CN101716277A - Method for determining active ingredients of rhizoma smilacis glabrae medicinal material - Google Patents
Method for determining active ingredients of rhizoma smilacis glabrae medicinal material Download PDFInfo
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- CN101716277A CN101716277A CN200910194384A CN200910194384A CN101716277A CN 101716277 A CN101716277 A CN 101716277A CN 200910194384 A CN200910194384 A CN 200910194384A CN 200910194384 A CN200910194384 A CN 200910194384A CN 101716277 A CN101716277 A CN 101716277A
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Abstract
The invention discloses a method for determining active ingredients of a rhizoma smilacis glabrae medicinal material, relating to the filed of medicament analysis. In the invention, a fingerprint taking an astilbin chromatographic peak as an internal reference peak is established. The method comprises the following steps of: (a) preparing a test article solution; (b) taking astilbin as a reference substance and taking 0.10mg/mL astilbin solution prepared from methanol as a reference substance solution; (c) chromatographic conditions: taking a chromatographic column made of octadecylsilane chemically bonded silica as filler; adopting gradient elution, detecting the wavelength of 325+/-2 nm and the column temperature of 30+/-5DEG C; calculating the theoretical column plate number not less than 3,000 according to astilbin peak; and (d) determining. The fingerprint established by rhizoma smilacis glabrae ethanol-soluble extractives contains active ingredients with main activity of rhizoma smilacis glabrae. The fingerprint is used for judging the quality of rhizoma smilacis glabrae and the one sidedness for judging the quality of rhizoma smilacis glabrae by determining one or two chemical components can be avoided. The method has the advantages of simpleness, convenience, stability, high precision, favorable reproduction, easy grasp and capability of rapidly and accurately identifying the authenticity and merits of products.
Description
Affiliated technical field
The present invention relates to the pharmaceutical analysis field, specifically is to set up the fingerprint spectrum method of measuring active ingredients of rhizoma smilacis glabrae medicinal material.
Background technology
Rhizoma Smilacis Glabrae is the dry rhizome of liliaceous plant smilacis glabra (Smilax glabra Roxb.); Has dehumidifying, detoxifcation, easing joint movement.Be used for the limbs contracture due to damp and hot stranguria with turbid discharge, leukorrhagia, carbuncle, scrofula, scabies, syphilis and the mercurialism, bones and muscles pain.Because in the discriminating of gathering, links such as circulation, clinical practice are not too paid attention to kind, therefore kind is relatively more chaotic in the Rhizoma Smilacis Glabrae production and operation, quality has difference greatly.For example: it is adulterant that Smilax belongs to Rhizoma Smilacis Chinensis S.china L..Radix osteomelis schwerinais Rhizoma Smilacis Chinensis S.glauco-china Warb. makes RHIZOMA SMILACIS GLABRAE in the Nanchuan, Sichuan, and Zhenjiang, Jiangsu claims " bright native Siberian cocklebur ", for commonly using product in the place.Smilax lanceaefolia Roxb. Var.opaca A.DC. is equal Xiao smilax (Heterosmilax genus) plant China Xiao Rhizoma Smilacis Chinensis H chinensis Wang, short column Xiao Rhizoma Smilacis Chinensis H.yunnanensis Gagnep. and Xiao Rhizoma Smilacis Chinensis H japonica Kunth. rhizome.Preceding two kinds is the main flow kind of Smilax lanceaefolia Roxb. Var.opaca A.DC., in Sichuan, Hubei, Hebei, Shandong, Xinjiang, Ningxia, zhejiang and other places distributes and use.
