CN102735766A - Establishment method for polygonum perfoliatum medicinal material finger print, and standard finger print thereof - Google Patents
Establishment method for polygonum perfoliatum medicinal material finger print, and standard finger print thereof Download PDFInfo
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Abstract
The present invention discloses an establishment method for a polygonum perfoliatum medicinal material finger print, and a standard polygonum perfoliatum medicinal material finger print obtained by the method, wherein the method comprises the following steps: preparing a specimen solution, preparing a reference solution, measuring by a high performance liquid chromatograph, and processing data and map. Compared with the method in the prior art, the method of the present invention has the following advantages that: the method has characteristics of simpleness, stability, high precision, and good reproducibility; the obtained polygonum perfoliatum finger print has characteristics of more peaks, good peak shape, good peak separation effect, large peak area, easy identification, high similarity, accuracy and reliability, and can be adopted as the standard polygonum perfoliatum medicinal material finger print; a scientific approach capable of comprehensively evaluating the polygonum perfoliatum medicinal material is provided, the quality of the polygonum perfoliatum medicinal material can be effectively characterized, and the finger print can be adopted as one of standard indicators of quality detection and control of the polygonum perfoliatum medicinal material, and provides contributions for increase of qualities and therapeutic effects of polygonum perfoliatum and historical preparations thereof.
Description
Technical field
The present invention relates to the method for building up of the finger-print of Chinese crude drug, the method for building up of polygonum perfoliatum medicinal material high performance liquid chromatography (HPLC) finger-print especially, and the standard finger-print of the resulting polygonum perfoliatum medicinal material of method thus.
Background technology
China's traditional Chinese medicines and preparation thereof lack the tight quality standard and the detection means of science mostly; Be difficult to effectively control its inherent quality; Can not guarantee the safe, effective of medication, also not meet the requirement of international medical market, seriously restrict the development of China's Chinese medicine industry.Strengthening the Chinese crude drug quality controling research is Chinese medicine standardization, standardized key issue.Have only the quality control of science that Chinese crude drug is carried out, could guarantee traditional Chinese medicine quality, realize " safety, effective, stable, controlled " of Chinese medicine.
Polygonum perfoliatum (Polygonum perfoliatum L.) is the polygonaceae arsesmart, and it is with all herbal medicine, and nature and flavor acid, bitter, cold, slightly poisonous has effects such as clearing heat and detoxicating, inducing diuresis for removing edema, heat clearing and blood circulation promoting; Another name: snake only, Herba Lespedezae Cuneatae, ram's horn thorn, pears head thorn etc.; Be widely distributed in ground such as Sichuan, Hunan, Guizhou, be born in mountain valley, the bushes or by the ditch; Be Guizhou Miao ethnic group's medicinal material commonly used, be widely used aspect the treatment inflammation especially gynaecological imflammation better curative effect is being arranged among the people.It has higher medical value, and complete stool can be used as medicine, and determined curative effect, has very big potentiality to be exploited.The polygonum perfoliatum medicinal ingredient is complicated, mainly comprises following several types: the derivant of flavonoids, anthraquinone analog compound, phenylpropyl alcohol chlorins compound, Coumarins, plain type of phenylpropyl alcohol and some other common materials.Research shows that the polygonum perfoliatum medicinal material has certain pharmacological action.Therefore, a certain or several kinds of active components detect to it, just lose comprehensively, can not truly reflect the globality of polygonum perfoliatum drug effect.To control the quality of polygonum perfoliatum and prescribed preparation thereof fully and effectively, must control its material crowd integral body.
