CN100370251C - Quality control method for liuwei Dihuang soft extract - Google Patents
Quality control method for liuwei Dihuang soft extract Download PDFInfo
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Abstract
The present invention relates to a novel quality control method for Liuwei Dihuang cream of the Yin nourishing and kidney reinforcing medicine in the third volume (WS3-B-2489-97) of <<the medicine standard of the sanitary department of the People's Republic of China>>(the formula preparation of the traditional Chinese medicine). The medicine of the present invention is prepared from prepared rehmannia root, dogwood (prepared), Chinese yam, tree peony bark, tuckahoe and water plantain. Concretely, the high-efficiency liquid chromatography is used for measuring the content of the paeoniflorin in the Liuwei Dihuang cream; the method of the present invention has the advantages of simplicity, convenience, good reproducibility and accurate result; the thin-layer chromatography is used for identifying the dogwood in the traditional Chinese medicines in the Liuwei Dihuang cream, and thus, the method is exclusive and feasible; the accuracy and the progress of the quality detection standard are ensured, and the quality of the Liuwei Dihuang cream can be effectively controlled.
Description
Technical field
The present invention relates to the method for quality control of the enriching yin and nourishing kidney medicine liuwei Dihuang soft extract made by Chinese crude drug prepared rhizome of rehmannia, the fruit of medicinal cornel (system), Chinese yam, moutan bark, Poria cocos, rhizoma alismatis, be specifically related to differentiate the method for quality control of the liuwei Dihuang soft extract of the Chinese crude drug fruit of medicinal cornel with content of paeoniflorin and thin-layered chromatography in this medicine of high effective liquid chromatography for measuring.
Background technology
The 3rd (WS of " Drug Standard of Ministry of Public Health of the Peoples Republic of China " (Chinese traditional patent formulation preparation)
3-B-2489-97) in the medicine liuwei Dihuang soft extract of enriching yin and nourishing kidney, by Chinese crude drug prepared rhizome of rehmannia 320g, the fruit of medicinal cornel (system) 160g, Chinese yam 160g, moutan bark 120g, Poria cocos 120g, the paste nourishing agent that rhizoma alismatis 120g makes, and issued the liuwei Dihuang soft extract quality control standard, but in the existing liuwei Dihuang soft extract method of quality control that the Ministry of Public Health issues, have only the thin-layer chromatography discriminating of Paeonol in the moutan bark and do not have content assaying method, limitation is very big, the quality of restive liuwei Dihuang soft extract; Paeoniflorin is another active component in the moutan bark, has analgesia, and spasmolysis is antibiotic, antiinflammatory action; Other Liuwei Dihuang preparations in existing pharmacopeia and the ministerial standard (assay is arranged) are the Paeonol measured in the moutan bark and the ursolic acid in the fruit of medicinal cornel; In addition, proposing with the Catalpol in the article " assay of Catalpol in the Liuwei Dihuang Wan " that deliver in 2000 13 volumes of traditional Chinese medical science research, 3 phase Liu long rivers etc. is the quality of such preparation of control indexes, and points out that Catalpol has the character of case of thermal instability; The time propose to adopt the high effective liquid chromatography for measuring Paeonol in " paeonol content in the high effective liquid chromatography for measuring LIUWEIDIHUANG JIAONANG " literary composition of delivering such as precious traditional Chinese medical science traditional Chinese medicines 1999 10 volumes 12 phase Zhang Zhongchun; Test and inquire at the problem that exists during the operation of tlc scanning determination ursolic acid content in the pharmacopeia in " improvement of Liuwei Dihuang Wan ursolic acid content assay method " literary composition that Chinese patent drug calendar year 2001 23 volumes, 1 phase You Huilian etc. delivers; " the RP-HPLC method is measured the content of loganin in the Liuwei Dihuang Wan " that 1999 8 volumes of China's medicine company, 11 phase Chen Hexins etc. are delivered provides new reliable method for the inherent quality of control said preparation.The present invention is the extraction process by water that adopts according to this preparation, and ursolic acid is slightly soluble in water, and the extraction ratio that water is carried is low, and the fusing point of Paeonol is low, the tool volatility, and it is low that water is carried content, is unfavorable for the control of quality; The Paeoniflorin tool is water-soluble, decocting extraction ratio height, so with the Paeoniflorin in the moutan bark as assay index in the preparation and set up its detection method, though Xiong Shanli etc. have set up morroniside, loganin, paeoniflorin content method for measuring in six drugs containing rehmanniae side's decocting liquid in " RP-HPLC measures the content of glycoside composition in the six drugs containing rehmanniae side " literary composition that CHINA JOURNAL OF CHINESE MATERIA MEDICA was delivered on 2003 28 8 phases of volume, its moving phase is all different with the present invention with the detection wavelength; The present invention utilizes content of paeoniflorin in this medicine of high effective liquid chromatography for measuring, and increase its thin-layer chromatography qualitative identification method by fruit of medicinal cornel medicinal material, confirmation method is easy, the result is accurate, favorable reproducibility, adopting ursolic acid mostly in existing pharmacopeia and the document is reference substance, and the present invention adopts is fruit of medicinal cornel control medicinal material, and exclusive, feasible, negative noiseless through methodology test confirmation method; Therefore the present invention has improved the quality control standard of liuwei Dihuang soft extract, has guaranteed this compound preparation higher quality standard level better.
Summary of the invention
The objective of the invention is adopting the liuwei Dihuang soft extract quality of extraction process by water, formulate new liuwei Dihuang soft extract method of quality control, utilize content of paeoniflorin in this medicine of high effective liquid chromatography for measuring, and investigate test through methodology, confirmation method is easy, the result is accurate, favorable reproducibility; Differentiate this medicine Chinese crude drug fruit of medicinal cornel with thin-layered chromatography, through the methodology test, confirmation method is exclusive, feasible, negative noiseless; Guarantee the accuracy and the advance of quality inspection standard, can control the quality of liuwei Dihuang soft extract effectively.
