CN103558329B - Method for establishing fingerprint of leech and standard fingerprint of leech - Google Patents
Method for establishing fingerprint of leech and standard fingerprint of leech Download PDFInfo
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Abstract
The invention provides a method for establishing a fingerprint of leech. The method comprises the following specific steps: preparing the leech into a test solution; and performing separation detection through a high performance liquid chromatograph under certain conditions to obtain the fingerprint of the leech. The invention also provides a standard fingerprint of the leech, which is obtained through the method. The fingerprint can provide a reliable basis for leech identification and internal quality control. The method is simple and convenient to operate, high in stability, high in repeatability and large in quantity of characteristic peaks, and is capable of comprehensively and accurately evaluating the quality of leech medicine.
Description
Technical field
The present invention relates to a kind of method for building up of leech finger-print, and by the leech standard finger-print of the method gained, belong to Analysis of Chinese Traditional Medicine field.
Background technology
Leech is that the drying of Hirudinidae animal whitmania Whitmania pigra Whitman, leech Hirudo nipponica Whitman or willow leaf leech Whitmania acranulata Whitman is all, summer, season in autumn two catch, scald dead with boiling water, dry or low temperature drying, its taste is salty, bitter, property is put down, and returns Liver Channel, slightly poisonous.The traditional Chinese medical science thinks that leech is stasis-breaking drug traditional simply, has the effect by blood stasis, qualcomm meridian, dredging water passages.Leech has a wide range of applications clinically, mainly contains with the clinical disease of leech treatment: chronic nephritis, passages through which vital energy circulates varicose, prostatitis, infertile, diabetes, cancer, heart disease, cirrhosis, hypertension, hyperlipemia, vasculitis, woman's blood are closed, conjunctivitis and cataract, asthma etc.; Meanwhile, containing multiple effective components such as hirudin, histamine sample material, heparin, antithromboxins in leech body, for eliminating sequelae that Post operation causes, hinder blood clotting, stimulating circulation also plays an important role.Chinese patent CN101412726A discloses separation from leech and obtains 3 new heterocycle compound leech amine A, B, C, and carried out pharmacodynamics test, result shows that noval chemical compound has anti-cell anoxic and antitumor cell effect, wherein the antitumor cell effect of Compound C is comparatively strong, and anti-cell anoxic and the antitumor cell effect of compd A and B compound are all very strong.
At present, in the Chinese patent drug of domestic and international listing, some is exactly the preparation containing leech or hirudo extract, this kind of preparation is mainly used in treating cardiovascular and cerebrovascular disease, and achieve good effect, as brain blood recovering particles, leech dragon's blood open capsule, brain blood disease treating drop pills, Compound Leech Oral Liquor, QISHEN CAPSULE, Tongxinluo medicinal capsule etc., visible, leech has multiple pharmacological effect effect, has wide potential applicability in clinical practice.But, due to insufficient to chemical constitution study in leech, cause being mostly extract inspection, TLC distinguish etc. to the method for quality control of leech or hirudo extract in the existing drug standards.Such as, leech control medicinal material TLC distinguish is adopted in " Pharmacopoeia of People's Republic of China 2010 editions " leech medicinal material standard, its assay also just records the volume that leech extract consumes fibrin ferment roughly, be an antithrombin activity unit to neutralize the amount of the antithrombase of a unit, wherein specify, every 1 gram of leech contains antithrombin activity and should be not less than 16.0U, and leech, the whitmania acranulata Whitman U that should be not less than in 3.0U(formula is every 1 gram and contains thrombin activity unit, U/g).More than differentiate or measure the method for leech medicinal material, lack the Qualitive test to active chemical in leech and assay, specificity is poor, effectively can not control leech and the quality containing leech preparation.
