CN102279240A - Research technology of polygonum flaccidum meism medicinal material high performance liquid chromatography (HPLC) characteristic fingerprint spectrum - Google Patents
Research technology of polygonum flaccidum meism medicinal material high performance liquid chromatography (HPLC) characteristic fingerprint spectrum Download PDFInfo
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- CN102279240A CN102279240A CN2011101854091A CN201110185409A CN102279240A CN 102279240 A CN102279240 A CN 102279240A CN 2011101854091 A CN2011101854091 A CN 2011101854091A CN 201110185409 A CN201110185409 A CN 201110185409A CN 102279240 A CN102279240 A CN 102279240A
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Abstract
The invention provides a method for establishing a polygonum flaccidum meism medicinal material high performance liquid chromatography (HPLC) characteristic fingerprint spectrum. The method comprises the following steps of collecting eleven groups of polygonum flaccidum meism medicinal materials from different producing areas, preparing test samples, preparing reference objects, establishing chromatographic conditions, constructing fingerprint spectrum and the like. Through establishing a polygonum flaccidum meism medicinal material high performance liquid chromatography (HPLC) characteristic fingerprint spectrum, the method realizes a purpose of stabilizing and controlling polygonum flaccidum meism medicinal material quality.
Description
Technical field
The present invention relates to a kind of quality control process of using in the fields such as high performance liquid chromatography (HPLC) instrument, Chinese medicine, especially be applied to polygonum flaccidum medicinal material HPLC characteristic fingerprint pattern.
Background technology
Polygonum flaccidum is a polygonaceae plant water polygonum flaccidum
Polygonum hydropiperL. or non-irrigated polygonum flaccidum
P.flaccidumMeisn. dry herb
[1]Its flavor is sad, warm in nature, returns spleen, large intestine channel, leads stagnant dampness elimination; Polygonum flaccidum be China the very abundant traditional Chinese herbal medicine of a kind of resource, in temperate zone, the Northern Hemisphere or the subtropical zone distribution is all arranged, in the most of area of China growth is arranged all.All peppery few function is described in many medical books of Ancient Times in China: Compendium of Material Medica: " polygonum flaccidum, suffering, temperature, " " not Lu ": " the knotweed leaf is returned tongue, removes the intestine and small intestine perverse trend, beneficial will in the profit." Tang Materia Medica: " and main by snakebite, smash and apply it; Strand juice clothes, it is vexed to control snake venom people vital organs; Poach soaks pin and holds it, and the beriberi that disappears is swollen." supplement to the Herbal: " knotweed leaf, main Xuan addiction are got 60g every day and are boiled clothes; By choleraic myospasm, get more and boil the Tang Jire pin of fighting; Leaf is smash and is applied fox thorn mole; Also main children's's sore." " south of the Five Ridges gather medicinal herbs record ": " apply to fall and beat, wash the mole scabies, relieving itching and eliminating swelling." from the chemical constitution angle, polygonum flaccidum mainly contains flavone components such as Quercetin, rutin, Kaempferol, Hyperoside, catechin and epicatechin; Also has phenolic acid compositions such as (gallic acid, chlorogenic acid, caffeic acid and forulic acids).Be mainly used in dysentery, enterogastritis, diarrhoea, beriberi, mange, rheumatic arthritis, treating swelling and pain by traumatic injury, functional uterine bleeding; Symptoms such as venomous snake bite, skin eczema are controlled in external application
[2]External studies show that in recent years, that polygonum flaccidum has is antimicrobial, desinsection, multiple biological effect such as anti-oxidant, antitumor, because region and weather conditions is different, its chemical constitution is also variant, in the prior art, still there is not research method at polygonum flaccidum medicinal material HPLC characteristic fingerprint pattern.
List of references:
[1] Luo Jie, Lu Xia. the HPLC method is measured the content [J] of rutin in the water polygonum flaccidum. Chinese herbal medicine, 35 (3) 285-286.
[2] Li Anren. Chinese Plants will (the 25th volume first fascicle) [M]. Beijing: Science Press, 1998:1-l18..