That traditional Chinese medicine fingerprint is meant is common in certain Chinese crude drug or the Chinese patent drug, have distinctive certain type or the chromatogram of number constituents or the collection of illustrative plates of spectrum.Do not have under the clear and definite situation in the present stage Effective Components of Chinese Herb overwhelming majority, finger-print has great importance for the quality of effective control Chinese crude drug or Chinese patent drug.The Japan main manufacturing enterprise of Chinese prescription medicine just adopts the high-efficiency liquid-phase fingerprint control of quality in the eighties in 20th century.Germany, France also adopt the high-efficiency liquid-phase fingerprint method in to the quality control of ginkgo biloba p.e.In the plant herbal medicine guide that U.S. FDA was formulated in recent years clearly the method for quality control of finger-print as the compounding substances crowd.Finger-print is become a consensus of the international community as Chinese herbal medicine and extraction of substance amount control method thereof at present.In order effectively to control and estimate the quality of polygonum perfoliatum medicinal material; Make more perfect, the science of its Quality Control Technology; The applicant has carried out comprehensive and systematic finger-print research to the polygonum perfoliatum ethanol extract, has set up the finger print measuring method of polygonum perfoliatum medicinal material ethanol extract.
Summary of the invention
The objective of the invention is to: method for building up and standard finger-print thereof that a kind of polygonum perfoliatum medicinal materials fingerprint is provided.The present invention provides a kind of better polygonum perfoliatum quality determining method through to the research of polygonum perfoliatum medicinal material HPLC finger-print, has remedied the deficiency of existing Quality Control Technology, makes the Quality Control Technology of polygonum perfoliatum medicinal material more perfect, science.
Technical scheme of the present invention: the method for building up of polygonum perfoliatum medicinal materials fingerprint may further comprise the steps:
(1) preparation of need testing solution: get the polygonum perfoliatum pulverizing medicinal materials, sieve, take by weighing sample, extract 1～4 time with alcohol heating reflux, each 0.5-3 hour, concentrate then, use methanol constant volume, to filter, filtrating is as need testing solution;
(2) preparation of reference substance solution: get an amount of Quercetin, Quercetin-3-O-β-D-glucuronic acid reference substance, add methyl alcohol respectively and process reference substance solution;
(3) measure: accurate respectively need testing solution and the reference substance solution drawn, measure with high performance liquid chromatograph; The polygonum perfoliatum need testing solution is injected high performance liquid chromatograph, with 0.05%H
3PO
4The WS is that A phase, acetonitrile are that B carries out gradient elution mutually, and eluent gets into the DAD UV-detector, and detecting device is gathered the detection data of 85min;
(4) relative retention time and the peak area with the reference substance chromatographic peak is 1, calculates the relative retention time and the peak area of test sample chromatographic peak, with finger-print software the data and the collection of illustrative plates of gained handled, and promptly obtains polygonum perfoliatum medicinal material standard finger-print.
In the preceding method, the chromatographic condition during high-performance liquid chromatogram determination is: chromatographic column is filler with the octadecylsilane chemically bonded silica; Moving phase is B phase acetonitrile and A 0.05%H mutually
3PO
4The WS; Detect wavelength 340nm; Flow velocity 1mL/min.
In the preceding method, the preparation process of need testing solution is: get the polygonum perfoliatum pulverizing medicinal materials and cross 40 mesh sieves, accurately take by weighing sample 1 gram; Add 70% ethanolic solution 80mL, place 80 ℃ of water-baths, heating and refluxing extraction 1 time, 3h; Be concentrated into dried then;, filter to 25mL with methanol constant volume, promptly get need testing solution.
In the preceding method; Being prepared as of reference substance solution: get Quercetin, Quercetin-3-O-β-two kinds of reference substances of D-glucuronic acid; Add methyl alcohol respectively, process two kinds of reference substance solution that every 1ml contains 10 μ g Quercetins, 10 μ g Quercetins-3-O-β-D-glucuronic acid respectively.
Accurate respectively need testing solution and each 20 μ L of reference substance solution of drawing in the step of preceding method (3).