The 3rd (WS of " Drug Standard of Ministry of Public Health of the Peoples Republic of China " (Chinese traditional patent formulation preparation)
3-B-2489-97) in the medicine liuwei Dihuang soft extract of enriching yin and nourishing kidney, by Chinese crude drug prepared rhizome of rehmannia 320g, the fruit of medicinal cornel (system) 160g, Chinese yam 160g, moutan bark 120g, Poria cocos 120g, the paste nourishing agent that rhizoma alismatis 120g makes.Main flavour of a drug are the process of preparing Chinese medicine processed goods of radix rehmanniae recen in the cultivated land side of being, radix rehmanniae recen is the fresh or dried root of scrophulariaceae rehmannia glutinosa plant Rehmannia glutinosa Libosch., and rich sweet temperature has the effect of nourshing kidney replenishing essence; Mainly contain chemical constitutions such as iridoid glycosideses such as Catalpol, rehmannioside and polysaccharide; Moutan bark is the dry root skin of ranunculaceae peony Paeonia suffruticosa Andr., has clearing heat and cooling blood, and effect promoting blood circulation and removing blood stasis mainly contains chemical constitutions such as Paeonol, Paeoniflorin; The fruit of medicinal cornel is the drying and ripening pulp of Cornaceae plant fruit of medicinal cornel Cornus officinalis Sieb.et Zucc., has the effect of the puckery essence of nourishing the liver; Mainly contain chemical constitutions such as loganin, ursolic acid, oleanolic acid, the preparation method of enriching yin and nourishing kidney medicine liuwei Dihuang soft extract of the present invention is by the 3rd (WS of " Drug Standard of Ministry of Public Health of the Peoples Republic of China " (Chinese traditional patent formulation preparation)
3The paste nourishing agent that the extraction process by water that-preparation method in B-2489-97) adopts is made.Effective ingredient ursolic acid in the side is slightly soluble in water, and the extraction ratio that water is carried is low, and the fusing point of Paeonol is low, the tool volatility, and it is low that water is carried content, is unfavorable for the control of liuwei Dihuang soft extract quality; And another effective ingredient Paeoniflorin tool is water-soluble, decocting extraction ratio height, die with the Paeoniflorin in the moutan bark as assay index in the preparation, utilize content of paeoniflorin in this medicine of high effective liquid chromatography for measuring, method is easy, the result is accurate, favorable reproducibility, and increase the wherein thin-layer chromatography qualitative identification method of fruit of medicinal cornel medicinal material, and adopt fruit of medicinal cornel control medicinal material, and exclusive, feasible, negative noiseless through methodology test confirmation method; Improve the quality control standard of liuwei Dihuang soft extract, guaranteed this compound preparation higher quality standard level better.
Finishing the present invention is achieved by following technical proposals: one is with content of paeoniflorin in this medicine of high effective liquid chromatography for measuring; Its dual-purpose thin-layered chromatography is differentiated this medicine Chinese crude drug fruit of medicinal cornel.
The quality control detection method that concrete liuwei Dihuang soft extract is new, can pass through following steps in advance to realize:
Instrument, medicine and material
Instrument: Waters 600E type high performance liquid chromatograph, Waters 486 detecting devices: U.S. Waters company
Shen, river chromatographic work station: big Shen, Liaanjiang county separation science technology company
SK5200H ultrasonic cleaner: Shanghai High Kudos Science Instrument Co., Ltd.
Mettler AE 240 rigorous analysis balances (0.00001): Mettler instrument company
HH-4 digital display thermostat water bath: state China Electrical Appliances Co., Ltd
101-2 type electric drying oven with forced convection: Shanghai City experimental apparatus head factory
Reference substance and control medicinal material: the Paeoniflorin reference substance is purchased in Nat'l Pharmaceutical ﹠ Biological Products Control Institute.
The rhizoma alismatis control medicinal material is purchased in Nat'l Pharmaceutical ﹠ Biological Products Control Institute.
The self-control of fruit of medicinal cornel control medicinal material
Each single medicinal material: provide by letter medicine company Ltd in the city of Haozhou, Anhui.After testing, all meet 2000 editions one every regulation down of the Pharmacopoeia of the People's Republic of China.
Test sample: liuwei Dihuang soft extract
Silica gel g thin-layer plate: road and bridge tetramethyl biochemical plastic molding and processing plant in specification 10 * 20cm Taizhou city produces
Developer and test solution: sulfuric acid-aceticanhydride-ethanol solution (1: 1: 1), sulfuric acid-aceticanhydride-ethanol solution (1: 1: 5), it is pure that used reagent is analysis, and test solution is all according to the preparation of the every method down of 2000 editions one appendix XV B of the Pharmacopoeia of the People's Republic of China.
1, with content of paeoniflorin in this medicine of high effective liquid chromatography for measuring
A. chromatographic condition filling agent: octadecylsilane chemically bonded silica
Moving phase: acetonitrile--water
Detect wavelength: 230 ± 2nm
Number of theoretical plate calculates by the Paeoniflorin peak should be not less than 3000
B. the preparation of reference substance solution: it is an amount of that precision takes by weighing the Paeoniflorin reference substance, and solution is made in any dissolving that adds in organic solvent or the water;
C. the preparation of need testing solution: get this product, behind the thin up, extract with water-saturated n-butanol, behind the pure liquid evaporate to dryness, residue dilutes and constant volume with any dissolving in organic solvent or the water;
D. content of paeoniflorin assay method: precision is measured reference substance solution and need testing solution respectively, injects liquid chromatograph, measures content of paeoniflorin;
2, this medicine Chinese crude drug fruit of medicinal cornel is differentiated with thin-layered chromatography
A. the preparation of control medicinal material solution: get fruit of medicinal cornel control medicinal material, add alcohol reflux, the leaching extract, evaporate to dryness, residue is dissolved in water, and uses extracted with diethyl ether, and ether solution evaporate to dryness, residue are dissolved in the ethyl acetate, are control medicinal material solution;
B. the preparation of need testing solution: get this product, add ethanol jolting mixing, put refluxing extraction in the water-bath, extract steams to there not being the alcohol flavor, and thin up is used extracted with diethyl ether, ether solution 5%NaHCO
3Solution washing washes with water again, and ether solution evaporate to dryness, residue are dissolved in the ethyl acetate, are need testing solution;
C. test according to thin-layered chromatography, draw above-mentioned two kinds of solution, point is on same silica gel g thin-layer plate, with cyclohexane-chloroform-ethyl acetate is developping agent, spray is dried by the fire to clear spot with aceticanhydride-concentrated sulphuric acid-ethanol solution, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.
The quality control detection method that above-mentioned liuwei Dihuang soft extract is new, preferred following concrete steps:
1, measure content of paeoniflorin with high performance liquid chromatography (appendix VID of Chinese Pharmacopoeia version in 2000):
A. chromatographic condition: with octadecylsilane chemically bonded silica is filling agent, and (10-20: 90-80) be moving phase, the detection wavelength is 230 ± 2nm to acetonitrile--water, and number of theoretical plate calculates by the Paeoniflorin peak should be not less than 3000;
B. the preparation of reference substance solution: it is an amount of that precision takes by weighing the Paeoniflorin reference substance, adds in methyl alcohol or ethanol or the water any and make the solution that every 1ml contains 5--50 μ g;
C. the preparation of need testing solution: get this product 0.5--3g, the accurate title, decide, and adds water 10--25ml, agitation and dilution moves in the separating funnel, adds water-saturated n-butanol and extracts 3--6 time, each 10--25ml, merge pure liquid, put evaporate to dryness in the water-bath, residue is with any an amount of dissolving in methyl alcohol or ethanol or the water, in the dislocation 10--25ml measuring bottle, dilution also is settled to scale, shakes up, promptly;
D. content of paeoniflorin assay method: precision is measured reference substance solution and each 5--20 μ l of need testing solution respectively, injects liquid chromatograph, measures content of paeoniflorin;
E. every gram contains moutan bark with Paeoniflorin (C
23H
28O
11) meter, must not be lower than 0.08mg.
