CN102707003B - Moldavica dragonhead extractive and fingerprint spectrum detection method thereof - Google Patents
Moldavica dragonhead extractive and fingerprint spectrum detection method thereof Download PDFInfo
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- CN102707003B CN102707003B CN201210179993.4A CN201210179993A CN102707003B CN 102707003 B CN102707003 B CN 102707003B CN 201210179993 A CN201210179993 A CN 201210179993A CN 102707003 B CN102707003 B CN 102707003B
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Abstract
The invention provides a moldavica dragonhead extractive and a fingerprint spectrum detection method thereof. Specifically, a method for preparing the moldavica dragonhead extractive comprises the following steps: crushing a moldavica dragonhead medicinal material; adding water, of which the weight is 5-30 times of the weight of the medicinal material, and extracting for 30-90 minutes; adding 95% alcohol, of which the volume is 1-3 times of the volume of water, keeping on heating and performing backflow extraction for 30-90 minutes; and recycling alcohol from an extracting solution, concentrating and drying, thereby obtaining the moldavica dragonhead extractive. The invention also provides the moldavica dragonhead extractive prepared according to the method, the method for detecting and evaluating the moldavica dragonhead medicinal material or the moldavica dragonhead extractive according to a fingerprint spectrum method, and the method for detecting and evaluating the moldavica dragonhead medicinal material or the moldavica dragonhead extractive according to the fingerprint spectrum method by taking a prototype absorbing component and/or a metabolism component as indexes. The fingerprint spectrum detection method provided by the invention can be effectively applied to the quality control of the moldavica dragonhead or the moldavica dragonhead extractive.
Description
Technical field
The invention belongs to medicine preparation, Pharmaceutical Analysis and drug metabolism technical field.Be specifically related to the preparation of Chinese medical extract, and carry out traditional Chinese medicine quality control by traditional Chinese medicine fingerprint method; By high performance liquid chromatography (HPLC) finger-print, carry out multicomponent total quality control on the one hand, by locking prototype, absorb composition on the other hand, carry out the quality control of concrete composition.
Background technology
In Chinese medicine, contain number of chemical composition, and taken in body simultaneously, produce therapeutic action.But it is remarkable that Chinese medicine includes the polarity gap of chemical composition, be often not only to contain the chemical composition that polarity is very little but also contain the chemical composition that polarity is very large, single solvent is difficult to extract all kinds of polar components conventionally.So adopt opposed polarity reagent can extract all kinds of formulation chemical compositions through two steps or multistep extraction, in assurance extract, each constituents fully comprises as far as possible.
The application of fingerprint pattern technology in the autonomic drug quality controls such as Chinese medicine, has become one of effective means of generally acknowledging both at home and abroad.U.S. FDA is at the < < FDA promulgating for 1996 in the industry guide > > about the < < autonomic drug product of the guide > > of vegetable products and 2004, requirement is carried out quality control to product and autonomic drug product in the middle of plant material, autonomic drug with finger-print, has proposed constructive suggestions.Medicinal and the fragrant plant association of British Herbal Pharmacopoeia, India herbal medicine allusion quotation and Canada, German medicinal plant association also accept chromatographic fingerprinting.State food and drug administration has issued technical requirement (provisional) the > > of the finger-print research of < < traditional Chinese medicine in 2000, explicitly call for the traditional Chinese medicine of new application and the traditional Chinese medicine that gone on the market are carried out to finger-print standard, China national pharmacopoeia commission has issued < < traditional Chinese medicine chromatographic fingerprinting experimental study technical manual (trying) > > in 2002, the technology contents of chromatographic fingerprinting has been proposed to guiding suggestion.Chemical composition chromatographic fingerprinting, especially high performance liquid chromatography (HPLC) finger-print directly shows the fingerprint characteristic information of multiple specific chemical composition, be convenient to carry out on the whole multicomponent quality control from composition, easily obtain in the world approval, therefore Chinese medicine adopts HPLC finger-print to carry out quality control, is the rational effective mass control device of international endorsement, technology.
Adopt concrete certain Chinese medicine component content to carry out traditional Chinese medicine quality control, the selection of composition directly determines the height of traditional Chinese medicine quality level of control.Optimal alternative composition is that drug effect is clear and definite, and mechanism is clear, prototype absorbs and reach target spot long-time, high concentration is resident, but it is normal clearly difficult to meet the composition of this high request, so indirectly hindered the lifting of traditional Chinese medicine quality level of control.But our practice innovation method of the Quality Control target call of drug effect-quality association, moves closer to target; So the situation that mainly produces drug effect by absorbed into serum in Chinese medicine multicomponent is conducted a research, can prototype absorbed into serum as the screening principle of Quality Control composition, can more approach the Quality Control target of drug effect-quality association.
Dracocephalum moldavica is Xinjiang Uygur medicine, is the dry aerial parts of labiate Dracocephalum moldavica, has purge the green, clearing away the stomach-heat, hemostasia effect, for diseases such as the pharyngalgia of having a headache, jaundice, haematemesis.Chemical composition in Dracocephalum moldavica and its efficacy effect are closely related.It absorbs and enters after blood circulation system mainly with appropriate format greatly, is transported to corresponding site of action and could produces curative effect.So by the comparative analysis of extract finger-print and oral extract artifact sample finger-print, the prototype defining in Dracocephalum moldavica absorbs composition as the concrete composition of Quality Control, and comprehensively carry out quality control in conjunction with the overall ingredients fingerprint of Dracocephalum moldavica, be the quality control method of novelty.
