CN101091749A - Medicinal material of polygonum capilalum, extractive, and quality control method - Google Patents
Medicinal material of polygonum capilalum, extractive, and quality control method Download PDFInfo
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Abstract
The present invention provides a Chinese medicinal material polygonum capitatum. Said invention provides its HPLC fingerprint chromatogram map, it contains 13 characteristic peaks. Its chromatographic condition is as flows: chromatographic column: Hypersil ODS chromatographic column (4.6mmX150mm, 5micrometers); column temperature: 25deg.C; mobile phase: acetonitrile (A)-0.4% phosphoric acid (B) solution, binary gradient elution, weight percentage is 0-30%: 100-70%; flowing rate: 0.8ml.min-1; detection wavelength: 310nm; and sample size: 20 microliters. Besides, said invention also provides its extract and its quality control method.
Description
Technical field
The present invention relates to medicinal material of polygonum capilalum and extract thereof and method of quality control.
Background technology
Herba Polygoni Capitati is dry herb or the aerial parts of polygonaceae plant Herba Polygoni Capitati (Polygonum capitatum Buch.-Han.ex D.Don).Herba Polygoni Capitati is as medicinal, record the earliest in 1963 version " Guangxi Chinese medicinal herbal " be called Herba Polygoni Capitati, Polygonum capitatum Buch, " acrid in the mouth, little puckery, warm in nature, eliminating stasis to stop pain, dispelling the wind and dampness pathogens, traumatic injury." " Guangxi Chinese herbal medicine " put down in writing its flavor " property is flat for hardship, suffering ", detoxicating, relieving inflammation is controlled dysentery, skin ulcer, innominate toxic swelling." Yunnan Chinese herbal medicine " is called Herba Polygoni Capitati, and sour in the mouth is cool in nature.Clearing away heat and promoting diuresis, treating stranguria, control hematuria, cystitis." mountain of papers Chinese herbal medicine " is called red fullly, fire slide grass, treatment cloth rash, impetigo." Guizhou Chinese herbal medicine register " is called province's fourth grass, Herba Polygoni Capitati, and its sour in the mouth is cool in nature, heat-clearing and toxic substances removing, and inducing diuresis to remove edema is controlled oedema due to nephritis, parotitis, treating swelling and pain by traumatic injury, innominate toxic swelling." national Chinese herbal medicine compilation " controls " urinary system infection, dysentery, diarrhoea, hematuria; Diaper rash, impetigo "." Guizhou Province's Chinese crude drug, national quality of medicinal material standard ": bitter in the mouth, suffering, cool in nature, clearing away heat-damp and promoting diuresis, detoxifcation pain relieving, promoting blood circulation to remove blood stasis, inducing diuresis for treating stranguria syndrome.Be used for dysentery, pyelonephritis, cystitis, lithangiuria, pelvic inflammatory disease, prostatitis, rheumatalgia, traumatic injury, skin infection eczema.In recent years, be that raw material is successfully developed and secreted the clever dry syrup of sense, the clear granule of pyretic stranguria, pyretic stranguria clearing capsule with the Herba Polygoni Capitati, the pyretic stranguria clearing dropping pill, RELINQING TANGJIANG is secreted the pouring capsule, secretes the pouring granule, NINGBITAI JIAONANG, TONGLINSHU KELI is secreted a series of Miao Ethnomedicine preparations such as drenching clearing capsule, has brought into play therapeutical effect preferably clinically.
Further investigation reveals that, equally belong on the same group that plant Nepal knotweed (head knotweed) Polygonum nepalense Meisn easily is construed to Herba Polygoni Capitati together.The head knotweed has heat-clearing and toxic substances removing, the solid intestinal of convergence, and the effect of damp eliminating, different with the Herba Polygoni Capitati effect, both can not use with.But two kinds of medical materials are difficult to difference on character, the also difficult difference of powder microscopical identification.In 2003 version " Guizhou Province's Chinese crude drug, national quality of medicinal material standards " is with the index of Quercetin as quality evaluation.Herba Polygoni Capitati for complicated component is incomplete with Quercetin as quality control index only.
Summary of the invention
Technical problem to be solved by this invention provides a kind of steady quality, homogeneous, medicinal material of polygonum capilalum that controllability is strong, and another technical problem of solution of the present invention has provided the method for quality control of this medical material.
The invention provides a kind of medicinal material of polygonum capilalum, it has the HPLC finger printing shown in Fig. 1 D, contains 13 characteristic peaks,
Wherein, chromatographic condition is: chromatographic column: Yi Lite Hypersil ODS chromatographic column (4.6mm * 150mm, 5 μ m); Column temperature: 25 ℃; Mobile phase: acetonitrile (A)-0.4% phosphoric acid (B) solution binary gradient elution, percentage by weight is: 0-30%: 100-70%; Flow velocity: 0.8mLmin
-1Detect wavelength: 310nm; Sample size 20 μ l.