Traditional Chinese medicine fingerprint technology be being used for of generally acknowledging at present both at home and abroad control Chinese medicine, natural drug or Chinese crude drug total quality the most effectively, the most accurately, the most direct means, be a kind of comprehensively, macroscopic view with quantifiable discrimination method, its base attribute is " globality " and " ambiguity "." globality " refers to the feature " looks " intactly comparing chromatogram; " ambiguity " is it is emphasised that the similarity of tester and testing sample finger-print.Use fingerprint pattern technology can differentiate the true and false of Chinese crude drug and Chinese patent drug, evaluate homogeneity and the stability of raw medicinal material, semi-manufacture and end product quality.For the quality controlling leech medicinal material with finger-print, domestic existing patent and bibliographical information, such as, Chinese patent CN1844911A discloses the method for building up of leech finger-print and discloses characteristic fingerprint pattern, but it extracts the composition in leech for 16 hours with physiological saline soaking at room temperature, the test period is long, and physiological saline or water effectively can not propose the pteridine constituents in leech, the irrelevant impurity of gained is many, affects the comprehensive of finger-print and accuracy; Only using hypoxanthine as object of reference, and to other important drug effect and quality mark composition in leech, as uracil, xanthine, uridine, leech amine C, leech amine B, leech amine A etc. all do not relate to, the quality of leech medicinal material can not be reflected accurately, all sidedly; In gradient elution process, at mobile phase not containing the time period of organic phase, common C
18c in chromatographic column filler
18the random coils of chain will cause component retention to change, and cause chromatographic system unstable.Yuan Xiaohuan etc. control the quality of leech injection and compound leech injection with HPLC finger-print, and establish the finger-print of leech injection, but the limitation of two sections of document leech fingerprints is identical with Chinese patent CN1844911A, its parenteral solution or compound injection are only using hypoxanthine as object of reference, and to other important effective component and quality mark composition in leech, as uracil, xanthine, uridine, leech amine C, leech amine B, leech amine A etc. does not all relate to, can not be accurate, reflect the quality of leech injection or compound leech injection all sidedly, and in gradient elution process, at mobile phase not containing the time period of organic phase, common C
18c in filler
18the random coils of chain will cause component retention to change, cause chromatographic system instability (Yang Xiaohuan, Yang Xudong, Cui Rongjun, Deng. the HPLC finger-print [J] of leech injection. Chinese Journal of Pharmaceuticals, 2008,39(7): 530 ~ 532.), (Yang Xiaohuan, Yang Xudong, Wang Chuntao, Deng. compound leech injection HPLC finger-print research [J]. CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2008,33(15): 1825 ~ 1827.).The method for building up to leech freeze drying powder injection finger-print such as Xu Changgen is studied, and obtain its finger-print, but it is only using hypoxanthine solution as object of reference, the quality of other important leech effective component in leech freeze drying powder injection comprehensively, accurately can not be reflected; In gradient elution process, in the time period of mobile phase containing minimum ratio (being less than 0.15%) organic phase, common C
18c in filler
18the random coils of chain will cause component retention to change, and cause chromatographic system unstable; And the characteristic peak on its finger-print is very little, and be separated not good (Xu Changgen, Luo Shishu, Xie Yanhua. leech freeze drying powder injection finger-print evaluate [J]. Journal of the Fourth Military Medical University, 2009,30(22): 2667 ~ 2669.).
In view of above deficiency, be necessary to invent a kind of new method setting up leech finger-print, making it can be easy and simple to handle, stability and favorable reproducibility, the quality of leech medicinal material can be evaluated again comprehensively, exactly, thus the discriminating to the leech medicinal material true and false, the place of production can be strengthened, thoroughly evaluating and control can be carried out to the quality of leech medicinal material and quality again, compensate for the deficiency of existing leech method of quality control, make it more improve and science.
Summary of the invention
The object of the invention is the deficiency for existing leech quality of medicinal material control method and a kind of method for building up of leech finger-print is provided, after being characterized in that leech is prepared into need testing solution, be separated through high performance liquid chromatograph and detect, obtain the finger-print of leech, thus to differentiate for leech and inherent quality control provides reliable foundation.
The method for building up of a kind of leech finger-print of the present invention specifically comprises the steps:
1) preparation of need testing solution: water intaking leech meal, with methanol solution refluxing extraction, centrifugal, get supernatant and be added on alkali alumina chromatographic column, collected post liquid, then with water elution, merged post liquid and water elution liquid, filter, as need testing solution;
2) preparation of object of reference solution: get uracil, hypoxanthine, xanthine, uridine, leech amine C, leech amine B, leech amine A reference substance, be dissolved in methanol solution as object of reference solution;
3) leech finger-print is made: accurate absorption need testing solution and object of reference solution respectively, is separated through high performance liquid chromatograph and detects, wherein, the potassium dihydrogen phosphate-acetonitrile consisting of 10mmol/L of mobile phase; Adopt gradient elution; Obtain leech finger-print.