In the eluent gradient elution process of preceding method, the A of eluent, B phase transformation turn to: 0～10min, B phase 10%～15%; 10～40min, B phase 15%～25%; 40～55min, B phase 25%～35%; 55～75min, B phase 35%～50%; 75～76min, B phase 50%～90%; 76～85min, B phase 90%.
The polygonum perfoliatum medicinal material standard finger-print that obtains of the method for building up of polygonum perfoliatum medicinal materials fingerprint as stated; Shared peak in this collection of illustrative plates must not be less than 15.
Compared with prior art, the present invention has the following advantages:
1. research shows that the polygonum perfoliatum medicinal material has various concrete pharmacological actions, if its a certain active component is detected, just loses comprehensively.The HPLC finger-print that the present invention sets up with the polygonum perfoliatum ethanol extract; But a kind of science approach of thoroughly evaluating polygonum perfoliatum medicinal material is provided; Can characterize the quality of polygonum perfoliatum medicinal material effectively, can be used as one of standard index of detection of polygonum perfoliatum quality of medicinal material and quality control.
2. the polygonum perfoliatum medicinal materials fingerprint is done as a wholely to treat, pay attention to its whole facial feature, both avoided only measuring the one-sidedness that one, two chemical constitution is judged the polygonum perfoliatum total quality, having reduced again is the artificial possibility of handling of requisite quality.The present invention provides new reference standard for quality complete, that accurately estimate the polygonum perfoliatum medicinal material, for quality and the curative effect that improves polygonum perfoliatum and prescribed preparation thereof contributes.
3. the present invention has that method is easy, stable, precision is high and characteristics such as favorable reproducibility.Because finger-print is not in order to measure the accurate content of certain composition, but will fully reflect the information of chemical constitution.Therefore, the present invention is chosen in the 340nm wavelength and measures, and it is more that it goes out the peak, and the information of reflection is more complete; Each peak absorption value is good, and baseline is steady, has also avoided the big absorbing state of near ultraviolet impurity peaks.
4. in the past for measuring the content of certain chemical constitution in the polygonum perfoliatum, often will sample be carried out than complex processing, and the present invention selects its composition of heating and refluxing extraction for use, method for distilling is simple, has practiced thrift the cost and the time of check.
5. the peak of the inventive method gained polygonum perfoliatum finger-print is many, and peak shape is good, is easy to differentiate, similarity is high, accurately and reliably.
Below in conjunction with embodiment and accompanying drawing the present invention is done further explanation.
Description of drawings
Fig. 1 is the polygonum perfoliatum medicinal materials fingerprint of heating and refluxing extraction.
Fig. 2 is the polygonum perfoliatum medicinal materials fingerprint of ultrasonic Extraction.
Fig. 3 is that methyl alcohol is as the polygonum perfoliatum HPLC finger-print that extracts solvent.
Fig. 4 is that 90% ethanol is as the polygonum perfoliatum HPLC finger-print that extracts solvent.
Fig. 5 is that 70% ethanol is as the polygonum perfoliatum HPLC finger-print that extracts solvent.
Fig. 6 is that ethyl acetate is as the polygonum perfoliatum HPLC finger-print that extracts solvent.
Fig. 7 is with CH
3CN-0.2%HCOOH is the polygonum perfoliatum finger-print that moving phase is measured by different gradients.
Fig. 8 is with CH
3CN-0.2%HAc is the polygonum perfoliatum finger-print that moving phase is measured by different gradients.
Fig. 9 is with CH
3CN-0.02%H
3PO
4The polygonum perfoliatum finger-print of measuring by different gradients for moving phase.
Figure 10 is with CH
3CN-0.05%H
3PO
4The polygonum perfoliatum finger-print of measuring by different gradients for moving phase.
Figure 11 is the polygonum perfoliatum medicinal materials fingerprint that records with chromatographic column ZORBAX SB-C18 (4.6 * 250mm, 5 μ m).
Figure 12 is the polygonum perfoliatum medicinal materials fingerprint that records with chromatographic column Eclipse XDB-C8 (4.6 * 250mm, 5 μ m).