2. this medicine Chinese crude drug fruit of medicinal cornel is differentiated with thin-layered chromatography
A. the preparation of control medicinal material solution: get fruit of medicinal cornel control medicinal material 0.5-4g, add ethanol 20-100ml backflow 0.5-2 hour, filter, filtrate evaporate to dryness, residue add water 10-30ml dissolving, with ether 30-90ml gradation extraction, merge diethyl ether solution, evaporate to dryness, residue are dissolved in the 0.5ml ethyl acetate, are control medicinal material solution;
B. the preparation of need testing solution: get this product 10-50g, add 95% ethanol 20-100ml, jolting made mixing in 10 minutes, put in the water-bath backflow 0.5-2 hour, filtered while hot, filtrate is steamed to there not being the alcohol flavor, adds water 10-50ml agitation and dilution, moves in the separating funnel, with extracted with diethyl ether 2-5 time, each 10-50ml merges diethyl ether solution, uses 5%NaHCO
3Solution 20-50ml gradation washing, the about 10-50ml washing of water again, diethyl ether solution evaporate to dryness, residue are dissolved in the 0.5ml ethyl acetate, are need testing solution;
C. test according to thin-layered chromatography (appendix VIB of Chinese Pharmacopoeia version in 2000), drawing above-mentioned two kinds of each 5-20ul of solution, put on same silica gel g thin-layer plate, is developping agent with cyclohexane-chloroform-ethyl acetate (20: 5: 5), launch, take out, dry, spray is with aceticanhydride-concentrated sulphuric acid-absolute ethyl alcohol (1: 1: 5) solution, dry by the fire to clear spot at 105 ℃, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.
The quality control detection method that liuwei Dihuang soft extract of the present invention is new, preferred technical scheme can be undertaken by following determination step:
1, measure content of paeoniflorin with high performance liquid chromatography (appendix VID of Chinese Pharmacopoeia version in 2000):
A. chromatographic condition: with octadecylsilane chemically bonded silica is filling agent; Acetonitrile--water (12: 88) is moving phase, and the detection wavelength is 230nm, and number of theoretical plate calculates by the Paeoniflorin peak should be not less than 3000;
B. the preparation of reference substance solution: it is an amount of that precision takes by weighing the Paeoniflorin reference substance, adds methyl alcohol and make the solution that every 1ml contains 20 μ g;
C. the preparation of need testing solution: get this product 1g, the accurate title, decide, and adds water 10ml, agitation and dilution moves in the separating funnel, adds water-saturated n-butanol and extracts 4 times, each 10ml, merge pure liquid, put evaporate to dryness in the water-bath, residue dissolves in right amount with methyl alcohol, in the dislocation 10ml measuring bottle, dilution also is settled to scale, shakes up, promptly;
D. determination method: precision is measured reference substance solution and each 5 μ l of need testing solution respectively, injects liquid chromatograph, measures content of paeoniflorin; Every gram contains moutan bark with Paeoniflorin (C
23H
28O
11) meter, must not be lower than 0.08mg.
2, this medicine Chinese crude drug fruit of medicinal cornel is differentiated with thin-layered chromatography
A. the preparation of control medicinal material solution: take by weighing fruit of medicinal cornel control medicinal material 2g, add ethanol 50ml and refluxed 1 hour, filter the filtrate evaporate to dryness, residue adds water 10ml dissolving, with ether 30ml gradation extraction, merges diethyl ether solution, evaporate to dryness, residue are dissolved in the 0.5ml ethyl acetate, are control medicinal material solution;
B. the preparation of need testing solution: get this product 30g, add 95% ethanol 60ml, jolting made mixing in 10 minutes, put in the water-bath and refluxed 1 hour, filtered while hot, filtrate is steamed to there not being the alcohol flavor, adds water 30ml agitation and dilution, moves in the separating funnel, with extracted with diethyl ether three times, each 20ml merges diethyl ether solution, uses 5%NaHCO
3Solution 30ml gradation washing, the about 15ml washing of water again, diethyl ether solution evaporate to dryness, residue are dissolved in the 0.5ml ethyl acetate, are need testing solution;
C. test according to thin-layered chromatography (appendix VIB of Chinese Pharmacopoeia version in 2000), drawing above-mentioned two kinds of each 10ul of solution, put on same silica gel g thin-layer plate, is developping agent with cyclohexane-chloroform-ethyl acetate (20: 5: 5), launch, take out, dry, spray is with aceticanhydride-concentrated sulphuric acid-absolute ethyl alcohol (1: 1: 5) solution, dry by the fire to clear spot at 105 ℃, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.
The preparation method of enriching yin and nourishing kidney medicine liuwei Dihuang soft extract of the present invention is by the 3rd (WS of " Drug Standard of Ministry of Public Health of the Peoples Republic of China " (Chinese traditional patent formulation preparation)
3The paste nourishing agent that-preparation method in B-2489-97) makes.
The TCL that the invention provides the fruit of medicinal cornel differentiates, and is feasible, negative noiseless through methodology test confirmation method; Having selected Paeoniflorin is testing index, and has carried out following methodological study test, has set up the HPLC content assaying method of liuwei Dihuang soft extract; Through revision test repeatedly, confirmation method is easy, the result is accurate, and favorable reproducibility can be used as the liuwei Dihuang soft extract quality control and investigates the index of the stability of technology.
The paeoniflorin content method for measuring is investigated:
1, linearity curve, regression equation, related coefficient and the range of linearity
1. precision takes by weighing 36 hours Paeoniflorin reference substance 6.02mg of drying in the phosphorus pentoxide vacuum drying apparatus, puts in the 25ml measuring bottle, adds methyl alcohol and makes dissolving in right amount, be diluted to scale, shake up, get Paeoniflorin stock solution (0.2408mg/ml), accurate respectively absorption 0.00,0.25,0.50,1.00,1.50,2.00ml, put in the 10ml measuring bottle, add methyl alcohol and be diluted to scale, shake up, promptly get each reference substance solution, accurate respectively above-mentioned each reference substance solution 5 μ l, injection liquid chromatograph, the mensuration drawn.As shown in Figure 1: with peak area (A) is horizontal ordinate, and paeoniflorin content (M) is an ordinate, gets regression equation: M=8.892 * 10
-8A+3.208 * 10
-3(in the formula: A is a peak area, and M is paeoniflorin content μ g), r=0.9994 shows paeoniflorin content between 0-0.2408 μ g, is the good linear relation, the results are shown in Table 1.
Table 1 Paeoniflorin linear relationship measurement result
| Numbering | Reference substance sampling amount (ml) | Reference substance content (μ g) | Peak area (A) | Linear equation | Related coefficient | The range of linearity (μ g) |
| 1 | 0.0 | 0.0000 | 0 | |||
| 2 | 0.25 | 0.0301 | 304461 | |||
| 3 | 0.50 | 0.0602 | 628361 | |||
| 4 | 1.00 | 0.1204 | 1252235 | M＝8.892×10 -8A+3.208 ×10 -3 | 0.9994 | 0-0.240 8 |
| 5 | 1.50 | 0.1806 | 2023004 | |||
| 6 | 2.00 | 0.2408 | 2684427 |
2, reappearance test
Precision takes by weighing the about 1g of same lot number sample, and totally 5 parts, according to method operation of the present invention, measure peak area A, and calculate content of paeoniflorin in each duplicate samples, the results are shown in Table 2.