Summary of the invention
The object of this invention is to provide a kind of preparation method of Dracocephalum moldavica extract, and use the method for HPLC finger-print to carry out quality control to it.For realizing this object, by high performance liquid chromatography (HPLC) finger-print, carry out multicomponent total quality control on the one hand, by metabolism of Chinese medicine method locking prototype, absorb composition on the other hand, carry out the quality control of concrete composition, make the effective substance of Dracocephalum moldavica clearer and more definite.
For this reason, first aspect present invention provides the method for preparing Dracocephalum moldavica extract, and it comprises the following steps: by Dracocephalum moldavica pulverizing medicinal materials; Add medicinal material weight 5-30 water extraction doubly to get 30-90 minute; Add again water volume 1-3 95% ethanol doubly to continue heating and refluxing extraction 30-90 minute; Extract reclaims ethanol concentrated, dry, obtains.
According to the method for first aspect present invention, when wherein water extraction is got, the consumption of water is 5-20 times of medicinal material weight, and for example doubly, for example 5-10 doubly for 5-15.
According to the method for first aspect present invention, when wherein water extraction is got, be that the mode decocting is extracted.In one embodiment, the time of decoction is 30-90 minute, 30-60 minute for example, for example approximately 45 minutes.
According to the method for first aspect present invention, while wherein adding alcohol extract, the amount of ethanol used is 1-2 times of water volume, preferably 1 times.
According to the method for first aspect present invention, during alcohol extract, be wherein that the mode refluxing is extracted.In one embodiment, the time of backflow is 30-90 minute, 30-60 minute for example, for example approximately 45 minutes.
Second aspect present invention provides a kind of Dracocephalum moldavica extract, and it is substantially according to the method for first aspect present invention, to extract and obtain.
Second aspect present invention provides a kind of Dracocephalum moldavica extract, and it is to take Dracocephalum moldavica through the method for following steps, to extract and obtain as medicinal material: by Dracocephalum moldavica pulverizing medicinal materials; Add medicinal material weight 5-30 water extraction doubly to get 30-90 minute; Add again water volume 1-3 95% ethanol doubly to continue heating and refluxing extraction 30-90 minute; Extract reclaims ethanol concentrated, dry, obtains.
According to the Dracocephalum moldavica extract of second aspect present invention, when wherein water extraction is got, the consumption of water is 5-20 times of medicinal material weight, and for example doubly, for example 5-10 doubly for 5-15.
According to the Dracocephalum moldavica extract of second aspect present invention, when wherein water extraction is got, be that the mode decocting is extracted.In one embodiment, the time of decoction is 30-90 minute, 30-60 minute for example, for example approximately 45 minutes.
According to the Dracocephalum moldavica extract of second aspect present invention, while wherein adding alcohol extract, the amount of ethanol used is 1-2 times of water volume, preferably 1 times.
According to the Dracocephalum moldavica extract of second aspect present invention, during alcohol extract, be wherein that the mode refluxing is extracted.In one embodiment, the time of backflow is 30-90 minute, 30-60 minute for example, for example approximately 45 minutes.
Third aspect present invention provides the method with fingerprint spectrum method detecting and assessing Dracocephalum moldavica medicinal material or Dracocephalum moldavica extract, and the method comprises the steps:
(a) get for the Dracocephalum moldavica medicinal material of test or for the Dracocephalum moldavica extract of testing, the described Dracocephalum moldavica medicinal material for test is processed and is obtained Dracocephalum moldavica extract (thus obtained Dracocephalum moldavica extract or the aforementioned Dracocephalum moldavica extract for test according to method described in first aspect present invention, the two can be described as test sample, or can be described as test sample extract);
(b) separately get the Dracocephalum moldavica medicinal material of contrast or the Dracocephalum moldavica extract of contrast, the Dracocephalum moldavica medicinal material of described contrast is processed and is obtained Dracocephalum moldavica extract (the Dracocephalum moldavica extract of thus obtained Dracocephalum moldavica extract or aforementioned contrast according to method described in first aspect present invention, the two can be described as reference substance, or can be described as reference substance extract);
(c) precision takes test sample extract and reference substance extract is appropriate respectively, with the methyl alcohol of 50-100 times of volume, dissolve, through 0.45 μ m filtering with microporous membrane, filtrate is called need testing solution and reference substance solution, for high performance liquid chromatography (HPLC), analyzes;
(d) high-efficient liquid phase chromatogram condition is: chromatographic column is C
18chromatographic column, phosphate aqueous solution-acetonitrile that mobile phase is 0.1%; Flow velocity 0.8 ~ 1.2ml/min; Adopt the mode of composition response under each wavelength of 200-400nm length ultraviolet detection record to detect; Gradient elution mode is:
(e) draw need testing solution and the reference substance solution of step (c), be injected into respectively in high performance liquid chromatography, according to (d) described chromatographic condition, carry out wash-out, obtain respectively test sample HPLC figure and reference substance HPLC figure;
(f) comparison step (e) gained test sample HPLC figure and reference substance HPLC figure, if two figure peak shapes are consistent, show that test sample is Dracocephalum moldavica certified products or Dracocephalum moldavica extract certified products, otherwise test sample is not Dracocephalum moldavica certified products or Dracocephalum moldavica extract certified products.