Figure 13 is the polygonum perfoliatum medicinal materials fingerprint that records with chromatographic column Lichrospher C18 (4.6 * 250mm, 5 μ m).
Figure 14 is the polygonum perfoliatum medicinal materials fingerprint of measuring in the 258nm wavelength.
Figure 15 is the polygonum perfoliatum medicinal materials fingerprint of measuring in the 310nm wavelength.
Figure 16 is the polygonum perfoliatum medicinal materials fingerprint of measuring in the 340nm wavelength.
Figure 17 is the polygonum perfoliatum medicinal materials fingerprint of measuring in the 370nm wavelength.
Figure 18 is the common pattern figure of polygonum perfoliatum medicinal materials fingerprint.
Figure 19 is the stacking diagram of polygonum perfoliatum medicinal materials fingerprint.
Figure 20 is a polygonum perfoliatum medicinal material HPLC standard finger-print.
Embodiment
Embodiments of the invention: the method for building up of polygonum perfoliatum medicinal material HPLC finger-print and polygonum perfoliatum medicinal material standard finger-print
1. instrument and reagent
1.1 instrument
Agilent 1100 high performance liquid chromatographs; Agilent DAD detecting device; The Agilent chromatographic work station.
1.2 reagent
Quercetin, Quercetin-3-O-β-D-glucuronic acid by laboratory member's separation and purification, are accredited as Quercetin, Quercetin-3-O-β-D-glucuronic acid through spectrum datas such as IR, 1H-NMR and 13C-NMR; Acetonitrile, chromatographically pure; The Robust pure water; The syringe-type nuclepore membrane filter, diameter 13mm, 0.45 μ m; The polygonum perfoliatum medicinal material.
2. method and result
2.1 the selection of method for distilling
In experimentation, select reflux and ultrasonic Extraction dual mode to measure, see accompanying drawing 1, Fig. 2 respectively, the result shows, heating and refluxing extraction can embody more chemical constitution.In order to choose the condition of finger-print better and more comprehensively; Experiment is measured methyl alcohol, 90% ethanol, 70% ethanol, ethyl acetate equal solvent; See accompanying drawing 3, Fig. 4, Fig. 5, Fig. 6 respectively, in conjunction with the comparison of several big peak-to-peak areas, 70% alcohol extract effect is best as a result.Experiment is investigated 70% consumption of ethanol, extracts the polygonum perfoliatum medicinal material with 40mL, 60mL, 70mL, 80mL respectively, and the result finds that the 80mL consumption is best.Also heating and refluxing extraction time 2h, 3h, 4h, 5h are measured, the result finds, the extraction time that reflux 3h is best suited for.
In sum, the preparation condition of need testing solution is: get each about 1g of different batches polygonum perfoliatum medicinal powder, the accurate title, decide, and places 150mL tool plug conical flask; Add 70% ethanol 80mL, put reflux 3h in the water-bath (80 ℃), cooling filters; Get subsequent filtrate, water-bath volatilizes, medicinal extract with methanol constant volume to the 25mL volumetric flask; Filter with 0.45 μ m filter membrane, get subsequent filtrate, promptly get.
2.2 chromatographic condition
2.2.1 the selection of moving phase
The applicant has used four groups of moving phases to compare, i.e. CH
3CN-0.2%HCOOH water system, CH
3CN-0.2% HAc water system, CH
3CN-0.02%H
3PO
4Water system, CH
3CN-0.05%H
3PO
4Water system.The gained result sees accompanying drawing 7, Fig. 8, Fig. 9, Figure 10 respectively.Visible by figure, the spectrogram of Figure 10 is best.CH just
3CN-0.05%H
3PO
4Water system is obviously better than other 3 kinds of systems as the moving phase effect, the gradient elution best results, and the degree of separation of each chromatographic peak is good on the spectrogram, and retention time is moderate, therefore selects CH
3CN-0.05%H
3PO
4Water system is as the eluent gradient elution system.