The test of table 2 reappearance
| Sample number into |
1 | 2 | 3 | 4 | 5 |
| Content (mg/g) | 0.148 | 0.148 | 0.152 | 0.149 | 0.147 |
| Mean value (mg/g) RSD (%) | 0.149 1.26 |
3, precision test
Get same Paeoniflorin reference substance solution, survey continuously 6 times, each accurate 5 μ l that draw inject liquid chromatograph, measure peak area A, and test shows: its precision RSD=1.71%, and up to specification, the results are shown in Table 3.
The test of table 3 precision
| Numbering | 1 | 2 | 3 | 4 | 5 | 6 |
| Peak area | 1226384 | 1257754 | 1275782 | 1253258 | 1287385 | 1248002 |
| Mean value RSD (%) | 1258094.17 1.71 |
4, stability test
Get precision test Paeoniflorin reference substance solution and need testing solution down, accurate absorption 5 μ l are every 1.5 hours, after 6 hours, inject liquid chromatograph respectively, measure peak area, the result shows: Paeoniflorin reference substance solution and need testing solution are all stable in 6 hours, the results are shown in Table 4.
Table 4 stability test
| Time (hour) | The reference substance peak area | The test |
| 0 0.5 6.0 | 1226384 1258940 1260187 | 835469 837773 833106 |
| Mean value RSD (%) | 1248503.67 1.53 | 835449.3 0.28 |
5. average recovery test
Get this product 0.3-0.7g of known content, the accurate title, decide, and totally 6 parts, every part each add the accurate fixed Paeoniflorin reference substance 30-120 μ g that claims, shines method operation of the present invention, measures content, and calculate recovery rate the results are shown in Table 5.
Table 5 average recovery measurement result
| Numbering | Sample contains Paeoniflorin amount (mg) | Add Paeoniflorin amount (mg) | Record Paeoniflorin amount (mg) | The recovery (%) | Average recovery rate (%) |
| 1 2 3 4 5 6 | 0.1084 0.1014 0.07507 0.09196 0.05561 0.06636 | 0.03612 0.03612 0.07224 0.07224 0.1084 0.1084 | 0.1437 0.1365 0.1473 0.1651 0.1645 0.1756 | 97.73 97.18 99.99 101.25 100.45 100.77 | 99.56 RSD＝1.57％ |
Description of drawings
Fig. 1 is the linear relationship chart of paeoniflorin content and peak area.
Embodiment
Embodiment 1: get Chinese crude drug prepared rhizome of rehmannia 320g, the fruit of medicinal cornel (system) 160g, Chinese yam 160g, moutan bark 120g, Poria cocos 120g, rhizoma alismatis 120g, above-mentioned raw materials boiling three times, first and second time respectively add 9 times of water gagings, decocted 2 hours, add 7 times of water gagings for the third time, decocted collecting decoction 1 hour, filter, filtrate is left standstill, and gets supernatant and is evaporated to the clear cream that relative density is 1.28～1.32 (85 ℃), and the clear cream of every 100g adds refined honey 300g, mixing, promptly.
1, measuring content of paeoniflorin with high performance liquid chromatography (appendix VID of Chinese Pharmacopoeia version in 2000) measures and standard:
A. chromatographic condition: with octadecylsilane chemically bonded silica is filling agent; Acetonitrile--water (12: 88) is moving phase, and the detection wavelength is 230nm, and number of theoretical plate calculates by the Paeoniflorin peak should be not less than 3000;
B. the preparation of reference substance solution: it is an amount of that precision takes by weighing the Paeoniflorin reference substance, adds methyl alcohol and make the solution that every 1ml contains 20 μ g;
C. the preparation of need testing solution: get this product 1g, the accurate title, decide, and adds water 10ml, agitation and dilution moves in the separating funnel, adds water-saturated n-butanol and extracts 4 times, each 10ml, merge pure liquid, put evaporate to dryness in the water-bath, residue dissolves in right amount with methyl alcohol, in the dislocation 10ml measuring bottle, dilution also is settled to scale, shakes up, promptly.
D. determination method: precision is measured reference substance solution and each 5 μ l of need testing solution respectively, injects liquid chromatograph, measures content of paeoniflorin.Every gram contains moutan bark with Paeoniflorin (C
23H
28O
11) meter, must not be lower than 0.08mg.
2, this medicine Chinese crude drug fruit of medicinal cornel is differentiated with thin-layered chromatography
A. the preparation of control medicinal material solution: take by weighing fruit of medicinal cornel control medicinal material 2g, add ethanol 50ml and refluxed 1 hour, filter the filtrate evaporate to dryness, residue adds water 10ml dissolving, with ether 30ml gradation extraction, merges diethyl ether solution, evaporate to dryness, residue are dissolved in the 0.5ml ethyl acetate, are control medicinal material solution.
B. the preparation of need testing solution: get this product 30g, add 95% ethanol 60ml, jolting made mixing in 10 minutes, put in the water-bath and refluxed 1 hour, filtered while hot, filtrate is steamed to there not being the alcohol flavor, adds water 30ml agitation and dilution, moves in the separating funnel, with extracted with diethyl ether three times, each 20ml merges diethyl ether solution, uses 5%NaHCO
3Solution 30ml gradation washing, the about 15ml washing of water again, diethyl ether solution evaporate to dryness, residue are dissolved in the 0.5ml ethyl acetate, are need testing solution;
C. test according to thin-layered chromatography (appendix VIB of Chinese Pharmacopoeia version in 2000), draw above-mentioned two kinds of each 10ul of solution, point is on same silica gel g thin-layer plate, with cyclohexane-chloroform-ethyl acetate (20: 5: 5) is developping agent, launch, take out, dry, spray is dried by the fire to clear spot at 105 ℃ with aceticanhydride-concentrated sulphuric acid-absolute ethyl alcohol (1: 1: 5) solution.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.With the index of said method as the stability of this drug quality control and investigation technology.
Embodiment 2: get Chinese crude drug prepared rhizome of rehmannia 320g, the fruit of medicinal cornel (system) 160g, Chinese yam 160g, moutan bark 120g, Poria cocos 120g, rhizoma alismatis 120g, above-mentioned raw materials boiling three times, first and second time respectively adds 9 times of water gagings, decocted 2 hours, and added 7 times of water gagings for the third time, decocted 1 hour, collecting decoction, filter, filtrate is left standstill, and gets supernatant and is evaporated to the clear cream that relative density is 1.28～1.32 (85 ℃).The clear cream of every 100g adds refined honey 300g, mixing, promptly.