Wherein, need testing solution is prepared as follows: precision takes leech meal 5g, put in tool plug container, precision adds 50% methanol solution 50ml, water-bath refluxing extraction 30min, with the speed high speed centrifugation 10min of 10000rpm, precision measures supernatant 20ml, be added on alkali alumina post (aluminium oxide is 6g, 100 ~ 200 orders, and chromatographic column internal diameter is 1.5cm), collected post liquid, use water 30ml wash-out again, merged post liquid and water elution liquid, put in 50ml volumetric flask, add water to scale, shake up, through 0.45 μm of filtering with microporous membrane, as need testing solution.Matching while using.
Tester solution is prepared as follows: get uracil, hypoxanthine, xanthine, uridine, leech amine C, leech amine B, leech amine A reference substance are appropriate, accurately weighed, add 50% methyl alcohol dissolve and be diluted to the mixed solution containing uracil 30 μ g, hypoxanthine 80 μ g, xanthine 20 μ g, uridine 20 μ g, leech amine C30 μ g, leech amine B30 μ g and leech amine A30 μ g in every 1ml, shake up, to obtain final product.
Chromatographic condition is: chromatographic column is YMC-Pack-ODS-AQ C
18post (250x4.6mm, 5 μm); Mobile phase is the potassium dihydrogen phosphate-acetonitrile of 10mmol/L; Adopt gradient elution: 0min → 15min → 40min → 60min, acetonitrile 0% → 0% → 20% → 20%; Flow velocity 0.8ml/min; Determined wavelength 254nm; Sample size 10 μ l; Column temperature 30 DEG C.
The present invention also provides the leech standard finger-print obtained by above-mentioned leech fingerprint.
10 parts of leech samples are prepared into need testing solution as stated above, high performance liquid chromatograph are separated and detect, use " similarity evaluation 2004 editions " software analysis of Chinese Pharmacopoeia Commission, obtain leech standard finger-print.
Described standard finger-print has 13 common characteristic peaks, 3rd, 5,6,7,9,10, No. 12 chromatographic peaks are corresponding in turn to uracil, hypoxanthine, xanthine, uridine, leech amine C, leech amine B and leech amine A, wherein 5 number xanthine peaks are the strongest, collection of illustrative plates total length is 60min, specific as follows:
No. 1 peak, Average residence time t is 4.6min;
No. 2 peaks, Average residence time t is 7.3min;
No. 3 peaks, Average residence time t is 8.4min;
No. 4 peaks, Average residence time t is 10.3min;
No. 5 peaks, Average residence time t is 14.9min;
No. 6 peaks, Average residence time t is 18.0min;
No. 7 peaks, Average residence time t is 20.2min;
No. 8 peaks, Average residence time t is 26.8min;
No. 9 peaks, Average residence time t is 30.7min;
No. 10 peaks, Average residence time t is 33.2min;
No. 11 peaks, Average residence time t is 35.4min;
No. 12 peaks, Average residence time t is 37.1min;
No. 13 peaks, Average residence time t is 40.9min.
Leech of the present invention refers to " Chinese name republic pharmacopeia one " (2010 editions) record the drying of Hirudinidae animal whitmania Whitmania pigra Whitman all.