At present, the net of Chinese Pharmacopoeia Commission (
Http:// www.chp.org.cn/) on, " Chinese pharmacopoeia consults on version in 2010, Chinese medicine (kind), version pharmacopeia (one one) part in 2010 increases revision and newly-increased kind list (the 4th batch) public notification period (2009.8.7～2009.8.31), to the quality standard of disclosed Chinese crude drug Rhizoma Smilacis Glabrae in the version Chinese Pharmacopoeia exposure draft in 2010, adopt the content of astilbin in the high effective liquid chromatography for measuring rhizoma smilacis glabrae medicinal material.Method in the pharmacopeia exposure draft just detects a chemical constituent in the rhizoma smilacis glabrae medicinal material, because the component complexity of Chinese medicine, therefore estimates its quality and should adopt and adapt with it, and the detection method of enriching authentication information can be provided.Analytical method in the pharmacopeia exposure draft is only carried out qualitative, quantitative analysis at certain effective ingredient of Chinese crude drug, can not control the Chinese crude drug quality effectively, produces the needs of going up quality of medicinal material control so can't satisfy.
Need study a kind of detection method that can effectively control the rhizoma smilacis glabrae medicinal material quality for this reason.
Summary of the invention
The objective of the invention is to set up the standard finger-print of rhizoma smilacis glabrae medicinal material, and measure effective ingredient in the Rhizoma Smilacis Glabrae, as one of index of quality control and real and fake discrimination with the method.
Is the finger printing at interior reference peak for reaching the foundation of the object of the invention employing high performance liquid chromatography with the astilbin chromatographic peak, specifically is to take following method:
(a) preparation of need testing solution: precision takes by weighing exsiccant rhizoma smilacis glabrae medicinal material powder 1g, places the 25mL volumetric flask, adds the ethanol water of 60% (V/V), supersound extraction, and standardize solution shakes up, and gets supernatant liquid filtering, promptly gets need testing solution;
(b) preparation of reference substance solution: get the astilbin reference substance, be made into the solution that every 1mL contains the 0.1mg astilbin with dissolve with methanol;
(c) chromatographic condition: chromatographic column is that octadecylsilane chemically bonded silica is a filler; Adopt gradient elution, mobile phase: by A is methanol mutually, and B is the gradient eluent that the acetic acid aqueous solution of 0.3% (V/V) is formed mutually; Flow velocity is 0.8mL/min; Detect wavelength 325 ± 2nm; Column temperature is 30 ± 5 ℃; Theoretical cam curve is calculated with the astilbin peak, is not less than 3000;
(d) measure: draw astilbin reference substance solution and need testing solution 5 μ L respectively, inject high performance liquid chromatograph, measure in accordance with the law, the record chromatogram.
Measuring rhizoma smilacis glabrae medicinal material according to method provided by the present invention, is interior reference peak with the chromatographic peak of astilbin, and its retention time is 1, calculates relative retention time, the standard finger-print of rhizoma smilacis glabrae medicinal material has at least 12 features to have the peak:
No. 1 average relative retention time in peak (being called for short RT, as follows) is 0.113, and RSD is 1.55%
No. 2 peak RT is 0.575, and RSD is 0.95%
No. 3 peak RT is 0.648, and RSD is 0.50%
No. 4 peak RT is 0.663, and RSD is 0.50%
No. 5 peak RT is 0.728, and RSD is 0.40%
No. 6 peak RT is 0.829, and RSD is 0.29%
No. 7 peak RT is 0.863, and RSD is 1.92%
No. 8 peak RT is 0.964, and RSD is 0.03%
No. 9 peak RT is 1.00, and this peak is interior reference peak
No. 10 peak RT is 1.085, and RSD is 0.39%
No. 11 peak RT is 1.117, and RSD is 0.11%
No. 12 peak RT is 1.165, and RSD is 0.13%
Advantage of the present invention is as follows:
1) the HPLC finger printing of setting up with the rhizoma smilacis glabrae medicinal material ethanol-soluble extractives is comprising the active substance of the main pharmacologically active of rhizoma smilacis glabrae medicinal material.
2), avoided judging the one-sidedness of rhizoma smilacis glabrae medicinal material quality because of only measuring one, two chemical constituent with the quality of finger printing do judgement rhizoma smilacis glabrae medicinal material.
3) the inventive method is easy, stable, precision is high, favorable reproducibility, be easy to grasp, and can differentiate the true and false quality of product quickly and accurately.