According to the method for third aspect present invention, wherein said C
18chromatographic column is Thermo scientific ODS-2 Hypersil C
18chromatographic column.In one embodiment, described Thermo scientific ODS-2 Hypersil C
18chromatographic column specification is 4.6mm * 250mm, 5 μ m.
According to the method for third aspect present invention, wherein the flow velocity of (d) described mobile phase is 1ml/min.
According to the method for third aspect present invention, wherein (d) described wavelength is 277nm.
According to the method for third aspect present invention, wherein described in (d) item, the column temperature of chromatographic column is controlled at 30 ℃.
Fourth aspect present invention provides the method with fingerprint spectrum method detecting and assessing Dracocephalum moldavica medicinal material or Dracocephalum moldavica extract, and the method comprises the steps:
(i) get for the Dracocephalum moldavica medicinal material of test or for the Dracocephalum moldavica extract of testing, the described Dracocephalum moldavica medicinal material for test is processed and is obtained Dracocephalum moldavica extract (thus obtained Dracocephalum moldavica extract or the aforementioned Dracocephalum moldavica extract for test according to method described in first aspect present invention, the two can be described as test sample, or can be described as test sample extract);
(ii) separately get the Dracocephalum moldavica medicinal material of contrast or the Dracocephalum moldavica extract of contrast, the Dracocephalum moldavica medicinal material of described contrast is processed and is obtained Dracocephalum moldavica extract (the Dracocephalum moldavica extract of thus obtained Dracocephalum moldavica extract or aforementioned contrast according to method described in first aspect present invention, the two can be described as reference substance, or can be described as reference substance extract);
(iii) precision takes test sample extract and reference substance extract is appropriate respectively, with the methyl alcohol of 50-100 times of volume, dissolve, through 0.45 μ m filtering with microporous membrane, filtrate is called need testing solution and reference substance solution, for high performance liquid chromatography (HPLC), analyzes;
(iv) high-efficient liquid phase chromatogram condition is: chromatographic column is C
18chromatographic column, phosphate aqueous solution-acetonitrile that mobile phase is 0.1%; Flow velocity 0.8 ~ 1.2ml/min; Adopt the mode of composition response under each wavelength of 200-400nm length ultraviolet detection record to detect; Gradient elution mode is:
(v) draw need testing solution and the reference substance solution of step (iii), be injected into respectively in high performance liquid chromatography, according to (iv) described chromatographic condition, carry out wash-out, obtain respectively test sample HPLC figure and reference substance HPLC figure;
(vi) comparison step (v) gained test sample HPLC figure and reference substance HPLC figure, if two figure peak shapes are consistent, show that test sample is Dracocephalum moldavica certified products or Dracocephalum moldavica extract certified products, otherwise test sample is not Dracocephalum moldavica certified products or Dracocephalum moldavica extract certified products;
(vii) make the test sample extract of the mammal oral step of difference (i) or the reference substance extract of step (ii), in oral rear 0.5-10 hour, get biological sample, extract this biological sample, obtain biological need testing solution (obtaining after the test sample extract oral by step (i)) or biological reference substance solution (obtaining after the reference substance extract oral by step (ii));
(viii) draw biological need testing solution and the biological reference substance solution of step (vii), be injected into respectively in high performance liquid chromatography, according to (iv) described chromatographic condition, carry out wash-out, obtain respectively biological test sample HPLC figure and biological reference substance HPLC figure;
(ix) the biological test sample HPLC figure of comparison step (viii) gained and biological reference substance HPLC figure, if two figure peak shapes are consistent, show that test sample is Dracocephalum moldavica certified products or Dracocephalum moldavica extract certified products, otherwise test sample not Dracocephalum moldavica certified products or Dracocephalum moldavica extract certified products.
The method of fourth aspect present invention can be verified the true and false or the quality of medicine or extract by step (vi) and two kinds of modes of step (ix) with vitro samples and vivo sample.
According to the method for fourth aspect present invention, wherein said C
18chromatographic column is Thermoscientific ODS-2 Hypersil C
18chromatographic column.In one embodiment, described Thermo scientific ODS-2 Hypersil C
18chromatographic column specification is 4.6mm * 250mm, 5 μ m.
According to the method for fourth aspect present invention, wherein the flow velocity of (iv) described mobile phase is 1ml/min.
According to the method for fourth aspect present invention, wherein (iv) described wavelength is 277nm.
According to the method for fourth aspect present invention, wherein described in (iv) item, the column temperature of chromatographic column is controlled at 30 ℃.
According to the method for fourth aspect present invention, wherein described in step (vii), mammal is selected from people, rat, mouse, dog, monkey etc.In one embodiment, described mammal is rat.