Compared with prior art, the method for building up of leech finger-print of the present invention and characteristic fingerprint pattern thereof have following significant progress:
(1) preparation method of need testing solution is easy, and be easy to operation, the test period is short, and the need testing solution impurity of preparation is few, more totally, not easily pollutes chromatographic column, and the chromatogram impurity signal interference obtained is few;
(2) chromatographic condition easily realizes, and acetonitrile system post pressure is lower, be conducive to the stable of system, and chromatographic column selects water-fast YMC-Pack-ODS-AQ C
18filler, prevents common C
18filler chromatographic column uses water as and causes the instability of chromatographic system for column cap during mobile phase subsides;
(3) document shows, the suppression Blood coagulation of leech alcohol extract be better than water extract (yellow center of percussion. the clinical practice [J] of leech. Hubei Journal of Traditional Chinese Medicine, 1985(3): 13 ~ 15.), so, the present invention is with the composition in the methyl alcohol extraction leech of 50%, can Small molecular effective component at utmost in extracting and developing leech, as uracil, hypoxanthine, xanthine, uridine, leech amine C, leech amine B and leech amine A etc., and set up the fingerprint spectrum method of leech on this basis, make it have more specific aim and practicality to the quality control of leech;
(4) each Characteristic chromatographic peak under chromatographic condition of the present invention all achieves baseline separation, can not only carry out fuzzy diagnosis on the whole to leech, also can contrast object of reference chromatogram carries out clear discriminating and leech polycomponent quantitative analysis to leech;
(5) the inventive method stability is high, favorable reproducibility, characteristic peak are many, can evaluate the quality of leech medicinal material comprehensively, exactly, is suitable for the identify and control to the leech true and false, the place of production and quality.
Accompanying drawing explanation
Fig. 1 is leech characteristic fingerprint pattern, is from left to right the 1 to No. 13 total characteristic peak in figure respectively;
Fig. 2 is the object of reference collection of illustrative plates of leech, from left to right distinguishes the 3rd, 5,6,7,9,10, No. 12 peak in corresponding diagram 1, respectively be uracil, hypoxanthine, xanthine, uridine, leech amine C, leech amine B and leech amine A in figure.
Embodiment
The foundation of embodiment 1 leech finger-print
1) medicine, reagent and instrument
Leech medicinal material totally 10 batches, is all collected in Jiangsu Baoying County; Use Agilent1100 high performance liquid chromatograph; Reagent is chromatographically pure or analyzes pure;
2) preparation of need testing solution
Precision takes leech meal 5g, put in tool plug container, precision adds 50% methanol solution 50ml, water-bath refluxing extraction 30min, with the speed high speed centrifugation 10min of 10000rpm, precision measures supernatant 20ml, and (aluminium oxide is 6g to be added in alkali alumina post, 100 ~ 200 orders, chromatographic column internal diameter is 1.5cm) on, collect post liquid, then use water 30ml wash-out, merged post liquid and water elution liquid, put in 50ml volumetric flask, add water to scale, shake up, through 0.45 μm of filtering with microporous membrane, as need testing solution;
3) preparation of object of reference solution
Get uracil, hypoxanthine, xanthine, uridine, leech amine C, leech amine B, leech amine A reference substance are appropriate, accurately weighed, add 50% methyl alcohol dissolve and be diluted to the mixed solution containing uracil 30 μ g, hypoxanthine 80 μ g, xanthine 20 μ g, uridine 20 μ g, leech amine C30 μ g, leech amine B30 μ g and leech amine A30 μ g in every 1ml, shake up, to obtain final product;
4) leech finger-print is made
Chromatographic column is YMC-Pack-ODS-AQ C
18post (250x4.6mm, 5 μm); Mobile phase is the potassium dihydrogen phosphate-acetonitrile of 10mmol/L; Adopt gradient elution: 0min → 15min → 40min → 60min, acetonitrile 0% → 0% → 20% → 20%; Flow velocity 0.8ml/min; Determined wavelength 254nm; Sample size 10 μ l; Column temperature 30 DEG C; Accurate absorption need testing solution and object of reference solution respectively, is separated through high performance liquid chromatograph and detects, obtain leech finger-print, see Fig. 1 under above chromatographic condition; Obtain the collection of illustrative plates of object of reference by identical chromatographic conditions, see Fig. 2.
Embodiment 2 leech standard finger-print
Leech medicinal material totally 10 batches, is all collected in Jiangsu Baoying County.Each batch of leech finger-print is obtained by method described in the embodiment of the present invention 1, use " similarity evaluation 2004 editions " software analysis of Chinese Pharmacopoeia Commission, obtain leech standard finger-print, determine 13 common characteristic peaks, see Fig. 1.