Wherein, the relative retention time of described 13 characteristic peaks is respectively:
No. 1 peak: 0.083 ± 0.001, No. 2 peaks: 0.178 ± 0.001, No. 3 peaks: 0.357 ± 0.001, No. 4 peaks: 0.388 ± 0.001, No. 5 peaks: 0.460 ± 0.001, No. 6 peaks: 0.520 ± 0.001, No. 7 peaks: 0.544 ± 0.002, No. 8 peaks: 0.792 ± 0.002, No. 9 peaks: 0.848 ± 0.002, No. 10 peaks: 0.888 ± 0.001,1, No. 12 peaks, No. 11 peaks: 1.266 ± 0.002, No. 13 peaks: 1.296 ± 0.002.
The present invention also provides a kind of Herba Polygoni Capitati extract, and it is to be raw material by medicinal material of polygonum capilalum, is prepared from through water or organic solvent extraction.
The invention provides a kind of pharmaceutical composition, it is to be active component by described Herba Polygoni Capitati primary crude drug or described Herba Polygoni Capitati extract, adds the medicament that acceptable accessories or complementary composition are prepared from.
Described medicament is oral formulations, ejection preparation.
The present invention also provides a kind of method of quality control of controlling medicinal material of polygonum capilalum, and it comprises the steps:
A, preparation gallic acid object of reference solution: get gallic acid and add 50% dissolve with methanol and make 0.0204mgmL
-1Solution;
B, preparation Quercetin object of reference solution: it is an amount of to get the Quercetin reference substance, adds dissolve with methanol and makes 0.0216mgmL
-1Solution;
C, preparation Quercitroside object of reference solution: it is an amount of to get the Quercitroside reference substance, adds dissolve with methanol and makes 0.0992mgmL
-1Solution;
The preparation of d, need testing solution: get the Herba Polygoni Capitati fine powder, add 100ml80% ethanol, put reflux in the water-bath, filter, residue reenters uses 80% alcohol reflux, filters merging filtrate.Filtrate is put in the evaporating dish and is volatilized, and residue adds 80% ethanol standardize solution, filters with microporous filter membrane, gets subsequent filtrate, promptly;
E, chromatographic condition:
Chromatographic condition is: chromatographic column: Yi Lite Hypersil ODS chromatographic column (4.6mm * 150mm, 5 μ m); Column temperature: 25 ℃; Mobile phase: acetonitrile (A)-0.4% phosphoric acid (B) solution binary gradient elution, percentage by weight is: 0-30%:100-70%; Flow velocity: 0.8mLmin
-1Detect wavelength: 310nm; Sample size 20 μ l.
F, algoscopy: get each 15ul of reference substance and need testing solution, inject high performance liquid chromatograph, the record chromatogram.
Method that the present invention set up and the finger printing that draws thereof have three chromatographic peaks to belong in the finger printing, it is stable in the finger printing 13 peaks being arranged simultaneously, can be used as the fingerprint characteristic peak of Herba Polygoni Capitati.Chromatographic fingerprinting has globality, ambiguity and can quantized characteristics, can reflect the total quality of Chinese medicine fully, all sidedly, is the pattern of best at present control Chinese medicine quality, also is the method for the evaluation Chinese medicine quality of comparison science.In order to remedy the deficiency of Herba Polygoni Capitati Quality Control Technology, make its more perfect, science.Method of quality control of the present invention has carried out investigating relatively to different places of production Herba Polygoni Capitati, made up the finger printing of Herba Polygoni Capitati HPLC, but utilize the quality of HPLC fingerprint characteristic overall monitor medicinal material of polygonum capilalum, guarantee its stable, homogeneous, controlled, the HPLC finger printing of foundation can significantly be distinguished qualified and defective medical material.Simultaneously, this method of quality control can be controlled the quality of medical material effectively, reaches the purpose of the whole pharmaceutical preparation of control.
Obviously, according to foregoing of the present invention,,, can also make modification, replacement or the change of other various ways not breaking away under the above-mentioned basic fundamental thought of the present invention prerequisite according to the ordinary skill knowledge and the customary means of this area.