Description of drawings
The HPLC collection of illustrative plates of Fig. 1 astilbin
Fig. 2 rhizoma smilacis glabrae medicinal material standard finger-print
The constitutional diagram of the finger printing of the rhizoma smilacis glabrae medicinal material of 0 different batches of Figure 31
Fig. 4 discloses disclosed method " the astilbin content assaying method of rhizoma smilacis glabrae medicinal material " in the exposure draft by version pharmacopeia (an one) Chinese Pharmacopoeia Commission in 2010, criticizes the spectrogram of medical material gained with high effective liquid chromatography for measuring S2.
Further set forth technical scheme of the present invention below in conjunction with specific embodiments and the drawings
The specific embodiment
Embodiment 1 high effective liquid chromatography for measuring active ingredients of rhizoma smilacis glabrae medicinal material, setting up with the astilbin chromatographic peak is the standard finger-print at interior reference peak
1 instrument and reagent
1.1 instrument
High performance liquid chromatograph: Agilent1100 high performance liquid chromatograph, diode array detector.
1.2 reagent
The reference substance astilbin, self-control, colourless acicular crystal, mp 182-186 ℃ is a single speckle on the thin layer collection of illustrative plates, and it is unimodal (as shown in Figure 1) that HPLC detects substantially, and area normalization method is measured content greater than 99%.Rhizoma smilacis glabrae medicinal material is purchased in medical material market.
2 methods and result
Chromatographic condition: chromatographic column: octadecylsilane chemically bonded silica post, model are Lichrospher C-18 (4.6mm * 250mm, 5 μ m); Mobile phase: A is methanol mutually, and B is that the acetic acid aqueous solution composition gradient eluent of 0.3% (V/V) carries out gradient elution mutually, and elution requirement sees Table 1; Flow velocity: 0.8mLmin
-1Detect wavelength: 325 ± 2nm; Detection time: 105min; Column temperature: 35 ℃; Sample size: 5 μ L.Theoretical cam curve is calculated with the astilbin peak, should be not less than 3000.
Table 1 finger printing condition of gradient elution
Annotate: A is methanol mutually in the table, and B is the acetic acid aqueous solution of 0.3% (V/V) mutually.
The astilbin reference substance is got in the preparation of reference substance solution, is made into the solution that every 1mL contains the 0.10mg astilbin with dissolve with methanol, in contrast product solution;
Need testing solution prepares precision and takes by weighing rhizoma smilacis glabrae medicinal material dried powder 1 gram, places the 25mL volumetric flask, adds 60% alcoholic acid aqueous solution, fully soaks the back supersound extraction 3 hours, extracts to put after finishing coldly, and standardize solution shakes up, and leaves standstill; Get supernatant liquid filtering, promptly get the medical material need testing solution.
Measure
Draw astilbin reference substance solution and need testing solution 5 μ L respectively, inject high performance liquid chromatograph, measure in accordance with the law, the record chromatogram is seen accompanying drawing 1,2; Chromatographic peak with astilbin is interior reference peak, and its retention time is 1 calculating relative retention time.
Test method is with embodiment 1.The result as shown in Figure 3.10 batches of medical materials are purchased in Guangxi (lot number is respectively S1, S2, S3, S4) respectively, Hunan (lot number is respectively S5, S6, S7), three ground, Zhejiang (lot number is respectively S8, S9, S 10).
More than the total peak relative retention time statistical results of the 10 batches of medical materials see Table 2.From the result of table 2, the relative standard deviation (being called for short RSD, as follows) of the relative retention time at 12 total peaks (being called for short RT, as follows) is all less than 2%, and is more stable.