1, measuring content of paeoniflorin with high performance liquid chromatography (appendix VID of Chinese Pharmacopoeia version in 2000) measures and standard:
A. chromatographic condition: with octadecylsilane chemically bonded silica is filling agent; Acetonitrile--water (10: 90) is moving phase, and the detection wavelength is 230nm;
B. the preparation of reference substance solution: it is an amount of that precision takes by weighing the Paeoniflorin reference substance, adds methyl alcohol and make the solution that every 1ml contains 5 μ g;
C. the preparation of need testing solution: get this product 0.5g, the accurate title, decide, and adds water 10ml, agitation and dilution moves in the separating funnel, adds water-saturated n-butanol and extracts 6 times, each 10ml, merge pure liquid, put evaporate to dryness in the water-bath, residue dissolves in right amount with methyl alcohol, in the dislocation 10ml measuring bottle, dilution also is settled to scale, shakes up, promptly.
D. determination method: precision is measured reference substance solution and each 20 μ l of need testing solution respectively, injects liquid chromatograph, measures content of paeoniflorin.Every gram contains moutan bark with Paeoniflorin (C
23H
28O
11) meter, must not be lower than 0.08mg.
2, this medicine Chinese crude drug fruit of medicinal cornel is differentiated with thin-layered chromatography
A. the preparation of control medicinal material solution: take by weighing fruit of medicinal cornel control medicinal material 0.5g, add ethanol 20ml and refluxed 2 hours, filter the filtrate evaporate to dryness, residue adds water 10ml dissolving, with ether 30ml gradation extraction, merges diethyl ether solution, evaporate to dryness, residue are dissolved in the 0.5ml ethyl acetate, are control medicinal material solution.
B. the preparation of need testing solution: get this product 10g, add 95% ethanol 20ml, jolting made mixing in 10 minutes, put in the water-bath and refluxed 2 hours, filtered while hot, filtrate is steamed to there not being the alcohol flavor, adds water 10ml agitation and dilution, moves in the separating funnel, with extracted with diethyl ether 5 times, each 10ml merges diethyl ether solution, uses 5%NaHCO
3Solution 20ml gradation washing, the about 10ml washing of water again, diethyl ether solution evaporate to dryness, residue are dissolved in the 0.5ml ethyl acetate, are need testing solution;
C. test according to thin-layered chromatography (appendix VIB of Chinese Pharmacopoeia version in 2000), draw above-mentioned two kinds of each 20ul of solution, point is on same silica gel g thin-layer plate, with cyclohexane-chloroform-ethyl acetate (20: 5: 5) is developping agent, launch, take out, dry, spray is dried by the fire to clear spot at 105 ℃ with aceticanhydride-concentrated sulphuric acid-absolute ethyl alcohol (1: 1: 5) solution.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.With the index of said method as the stability of this drug quality control and investigation technology.
Embodiment 3: get Chinese crude drug prepared rhizome of rehmannia 320g, the fruit of medicinal cornel (system) 160g, Chinese yam 160g, moutan bark 120g, Poria cocos 120g, rhizoma alismatis 120g, above-mentioned raw materials boiling three times, first and second time respectively adds 9 times of water gagings, decocted 2 hours, and added 7 times of water gagings for the third time, decocted 1 hour, collecting decoction, filter, filtrate is left standstill, and gets supernatant and is evaporated to the clear cream that relative density is 1.28～1.32 (85 ℃).The clear cream of every 100g adds refined honey 300g, mixing, promptly.
1, measuring content of paeoniflorin with high performance liquid chromatography (appendix VID of Chinese Pharmacopoeia version in 2000) measures and standard:
A. chromatographic condition: with octadecylsilane chemically bonded silica is filling agent; Acetonitrile--water (15: 85) is moving phase, and the detection wavelength is 230nm;
B. the preparation of reference substance solution: it is an amount of that precision takes by weighing the Paeoniflorin reference substance, adds ethanol and make the solution that every 1ml contains 50 μ g;
C. the preparation of need testing solution: get this product 3.0g, the accurate title, decide, and adds water 25ml, agitation and dilution moves in the separating funnel, adds water-saturated n-butanol and extracts 3 times, each 25ml, merge pure liquid, put evaporate to dryness in the water-bath, residue dissolves in right amount with ethanol, in the dislocation 25ml measuring bottle, dilution also is settled to scale, shakes up, promptly.
D. determination method: precision is measured reference substance solution and each 10 μ l of need testing solution respectively, injects liquid chromatograph, measures content of paeoniflorin.
Every gram contains moutan bark with Paeoniflorin (C
23H
28O
11) meter, must not be lower than 0.08mg.
2, this medicine Chinese crude drug fruit of medicinal cornel is differentiated with thin-layered chromatography
A. the preparation of control medicinal material solution: take by weighing fruit of medicinal cornel control medicinal material 4g, add ethanol 100ml and refluxed 0.5 hour, filter the filtrate evaporate to dryness, residue adds water 30ml dissolving, with ether 90ml gradation extraction, merges diethyl ether solution, evaporate to dryness, residue are dissolved in the 0.5ml ethyl acetate, are control medicinal material solution.
B. the preparation of need testing solution: get this product 50g, add 95% ethanol 100ml, jolting made mixing in 10 minutes, put in the water-bath and refluxed 0.5 hour, filtered while hot, filtrate is steamed to there not being the alcohol flavor, adds water 50ml agitation and dilution, moves in the separating funnel, with extracted with diethyl ether 2 times, each 50ml merges diethyl ether solution, uses 5%NaHCO
3Solution 50ml gradation washing, the about 50ml washing of water again, diethyl ether solution evaporate to dryness, residue are dissolved in the 0.5ml ethyl acetate, are need testing solution;
C. test according to thin-layered chromatography (appendix VIB of Chinese Pharmacopoeia version in 2000), draw above-mentioned two kinds of each 5ul of solution, point is on same silica gel g thin-layer plate, with cyclohexane-chloroform-ethyl acetate (20: 5: 5) is developping agent, launch, take out, dry, spray is dried by the fire to clear spot at 105 ℃ with aceticanhydride-concentrated sulphuric acid-absolute ethyl alcohol (1: 1: 5) solution.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.With the index of said method as the stability of this drug quality control and investigation technology.
Embodiment 4: get Chinese crude drug prepared rhizome of rehmannia 320g, the fruit of medicinal cornel (system) 160g, Chinese yam 160g, moutan bark 120g, Poria cocos 120g, rhizoma alismatis 120g, above-mentioned raw materials boiling three times, first and second time respectively adds 9 times of water gagings, decocted 2 hours, and added 7 times of water gagings for the third time, decocted 1 hour, collecting decoction, filter, filtrate is left standstill, and gets supernatant and is evaporated to the clear cream that relative density is 1.28～1.32 (85 ℃).The clear cream of every 100g adds refined honey 300g, mixing, promptly.