The specific embodiment of form is described in further detail foregoing of the present invention again by the following examples.But this should be interpreted as that the scope of the above-mentioned theme 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
The HPLC finger printing of Fig. 1 Herba Polygoni Capitati and head knotweed (wherein, Figure 1A-gallic acid Figure 1B-Quercitroside Fig. 1 C-Quercetin Fig. 1 D-Herba Polygoni Capitati Fig. 1 E-head knotweed)
Fig. 2 stability test similarity evaluation result
Fig. 3 precision test similarity evaluation result
Fig. 4 replica test similarity evaluation is figure as a result
The comparison diagram of Fig. 5 head knotweed and medicinal material of polygonum capilalum reference fingerprint (S1-Herba Polygoni Capitati S2-head knotweed)
HPLC finger printing (the S of the different places of production of Fig. 6 Herba Polygoni Capitati
1Kweiyang ,-Guizhou S
2-Guizhou Jianhe S
3-Guizhou silk screen S
4Anshun ,-Guizhou S
5Shibing ,-Guizhou S
6Miyi ,-Sichuan S
7The high eyebrow in-Sichuan (area without shade) S
8The high eyebrow in-Sichuan (shady place) S
9Chengdu ,-Sichuan S
10Kunming ,-Yunnan S
11-Dali S
12-Tibet Motuo)
The HPLC finger printing of Fig. 7 Different Harvesting Time Herba Polygoni Capitati
Fig. 8 organic solvent reflux, extract, chromatogram (wherein, Fig. 8 A, methanol supersound extraction chromatogram; Fig. 8 B, 80% ethanol ultrasonic extraction chromatogram; Fig. 8 C, 80% methanol supersound extraction chromatogram; Fig. 8 D, methanol eddy extraction chromatography figure; Fig. 8 E, 80% alcohol reflux chromatogram; Fig. 8 F, 80% methanol eddy extraction chromatography figure)
Fig. 9 methanol-0.4% phosphoric acid gradient elution Herba Polygoni Capitati HPLC chromatogram
Figure 10 acetonitrile-0.4% phosphoric acid gradient elution Herba Polygoni Capitati HPLC chromatogram
The specific embodiment
The preparation of embodiment 1 medicine of the present invention
[method for making] gets the Herba Polygoni Capitati cutting, adds the decocting in water secondary, and each 1.5 hours, collecting decoction left standstill, and filters, and filtrate is condensed into thick paste, and drying is pulverized, and adds right amount of auxiliary materials, and mixing is encapsulated, promptly.
[character] this product is a capsule, and content is a chocolate brown powder; Feeble QI, it is little puckery to distinguish the flavor of.
[discriminating] got this product 3g and added methanol 20ml, water-bath refluxed 1 hour, filtered, and filtrate is concentrated into about 2ml, last alumina column (neutral alumina 3g, 100～200 orders, 105 ℃ of dryings 30 minutes, column internal diameter 1cm), with methanol 50ml eluting, liquid is taken off in collection, is concentrated into about 1ml in the water-bath, as need testing solution.Other gets Herba Polygoni Capitati control medicinal material 1.5g, adds water 20ml, boils 30 minutes, filters, and filtrate is concentrated into dried, adds methanol 2ml dissolving, makes reference substance solution with method.According to thin layer chromatography (appendix VI B).Each 5 μ l of above-mentioned two kinds of solution are drawn in test, put respectively on same polyamide lamellae, and (5: 3: 1: 1) be developing solvent, expansion was taken out, and dries, and smokes in the ammonia 1 minute, puts under the uviol lamp (365nm) and inspects with ethyl acetate-butanone-formic acid-water.In the test sample chromatograph, with control medicinal material chromatograph relevant position on, show the fluorescence speckle of same color.
[inspection] should meet every regulation relevant under the capsule item (appendix I L).
[function with cure mainly] clearing away heat-fire, relieving stranguria by diuresis.Cure mainly pyretic stranguria.
[usage and consumption] is oral, one time 4～6,3 times on the one.
The preparation of embodiment 2 medicinal granules of the present invention
[method for making] got Herba Polygoni Capitati 1000g and decocted with water secondary, and each 1.5 hours, collecting decoction, filter, filtrate is concentrated into the thick paste that relative density is 1.38 (80 ℃), drying, pulverize, make granule, drying, make 400g, or add suitable amount of sucrose powder, mixing, make granule, drying is made 800g, promptly.
[character] this product is that sepia is to auburn granule; Gas perfume (or spice), it is sweet, slightly puckery to distinguish the flavor of.
[discriminating] gets this product 3g (Sugarless type) or 6g (sugar-containing type), adds methanol 20ml, and water-bath refluxed 1 hour, filters, and filtrate is concentrated into about 2ml, is added in alumina column (neutral alumina 3g, 100～200 orders, 105 ℃ of dryings 30 minutes; Column internal diameter is 1cm) on, with methanol 50ml eluting, collect eluent, to put and be concentrated into driedly in the water-bath, residue adds methanol 1ml makes dissolving, as need testing solution.
Other gets Herba Polygoni Capitati control medicinal material 1.5g, adds water 20ml, boils 30 minutes, filters, and filtrate is concentrated into dried, and residue adds methanol 2ml makes dissolving, according to the need testing solution preparation method, makes control medicinal material solution on the alumina column from being added in accordance with the law.
According to thin layer chromatography (appendix VI B) test, draw each 5 μ 1 of above-mentioned two kinds of solution, put respectively on same polyamide film, (5: 3: 1: 1) be developing solvent, expansion was taken out with ethyl acetate-butanone-formic acid-water, dry, put in the ammonia and smoked 1 minute, put under the ultra-violet lamp (365nm) and inspect.
In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence speckle of same color.
[inspection]
Should meet every regulation relevant under the granule item (appendix I C).
[function with cure mainly]
Heat-clearing and toxic substances removing, inducing diuresis for treating stranguria syndrome.