Fifth aspect present invention provides take that prototype absorbs composition and/or Metabolite is the method for fingerprint spectrum method detecting and assessing Dracocephalum moldavica medicinal material or Dracocephalum moldavica extract for index, and the method comprises the steps:
(1) get for the Dracocephalum moldavica medicinal material of test or for the Dracocephalum moldavica extract of testing, the described Dracocephalum moldavica medicinal material for test is processed and is obtained Dracocephalum moldavica extract (thus obtained Dracocephalum moldavica extract or the aforementioned Dracocephalum moldavica extract for test according to method described in first aspect present invention, the two can be described as test sample, or can be described as test sample extract);
(2) separately get the Dracocephalum moldavica medicinal material of contrast or the Dracocephalum moldavica extract of contrast, the Dracocephalum moldavica medicinal material of described contrast is processed and is obtained Dracocephalum moldavica extract (the Dracocephalum moldavica extract of thus obtained Dracocephalum moldavica extract or aforementioned contrast according to method described in first aspect present invention, the two can be described as reference substance, or can be described as reference substance extract);
(3) precision takes test sample extract and reference substance extract is appropriate respectively, with the methyl alcohol of 50-100 times of volume, dissolve, through 0.45 μ m filtering with microporous membrane, filtrate is called need testing solution and reference substance solution, for high performance liquid chromatography (HPLC), analyzes;
(4) high-efficient liquid phase chromatogram condition is: chromatographic column is C
18chromatographic column, phosphate aqueous solution-acetonitrile that mobile phase is 0.1%; Flow velocity 0.8 ~ 1.2ml/min; Adopt the mode of composition response under each wavelength of 200-400nm length ultraviolet detection record to detect; Gradient elution mode is:
(5) draw need testing solution and the reference substance solution of step (3), be injected into respectively in high performance liquid chromatography, according to (4) described chromatographic condition, carry out wash-out, obtain respectively test sample HPLC figure and reference substance HPLC figure;
(6) comparison step (5) gained test sample HPLC figure and reference substance HPLC figure, if two figure peak shapes are consistent, show that test sample is Dracocephalum moldavica certified products or Dracocephalum moldavica extract certified products, otherwise test sample is not Dracocephalum moldavica certified products or Dracocephalum moldavica extract certified products;
(7) make the test sample extract of the mammal oral step of difference (1) or the reference substance extract of step (2), in oral rear 0.5-10 hour, get biological sample, extract this biological sample, obtain biological need testing solution (obtaining after the test sample extract oral by step (1)) or biological reference substance solution (obtaining after the reference substance extract oral by step (2));
(8) draw biological need testing solution and the biological reference substance solution of step (7), be injected into respectively in high performance liquid chromatography, according to (4) described chromatographic condition, carry out wash-out, obtain respectively biological test sample HPLC figure and biological reference substance HPLC figure;
(9) the biological test sample HPLC figure of comparison step (8) gained and biological reference substance HPLC figure, if two figure peak shapes are consistent, show that test sample is Dracocephalum moldavica certified products or Dracocephalum moldavica extract certified products, otherwise test sample not Dracocephalum moldavica certified products or Dracocephalum moldavica extract certified products;
(10) the reference substance solution HPLC of comparison step (5) figure and the biological reference substance HPLC figure of step (8) gained, determine that the prototype that reference substance characterizes absorbs composition and/or Metabolite;
(11) the need testing solution HPLC of comparison step (5) figure and the biological test sample HPLC figure of step (8) gained, if showing to have with the described prototype of step (10), this test sample absorbs composition and/or Metabolite chromatographic peak, show that test sample is Dracocephalum moldavica certified products or Dracocephalum moldavica extract certified products, otherwise test sample not Dracocephalum moldavica certified products or Dracocephalum moldavica extract certified products.
The method of fifth aspect present invention can be verified the true and false or the quality of medicine or extract by step (6) and (9) two kinds of modes of step with vitro samples and vivo sample; Step (10) and step (11) complementally absorb composition and/or are metabolized to the true and false or the quality of assigning to verify medicine or extract with prototype simultaneously.
According to the method for fifth aspect present invention, wherein said C
18chromatographic column is Thermo scientific ODS-2 Hypersil C
18chromatographic column.In one embodiment, described Thermo scientific ODS-2 Hypersil C
18chromatographic column specification is 4.6mm * 250mm, 5 μ m.
According to the method for fifth aspect present invention, wherein the flow velocity of (4) described mobile phase is 1ml/min.
According to the method for fifth aspect present invention, wherein (4) described wavelength is 277nm.
According to the method for fifth aspect present invention, wherein described in (4) item, the column temperature of chromatographic column is controlled at 30 ℃.
According to the method for fifth aspect present invention, wherein described in step (7), mammal is selected from people, rat, mouse, dog, monkey etc.In one embodiment, described mammal is rat.
According to the method for fifth aspect present invention, wherein described in step (7), biological sample includes but not limited to blood sample, urine sample and saliva sample.
According to the method for fifth aspect present invention, wherein in step (10), obtain 1 prototype and absorb composition and 2 Metabolites.In one embodiment, the retention time that described 1 prototype absorbs composition is about 21.0min.In one embodiment, the retention time of described 2 Metabolites is about respectively 21.5min and 25.3min.
Arbitrary embodiment of applicable equally other the arbitrary embodiment of arbitrary technical characterictic that arbitrary embodiment of either side of the present invention or this either side has or other either side, as long as they can be not conflicting, certainly, at where applicable each other, necessary words can be done suitably to modify to individual features.Be further described with feature to various aspects of the present invention below.
All documents that the present invention quotes from, their full content is incorporated to herein by reference, and if when the expressed implication of these documents and the present invention are inconsistent, be as the criterion with statement of the present invention.In addition, various terms and phrase that the present invention uses have the general sense of well known to a person skilled in the art, nonetheless, the present invention still wishes at this, these terms and phrase to be described in more detail and to be explained, the term of mentioning and phrase, if any inconsistent with known implication, are as the criterion with the implication that the present invention was explained.
