamultin and/or multiflorin content; 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; In the step of the assay of described rosamultin and/or multiflorin, assay adopts following methods: precision is drawn reference substance solution 5 μ l, 10 μ l and need testing solution 10～20 μ l respectively, the injection liquid chromatography, measure, calculate respectively the content of rosamultin, multiflorin with external standard two-point method logarithmic equation, and get final product; Every of this product contains Radix Rosae Laevigatae with rosamultin (C ₃₆H ₅₈O ₁₀) meter, small pieces are no less than 0.10mg; Sheet is no less than 0.16mg; Contain Radix Rosae Laevigatae with multiflorin (C ₃₆H ₅₈O ₁₀) meter, small pieces are no less than 0.10mg; Sheet is no less than 0.16mg.

2. detection method according to claim 1, is characterized in that, described chromatographic condition and system suitability test are to be filler with octadecylsilane chemically bonded silica; Volume ratio is that the methanol-water of 48:52 is mobile phase; Detect with evaporative light scattering detector, number of theoretical plate is not less than 2000 by rosamultin peak calculating.

3. detection method according to claim 1, is characterized in that, following methods is adopted in the preparation of described reference substance solution: get respectively rosamultin and the multiflorin reference substance is appropriate, and accurately weighed, add methanol and make the mixed solution that every 1ml contains 0.5mg, and get final product.

4. detection method according to claim 3, it is characterized in that, described rosamultin and multiflorin reference substance adopt following method self-control: with the rhizome of Radix Rosae Laevigatae, pulverize, after extracting solvent extraction, with the extracting solution concentrating under reduced pressure, carry out solvent extraction or upper macroporous resin adsorption, and water, 30%, 40～90% ethanol elutions successively, collect 40～90% ethanol elution things, reclaim solvent, then 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 again, and collection contains the fraction of rosamultin and multiflorin, gets rosamultin and multiflorin coarse-grain, then adds recrystallization reagent recrystallization and get final product repeatedly.

5. detection method according to claim 1, it is characterized in that, following methods is adopted in the preparation of described need testing solution: get 30 small pieces of this product or 20 sheets, remove coating, accurately weighed, porphyrize, get approximately 1.5g, accurately weighed, precision adds methanol 50ml, weighed weight, at power 250W, under frequency 40kHz condition, ultrasonic place is 45 minutes, let cool, weighed weight again, supply the weight of less loss with methanol, shake up, filter, precision measures subsequent filtrate 25ml, reclaim solvent to doing, residue adds water 20ml makes dissolving, extract 3 times with water saturated n-butyl alcohol jolting, each 15ml, merge n-butanol extracting liquid, with ammonia solution washing 2 times, each 15ml, get n-butyl alcohol liquid, decompression and solvent recovery is to doing, the residue dissolve with methanol, be transferred in the 5ml measuring bottle, add methanol to scale, shake up, and get final product.

6. detection method according to claim 1, is characterized in that, described employing HPLC-ELSD method is measured asiaticoside and/or asiaticoside content comprises the steps:

Measure according to high performance liquid chromatography (appendix VI D of Chinese Pharmacopoeia version in 2010);

Chromatographic condition and system suitability: be filler with octadecylsilane chemically bonded silica; Volume ratio is that the methanol-water of 48:52 is mobile phase; Detect or use UV-detector to detect with evaporative light scattering detector, detecting wavelength 210nm, number of theoretical plate is not less than 2000 by asiaticoside peak calculating;

The preparation of reference substance solution: get respectively the asiaticoside reference substance appropriate, accurately weighed, add methanol and make solution and the every 1ml that every 1ml contains respectively 0.2mg and contain the solution of 0.6mg, and get final product;

the preparation of need testing solution: get this product 30 small pieces or 20 sheets, remove coating, accurately weighed, porphyrize, get approximately 1.5g, accurately weighed, precision adds methanol 50ml, weighed weight, at power 250W, under the condition of frequency 40kHz, supersound process is 45 minutes, let cool, weighed weight again, supply the weight of less loss with methanol, shake up, filter, precision measures subsequent filtrate 25ml, reclaim solvent to doing, residue adds water 20ml makes dissolving, extract 3 times with water saturated n-butyl alcohol jolting, each 15ml, merge n-butanol extracting liquid, with ammonia solution washing 2 times, each 15ml, get n-butyl alcohol liquid, decompression and solvent recovery is to doing, the residue dissolve with methanol, be transferred in the 5ml measuring bottle, add methanol to scale, shake up, and get final product,

Algoscopy: precision is drawn reference substance solution each 10 μ l and need testing solution 5～10 μ l of above-mentioned two kinds of concentration respectively, and the injection liquid chromatography is measured, and with the calculating of external standard two-point method logarithmic equation, and get final product; Every of this product contains Herba Centellae with asiaticoside (C ₄₈H ₇₈O ₂₀) meter, small pieces are no less than 0.22m _gSheet is no less than 0.35m _g

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