Be used for damp-heat accumulation, the disease of yellowish or reddish urine, odynuria, urinary tract infection, pyelonephritis is seen above-mentioned patient.
One, experiment material and instrument
1, medical material
Pick up from Guizhou, Sichuan, Yunnan, Tibet, totally 15 batches, be accredited as the dry herb of polygonaceae plant Herba Polygoni Capitati Polygonum capitatum Buch.-Ham.ex D.Don through professor Wan Deguang of Chengdu University of Traditional Chinese Medicine.The head knotweed is the dry herb of polygonaceae plant Nepal knotweed Polygonum nepalense Meisn, adopts to the Mount Emei, Sichuan.
2, instrument and reagent
The Waters2695 high performance liquid chromatograph, diode array detector, Empower chromatographic work station, SB2200 type ultrasonic cleaner.Gallic acid reference substance (Nat'l Pharmaceutical ﹠ Biological Products Control Institute, lot number: 0638-9501), Quercetin reference substance (Nat'l Pharmaceutical ﹠ Biological Products Control Institute, lot number: 081-90003), Quercitroside reference substance (self-control, purity＞98%), acetonitrile is a chromatograph alcohol, other reagent is analytical pure, the water distilled water of attaching most importance to.
Two, method and result
1, the preparation of object of reference solution
1.1 the preparation of gallic acid object of reference solution
It is an amount of that precision takes by weighing the gallic acid reference substance, adds 50% dissolve with methanol and make 0.0204mgmL
-1Solution, promptly.
1.2 the preparation of Quercetin object of reference solution
It is an amount of that precision takes by weighing the Quercetin reference substance, adds dissolve with methanol and make 0.0216mgmL
-1Solution, promptly.
1.3 the preparation of Quercitroside object of reference solution
It is an amount of that precision takes by weighing the Quercitroside reference substance, adds dissolve with methanol and make 0.0992mgmL
-1Solution, promptly.
2, the preparation of need testing solution
Get Herba Polygoni Capitati fine powder (No. 3 sieves) 2g, the accurate title, decide, and puts in the round-bottomed flask, adds 100ml80% ethanol respectively, puts reflux 2h in the water-bath, filters, and residue adds 100ml80% alcohol reflux 1h, filters merging filtrate.Filtrate is put in the evaporating dish and is volatilized, and residue adds 80% ethanol standardize solution and puts in the 10ml volumetric flask.Filter with microporous filter membrane (0.45 μ m), get subsequent filtrate, promptly.
Get head knotweed fine powder (No. 3 sieves) 2g, the accurate title, decide, and puts in the round-bottomed flask, adds 100ml80% ethanol, puts reflux 2h in the water-bath, filters, and residue adds 100ml80% alcohol reflux 1h, filters merging filtrate.Filtrate is put in the evaporating dish and is volatilized, and residue adds 80% ethanol standardize solution and puts in the 10ml volumetric flask.Filter with microporous filter membrane (0.45 μ m), get subsequent filtrate, promptly.
3, chromatographic condition
Chromatographic column: Yi Lite Hypersil ODS chromatographic column (4.6mm * 150mm, 5 μ m); Column temperature: 25 ℃; Mobile phase: acetonitrile (A)-0.4% phosphoric acid (B) solution binary gradient elution sees Table 1; Flow velocity: 0.8mLmin
-1Detect wavelength: 310nm; Sample size 20 μ l.
Table 1 linear gradient elution proportion of mobile phase changes
| Time (min) | Acetonitrile (%) | 0.4% phosphate aqueous solution (%) |
| 0 5 55 70 | 0 5 25 30 | 100 95 75 70 |
4, blank reagent test
Get the reagent (80% ethanol) of preparation need testing solution, sample introduction 15ul, the record chromatogram, from chromatogram as can be known, agents useful for same is noiseless substantially to determining fingerprint pattern.
5, algoscopy: get each 15ul of reference substance and need testing solution, inject high performance liquid chromatograph, the record chromatogram.See Fig. 1.
6, methodological study
Producing medicinal material of polygonum capilalum with the Guizhou silk screen is sample, and its stability, instrument precision, experimental technique, repeatability have been done corresponding investigation, and the result proves that stability and repeatability are good.
6.1 stability test is got with a need testing solution, respectively 0,2,6,12,20,30h detects, the overall picture of observing finger printing directly perceived does not have significant difference, uses the similarity computed in software, is respectively in the similarity of same Instrument measuring chromatographic fingerprinting: 0.984,0.994,0.995,0.961,0.992,0.982, all greater than 0.95.With the Quercitroside peak is with reference to the peak, investigates the concordance of chromatographic peak relative retention time and relative peak area, and the RSD of each main chromatographic peak relative retention time and relative peak area all is lower than 3.See Table 2, table 3, see Fig. 2.