1. measuring content of paeoniflorin with high performance liquid chromatography (appendix VID of Chinese Pharmacopoeia version in 2000) measures and standard
A. chromatographic condition: with octadecylsilane chemically bonded silica is filling agent; Acetonitrile--water (20: 80) is moving phase, and the detection wavelength is 230nm;
B. the preparation of reference substance solution: it is an amount of that precision takes by weighing the Paeoniflorin reference substance, adds water and make the solution that every 1ml contains 30 μ g;
C. the preparation of need testing solution: get this product 2g, the accurate title, decide, and adds water 20ml, agitation and dilution moves in the separating funnel, adds water-saturated n-butanol and extracts 5 times, each 20ml, merge pure liquid, put evaporate to dryness in the water-bath, the residue water dissolves in right amount, in the dislocation 10ml measuring bottle, dilution also is settled to scale, shakes up, promptly.
D. determination method: precision is measured reference substance solution and each 10 μ l of need testing solution respectively, injects liquid chromatograph, measures content of paeoniflorin.Every gram contains moutan bark with Paeoniflorin (C
23H
28O
11) meter, must not be lower than 0.08mg.
2, this medicine Chinese crude drug fruit of medicinal cornel is differentiated with thin-layered chromatography
A. the preparation of control medicinal material solution: take by weighing fruit of medicinal cornel control medicinal material 2g, add ethanol 50ml and refluxed 1.5 hours, filter the filtrate evaporate to dryness, residue adds water 10ml dissolving, with ether 30ml gradation extraction, merges diethyl ether solution, evaporate to dryness, residue are dissolved in the 0.5ml ethyl acetate, are control medicinal material solution.
B. the preparation of need testing solution: get this product 40g, add 95% ethanol 80ml, jolting made mixing in 10 minutes, put in the water-bath and refluxed 1.5 hours, filtered while hot, filtrate is steamed to there not being the alcohol flavor, adds water 30ml agitation and dilution, moves in the separating funnel, with extracted with diethyl ether three times, each 20ml merges diethyl ether solution, uses 5%NaHCO
3Solution 30ml gradation washing, the about 25ml washing of water again, diethyl ether solution evaporate to dryness, residue are dissolved in the 0.5ml ethyl acetate, are need testing solution;
C. test according to thin-layered chromatography (appendix VIB of Chinese Pharmacopoeia version in 2000), draw control medicinal material solution 10ul, draw need testing solution 15ul, point is a developping agent with cyclohexane-chloroform-ethyl acetate (20: 5: 5) on same silica gel g thin-layer plate, launches, take out, dry, spray is dried by the fire to clear spot at 105 ℃ with aceticanhydride-concentrated sulphuric acid-absolute ethyl alcohol (1: 1: 5) solution.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.With the index of said method as the stability of this drug quality control and investigation technology.
Embodiment 5: get Chinese crude drug prepared rhizome of rehmannia 320g, the fruit of medicinal cornel (system) 160g, Chinese yam 160g, moutan bark 120g, Poria cocos 120g, rhizoma alismatis 120g, above-mentioned raw materials boiling three times, first and second time respectively adds 9 times of water gagings, decocted 2 hours, and added 7 times of water gagings for the third time, decocted 1 hour, collecting decoction, filter, filtrate is left standstill, and gets supernatant and is evaporated to the clear cream that relative density is 1.28～1.32 (85 ℃).The clear cream of every 100g adds refined honey 300g, mixing, promptly.
1. measuring content of paeoniflorin with high performance liquid chromatography (appendix VID of Chinese Pharmacopoeia version in 2000) measures and standard
A. chromatographic condition: with octadecylsilane chemically bonded silica is filling agent; Acetonitrile--water (15: 85) is moving phase, and the detection wavelength is 230nm;
B. the preparation of reference substance solution: it is an amount of that precision takes by weighing the Paeoniflorin reference substance, adds water and make the solution that every 1ml contains 40 μ g;
C. the preparation of need testing solution: get this product 1.5g, the accurate title, decide, and adds water 20ml, agitation and dilution moves in the separating funnel, adds water-saturated n-butanol and extracts 4 times, each 20ml, merge pure liquid, put evaporate to dryness in the water-bath, the residue water dissolves in right amount, in the dislocation 10ml measuring bottle, dilution also is settled to scale, shakes up, promptly.
D. determination method: precision is measured reference substance solution and each 10 μ l of need testing solution respectively, injects liquid chromatograph, measures content of paeoniflorin.Every gram contains moutan bark with Paeoniflorin (C
23H
28O
11) meter, must not be lower than 0.08mg.
2, this medicine Chinese crude drug fruit of medicinal cornel is differentiated with thin-layered chromatography
A. the preparation of control medicinal material solution: take by weighing fruit of medicinal cornel control medicinal material 3g, add ethanol 50ml and refluxed 1.0 hours, filter the filtrate evaporate to dryness, residue adds water 20ml dissolving, with ether 60ml gradation extraction, merges diethyl ether solution, evaporate to dryness, residue are dissolved in the 0.5ml ethyl acetate, are control medicinal material solution.
B. the preparation of need testing solution: get this product 30g, add 95% ethanol 60ml, jolting made mixing in 10 minutes, put in the water-bath and refluxed 1.0 hours, filtered while hot, filtrate is steamed to there not being the alcohol flavor, adds water 20ml agitation and dilution, moves in the separating funnel, with extracted with diethyl ether 4 times, each 20ml merges diethyl ether solution, uses 5%NaHCO
3Solution 30ml gradation washing, the about 20ml washing of water again, diethyl ether solution evaporate to dryness, residue are dissolved in the 0.5ml ethyl acetate, are need testing solution;
C. test according to thin-layered chromatography (appendix VIB of Chinese Pharmacopoeia version in 2000), draw control medicinal material solution 10ul, draw need testing solution 15ul, point is a developping agent with cyclohexane-chloroform-ethyl acetate (20: 5: 5) on same silica gel g thin-layer plate, launches, take out, dry, spray is dried by the fire to clear spot at 105 ℃ with aceticanhydride-concentrated sulphuric acid-absolute ethyl alcohol (1: 1: 5) solution.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.
With the index of said method as the stability of this drug quality control and investigation technology.
Embodiment 6: get Chinese crude drug prepared rhizome of rehmannia 320g, the fruit of medicinal cornel (system) 160g, Chinese yam 160g, moutan bark 120g, Poria cocos 120g, rhizoma alismatis 120g, above-mentioned raw materials boiling three times, first and second time respectively adds 9 times of water gagings, decocted 2 hours, and added 7 times of water gagings for the third time, decocted 1 hour, collecting decoction, filter, filtrate is left standstill, and gets supernatant and is evaporated to the clear cream that relative density is 1.28～1.32 (85 ℃).The clear cream of every 100g adds refined honey 300g, mixing, promptly.
1. measuring content of paeoniflorin with high performance liquid chromatography (appendix VID of Chinese Pharmacopoeia version in 2000) measures and standard
A. chromatographic condition: with octadecylsilane chemically bonded silica is filling agent; Acetonitrile--water (12: 88) is moving phase, and the detection wavelength is 230nm;
B. the preparation of reference substance solution: it is an amount of that precision takes by weighing the Paeoniflorin reference substance, adds ethanol and make the solution that every 1ml contains 10 μ g;
C. the preparation of need testing solution: get this product 2.5g, the accurate title, decide, and adds water 20ml, agitation and dilution moves in the separating funnel, adds water-saturated n-butanol and extracts 3 times, each 20ml, merge pure liquid, put evaporate to dryness in the water-bath, residue dissolves in right amount with ethanol, in the dislocation 25ml measuring bottle, dilution also is settled to scale, shakes up, promptly.