In the present invention, when obtaining biological sample, for example, while obtaining the described biological sample of fifth aspect present invention step (7), can process with reference to the conventional method of this area.For example, biological sample (as: blood sample, urine sample and saliva sample) after animal used as test (such as rat) or human oral Dracocephalum moldavica extract etc. can be extracted to composition by adding 3-5 doubly to measure methyl alcohol, acetonitrile etc., also can be in conjunction with the method removal of impurities of the precipitation bioturbation things such as perchloric acid, vortex mixes 1-3min, centrifugal 3 ~ the 5min of 4000-9000r/min, get 0.45 μ m filtering with microporous membrane for supernatant, filtrate, as need testing solution, can be analyzed for HPLC thus.
In the present invention, the term such as " approximately ", " substantially " all can be understood according to the usual manner of this area.For example, spectrogram peak shape is basically identical, and the strong deviation of retention time, Relative Peak that is interpreted as spectrogram is all no more than 20%.
In the present invention, when carrying out high performance liquid chromatography (HPLC) analysis, can use C
18chromatographic column, phosphate aqueous solution-acetonitrile that mobile phase is 0.1%; Flow velocity 0.8 ~ 1.2ml/min; Adopt the mode of composition response under each wavelength of 200-400nm length ultraviolet detection record to detect; Gradient elution mode can be 0min → 10min → 15min → 20min → 30min → 40min → 50min → 70min, acetonitrile: 15% → 15% → 30% → 45% → 45% → 60% → 70% → 100%.The inventor finds, uses Thermo scientific ODS-2 Hypersil C
18chromatographic column (4.6mm * 250mm, 5 μ m) time, for example, if above-mentioned condition of gradient elution is done trickle change (when each time point acetonitrile concentration change 10% is above), be difficult to obtain prototype and absorb the effective chromatographic resolution between composition and Metabolite.
In one embodiment of the invention, particularly, in fifth aspect present invention embodiment, provide the authentication method of Dracocephalum moldavica medicinal material efficient liquid-phase chromatograph finger print atlas, comprised the following steps:
(1) preparation of Dracocephalum moldavica extract: adopt first water refluxing extraction, then add ethanol to carry out 2 step extraction methods of refluxing extraction for the second time, prepare Dracocephalum moldavica extract.After Dracocephalum moldavica pulverizing medicinal materials, add medicinal material weight 5-30 water extraction doubly to get 30-90 minute, then add water volume 1-3 95% ethanol doubly to continue heating and refluxing extraction 30-90 minute, extract reclaim ethanol and concentrated and Dracocephalum moldavica extract;
(2) evaluation of Dracocephalum moldavica extract and finger-print quality assessment: get extract a small amount of, with the methyl alcohol of 50-100 times of volume, dissolve, after 0.45 μ m filtering with microporous membrane, filtrate is carried out high performance liquid chromatography (HPLC) analysis as need testing solution, and chromatographic column is C
18chromatographic column, phosphate aqueous solution-acetonitrile that mobile phase is 0.1%; Flow velocity 0.8 ~ 1.2ml/min; Adopt the mode of composition response under each wavelength of 200-400nm length ultraviolet detection record to detect; Gradient elution mode is: 0min → 10min → 15min → 20min → 30min → 40min → 50min → 70min, acetonitrile: 15% → 15% → 30% → 45% → 45% → 60% → 70% → 100%; The HPLC finger-print of thus obtained Dracocephalum moldavica extract, can be used for evaluation and the quality control of medicinal material or its extract.
(3) Dracocephalum moldavica extract mesarcs absorbs the locking of composition, and for quality assessment:
Biological sample (as: blood sample, urine sample and saliva sample) after animal used as test and human oral Dracocephalum moldavica extract etc. is extracted to composition by adding 3-5 doubly to measure methyl alcohol, acetonitrile etc., also can be in conjunction with the method removal of impurities of the precipitation bioturbation things such as perchloric acid, vortex mixes 1-3min, centrifugal 3 ~ the 5min of 4000-9000r/min, get 0.45 μ m filtering with microporous membrane for supernatant, filtrate, as need testing solution, is carried out high performance liquid chromatography (HPLC) analysis, uses C
18chromatographic column, phosphate aqueous solution-acetonitrile that mobile phase is 0.1%; Flow velocity 0.8 ~ 1.2ml/min; Adopt the mode of composition response under each wavelength of 200-400nm length ultraviolet detection record to detect; Gradient elution mode is 0min → 10min → 15min → 20min → 30min → 40min → 50min → 70min, acetonitrile: 15% → 15% → 30% → 45% → 45% → 60% → 70% → 100%; Obtain thus the biological sample HPLC finger-print of Dracocephalum moldavica extract, and with the HPLC finger-print comparison of Dracocephalum moldavica extract, locking prototype absorbs composition and carries out quality control.