Table 2 stability test result
| Numbering | |
||||||||||||
| 1 2 3 4 5 6 RSD (％) | 0.083 0.084 0.082 0.084 0.083 0.084 0.98 | 0.178 0.179 0.177 0.179 0.176 0.179 0.71 | 0.356 0.358 0.357 0.357 0.356 0.357 0.21 | 0.389 0.388 0.388 0.387 0.388 0.388 0.16 | 0.461 0.461 0.460 0.461 0.460 0.460 0.12 | 0.521 0.520 0.521 0.521 0.520 0.521 0.10 | 0.545 0.543 0.544 0.544 0.544 0.544 0.12 | 0.792 0.791 0.791 0.792 0.792 0.792 0.07 | 0.845 0.848 0.848 0.846 0.848 0.848 0.16 | 0.888 0.889 0.888 0.886 0.879 0.889 0.44 | 1 1 1 1 1 1 0 | 1.266 1.265 1.267 1.266 1.266 1.266 0.05 | 1.299 1.297 1.296 1.298 1.296 1.296 0.10 |
Table 3 stability test result
| Numbering | |
||||||||||||
| 1 2 3 4 5 6 RSD (％) | 0.027 0.027 0.027 0.026 0.027 0.026 1.94 | 0.059 0.060 0.061 0.062 0.061 0.060 1.73 | 0.189 0.187 0.191 0.192 0.200 0.199 2.63 | 0.127 0.130 0.134 0.125 0.127 0.127 2.50 | 0.414 0.400 0.420 0.416 0.411 0.407 1.72 | 0.066 0.067 0.066 0.068 0.067 0.066 1.22 | 0.047 0.047 0.048 0.047 0.048 0.046 1.59 | 3.034 3.100 3.151 3.133 3.133 3.078 1.49 | 0.399 0.400 0.397 0.405 0.400 0.413 1.45 | 0.237 0.230 0.234 0.233 0.231 0.233 1.05 | 1 1 1 1 1 1 0 | 0.097 0.097 0.102 0.097 0.103 0.099 2.74 | 0.019 0.020 0.020 0.020 0.019 0.019 2.80 |
6.2 the precision test is got with a need testing solution, repeat sample introduction 6 times, the overall picture of observing finger printing directly perceived does not have significant difference, use the similarity computed in software, be respectively in the similarity of same Instrument measuring chromatographic fingerprinting: 0.999,0.996,0.998,0.999,0.996, all greater than 0.95.With the Quercitroside peak is with reference to the peak, investigates the concordance of chromatographic peak relative retention time and relative peak area, and the RSD of each main chromatographic peak relative retention time and relative peak area all is lower than 3.See Table 4,5, see Fig. 3.
Table 4 Precision test result
| Numbering | |
||||||||||||
| 1 2 3 4 5 RSD (％) | 0.083 0.083 0.084 0.083 0.084 0.66 | 0.178 0.179 0.179 0.179 0.179 0.25 | 0.356 0.357 0.357 0.357 0.357 0.12 | 0.389 0.388 0.388 0.388 0.388 0.11 | 0.461 0.460 0.460 0.460 0.460 0.10 | 0.521 0.521 0.521 0.521 0.521 0 | 0.545 0.544 0.544 0.543 0.544 0.13 | 0.792 0.792 0.792 0.792 0.791 0.05 | 0.845 0.848 0.848 0.848 0.848 0.16 | 0.888 0.889 0.889 0.889 0.889 0.05 | 1 1 1 1 1 0 | 1.266 1.265 1.266 1.266 1.266 0.03 | 1.299 1.296 1.296 1.297 1.296 0.10 |
Table 5 Precision test result
| Numbering | |
||||||||||||
| 1 2 3 4 5 RSD (％) | 0.027 0.027 0.026 0.027 0.027 1.67 | 0.059 0.060 0.060 0.060 O.057 2.20 | 0.189 0.187 0.196 0.196 0.196 2.55 | 0.127 0.130 0.131 0.128 0.127 1.41 | 0.414 0.400 0.411 0.415 0.400 1.83 | 0.066 0.067 0.068 0.068 0.066 1.49 | 0.047 0.047 0.048 0.047 0.047 0.95 | 3.034 3.100 3.130 3.135 3.098 1.30 | 0.399 0.400 0.397 0.405 0.400 0.74 | 0.237 0.230 0.234 0.234 0.233 1.07 | 1 1 1 1 1 0 | 0.097 0.097 0.097 0.102 0.101 2.52 | 0.019 0.020 0.020 0.020 0.019 2.79 |
6.3 5 parts of the test samples in the same place of production are got in repeatability test, detect with method, the overall picture of observing finger printing directly perceived does not have significant difference, use the similarity computed in software, be respectively in the similarity of same Instrument measuring chromatographic fingerprinting: 0.992,0.951,0.995,0.993,0.979, all greater than 0.95.With the Quercitroside peak is with reference to the peak, investigates the concordance of chromatographic peak relative retention time and relative peak area, and the RSD of each main chromatographic peak relative retention time and relative peak area all is lower than 3.See Table 6,7, see Fig. 4.