D. determination method: precision is measured reference substance solution and each 10 μ l of need testing solution respectively, injects liquid chromatograph, measures content of paeoniflorin.Every gram contains moutan bark with Paeoniflorin (C
23H
28O
11) meter, must not be lower than 0.08mg.
2, this medicine Chinese crude drug fruit of medicinal cornel is differentiated with thin-layered chromatography
A. the preparation of control medicinal material solution: take by weighing fruit of medicinal cornel control medicinal material 1g, add ethanol 50ml and refluxed 2.0 hours, filter the filtrate evaporate to dryness, residue adds water 10ml dissolving, with ether 30ml gradation extraction, merges diethyl ether solution, evaporate to dryness, residue are dissolved in the 0.5ml ethyl acetate, are control medicinal material solution.
B. the preparation of need testing solution: get this product 20g, add 95% ethanol 40ml, jolting made mixing in 10 minutes, put in the water-bath and refluxed 2.0 hours, filtered while hot, filtrate is steamed to there not being the alcohol flavor, adds water 20ml agitation and dilution, moves in the separating funnel, with extracted with diethyl ether three times, each 20ml merges diethyl ether solution, uses 5%NaHCO
3Solution 30ml gradation washing, the about 15ml washing of water again, diethyl ether solution evaporate to dryness, residue are dissolved in the 0.5ml ethyl acetate, are need testing solution;
C. test according to thin-layered chromatography (appendix VIB of Chinese Pharmacopoeia version in 2000), draw control medicinal material solution 15ul, draw need testing solution 20ul, point is a developping agent with cyclohexane-chloroform-ethyl acetate (20: 5: 5) on same silica gel g thin-layer plate, launches, take out, dry, spray is dried by the fire to clear spot at 105 ℃ with aceticanhydride-concentrated sulphuric acid-absolute ethyl alcohol (1: 1: 5) solution.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.With the index of said method as the stability of this drug quality control and investigation technology.
Embodiment 7: get Chinese crude drug prepared rhizome of rehmannia 320g, the fruit of medicinal cornel (system) 160g, Chinese yam 160g, moutan bark 120g, Poria cocos 120g, rhizoma alismatis 120g, above-mentioned raw materials boiling three times, first and second time respectively add 9 times of water gagings, decocted 2 hours, add 7 times of water gagings for the third time, decocted collecting decoction 1 hour, filter, filtrate is left standstill, and gets supernatant and is evaporated to the clear cream that relative density is 1.28～1.32 (85 ℃), and the clear cream of every 100g adds refined honey 300g, mixing, promptly.
1, measuring content of paeoniflorin with high performance liquid chromatography (appendix VID of Chinese Pharmacopoeia version in 2000) measures and standard:
A. chromatographic condition: with octadecylsilane chemically bonded silica is filling agent; Acetonitrile--water (12: 88) is moving phase, and the detection wavelength is 230nm, and number of theoretical plate calculates by the Paeoniflorin peak should be not less than 3000;
B. the preparation of reference substance solution: it is an amount of that precision takes by weighing the Paeoniflorin reference substance, adds methyl alcohol and make the solution that every 1ml contains 20 μ g;
C. the preparation of need testing solution: get this product 1g, the accurate title, decide, and adds water 10ml, agitation and dilution moves in the separating funnel, adds water-saturated n-butanol and extracts 4 times, each 10ml, merge pure liquid, put evaporate to dryness in the water-bath, residue dissolves in right amount with methyl alcohol, in the dislocation 10ml measuring bottle, dilution also is settled to scale, shakes up, promptly.
D. determination method: precision is measured reference substance solution and each 5 μ l of need testing solution respectively, injects liquid chromatograph, measures content of paeoniflorin.Every gram contains moutan bark with Paeoniflorin (C
23H
28O
11) meter, must not be lower than 0.08mg.
2, this medicine Chinese crude drug fruit of medicinal cornel is differentiated with thin-layered chromatography
A. the preparation of control medicinal material solution: take by weighing fruit of medicinal cornel control medicinal material 2g, add ethanol 50ml and refluxed 1 hour, filter the filtrate evaporate to dryness, residue adds water 10ml dissolving, with ether 30ml gradation extraction, merges diethyl ether solution, evaporate to dryness, residue are dissolved in the 0.5ml ethyl acetate, are control medicinal material solution.
B. the preparation of need testing solution: get this product 30g, add 95% ethanol 60ml, jolting made mixing in 10 minutes, put in the water-bath and refluxed 1 hour, filtered while hot, filtrate is steamed to there not being the alcohol flavor, adds water 30ml agitation and dilution, moves in the separating funnel, with extracted with diethyl ether three times, each 20ml merges diethyl ether solution, uses 5%NaHCO
3Solution 30ml gradation washing, the about 15ml washing of water again, diethyl ether solution evaporate to dryness, residue are dissolved in the 0.5ml ethyl acetate, are need testing solution;
C. test according to thin-layered chromatography (appendix VIB of Chinese Pharmacopoeia version in 2000), draw above-mentioned two kinds of each 10ul of solution, point is on same silica gel g thin-layer plate, with cyclohexane-chloroform-ethyl acetate (20: 5: 5) is developping agent, launch, take out, dry, spray is dried by the fire to clear spot at 105 ℃ with aceticanhydride-concentrated sulphuric acid-absolute ethyl alcohol (1: 1: 5) solution.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.
3, this medicine Chinese crude drug rhizoma alismatis is differentiated with thin-layered chromatography
A. the preparation of control medicinal material solution: get rhizoma alismatis control medicinal material 4g, add 60% ethanol 50ml refluxing extraction 1 hour, filter, filtrate is evaporate to dryness in water-bath, and residue adds water 10ml dissolving, extract 3 times with the ether jolting, each 10ml merges ether solution, evaporate to dryness, residue adds chloroform 1.0ml makes dissolving, as rhizoma alismatis control medicinal material solution.
B. the preparation of need testing solution: get this product 60g, add ethanol 120ml, put in the water-bath reflux 1 hour, put coldly, centrifugal 20 minutes (4000 rev/mins) get supernatant, in water-bath, steam to there not being the alcohol flavor, add water 30ml agitation and dilution, move in the separating funnel, the jolting that adds diethyl ether is extracted 3 times, and each 30ml merges ether solution, wash with water, ether solution evaporate to dryness, residue add chloroform 1.0ml makes dissolving, as need testing solution.
