CN112345679A - Fingerprint characteristic spectrum of Wuling preparation, establishing method and application thereof - Google Patents
Fingerprint characteristic spectrum of Wuling preparation, establishing method and application thereof Download PDFInfo
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Abstract
The invention provides a fingerprint characteristic spectrum of a Wuling preparation, an establishing method and application thereof. The method comprises the following steps: respectively preparing a mixed reference substance solution and a test substance solution of the Wuling preparation, wherein the Wuling preparation is a Wuling capsule; respectively preparing a first negative control solution, a second negative control solution, a third negative control solution and a fourth negative control solution which lack one medicine; and (3) sucking the mixed reference substance solution, the test article solution, the first negative reference solution, the second negative reference solution, the third negative reference solution and the fourth negative reference solution, injecting into a high performance liquid chromatograph, and detecting according to the high performance liquid chromatography to obtain the fingerprint characteristic spectrum of the Wuling preparation. The method comprehensively reflects the distribution and proportion relation of various components in the Wuling capsules, and is beneficial to comprehensively and effectively controlling the quality of the Wuling capsules.
Description
Technical Field
The invention relates to the field of traditional Chinese medicine fingerprint spectrums, and in particular relates to a fingerprint characteristic spectrum of a Wuling preparation, and an establishment method and application thereof.
Background
The Wuling capsule is developed by the Xijing Hospital of the fourth medical university and is exclusively produced by Xian Happy pharmacy Co. The Wuling capsule is prepared with 4 kinds of Chinese medicinal materials including bupleurum root, glossy ganoderma, red sage, schisandra and other five kinds of Chinese medicinal materials. The main function is to soothe the liver and strengthen the spleen. Can be used for treating chronic hepatitis B with symptoms of anorexia, abdominal distention, belch, hypochondrium distending pain, fatigue, asthenia, etc. In the formula, the bupleurum root is a monarch drug which has the effects of pungent flavor, bitter taste and diarrhea, good effect of regulating liver qi, soothing liver and relieving depression, and regulating qi and relieving pain; the lucid ganoderma is sweet in taste and neutral in nature, has the effects of tonifying qi and spleen, invigorating spleen and middle warmer, and promoting blood circulation and removing blood stasis of the salvia miltiorrhiza, and stimulating menstrual flow and relieving pain, and is used as a ministerial drug together; fructus Schisandrae is sour and sweet in nature and warm in nature, and has effects of invigorating qi and nourishing yin, assisting Ganoderma in invigorating qi and strengthening middle warmer, tranquilizing mind, and preventing bupleuri radix from dispersing and dispersing pathogen and consuming qi and impairing yin as adjuvant drugs. Modern medical research proves that the Wuling capsule also has special curative effect on fatty liver and hepatic fibrosis.
At present, few reports on the quality control research of the Wuling capsules exist, and the quality control research mainly takes the detection of the chemical components in the Chinese thorowax root, the lucid ganoderma, the red sage root and the Chinese magnoliavine fruit. According to the report of the literature, the main components of the bupleurum are saponins, volatile oils, flavonoids, polysaccharides, sterols and the like, and the main pharmacological effects are as follows: antipyretic, tranquilizing, analgesic, antitussive, antiinflammatory, antiviral, hepatoprotective, immunity regulating, and antitumor effects. The main components of the ganoderma lucidum are polysaccharides, triterpenes, sterols, nucleosides, alkaloids, amino acids, fatty acids and trace elements, and the main pharmacological actions are as follows: anti-tumor, immunoregulation, anti-aging, liver protection, central inhibition, blood lipid regulation, etc. The main components of the salvia miltiorrhiza are water-soluble salvianolic acids, fat-soluble tanshinone, polysaccharides, flavonoids, steroids, phenanthraquinone and the like, and the main pharmacological actions are as follows: myocardial protection, anticoagulant, anti-platelet aggregation, anti-Atherosclerosis (AS), blood lipid regulation, blood pressure reduction, brain injury improvement, anti-inflammatory, anti-tumor, antioxidant, immunoregulation, anti-fibrosis, etc. The schisandra chinensis mainly contains lignans, polysaccharides, volatile oils, organic acids and other components, and has the following main pharmacological effects: protecting cardiovascular system, protecting central nerve, tranquilizing, hypnotizing, resisting oxidation, protecting liver, lowering blood sugar, inhibiting bacteria, resisting inflammation, resisting tumor, etc. The active ingredients of the Wuling capsules are complex, and the simple detection of one or more types of active ingredients is not enough to comprehensively and effectively control the medicine quality of the Wuling capsules.