Table 6 reproducible test results
| Numbering | |
||||||||||||
| 1 2 3 4 5 RSD (％) | 0.083 0.084 0.086 0.086 0.087 1.92 | 0.178 0.181 0.179 0.179 0.178 0.68 | 0.356 0.357 0.357 0.357 0.356 0.15 | 0.389 0.389 0.386 0.388 0.388 0.31 | 0.461 0.460 0.461 0.463 0.460 0.27 | 0.521 0.522 0.521 0.521 0.521 0.09 | 0.545 0.544 0.543 0.545 0.544 0.15 | 0.792 0.791 0.792 0.801 0.791 0.54 | 0.845 0.848 0.848 0.848 0.848 0.16 | 0.888 0.889 0.888 0.889 0.889 0.06 | 1 1 1 1 1 0 | 1.266 1.266 1.266 1.263 1.265 0.10 | 1.299 1.297 1.296 1.299 1.296 0.12 |
Table 7 reproducible test results
| Numbering | |
||||||||||||
| 1 2 3 4 5 RSD (％) | 0.027 0.026 0.027 0.027 0.026 2.06 | 0.059 0.056 0.057 0.056 0.058 2.27 | 0.189 0.187 0.190 0.197 0.187 2.17 | 0.127 0.129 0.127 0.133 0.133 2.34 | 0.414 0.406 0.401 0.410 0.403 1.29 | 0.066 0.067 0.067 0.065 0.067 1.35 | 0.047 0.047 0.047 0.047 0.047 0.96 | 3.034 3.100 3.133 3.133 3.067 1.39 | 0.399 0.411 0.406 0.398 0.403 1.32 | 0.237 0.229 0.233 0.233 0.233 1.22 | 1 1 1 1 1 0 | 0.097 0.098 0.102 0.097 0.100 2.19 | 0.019 0.020 0.020 0.020 0.019 2.79 |
6.4 specificity experiment head knotweed is compared with the finger printing of Herba Polygoni Capitati, similarity is 0.304, shows that this finger printing can distinguish out other medical materials.The results are shown in Figure 5.
6.4 finger printing is set up accurate reference substance and each 20ul of need testing solution of drawing, and injects high performance liquid chromatograph, the chromatographic condition by selected detects.Under the same experiment condition, measure all test sample HPLC chromatograms.Analyze, compare according to the different places of production, given peak number, peak value (integrated value) and the peak position relevant parameters such as (relative retention time) of different collecting time test sample measurement result, formulate the finger printing of optimizing (seeing Fig. 6,7)
6.5 fingerprint map analyzing and date processing
6.5.1 the relevant parameter that total fingerprint peaks marked ratio more different places of production Herba Polygoni Capitati and Different Harvesting Time Herba Polygoni Capitati need testing solution provide, wherein 13 peaks are that each batch test sample is total, therefore determine that these 13 peaks are its total fingerprint peaks, wherein the Quercitroside peak is big than gallic acid peak and Quercetin peak, be elected to be and be the reference peak, the relative retention time at the total peak of the different places of production and Different Harvesting Time Herba Polygoni Capitati finger printing sees Table 8.
The relative retention time of table 8 Herba Polygoni Capitati sample
| Numbering | The place of production | Relative retention time | ||||||||||||
| 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 | Kweiyang, Guizhou, (wild) Guizhou Jianhe, (wild) nationality of Jinping in Guizhou, (wild) From Anshun of Guizhou, (wild) Shibing, Guizhou, (cultivation) Miyi, Sichuan, China, (wild) Emei, Sichuan Province, (wild) Emei, Sichuan Province, (wild) Chengdu, Sichuan, (cultivation) Kunming, Yunnan, (wild) Dali, (wild) Motuo In Xizang, (wild) Motuo In Xizang, (wild) Motuo In Xizang, (wild) Motuo In Xizang, (wild) | 0.083 0.083 0.083 0.083 0.084 0.083 0.083 0.083 0.083 0.083 0.083 0.083 0.083 0.083 0.083 | 0.178 0.178 0.179 0.178 0.179 0.178 0.179 0.179 0.178 0.179 0.178 0.178 0.178 0.178 0.178 | 0.357 0.357 0.357 0.356 0.357 0.356 0.357 0.357 0.357 0.357 0.356 0.357 0.357 0.357 0.357 | 0.388 0.388 0.388 0.388 0.388 0.388 0.388 0.388 0.388 0.388 0.387 0.388 0.389 0.388 0.389 | 0.460 0.460 0.460 0.460 0.460 0.460 0.460 0.460 0.460 0.460 0.459 0.460 0.461 0.460 0.461 | 0.521 0.520 0.521 0.520 0.521 0.520 0.521 0.520 0.521 0.521 0.520 0.521 0.521 0.521 0.521 | 0.544 0.544 0.544 0.543 0.544 0.544 0.543 0.543 0.545 0.544 0.542 0.545 0.545 0.544 0.544 | 0.792 0.792 0.792 0.792 0.792 0.792 0.791 0.792 0.792 0.792 0.793 0.793 0.794 0.793 0.794 | 0.848 0.847 0.848 0.846 0.848 0.847 0.847 0.847 0.848 0.848 0.849 0.848 0.848 0.848 0.849 | 0.888 0.888 0.889 0.888 0.889 0.888 0.889 0.888 0.889 0.889 0.888 0.888 0.889 0.888 0.889 | 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 | 1.266 1.266 1.265 1.266 1.266 1.266 1.266 1.266 1.266 1.266 1.265 1.266 1.266 1.266 1.264 | 1.297 1.297 1.296 1.298 1.296 1.298 1.297 1.297 1.297 1.297 1.296 1.296 1.297 1.297 1.295 |
Annotate: No. 11 for gathering product November; 13, be respectively 8,10 for 14, No. 15, gather product March, other is JIUYUE
6.5.2 Herba Polygoni Capitati finger printing similarity evaluation: with 12 different places of production, 4 Different Harvesting Time Herba Polygoni Capitati sample determination data importing Chinese medicine fingerprint similarity software for calculation, through selecting the peak, set matching template, the peak is mated automatically, the established standards template is composed the peak diversity and is estimated and the global similarity evaluation then.Draw Herba Polygoni Capitati HPLC finger printing common pattern by Chinese medicine fingerprint similarity software for calculation, compare with common pattern, the similarity result of 12 batches of different places of production Herba Polygoni Capitati sees Table, and the similarity result of 4 batches of Different Harvesting Time Herba Polygoni Capitati sees Table 9,10.