Visible, the present invention provides a kind of preparation method of Dracocephalum moldavica medicinal substances extract substantially, and uses the method for HPLC finger-print to carry out quality control to it.After Dracocephalum moldavica pulverizing medicinal materials, extracting in water 30-90 minute, then add water volume 1-3 95% ethanol doubly to continue heating and refluxing extraction 30-90 minute, extract reclaim ethanol and concentrated and Dracocephalum moldavica extract.Get extract a small amount of, with the methyl alcohol of 50-100 times of volume, dissolve, after filtering with microporous membrane, filtrate is carried out high performance liquid chromatography (HPLC) analysis as need testing solution, and chromatographic column is C
18chromatographic column, phosphate aqueous solution-acetonitrile that mobile phase is 0.1%; Adopt gradient elution mode; Adopt the mode of composition response under each wavelength of 200-400nm length ultraviolet detection record to detect; Obtain the HPLC finger-print of Dracocephalum moldavica extract, for its global quality control.Biological sample after animal used as test and human oral Dracocephalum moldavica extract is carried out after removal of impurities processing, carries out the experiment of finger-print under identical HPLC condition, and with the comparison of extract finger-print, locking prototype absorbs composition as medicinal material Quality Control composition.
The present invention has following significant advantage and purposes:
(1) in Dracocephalum moldavica extract provided by the invention, both comprised polarity small component, and also comprised the large composition of polarity, composition kind is comprehensive;
(2) the present invention, by HPLC chromatographic fingerprinting, had both carried out the comprehensive Quality Control of multicomponent, again to being absorbed the concrete Quality Control of composition.
Accompanying drawing explanation
Fig. 1 is Dracocephalum moldavica extract chromatographic fingerprinting provided by the invention, has shown that retention time is about the chromatographic peak at 21min place in figure.The chromatographic peak of this retention time appears in the biological sample of corresponding extract (be prototype absorbs composition or prototype enters blood component) equally; Yet at 21.47min, 25.26min place, there are two new chromatographic peaks (being Metabolite) that do not occur in extract in biological sample.
Embodiment
Below by specific embodiment/experimental example, further illustrate the present invention, still, should be understood to, these embodiment and experimental example are only used for the use specifically describing more in detail, and should not be construed as for limiting in any form the present invention.
The present invention carries out generality and/or concrete description to the material and the test method that use in test.Although be well known in the art for realizing many materials and the method for operating that the object of the invention used, the present invention still does to describe in detail as far as possible at this.It will be apparent to those skilled in the art that hereinafter, if not specified, material therefor of the present invention and method of operating are well known in the art.
The preferred embodiments of the present invention following (arbitrary technical characterictic of the preferred embodiment or parameter all can be independently of one another or be applicable in combination the present invention first to arbitrary embodiment of the 5th aspect):
(1) instrument and medicine: use Waters1525 high performance liquid chromatograph; LG16-W supercentrifuge; QL-901 turbine mixer; Dracocephalum moldavica medicinal material is purchased from Chinese herbal medicine shop; Other reagent are chromatographically pure or analyze pure.
(2) animal: clean level healthy SD rat, 240 ~ 260g, Beijing Vital River Experimental Animals Technology Co., Ltd. provides; Conformity certification number: SCXK (capital) 2006-2009.Raise in meeting the barrier environment of national standard, 22 ~ 24 ℃ of room temperatures, relative humidity 60%, conventional raising fed, and freely drinks water.
(3) preparation of need testing solution: precision takes medicinal powder 10g, be placed in volumetric flask, the distilled water solution that adds 70ml, add hot reflux 50min, add again 95% ethanol of 2 times of water volume to continue heating and refluxing extraction 60 minutes, after filtrate is cooling, get 0.45 μ m filtering with microporous membrane for supernatant, filtrate is as need testing solution;
(4) making of finger-print: chromatographic column is Thermo scientific ODS-2 Hypersil C
18chromatographic column (4.6mm * 250mm, 5 μ m), phosphate aqueous solution-acetonitrile that mobile phase is 0.1%; Adopt gradient elution mode 0min → 10min → 15min → 20min → 30min → 40min → 50min → 70min, acetonitrile 15% → 15% → 30% → 45% → 45% → 60% → 70% → 100%; Flow velocity 1ml/min; Detect wavelength 277nm; 30 ℃ of column temperatures; Analyze with this understanding need testing solution, obtain the finger-print of Dracocephalum moldavica medicinal material;
(5) preparation of Dracocephalum moldavica blood serum sample: SD rat, 240 ~ 260g, gavage gives Dracocephalum moldavica water extraction liquid 3 ~ 5ml, abdominal aortic blood 6 ~ 8ml after 1h, the centrifugal 3 ~ 5min of 9000r/min, get upper serum, add 3 ~ 5 times of amount methanol extraction albumen, vortex mixes 2min, the centrifugal 3 ~ 5min of 9000r/min, get 0.45 μ m filtering with microporous membrane for supernatant, filtrate is as need testing solution; Pressing Dracocephalum moldavica medicinal materials fingerprint condition measures;
(6) Dracocephalum moldavica enters determining of blood component: by Dracocephalum moldavica medicinal material and serum HPLC fingerprint comparison, after Dracocephalum moldavica medicinal material rat oral gavage, 1 prototype enters blood component, 2 Metabolites.Retention time is respectively: 21.03min, 21.47min, 25.26min.