2.2.2 the selection of liquid-phase chromatographic column
The research of finger-print generally need compare with chromatographic columns different more than three, after experiment relatively, fixes and finishes whole finger-print with same root chromatogram column.The applicant has investigated 3 different chromatographic columns [1. chromatographic column ZORBAX SB-C18 (4.6 * 250mm, 5 μ m); 2. EclipseXDB-C8 (4.6 * 250mm, 5 μ m) chromatographic column; 3. chromatographic column Lichrospher C18 (4.6 * 250mm, 5 μ m)], under selected identical chromatographic conditions, measure.
Best by the visible post separating effect 3. of gained spectrogram result (seeing accompanying drawing 11, Figure 12, Figure 13), post is imitated the highest, so select post 3. to carry out the detection of polygonum perfoliatum medicinal materials fingerprint.
2.2.3 detect the selection of wavelength
In order to obtain multi-level information, the applicant has selected 258nm, 310nm, 340nm and four wavelength of 370nm to measure simultaneously, and carries out full wavelength scanner; Choose optimal wavelength; See accompanying drawing 14, Figure 15, Figure 16 and Figure 17, the chromatographic peak that the result occurs under wavelength 340nm is more, and Quercetin-3-O-β-D-glucuronic acid absorbs the strongest; And baseline is steady, so select the detection wavelength of 340nm as polygonum perfoliatum HPLC finger-print.
Through above experiment, confirm that chromatographic condition is following:
Chromatographic column: Lichrospher C18 (4.6 * 250mm, 5 μ m); Moving phase: acetonitrile-0.05% phosphate aqueous solution; Flow velocity: 1mL/min; Detect wavelength 340nm; Column temperature: 30 ℃.
3. the mensuration of polygonum perfoliatum medicinal materials fingerprint
3.1.1 the preparation of polygonum perfoliatum finger-print
The preparation of reference substance solution: get Quercetin, Quercetin-3-O-β-two kinds of reference substances of D-glucuronic acid, add methyl alcohol respectively, process two kinds of reference substance solution that every 1ml contains 10 μ g Quercetins, 10 μ g Quercetins-3-O-β-D-glucuronic acid respectively.
Object of reference: select for use Quercetin, Quercetin-3-O-β-D-glucuronic acid as object of reference.
The preparation of need testing solution: get each about 1g of different batches polygonum perfoliatum medicinal powder, the accurate title, decide, and places 150mL tool plug conical flask, adds 70% ethanol 80mL; Put reflux 3h in the water-bath (80 ℃), cooling filters; Get subsequent filtrate, water-bath volatilizes, medicinal extract with methanol constant volume to the 25mL volumetric flask; Filter with 0.45 μ m filter membrane, get subsequent filtrate, promptly get.
Accurate respectively need testing solution and each 20 μ L of reference substance solution of drawing inject high performance liquid chromatograph respectively, measure, and write down 85 minutes chromatogram, promptly obtain required finger-print.With the relative retention time of Quercetin, Quercetin-3-O-β-D-glucuronic acid chromatographic peak and the relative retention time and the peak area of calculated by peak area test sample chromatographic peak.Simultaneously, with finger-print software gained data and collection of illustrative plates are handled and promptly obtained standard finger-print.
3.1.2 precision experiment
Get the polygonum perfoliatum medicinal material of the Zha Zuo that improves literature; Described need testing solution preparation method prepares test sample by technical scheme of the present invention, and continuous sample introduction 6 times serves as to detect index with Quercetin-3-O-β-D-glucuronic acid; The RSD that calculates relative peak area is 2.47%, and the result shows that instrument precision is good.