3rd, 5,6,7,9,10, No. 12 chromatographic peaks are corresponding in turn to uracil, hypoxanthine, xanthine, uridine, leech amine C, leech amine B and leech amine A, and wherein 5 number xanthine peaks are the strongest, and collection of illustrative plates total length is 60min, specific as follows:
No. 1 peak, Average residence time t is 4.6min;
No. 2 peaks, Average residence time t is 7.3min;
No. 3 peaks, Average residence time t is 8.4min;
No. 4 peaks, Average residence time t is 10.3min;
No. 5 peaks, Average residence time t is 14.9min;
No. 6 peaks, Average residence time t is 18.0min;
No. 7 peaks, Average residence time t is 20.2min;
No. 8 peaks, Average residence time t is 26.8min;
No. 9 peaks, Average residence time t is 30.7min;
No. 10 peaks, Average residence time t is 33.2min;
No. 11 peaks, Average residence time t is 35.4min;
No. 12 peaks, Average residence time t is 37.1min;
No. 13 peaks, Average residence time t is 40.9min.
Claims (3)
1. the method for building up of leech finger-print, is characterized in that leech to be prepared into need testing solution, is separated and detects, obtain the finger-print of leech through high performance liquid chromatograph;
Described method comprises the steps:
1) preparation of need testing solution: water intaking leech meal, with methanol solution refluxing extraction, centrifugal, get supernatant and be added on alkali alumina chromatographic column, collected post liquid, then with water elution, merged post liquid and water elution liquid, filter, as need testing solution;
2) preparation of object of reference solution: get uracil, hypoxanthine, xanthine, uridine, leech amine C, leech amine B, leech amine A reference substance, be dissolved in methanol solution as object of reference solution;
3) making of leech finger-print: accurate absorption need testing solution and object of reference solution respectively, be separated through high performance liquid chromatograph and detect, testing conditions is: chromatographic column is YMC-Pack-ODS-AQ C
18post, 250mm × 4.6mm, 5 μm; Mobile phase is the potassium dihydrogen phosphate-acetonitrile of 10mmol/L; Adopt gradient elution: 0min → 15min → 40min → 60min, acetonitrile 0% → 0% → 20% → 20%; Flow velocity 0.8ml/min; Determined wavelength 254nm; Sample size 10 μ l; Column temperature 30 DEG C; Record 60 minutes chromatograms, obtain the finger-print of leech.
2. the method for building up of leech finger-print as claimed in claim 1, it is characterized in that described need testing solution is prepared as follows: precision takes leech meal 5g, put in tool plug container, precision adds 50% methanol solution 50ml, water-bath refluxing extraction 30min, with the speed high speed centrifugation 10min of 10000rpm, precision measures supernatant 20ml, be added on alkali alumina post, alumina weight is 6g, granularity is 100 ~ 200 orders, chromatographic column internal diameter is 1.5cm, collected post liquid, use water 30ml wash-out again, merged post liquid and water elution liquid, put in 50ml volumetric flask, add water to scale, shake up, through 0.45 μm of filtering with microporous membrane, as need testing solution.
3. the method for building up of leech finger-print as claimed in claim 1 or 2, it is characterized in that described object of reference solution is prepared as follows: get uracil, hypoxanthine, xanthine, uridine, leech amine C, leech amine B, leech amine A reference substance are appropriate, accurately weighed, add 50% methyl alcohol dissolve and be diluted to the mixed solution containing uracil 30 μ g, hypoxanthine 80 μ g, xanthine 20 μ g, uridine 20 μ g, leech amine C30 μ g, leech amine B30 μ g and leech amine A30 μ g in every 1ml, shake up, to obtain final product.
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| CN105181845A (en) * | 2015-09-24 | 2015-12-23 | 广西壮族自治区药用植物园 | Gas chromatography fingerprint spectrum detection method for oleic acid of whitmania pigra |
| CN108845059B (en) * | 2018-08-24 | 2020-05-26 | 鲁南制药集团股份有限公司 | Method for establishing HPLC fingerprint of Mailuoshutong granules and standard spectrum thereof |
| CN113984936A (en) * | 2021-12-09 | 2022-01-28 | 车延柠 | LC-MS characteristic map of leech enzymolysis polypeptide, detection method and application thereof |
| CN115184490B (en) * | 2022-06-28 | 2023-10-24 | 鲁南制药集团股份有限公司 | Establishment method and application of HPLC standard fingerprint of Hirudo vein relaxing capsule |
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