Summary of the invention
Technical matters to be solved by this invention provides a kind of at polygonum flaccidum medicinal material HPLC characteristic fingerprint pattern technology; In order to solve the problems of the technologies described above, adopted following technical scheme among the present invention, promptly adopt the concrete grammar of polygonum flaccidum medicinal material HPLC characteristic fingerprint pattern: (1) collects medicinal material: comprise S1, S2 totally 2 batches be the Chongqing region collection, S3, S4, S5 and S6 totally 4 batches be that collect in Hainan area, S7, S8 totally 2 batches be that collect in Guangxi area, S9, S10 and S11 totally 3 batches derive from respectively Chengdu, Guangdong and Jiangxi each 1 batch, amount to the 11 knotweed medicinal materials of criticizing and struggle, pulverize processing and concoct into medicine materical crude slice (Fig. 1), the decompression low temperature drying, standby.(2) the main 2 class main active substances that contain in the polygonum flaccidum: flavone component (as: Quercetin, rutin, Kaempferol, Hyperoside, catechin and epicatechin etc.) and liposoluble ingredient (gallic acid, chlorogenic acid, caffeic acid and forulic acid); Because the polarity of its active substance also has difference, the difference of maximum absorption wavelength, adopt different solvents to extract to the polarity difference, difference to maximum absorption wavelength, adopt day island proper Tianjin HPLC-2010AHT high performance liquid chromatograph (to comprise automatic sampler, the quaternary gradient pump, online degasser, the Lcbsolution chem workstation), set up the finger-print of the corresponding 2 class active principles of 2 characteristic absorption wavelength, and to carrying out precision after the finger-print foundation, repeatability and stability analysis, simultaneously in conjunction with quantized data relative retention time and relative peak area, reach similarity analysis method in the Chemical Measurement, finally set up the chromatographic fingerprinting of polygonum flaccidum, to realize the comprehensive and overall evaluation of finger-print.
The embodiment of form is described in further detail foregoing of the present invention by the following examples.But this should be interpreted as that scope of the present invention only limits to following example.All technology that realizes based on foregoing of the present invention all belong to scope of the present invention.
Description of drawings
Fig. 1 polygonum flaccidum pharmaceutical decocting piece
Fig. 2 object of reference HPLC collection of illustrative plates
Fig. 3 polygonum flaccidum medicinal material reference fingerprint (annotate: No. 7 peaks are object of reference composition rutin)
11 batches of different places of production of Fig. 4 polygonum flaccidum finger-print match map
Fig. 5 11 non-total peak area sum of knotweed medicinal material of criticizing and struggle accounts for the number percent of total peak area.
Embodiment
The present invention controls polygonum flaccidum quality of medicinal material method
One, experiment material and instrument
1, medicinal material
Be collected as Chongqing, Hainan, Guangxi, Chengdu, Guangdong and Jiangxi polygonum flaccidum medicinal material, be accredited as polygonaceae plant water polygonum flaccidum through senior engineer Yang Xian
Polygonum hydropiperL. or non-irrigated polygonum flaccidum
P.flaccidumMeisn. dry herb.
2, instrument and reagent
Day island proper Tianjin HPLC-2010AHT high performance liquid chromatograph (comprising automatic sampler, quaternary gradient pump, online degasser, Lcbsolution chem workstation).Switzerland METTLER AE240 analytical balance; KQ-250E ultrasonic cleaning instrument (going up sea green space bio tech ltd).Control substance of Rutin (Nat'l Pharmaceutical ﹠ Biological Products Control Institute, lot number: 100080-200707).Gallic acid reference substance (Nat'l Pharmaceutical ﹠ Biological Products Control Institute, lot number: 110831-200803).Acetonitrile is a chromatographically pure, and it is pure that all the other reagent are analysis.
Two, method and result
1,The preparation of test sample is characterized in that
The polygonum flaccidum sample powder that gets after collection and the processing is an amount of, and precision takes by weighing the about 2.0g of polygonum flaccidum respectively, accurate adding 80% methyl alcohol 50ml, claim to decide weight, reflux 1 hour is put coldly, claims to decide weight again, supply the weight that subtracts mistake with 80% methyl alcohol respectively, shake up, filter merging filtrate with 0.45 μ m filter membrane, get subsequent filtrate, promptly get need testing solution;
2, the preparation of object of reference is characterized in that
Precision takes by weighing gallic acid and control substance of Rutin is an amount of respectively, adds 80% methyl alcohol and makes the object of reference solution that every 100ml contains gallate-based 4.21mg, rutin 5.16mg;
3, the foundation of chromatographic condition is characterized in that
Chromatographic column, Waters ODS C18 (250 mm * 4.6 mm, 5 μ m); Moving phase is A with 0.4% phosphate-buffered salt, and acetonitrile is B, gradient elution, and elution program is: 0-8min, the concentration of B (v/v) remains 9%; 8-20min, the concentration change 9%-14% of B; 20-40min, the concentration change 14%-20% of B; 40-50min, the concentration change of B is 20%-45%; 50-65min, the concentration change of B is 45%-80%; 65-88min, the concentration of B remains 80%; Flow velocity is: 0.9ml/min; Column temperature: 30 ℃; Sample size 10 μ l; Detect in wavelength: 0-25min and the 55-88min scope, be made as 273nm, in the 25-55min scope, detect wavelength and be made as 360nm.