3.1.3 stability experiment
Get the polygonum perfoliatum medicinal material of the Zha Zuo that improves literature, described need testing solution preparation method prepares test sample by technical scheme of the present invention, respectively 0,2,4,8,12, the 24h sample introduction; Sample size is 20 μ L, serves as to detect index with Quercetin-3-O-β-D-glucuronic acid, and the RSD that calculates relative peak area is 2.51%; The result was illustrated in 24 hours; The chromatogram of polygonum perfoliatum finger-print is basicly stable, does not have significant change, meets the finger-print requirement.
3.1.4 reappearance experiment
Get 6 parts of the polygonum perfoliatum medicinal materials of the Zha Zuo that improves literature; By 6 parts of test samples of the parallel preparation of the described need testing solution preparation method of technical scheme of the present invention; The difference sample introduction; With Quercetin-3-O-β-D-glucuronic acid serves as to detect index, and the RSD that calculates relative peak area is 2.51%, and the result shows that the reappearance of this method is good.
3.1.5 confirm and the similarity at the total peak of polygonum perfoliatum medicinal materials fingerprint are calculated
Through the mensuration of polygonum perfoliatum medicinal materials fingerprint to 51 batches of different places of production, the whole nation; Crude drug source is seen table 1; And through area of computer aided similarity evaluation system fingerprint collection of illustrative plates software (traditional Chinese medicine fingerprint similarity evaluation system of Chinese Pharmacopoeia Commission (2004 A)) data of chromatogram and mensuration are handled, confirm 15 at total peak.Obtained the finger-print stacking diagram (accompanying drawing 19) of 51 batches of medicinal materials; Finger-print common pattern figure (accompanying drawing 18); Similarity analysis result (the table 2 of medicinal materials fingerprint; Sequence number is with table 1), be object of reference wherein with Quercetin-3-O-β-D-glucuronic acid, its peak is No. 6 peaks (accompanying drawing 18).
Polygonum perfoliatum finger-print similarity calculating of the different places of production, the whole nation and similarity analysis result are following:
51 batches of polygonum perfoliatum crude drug sources of table 1
| Sequence number | The place of production | Acquisition time | Sequence number | The place of production | Acquisition time |
| 1 | All spare Jiangzhou | 2008.8 | 27 | Hua Xi | 2008.6 |
| 2 | The Hou Shan of teachers college, the south of Guizhou Province | 2008.8.7 | 28 | Close earth dam the Meng | 2008.8.9 |
| 3 | Campus, teachers college, the south of Guizhou Province | 2008.8.7 | 29 | Newly add the cattle farm, stockaded village | 2008.8.8 |
| 4 | The Majiang County | 2008.8.9 | 30 | The medicinal garden | 2009.1.8 |
| 5 | All spare the Majiang duck pool | 2010.10 | 31 | The expensive road 63km that abides by locates southwards | 2008.8.1 |
| 6 | The Guiding County | 2008.8 | 32 | Expensively abide by road 63km northwards | 2008.8.1 |
| 7 | The Guiding County | 2010.7.2 | 33 | Waterside town | 2008.8.1 |
| 8 | The Danzhai County | 2008.8 | 34 | Waterside town | 2010.8.25 |
| 9 | The Danzhai County | 2010.10 | 35 | Six four jiaos of fields | 2008.9 |
| 10 | Longli paddy pin | 2008.8.1 | 36 | Chishui | 2010.8.25 |
| 11 | The Longli | 2010.7.23 | 37 | The Renhuai | 2010.8.23 |