Claims (19)
1. with fingerprint spectrum method, detect the method for Dracocephalum moldavica medicinal material or Dracocephalum moldavica extract, the method comprises the steps:
(a) get for the Dracocephalum moldavica medicinal material of test or for the Dracocephalum moldavica extract of testing, the described treated Dracocephalum moldavica extract that obtains of Dracocephalum moldavica medicinal material for test, the two is called test sample extract thus obtained Dracocephalum moldavica extract or the aforementioned Dracocephalum moldavica extract for test;
(b) separately get the Dracocephalum moldavica medicinal material of contrast or the Dracocephalum moldavica extract of contrast, the treated Dracocephalum moldavica extract that obtains of Dracocephalum moldavica medicinal material of described contrast, the two is called reference substance extract the Dracocephalum moldavica extract of thus obtained Dracocephalum moldavica extract or aforementioned contrast;
(c) precision takes test sample extract and reference substance extract is appropriate respectively, with the methyl alcohol of 50-100 times volume, dissolves, and through 0.45 μ m filtering with microporous membrane, filtrate is called need testing solution and reference substance solution, for efficient liquid phase chromatographic analysis;
(d) high-efficient liquid phase chromatogram condition is: chromatographic column is C
18chromatographic column, phosphate aqueous solution-acetonitrile that mobile phase is 0.1%; Flow velocity 0.8～1.2ml/min; Adopt the mode of composition response under each wavelength of 200-400nm length ultraviolet detection record to detect; Gradient elution mode is:
(e) draw need testing solution and the reference substance solution of step (c), be injected into respectively in high performance liquid chromatography, according to (d) described chromatographic condition, carry out wash-out, obtain respectively test sample HPLC figure and reference substance HPLC figure;
(f) comparison step (e) gained test sample HPLC figure and reference substance HPLC figure;
Wherein, in step (a) and step (b), processing the described Dracocephalum moldavica medicinal material of test and the Dracocephalum moldavica medicinal material of described contrast of supplying comprises the following steps to obtain the method for Dracocephalum moldavica extract and reference substance extract: by Dracocephalum moldavica pulverizing medicinal materials; Add medicinal material weight 5-30 water extraction doubly to get 30-90 minute; Add again water volume 1-3 95% ethanol doubly to continue heating and refluxing extraction 30-90 minute; Extract reclaims ethanol concentrated, dry, obtains.
2. with fingerprint spectrum method, detect the method for Dracocephalum moldavica medicinal material or Dracocephalum moldavica extract, the method comprises the steps:
(i) get for the Dracocephalum moldavica medicinal material of test or for the Dracocephalum moldavica extract of testing, the described treated Dracocephalum moldavica extract that obtains of Dracocephalum moldavica medicinal material for test, the two is called test sample extract thus obtained Dracocephalum moldavica extract or the aforementioned Dracocephalum moldavica extract for test;
(ii) separately get the Dracocephalum moldavica medicinal material of contrast or the Dracocephalum moldavica extract of contrast, the treated Dracocephalum moldavica extract that obtains of Dracocephalum moldavica medicinal material of described contrast, the two is called reference substance extract the Dracocephalum moldavica extract of thus obtained Dracocephalum moldavica extract or aforementioned contrast;
(iii) precision takes test sample extract and reference substance extract is appropriate respectively, with the methyl alcohol of 50-100 times volume, dissolves, and through 0.45 μ m filtering with microporous membrane, filtrate is called need testing solution and reference substance solution, for efficient liquid phase chromatographic analysis;
(iv) high-efficient liquid phase chromatogram condition is: chromatographic column is C
18chromatographic column, phosphate aqueous solution-acetonitrile that mobile phase is 0.1%; Flow velocity 0.8～1.2ml/min; Adopt the mode of composition response under each wavelength of 200-400nm length ultraviolet detection record to detect; Gradient elution mode is:
(v) draw need testing solution and the reference substance solution of step (iii), be injected into respectively in high performance liquid chromatography, according to (iv) described chromatographic condition, carry out wash-out, obtain respectively test sample HPLC figure and reference substance HPLC figure;
(vi) comparison step (v) gained test sample HPLC figure and reference substance HPLC figure;
(vii) make the test sample extract of the mammal oral step of difference (i) or the reference substance extract of step (ii), in oral rear 0.5-10 hour, get biological sample, extract this biological sample, obtain the biological reference substance solution obtaining after the biological need testing solution that obtains after the test sample extract oral by step (i) or the reference substance extract oral by step (ii);
(viii) draw biological need testing solution and the biological reference substance solution of step (vii), be injected into respectively in high performance liquid chromatography, according to (iv) described chromatographic condition, carry out wash-out, obtain respectively biological test sample HPLC figure and biological reference substance HPLC figure;
(ix) the biological test sample HPLC figure of comparison step (viii) gained and biological reference substance HPLC figure;
Wherein, in step (i) and step (ii), processing the described Dracocephalum moldavica medicinal material of test and the Dracocephalum moldavica medicinal material of described contrast of supplying comprises the following steps to obtain the method for Dracocephalum moldavica extract and reference substance extract: by Dracocephalum moldavica pulverizing medicinal materials; Add medicinal material weight 5-30 water extraction doubly to get 30-90 minute; Add again water volume 1-3 95% ethanol doubly to continue heating and refluxing extraction 30-90 minute; Extract reclaims ethanol concentrated, dry, obtains.
3. according to the method for claim 2, wherein said C
18chromatographic column is Thermo scientific ODS-2Hypersil C
18chromatographic column.
4. according to the method for claim 2, wherein said C
18chromatographic column is Thermo scientific ODS-2Hypersil C
18chromatographic column, specification is 4.6mm * 250mm, 5 μ m.
5. according to the method for claim 2, wherein the flow velocity of (iv) described mobile phase is 1ml/min.