Fingerprint spectrum method is learned and is investigated
Precision test, stability test, replica test measurement result: the total peak of polygonum flaccidum is 15 (Fig. 3), each total peak relative retention time result shows that the relative retention time relative standard deviation (RSD) of total chromatographic peak all is not more than 5.0%, and total peak relative peak area is all less than 3.0%; Test shows instrument precision, repeats well, and need testing solution is basicly stable in 48h.
5, the structure of finger-print
5.1 selection with reference to chromatographic peak
By chromatogram relatively, on definite basis, total peak, (see that 5.2 describe), press strong signal peak in each sample chromatogram of the descending statistics of peak area, composition situation according to each medicinal material, select appearance time composition more placed in the middle as reference peak (s) at random, contain gallic acid composition (Fig. 2) and rutin composition (among Fig. 2 and No. 7 peaks among Fig. 3) in the polygonum flaccidum medicinal material, and being medicinal herbs most in use, rutin quantitatively controls composition, it is also placed in the middle to go out the peak, be suitable as with reference to the peak, the present invention is decided to be the rutin peak with reference to peak (s).
5.2 total chromatographic peak is determined
According to 11 batches of each test sample HPLC collection of illustrative plates, adopt software " the chromatographic fingerprints of Chinese materia medica similarity evaluation 2004A of system version " (Chinese Pharmacopoeia Commission), adopt the automatic coupling of the capable chromatographic peak of multi-point correcting method by chromatographic peak, with the 1st batch of polygonum flaccidum medicinal material as with reference to figure, with the average method generation method of finger-print in contrast, the time window width parameter of setting is 0.50min.The contrast color spectrogram of polygonum flaccidum medicinal material and 11 batches of medicinal materials fingerprint match map are seen Fig. 3 and Fig. 4 respectively.By peak match, the 11 total peaks of knotweed medicinal material of criticizing and struggle are 15 (Fig. 3);
5.3 relative retention time (t
R) and relative peak area (A
R) determine
Is 1 with selected rutin composition at the retention time and the peak area of each batch medicinal materials fingerprint, each fingerprint peaks retention time is compared with the retention time of object of reference in the same collection of illustrative plates, ratio is the relative retention time of each fingerprint peaks, in like manner, the peak area at each total peak is compared with the peak area of object of reference in the same collection of illustrative plates, and ratio is the relative peak area at each total peak.11 the criticize and struggle relative retention time of knotweed medicinal material and relative peak areas see Table 1 and table 2.
5.4 non-total peak accounts for the number percent of total peak area
By 11 polygonum flaccidum medicinal material test samples are measured, add up the number percent that the non-total peak of each batch accounts for corresponding collection of illustrative plates total peak area, the result shows: in the finger-print of 11 polygonum flaccidum medicinal materials, the number percent (Fig. 5) that non-total peak area sum accounts for total peak area is: 5.88-8.22% meets " technical requirement of traditional Chinese medicine finger-print research " and stipulates the detection requirement less than 10%.Thereby also side light, the mass change of crude drug is by factor affecting such as the places of production, and therefore, by the research to finger-print, the quality of crude drug is controlled, and meets the requirements, and the quality of the Chinese medicine of being made by them gets also and could keep stability and consistance.