| 12 | All spare the Gan Tang town | 2010.10 | 38 | The bright rock of Bijie | 2008.8 |
| 13 | Song Tao group stockaded village | 2010.8.22 | 39 | The Bijie Prefecture | 2010.9.3 |
| 14 | Jiangkou De Wang | 2008.8.27 | 40 | The Zhenfeng County | 2007.8 |
| 15 | Along the river county | 2008.8.20 | 41 | Long-range drugmaker | 2011.4 purchase |
| 16 | Paddy field, Guiyang town | 2010.9.3 | 42 | Tongrentang | 2011.4 purchase |
| 17 | Gardens and orchards medicinal material market | 2011.3 purchase | 43 | The Jinping County | 2008.9.3 |
| 18 | Medicinal material market | 2008.8 | 44 | The Dafang County | 2010.8.1 |
| 19 | The Zha Zuo town of improving literature | 2010.8.22 | 45 | Dabie Mountain, Anhui | 2008.8 |
| 20 | The Xiuwen County | 2010.8.10 | 46 | Manjusri Guanshan Mountain, Henan | 2008.8 |
| 21 | The length of a specified duration of improving literature town | 2008.8.2 | 47 | The Dao County, Hunan | 2010.5 |
| 22 | Xifeng County | 2010.7.29 | 48 | Chunan, Zhejiang | 2008.8 |
| 23 | Persimmon flower level ground, Xifeng | 2008.8.2 | 49 | The Chenxi, Hunan | 2008.8.15 |
| 24 | The Xifeng | 2008.8.11 | 50 | The unknown time place of production 2 | |
| 25 | Kweiyang Guan Gongpo | 2008.8.9 | 51 | The unknown time place of production 1 | |
| 26 | Herd-boy Guan Daxing field | 2008.8.9 |
The similarity analysis result of table 2 polygonum perfoliatum medicinal materials fingerprint
| Sequence number | Similarity | Sequence number | Similarity | | Similarity | |
| 1 | 0.993 | 18 | 0.984 | 35 | 0.432 | |
| 2 | 0.972 | 19 | 0.977 | 36 | 0.974 | |
| 3 | 0.988 | 20 | 0.981 | 37 | 0.955 | |
| 4 | 0.928 | 21 | 0.976 | 38 | 0.951 | |
| 5 | 0.953 | 22 | 0.984 | 39 | 0.933 | |
| 6 | 0.946 | 23 | 0.992 | 40 | 0.968 | |
| 7 | 0.993 | 24 | 0.996 | 41 | 0.982 | |
| 8 | 0.893 | 25 | 0.980 | 42 | 0.942 | |
| 9 | 0.993 | 26 | 0.991 | 43 | 0.74 | |
| 10 | 0.998 | 27 | 0.962 | 44 | 0.991 | |
| 11 | 0.978 | 28 | 0.944 | 45 | 0.967 | |
| 12 | 0.994 | 29 | 0.992 | 46 | 0.994 | |
| 13 | 0.967 | 30 | 0.988 | 47 | 0.866 | |
| 14 | 0.883 | 31 | 0.991 | 48 | 0.993 | |
| 15 | 0.841 | 32 | 0.962 | 49 | 0.553 | |
| 16 | 0.911 | 33 | 0.969 | 50 | 0.990 | |
| 17 | 0.988 | 34 | 0.933 | 51 | 0.895 |
3.1.6 the description at common characteristic peak: (seeing accompanying drawing 18)
(1) characteristic peak has 15, and is wherein maximum, the highest with the peak area of Quercetin-3-O-β-D-glucuronic acid.
(2) 2,3,4,5,6,8,9,10,13 the peak area and the peak height at totally nine peaks is bigger.
(3) reference substance Quercetin-3-O-β-D-glucuronic acid is No. 6 peaks, and its peak height, peak area and peak shape are all fine.The total finger-print of gained polygonum perfoliatum medicinal material such as accompanying drawing 18.
(4) similarity coefficient at common characteristic peak is totally higher.The similarity that finger-print is described is higher, can be used as the standard finger-print of polygonum perfoliatum medicinal material.
4. above experimental result shows, uses this method the polygonum perfoliatum medicinal material is measured, and method is simple and reliable; The finger-print peak shape of being set up is good, the peak good separating effect, and peak area is bigger; Can be used as the standard finger-print of polygonum perfoliatum medicinal material; To polygonum perfoliatum plantation, the quality control of producing and the discriminating of polygonum perfoliatum medicinal material a good approach is provided.