The different place of production of table 9 Herba Polygoni Capitati result
| Numbering | The place of production | Collecting time | Similarity | Gallic acid peak area integrated value | Quercitroside peak area integrated value | Quercetin peak area integrated |
| 1 2 3 4 5 6 7 8 9 10 11 12 | Kweiyang, Guizhou (wild) Guizhou Jianhe (wild) nationality of Jinping in Guizhou (wild) From Anshun of Guizhou (wild) Shibing, Guizhou (cultivation) Miyi, Sichuan, China (wild) Emei, Sichuan Province (wild) Emei, Sichuan Province (wild) Chengdu, Sichuan (cultivation) Kunming, Yunnan (wild) Dali (wild) Motuo In Xizang (wild) | 2005-9 2005-9 2005-9 2005-9 2005-9 2005-9 2005-9 2005-9 2005-9 2005-9 2005-11 2005-9 | 0.988 0.993 0.916 0.985 0.130 0.992 0.993 0.991 0.992 0.988 0.958 0.936 | 4.586E-1 3.591E-1 1.879E-1 1.974E-1 2.029E-1 3.303E-1 2.873E-1 1.978E-1 3.198E-1 1.988E-1 2.945E-1 2.570E-1 | 3.153 2.793 2.967 2.369 3.198 2.902 4.061 2.967 2.898 4.175 1.374 2.483 | 8.789E-2 6.384E-2 5.836E-2 2.172E-2 1.299E-1 5.505E-2 5.547E-2 3.659E-2 6.183E-2 5.467E-2 1.669E-1 1.087E-1 |
Annotate: No. 7 samples are Mount Emei area without shade plant; No. 8 samples are shady and cool place, Mount Emei plant;
Table 10 Different Harvesting Time result
| Sample number into spectrum | The place of production | Collecting time | Similarity | Gallic acid peak area integrated value | Quercitroside peak area integrated value | Quercetin peak area integrated |
| 1 2 3 4 | Tibet Motuo (wild) Tibet Motuo (wild) Tibet Motuo (wild) Tibet Motuo (wild) | 2005-3 2005-8 2005-9 2005-10 | 0.328 0.886 0.960 0.950 | 1.596E-1 3.686E-1 2.570E-1 1.853E-1 | 1.197 2.491 2.483 3.023 | 6.981E-2 5.479E-2 1.087E-1 1.560E-1 |
The conditional filtering of embodiment 4 quality of medicinal material control methods of the present invention
1, the selection of sample extraction condition
In order to embody the feature of the contained chemical constituent of Herba Polygoni Capitati as much as possible.According to the character of Herba Polygoni Capitati ingredient, selected 80% methanol, methanol, 80% ethanol for extracting solvent, extracting method has been investigated hot reflux, supersound extraction, and is wherein more complete with 80% alcohol reflux, and 80% ethanol is than methanol safety.See Fig. 8.
2, detect the selection of wavelength
Adopt diode array 200～400nm scanning, take all factors into consideration the absorption spectrum of each main component in the Herba Polygoni Capitati, make each tested component that bigger absorption all be arranged, selecting 310nm for use is that finger printing detects wavelength.
3, the selection of mobile phase
Compared the chromatogram under acetonitrile-0.4% phosphoric acid solution system and the methanol-0.4% phosphoric acid solution system, the post pressure of finding acetonitrile-0.4% phosphoric acid solution system gradient elution is lower, the change of finding mobile phase simultaneously is less to its baseline influence, is mobile phase so select acetonitrile-0.4% phosphoric acid solution system.See Fig. 9,10.