6. according to the method for claim 2, wherein (iv) described wavelength is 277nm.
7. according to the method for claim 2, wherein described in (iv) item, the column temperature of chromatographic column is controlled at 30 ℃.
8. according to the method for claim 2, wherein described in step (vii), mammal is selected from people, rat, mouse, dog, monkey.
9. the prototype of take absorbs composition and/or Metabolite is the method for index with fingerprint spectrum method detection Dracocephalum moldavica medicinal material or Dracocephalum moldavica extract, and the method comprises the steps:
(1) get for the Dracocephalum moldavica medicinal material of test or for the Dracocephalum moldavica extract of testing, the described treated Dracocephalum moldavica extract that obtains of Dracocephalum moldavica medicinal material for test, the two is called test sample extract thus obtained Dracocephalum moldavica extract or the aforementioned Dracocephalum moldavica extract for test;
(2) separately get the Dracocephalum moldavica medicinal material of contrast or the Dracocephalum moldavica extract of contrast, the treated Dracocephalum moldavica extract that obtains of Dracocephalum moldavica medicinal material of described contrast, the two is called reference substance extract the Dracocephalum moldavica extract of thus obtained Dracocephalum moldavica extract or aforementioned contrast;
(3) precision takes test sample extract and reference substance extract is appropriate respectively, with the methyl alcohol of 50-100 times volume, dissolves, and through 0.45 μ m filtering with microporous membrane, filtrate is called need testing solution and reference substance solution, for efficient liquid phase chromatographic analysis;
(4) high-efficient liquid phase chromatogram condition is: chromatographic column is C
18chromatographic column, phosphate aqueous solution-acetonitrile that mobile phase is 0.1%; Flow velocity 0.8～1.2ml/min; Adopt the mode of composition response under each wavelength of 200-400nm length ultraviolet detection record to detect; Gradient elution mode is:
(5) draw need testing solution and the reference substance solution of step (3), be injected into respectively in high performance liquid chromatography, according to (4) described chromatographic condition, carry out wash-out, obtain respectively test sample HPLC figure and reference substance HPLC figure;
(6) comparison step (5) gained test sample HPLC figure and reference substance HPLC figure;
(7) make the test sample extract of the mammal oral step of difference (1) or the reference substance extract of step (2), in oral rear 0.5-10 hour, get biological sample, extract this biological sample, obtain the biological reference substance solution obtaining after the biological need testing solution that obtains after the test sample extract oral by step (1) or the reference substance extract oral by step (2);
(8) draw biological need testing solution and the biological reference substance solution of step (7), be injected into respectively in high performance liquid chromatography, according to (4) described chromatographic condition, carry out wash-out, obtain respectively biological test sample HPLC figure and biological reference substance HPLC figure;
(9) the biological test sample HPLC figure of comparison step (8) gained and biological reference substance HPLC figure;
(10) the reference substance solution HPLC of comparison step (5) figure and the biological reference substance HPLC figure of step (8) gained, determine that the prototype that reference substance characterizes absorbs composition and/or Metabolite;
(11) the need testing solution HPLC of comparison step (5) figure and the biological test sample HPLC figure of step (8) gained;
Wherein, in step (1) and step (2), processing the described Dracocephalum moldavica medicinal material of test and the Dracocephalum moldavica medicinal material of described contrast of supplying comprises the following steps to obtain the method for Dracocephalum moldavica extract and reference substance extract: by Dracocephalum moldavica pulverizing medicinal materials; Add medicinal material weight 5-30 water extraction doubly to get 30-90 minute; Add again water volume 1-3 95% ethanol doubly to continue heating and refluxing extraction 30-90 minute; Extract reclaims ethanol concentrated, dry, obtains.
10. according to the method for claim 9, wherein said C
18chromatographic column is Thermo scientific ODS-2Hypersil C
18chromatographic column.
11. according to the method for claim 9, wherein said C
18chromatographic column is Thermo scientific ODS-2Hypersil C
18chromatographic column, specification is 4.6mm * 250mm, 5 μ m.
12. according to the method for claim 9, and wherein the flow velocity of (4) described mobile phase is 1ml/min.
13. according to the method for claim 9, and wherein (4) described wavelength is 277nm.
14. according to the method for claim 9, and wherein described in (4) item, the column temperature of chromatographic column is controlled at 30 ℃.
15. according to the method for claim 9, and wherein described in step (7), mammal is selected from people, rat, mouse, dog, monkey.
16. according to the method for claim 9, and wherein described in step (7), biological sample is selected from blood sample, urine sample and saliva sample.
17. according to the method for claim 9, wherein in step (10), obtains 1 prototype and absorbs composition and 2 Metabolites.
18. according to the method for claim 17, and the retention time that described 1 prototype absorbs composition is 21.0min.
19. according to the method for claim 17, and the retention time of described 2 Metabolites is respectively 21.5min and 25.3min.
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| CN103948676A (en) * | 2014-02-17 | 2014-07-30 | 芜湖绿叶制药有限公司 | Heart benefiting badiranjibuya particle drug composition and preparation method thereof |
| RU2696867C1 (en) * | 2019-06-04 | 2019-08-07 | Федеральное государственное бюджетное научное учреждение Всероссийский научно-исследовательский институт лекарственных и ароматических растений (ФГБНУ ВИЛАР) | Method for preparing medicinal substances from dracocephalum moldavica l. |
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