Claims (8)
1. the method for building up of a polygonum perfoliatum medicinal materials fingerprint is characterized in that: may further comprise the steps:
(1) preparation of need testing solution: get the polygonum perfoliatum pulverizing medicinal materials, sieve, take by weighing sample, extract 1～4 time with alcohol heating reflux, each 0.5-3 hour, concentrate then, use methanol constant volume, to filter, filtrating is as need testing solution;
(2) preparation of reference substance solution: get an amount of Quercetin, Quercetin-3-O-β-D-glucuronic acid reference substance, add methyl alcohol respectively and process reference substance solution;
(3) measure: accurate respectively need testing solution and the reference substance solution drawn, measure with high performance liquid chromatograph; The polygonum perfoliatum need testing solution is injected high performance liquid chromatograph, with 0.05%H
3PO
4The WS is that A phase, acetonitrile are that B carries out gradient elution mutually, and eluent gets into the DAD UV-detector, and detecting device is gathered the detection data of 85min;
(4) relative retention time and the peak area with the reference substance chromatographic peak is 1, calculates the relative retention time and the peak area of test sample chromatographic peak, with finger-print software the data and the collection of illustrative plates of gained handled, and promptly obtains polygonum perfoliatum medicinal material standard finger-print.
2. according to the method for building up of the said polygonum perfoliatum medicinal materials fingerprint of claim 1, it is characterized in that: the chromatographic condition during high-performance liquid chromatogram determination is: chromatographic column is filler with the octadecylsilane chemically bonded silica; Moving phase is B phase acetonitrile and A 0.05%H mutually
3PO
4The WS; Detect wavelength 340nm; Flow velocity 1mL/min.
3. according to the method for building up of the said polygonum perfoliatum medicinal materials fingerprint of claim 1, it is characterized in that: the preparation process of need testing solution is: get the polygonum perfoliatum pulverizing medicinal materials and cross 40 mesh sieves, accurately take by weighing sample 1 gram; Add 70% ethanolic solution 80mL, place 80 ℃ of water-baths, heating and refluxing extraction 1 time, 3h; Be concentrated into dried then;, filter to 25mL with methanol constant volume, promptly get need testing solution.
4. according to the method for building up of the said polygonum perfoliatum medicinal materials fingerprint of claim 1; It is characterized in that: being prepared as of reference substance solution: get Quercetin, Quercetin-3-O-β-two kinds of reference substances of D-glucuronic acid; Add methyl alcohol respectively, process two kinds of reference substance solution that every 1ml contains 10 μ g Quercetins, 10 μ g Quercetins-3-O-β-D-glucuronic acid respectively.
5. according to the method for building up of the said polygonum perfoliatum medicinal materials fingerprint of claim 1, it is characterized in that: accurate respectively need testing solution and each 20 μ L of reference substance solution of drawing in the step (3).
6. according to the method for building up of the said polygonum perfoliatum medicinal materials fingerprint of claim 1, it is characterized in that: in the eluent gradient elution process, the A of eluent, B phase transformation turn to: 0～10min, B phase 10%～15%; 10～40min, B phase 15%～25%; 40～55min, B phase 25%～35%; 55～75min, B phase 35%～50%; 75～76min, B phase 50%～90%; 76～85min, B phase 90%.
7. the polygonum perfoliatum medicinal material standard finger-print that obtains of the method for building up of polygonum perfoliatum medicinal materials fingerprint according to claim 1.
8. polygonum perfoliatum medicinal material standard finger-print according to claim 7 is characterized in that: the shared peak in the said collection of illustrative plates must not be less than 15.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012101612805A CN102735766A (en) | 2012-05-23 | 2012-05-23 | Establishment method for polygonum perfoliatum medicinal material finger print, and standard finger print thereof |
Applications Claiming Priority (1)
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