4, the discriminating of medicinal material of polygonum capilalum and evaluation
The finger printing that sample preparation methods that this experiment is determined and chromatographic condition are measured can carry out the discriminating of Herba Polygoni Capitati and head knotweed equally.Can find out that from collection of illustrative plates the Quercitroside peak of head knotweed can not detect substantially, the overall picture of chromatogram also obviously is different from Herba Polygoni Capitati, and the finger printing of its chromatogram and Herba Polygoni Capitati is carried out similarity evaluation, and similarity only is 0.304.
5, the evaluation of different place of production medicinal material of polygonum capilalum
By finger printing similarity software 12 batches of different places of production Herba Polygoni Capitati are estimated and to be drawn, the Herba Polygoni Capitati of Shibing, Guizhou cultivation and common pattern are relatively, similarity only is 0.130, and other place of production Herba Polygoni Capitati samples and common pattern compare similarity all greater than 0.910, illustrate in 12 batches of different places of production medicinal material of polygonum capilalum except that Shibing, Guizhou cultivation sample to have similarity preferably between other place of production Herba Polygoni Capitati.
Claims (8)
1, medicinal material of polygonum capilalum is characterized in that: it has the HPLC finger printing shown in Fig. 1 D, contains 13 characteristic peaks,
Wherein, chromatographic condition is: chromatographic column: Yi Lite Hypersil ODS chromatographic column (4.6mm * 150mm, 5 μ m); Column temperature: 25 ℃; Mobile phase: acetonitrile (A)-0.4% phosphoric acid (B) solution binary gradient elution, percentage by weight is: 0-30%: 100-70%; Flow velocity: 0.8mLmin
-1Detect wavelength: 310nm; Sample size 20 μ l.
2, medicinal material of polygonum capilalum according to claim 1 is characterized in that: the relative retention time of described 13 characteristic peaks is respectively:
No. 1 peak: 0.083 ± 0.001, No. 2 peaks: 0.178 ± 0.001, No. 3 peaks: 0.357 ± 0.001, No. 4 peaks: 0.388 ± 0.001, No. 5 peaks: 0.460 ± 0.001, No. 6 peaks: 0.520 ± 0.001, No. 7 peaks: 0.544 ± 0.002, No. 8 peaks: 0.792 ± 0.002, No. 9 peaks: 0.848 ± 0.002, No. 10 peaks: 0.888 ± 0.001,1, No. 12 peaks, No. 11 peaks: 1.266 ± 0.002, No. 13 peaks: 1.296 ± 0.002.
3, a kind of Herba Polygoni Capitati extract, it is to be raw material by claim 1 or 2 described medicinal material of polygonum capilalum, is prepared from through water or organic solvent extraction.
4, a kind of pharmaceutical composition, it is to be active component by claim 1 or 2 described Herba Polygoni Capitati primary crude drugs or the described Herba Polygoni Capitati extract of claim 3, adds the medicament that acceptable accessories or complementary composition are prepared from.
5, pharmaceutical composition according to claim 4 is characterized in that: described medicament is oral formulations, ejection preparation.
6, a kind of control claim 1 or 2 described medicinal material of polygonum capilalum method for quality, it comprises the steps:
A, preparation gallic acid object of reference solution: get gallic acid and add 50% dissolve with methanol and make 0.0204mgmL
-1Solution;
B, preparation Quercetin object of reference solution: it is an amount of to get the Quercetin reference substance, adds dissolve with methanol and makes 0.0216mgmL
-1Solution;
C, preparation Quercitroside object of reference solution: it is an amount of to get the Quercitroside reference substance, adds dissolve with methanol and makes 0.0992mgmL
-1Solution;
The preparation of d, need testing solution: get the Herba Polygoni Capitati fine powder, add 100ml 80% ethanol or methanol, put reflux in the water-bath, filter, residue reenters uses 80% alcohol reflux, filters merging filtrate.Filtrate is put in the evaporating dish and is volatilized, and residue adds 80% ethanol standardize solution, filters with microporous filter membrane, gets subsequent filtrate, promptly;
E, chromatographic condition:
Chromatographic condition is: chromatographic column: Yi Lite Hypersil ODS chromatographic column (4.6mm * 150mm, 5 μ m); Column temperature: 25 ℃; Mobile phase: acetonitrile-0.4% phosphoric acid solution or methanol-0.4% phosphoric acid solution binary gradient elution, percentage by weight is: 0-30%: 100-70%; Flow velocity: 0.8mLmin
-1Detect wavelength: 310nm; Sample size 20 μ l.
F, algoscopy: get each 15ul of reference substance and need testing solution, inject high performance liquid chromatograph, the record chromatogram.
7, the method for quality control of medicinal material of polygonum capilalum according to claim 6 is characterized in that: the described solvent of steps d is 80% ethanol.
8, the method for quality control of medicinal material of polygonum capilalum according to claim 6 is characterized in that: the described mobile phase of step e is: acetonitrile-0.4% phosphoric acid solution.
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