By to 10 batches of rhizoma smilacis glabrae medicinal material determining fingerprint patterns, compare its chromatogram, determine that total peak is 12:
No. 1 the average relative retention time in peak is 0.113, and RSD is 1.55%
No. 2 peak RT is 0.575, and RSD is 0.95%
No. 3 peak RT is 0.648, and RSD is 0.50%
No. 4 peak RT is 0.663, and RSD is 0.50%
No. 5 peak RT is 0.728, and RSD is 0.40%
No. 6 peak RT is 0.829, and RSD is 0.29%
No. 7 peak RT is 0.863, and RSD is 1.92%
No. 8 peak RT is 0.964, and RSD is 0.03%
No. 9 peak RT is 1.00, and this peak is interior reference peak
No. 10 peak RT is 1.085, and RSD is 0.39%
No. 11 peak RT is 1.117, and RSD is 0.11%
No. 12 peak RT is 1.165, and RSD is 0.13%
The chromatographic fingerprints of Chinese materia medica similarity evaluation 2004A of the system version that similarity evaluation adopts Chinese Pharmacopoeia committee to recommend is calculated similarity.The rhizoma smilacis glabrae medicinal material similarity that the results are shown in Table 3, ten different batches is all greater than 0.9.Finger printing after coupling is seen accompanying drawing 3.
Table 3 rhizoma smilacis glabrae medicinal material finger printing similarity result of calculation
The precision test of embodiment 3 methods of the present invention:
Criticizing with rhizoma smilacis glabrae medicinal material S2 is test sample, gets with a need testing solution, repeats sample introduction 6 times under above-mentioned chromatographic condition, writes down the retention time and the peak area of each characteristic peak, is reference with the astilbin, calculates the relative retention time and the peak area of each characteristic peak.The result shows that the RSD of each total peak relative retention time is not more than 1.0%, and the RSD of relative peak area is not more than 1.5%.The results are shown in Table 4, table 5.
The total peak of table 4 precision test relative retention time statistics
The total peak of table 5 precision test relative peak area statistics
The test of finger printing repeatability:
Precision takes by weighing exsiccant rhizoma smilacis glabrae medicinal material S2 and criticizes 6 parts in powder, every part of 1.0g, prepare need testing solution down by " 2.1 " item, carrying out HPLC under above-mentioned chromatographic condition analyzes, write down the retention time and the peak area of each characteristic peak, with the astilbin is reference, calculates the relative retention time and the peak area of each characteristic peak.The result shows that the RSD of each total peak relative retention time is not more than 1.0%, and the RSD of relative peak area is not more than 2.0%.The results are shown in Table 6, table 7.
The total peak of table 6 repeatability test relative retention time statistics
The total peak of table 7 repeatability test relative peak area statistics
The finger printing stability test:
The Poria medical material S2 that fetches earth criticizes need testing solution respectively 0,2,4,8,12, and the 24h sample introduction is analyzed, and writes down the retention time and the peak area of each characteristic peak, is reference with the astilbin, calculates the relative retention time and the peak area of each characteristic peak.The result shows that the RSD of each total peak relative retention time is not more than 1.75%, and the RSD of relative peak area is not more than 2.5%, illustrates that need testing solution is stable in 24h.The results are shown in Table 8, table 9.
The total peak of table 8 stability test relative retention time statistics
The total peak of table 9 stability test relative peak area statistics
More than test shows that this method is reliable and stable, meets the requirement of methodology checking.
Concrete assay method:
Chromatographic condition and system suitability test are filler with the octadecylsilane chemically bonded silica; With methanol-0.1% glacial acetic acid solution (39: 61) is mobile phase; The detection wavelength is 291nm.Flow velocity is 0.8ml/ minute; Number of theoretical plate calculates by the astilbin peak should be not less than 5000.
It is an amount of that the astilbin reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds 60% methanol and makes the solution that every 1ml contains 0.2mg, promptly.
This product powder (crossing sieve No. two) 0.8g is got in the preparation of need testing solution, and accurate the title decides, and puts in the round-bottomed flask, and the accurate 60% methanol 100ml that adds claims to decide weight, reflux 1 hour is put coldly, claims to decide weight again, supplies the weight that subtracts mistake with 60% methanol, shake up, filter, get subsequent filtrate, promptly.
Accurate respectively reference substance solution and each the 10 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, promptly.