5.5 similarity analysis
Choose the total peak in the chromatogram, automatically carry out peak match by " chromatographic fingerprints of Chinese materia medica similarity evaluation system A version in 2004 " Chinese medicine chromatogram analysis and data management software are auxiliary, adopting the correlation coefficient process (see figure 3) to carry out similarity calculates, the results are shown in Table 3, polygonum flaccidum medicinal material similarity is greater than 0.95, show to have consistance preferably between each batch medicinal material, this method can be used for the total quality analysis of comprehensive evaluation polygonum flaccidum medicinal material
The criticize and struggle relative retention time (t of knotweed medicinal material of table 1 11
R) result
The criticize and struggle relative peak area (A of knotweed medicinal material of table 2 11
R)
Table 3 polygonum flaccidum similarity result of calculation
Claims (3)
1. a polygonum flaccidum medicinal material HPLC characteristic fingerprint pattern method for building up comprises the steps:
A. the preparation of test sample: the polygonum flaccidum sample powder that gets after collection and the processing is an amount of, and precision takes by weighing the about 2.0g of polygonum flaccidum respectively, accurate adding 80% methyl alcohol 50ml, claim to decide weight, reflux 1 hour is put coldly, claims to decide weight again, supply the weight that subtracts mistake with 80% methyl alcohol respectively, shake up, filter merging filtrate with 0.45 μ m filter membrane, get subsequent filtrate, promptly get need testing solution;
B. the preparation of object of reference: precision takes by weighing gallic acid and control substance of Rutin is an amount of respectively, adds 80% methyl alcohol and makes the object of reference solution that every 100ml contains gallate-based 4.21mg, rutin 5.16mg;
C. the foundation of chromatographic condition: chromatographic column, Waters ODS C18 (250 mm * 4.6 mm, 5 μ m; Moving phase, the phosphate-buffered salt with 0.4% are A, and acetonitrile is B, gradient elution, and elution program is: 0-8min, the concentration of B (v/v) remains 9%; 8-20min, the concentration change 9%-14% of B; 20-40min, the concentration change 14%-20% of B; 40-50min, the concentration change of B is 20%-45%; 50-65min, the concentration change of B is 45%-80%; 65-88min, the concentration of B remains 80%; Flow velocity is: 0.9ml/min; Column temperature: 30 ℃; Flow velocity, 0.9mL/min; Sample size 10 μ l; Detect in wavelength: 0-25min and the 55-88min scope, be made as 273nm, in the 25-55min scope, detect wavelength and be made as 360nm.
2. the detection method of polygonum flaccidum medicinal material according to claim 1 is characterized in that the described moving phase of step c is phosphoric acid-acetonitrile of 0.4%.
3. the detection method of polygonum flaccidum medicinal material according to claim 1 is characterized in that the described detection wavelength of step c: in 0-25min and the 55-88min scope, be made as 273nm, in the 25-55min scope, detect wavelength and be made as 360nm.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106525997A (en) * | 2016-09-22 | 2017-03-22 | 广东东阳光药业有限公司 | Method for determination of organic acids and flavone components in polygonum viviparum |
| CN107976494A (en) * | 2017-11-10 | 2018-05-01 | 安徽安科余良卿药业有限公司 | The structure and its quality determining method of health skin tincture standard feature collection of illustrative plates |
| CN113419016A (en) * | 2021-08-10 | 2021-09-21 | 四川辅正药业股份有限公司 | Polygonum hydropiper extract and quality detection method of polygonum hydropiper |
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2011
- 2011-07-04 CN CN2011101854091A patent/CN102279240A/en active Pending
Non-Patent Citations (3)
| Title |
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| 王祥培等: "HPLC测定不同产地的头花蓼中没食子酸的含量", 《华西药学杂志》 * |
| 罗杰等: "HPLC法测定水辣藋中芦丁的含量", 《中草药》 * |
| 赖东美等: "RP-HPLC法测定辣蓼中没食子酸的含量", 《海峡药学》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106525997A (en) * | 2016-09-22 | 2017-03-22 | 广东东阳光药业有限公司 | Method for determination of organic acids and flavone components in polygonum viviparum |
| CN106525997B (en) * | 2016-09-22 | 2019-04-19 | 广东东阳光药业有限公司 | A method of organic acid and flavones ingredient in measurement serpentgrass |
| CN107976494A (en) * | 2017-11-10 | 2018-05-01 | 安徽安科余良卿药业有限公司 | The structure and its quality determining method of health skin tincture standard feature collection of illustrative plates |
| CN113419016A (en) * | 2021-08-10 | 2021-09-21 | 四川辅正药业股份有限公司 | Polygonum hydropiper extract and quality detection method of polygonum hydropiper |
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