C. test according to thin-layered chromatography (appendix VI B), draw need testing solution 15ul, reference substance solution 10ul, putting respectively on same silica gel thin-layer plate, is developping agent with sherwood oil-ethyl acetate-formic acid (24: 6: 0.5), launches, take out, dry, spray is with sulfuric acid-aceticanhydride-ethanol solution (1: 1: 1), 105 ℃ dry by the fire to spot colour developing clear.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.With the index of said method as the stability of this drug quality control and investigation technology.
Claims (2)
1. the method for quality control of the liuwei Dihuang soft extract of being made by Chinese crude drug prepared rhizome of rehmannia 320g, the fruit of medicinal cornel (system) 160g, Chinese yam 160g, moutan bark 120g, Poria cocos 120g, rhizoma alismatis 120g is characterized in that this method comprises the following steps:
(1), with content of paeoniflorin in this medicine of high effective liquid chromatography for measuring
A. chromatographic condition
Filling agent: octadecylsilane chemically bonded silica
Moving phase is that proportioning is 10-20: 90-80 acetonitrile--water
Detect wavelength: 230 ± 2nm;
The preparation of b reference substance solution: it is an amount of that precision takes by weighing the Paeoniflorin reference substance, adds in methyl alcohol or ethanol or the water any and make the solution that every 1ml contains 5-50 μ g;
C. the preparation of need testing solution: get this product 0.5--3g, the accurate title, decide, and adds water 10--25ml, agitation and dilution moves in the separating funnel, adds water-saturated n-butanol and extracts 3--6 time, each 10--25ml, merge pure liquid, put evaporate to dryness in the water-bath, residue is with any an amount of dissolving in methyl alcohol or ethanol or the water, in the dislocation 10--25ml measuring bottle, dilution also is settled to scale, shakes up, promptly;
D. content of paeoniflorin assay method: precision is measured reference substance solution and each 5-20 μ l of need testing solution respectively, injects liquid chromatograph, measures content of paeoniflorin;
E. every gram contains moutan bark with Paeoniflorin C
23H
28O
11Meter must not be lower than 0.08mg;
(2), this medicine Chinese crude drug fruit of medicinal cornel is differentiated with thin-layered chromatography
A. the preparation of control medicinal material solution: get fruit of medicinal cornel control medicinal material 0.5-4g, add ethanol 20-100ml backflow 0.5-2 hour, filter, filtrate evaporate to dryness, residue add water 10-30ml dissolving, with ether 30-90ml gradation extraction, merge diethyl ether solution, evaporate to dryness, residue are dissolved in the 0.5ml ethyl acetate, are control medicinal material solution;
B. the preparation of need testing solution: get this product 10-50g, add 95% ethanol 20-100ml, jolting made mixing in 10 minutes, put in the water-bath backflow 0.5-2 hour, filtered while hot, filtrate is steamed to there not being the alcohol flavor, adds water 10-50ml agitation and dilution, moves in the separating funnel, with extracted with diethyl ether 2-5 time, each 10-50ml merges diethyl ether solution, uses 5%NaHCO
3Solution 20-50ml gradation washing, the about 10-50ml washing of water again, diethyl ether solution evaporate to dryness, residue are dissolved in the 0.5ml ethyl acetate, are need testing solution;
C. test according to thin-layered chromatography, drawing above-mentioned two kinds of each 5-20ul of solution, put on same silica gel g thin-layer plate, is that cyclohexane-chloroform-ethyl acetate of 20: 5: 5 is a developping agent with proportioning, launch, take out, dry, spray is aceticanhydride-concentrated sulphuric acid-ethanol solution of 1: 1: 5 with proportioning, dry by the fire to clear spot at 105 ℃, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.
2. the method for quality control of liuwei Dihuang soft extract according to claim 1 is characterized in that:
(1)、
A. the described moving phase of chromatographic condition is that proportioning is 12: 88 acetonitrile a--water;
B. the concrete operations preparation process of reference substance solution is: it is an amount of that precision takes by weighing the Paeoniflorin reference substance, adds methyl alcohol and make the solution that every 1ml contains 20 μ g;
C. the concrete operations preparation process of need testing solution is: get this product 1g, the accurate title, decide, and adds water 10ml, agitation and dilution, move in the separating funnel, add water-saturated n-butanol and extract 4 times, each 10ml, merge pure liquid, put evaporate to dryness in the water-bath, residue dissolves in right amount with methyl alcohol, in the dislocation 10ml measuring bottle, dilution also is settled to scale, shakes up;
D. content of paeoniflorin mensuration concrete operation method process is: precision is measured reference substance solution and each 5 μ l of need testing solution respectively, injects liquid chromatograph, measures content of paeoniflorin;
E. every gram contains moutan bark with Paeoniflorin C
23H
28O
11Meter must not be lower than 0.08mg;
(2), this medicine Chinese crude drug fruit of medicinal cornel is differentiated with thin-layered chromatography:
A. the concrete operations preparation process of control medicinal material solution is: take by weighing fruit of medicinal cornel control medicinal material 2g, adding ethanol 50ml refluxed 1 hour, filter, filtrate evaporate to dryness, residue add water 10ml dissolving, with ether 30ml gradation extraction, merge diethyl ether solution, evaporate to dryness, residue are dissolved in the 0.5ml ethyl acetate, are control medicinal material solution;
B. the preparation of need testing solution: get this product 30g, add 95% ethanol 60ml, jolting made mixing in 10 minutes, put in the water-bath and refluxed 1 hour, filtered while hot, filtrate is steamed to there not being the alcohol flavor, adds water 30ml agitation and dilution, moves in the separating funnel, with extracted with diethyl ether three times, each 20ml merges diethyl ether solution, uses 5%NaHCO
3Solution 30ml gradation washing, the about 15ml washing of water again, diethyl ether solution evaporate to dryness, residue are dissolved in the 0.5ml ethyl acetate;
C. test according to thin-layered chromatography, draw above-mentioned two kinds of each 10ul of solution, point is on same silica gel g thin-layer plate, with proportioning is that cyclohexane-chloroform-ethyl acetate of 20: 5: 5 is a developping agent, launch, take out, dry, spray is aceticanhydride-concentrated sulphuric acid-ethanol solution of 1: 1: 5 with proportioning, dries by the fire to clear spot at 105 ℃.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.
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| CN101229308B (en) * | 2008-01-11 | 2010-12-15 | 天津海世达检测技术有限公司 | Detection method of liu-wei tonic tablets dual-wavelength fingerprint chromatogram |
| CN101773592B (en) * | 2009-01-13 | 2012-04-25 | 北京亚东生物制药有限公司 | Method fro controlling quality of pill of six ingredients with rehmannia |
| CN102520103B (en) * | 2011-12-19 | 2014-03-12 | 辽宁省食品药品检验所 | Quantitative determination method of component in pill of six ingredients with rehmannia |
| CN104865319B (en) * | 2015-03-31 | 2017-03-22 | 湖南中医药大学 | Ultra performance liquid chromatographic dual-wavelength multi-index content determination method for six-ingredient glutinous rehmannia preparation |
| CN113820403B (en) * | 2020-06-19 | 2023-11-07 | 九芝堂股份有限公司 | Method for detecting content of compound medicine residues |
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