Claims (2)
1. method of measuring active ingredients of rhizoma smilacis glabrae medicinal material, it is characterized in that adopting high performance liquid chromatography to set up with the astilbin chromatographic peak is that interior reference peak is the finger printing of characteristic peak, specifically is to take following method:
(a) preparation of need testing solution: precision takes by weighing exsiccant rhizoma smilacis glabrae medicinal material powder 1g, places the 25mL volumetric flask, adds 60% ethanol water, supersound extraction, and standardize solution shakes up, and gets supernatant liquid filtering, promptly gets need testing solution;
(b) preparation of reference substance solution: get the astilbin reference substance, be made into the solution that every 1mL contains the 0.10mg astilbin with dissolve with methanol;
(c) chromatographic condition: chromatographic column is that octadecylsilane chemically bonded silica is a filler; Adopt gradient elution, mobile phase: by A is methanol mutually, and B is the gradient eluent that the acetic acid aqueous solution of 0.3% (V/V) is formed mutually; Flow velocity is 0.8mL/min; Detect wavelength 325 ± 2nm; Column temperature is 30 ± 5 ℃; Theoretical cam curve is calculated with the astilbin peak, is not less than 3000;
(d) measure: draw astilbin reference substance solution and need testing solution 5 μ L respectively, inject high performance liquid chromatograph, measure in accordance with the law, the record chromatogram.
2. the method for mensuration active ingredients of rhizoma smilacis glabrae medicinal material as claimed in claim 1 is characterized in that: said is that interior reference peak is the finger printing of characteristic peak with the astilbin chromatographic peak, and the standard finger-print of rhizoma smilacis glabrae medicinal material has at least 12 features to have the peak:
No. 1 the average relative retention time in peak is 0.113, and RSD is 1.55%
No. 2 peak RT is 0.575, and RSD is 0.95%
No. 3 peak RT is 0.648, and RSD is 0.50%
No. 4 peak RT is 0.663, and RSD is 0.50%
No. 5 peak RT is 0.728, and RSD is 0.40%
No. 6 peak RT is 0.829, and RSD is 0.29%
No. 7 peak RT is 0.863, and RSD is 1.92%
No. 8 peak RT is 0.964, and RSD is 0.03%
No. 9 peak RT is 1.00, and this peak is interior reference peak
No. 10 peak RT is 1.085, and RSD is 0.39%
No. 11 peak RT is 1.117, and RSD is 0.11%
No. 12 peak RT is 1.165, and RSD is 0.13%
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109187820A (en) * | 2018-11-09 | 2019-01-11 | 湖南国华制药有限公司 | The method for building up and its finger-print of a kind of compound formula chinaroot greenbrier granules UPLC finger-print and application |
| CN110090274A (en) * | 2019-05-28 | 2019-08-06 | 青岛蔚蓝生物股份有限公司 | A kind of Chinese medicine composition and preparation method and application for treating the swollen humidifier fever of chicken kidney |
-
2009
- 2009-12-04 CN CN2009101943844A patent/CN101716277B/en active Active
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109187820A (en) * | 2018-11-09 | 2019-01-11 | 湖南国华制药有限公司 | The method for building up and its finger-print of a kind of compound formula chinaroot greenbrier granules UPLC finger-print and application |
| CN109187820B (en) * | 2018-11-09 | 2021-07-23 | 湖南国华制药有限公司 | Method for establishing UPLC fingerprint of compound formula chinaroot greenbrier granules |
| CN110090274A (en) * | 2019-05-28 | 2019-08-06 | 青岛蔚蓝生物股份有限公司 | A kind of Chinese medicine composition and preparation method and application for treating the swollen humidifier fever of chicken kidney |
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Owner name: GUANGZHOU BAIYUNSHAN JINGXIUTANG PHARMACEUTICAL CO Free format text: FORMER NAME: JINGXIUTANG (PHARMACEUTICAL) CO., LTD., GUANGZHOU;JINGXIUTANG (PHARMACEUTICAL) CO., LTD., GUANGZHOU |
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Address after: 510130 No. 179 Renmin South Road, Guangzhou, Guangdong, Liwan District Patentee after: Guangzhou Baiyunshan Jingxiutang Pharmaceutical Company Limited Address before: 510130 No. 179 Renmin South Road, Guangzhou, Guangdong, Liwan District Patentee before: Jingxiutang (Pharmacy) Co., Ltd., Guangzhou |