Compared with chemical preparations, the overall view of the traditional Chinese medicine determines that comprehensive evaluation needs to be carried out on various components in the traditional Chinese medicine, but the diversity, complexity and mutual synergistic effect of the chemical components in the traditional Chinese medicine and the preparation thereof determine that the quality index is difficult to accurately and comprehensively express the quality and curative effect of the traditional Chinese medicine by using a single component or a plurality of components.
The fingerprint spectrum technology is a quality control technology which is gradually developed in recent years, and has the characteristics of large information quantity, strong characteristics and capability of integrally controlling various components. Therefore, it is very necessary to establish a fingerprint of the wuling capsule to control the medicine quality of the wuling capsule from the perspective of the whole medicine material.
Disclosure of Invention
The invention mainly aims to provide a fingerprint characteristic spectrum of a Wuling preparation, an establishment method and application thereof, and aims to solve the problem that the prior art is not enough to comprehensively and effectively control the medicine quality of the Wuling capsule only by simply detecting one or more active ingredients during the quality control of the Wuling capsule.
In order to achieve the above object, according to an aspect of the present invention, there is provided a method for establishing a fingerprint characteristic spectrum of a wuling preparation, comprising the steps of: respectively preparing a mixed reference substance solution and a test substance solution of the Wuling preparation, wherein the Wuling preparation is a Wuling capsule; respectively preparing a first negative control solution, a second negative control solution, a third negative control solution and a fourth negative control solution; wherein, the first negative control solution is a Wuling capsule without bupleurum, and the second negative control solution is a Wuling capsule without lucid ganoderma; the third negative control solution is a Wuling capsule without salvia miltiorrhiza; the fourth negative control solution is a wuling capsule without schisandra chinensis; and (3) sucking the mixed reference substance solution, the test article solution, the first negative reference solution, the second negative reference solution, the third negative reference solution and the fourth negative reference solution, injecting into a high performance liquid chromatograph, and detecting according to the high performance liquid chromatography to obtain the fingerprint characteristic spectrum of the Wuling preparation.
Further, the chromatographic column used in the high performance liquid chromatography is a C18 reversed phase chromatographic column; preferably, in the detection of the high performance liquid chromatography, acetonitrile is taken as a mobile phase A, and phosphoric acid with the volume percentage content of 0.1-0.4% is taken as a mobile phase B for gradient elution; preferably, the gradient elution time in the detection of the high performance liquid chromatography is less than or equal to 90 min; preferably, the flow of gradient elution is: 0-15min, 20-50% of mobile phase A and 80-50% of mobile phase B; 15-40min, 50-70% of mobile phase A and 50-30% of mobile phase B; 40-60min, 70-100% of mobile phase A and 30-0% of mobile phase B; 60-80min, 100-100% of mobile phase A and 0-0% of mobile phase B; 80-85min, 100-20% of mobile phase A and 0-80% of mobile phase B; 85-90min, 20-20% of mobile phase A and 80-80% of mobile phase B.
Further, the flow rate of gradient elution is 0.6ml/min to 1.4 ml/min; preferably, in the detection of the high performance liquid chromatography, the column temperature of a chromatographic column is 25-35 ℃, and more preferably 28-32 ℃; preferably, the detection wavelength is 210nm in the high performance liquid chromatography detection.
Further, the mixed control solution is prepared by the following steps: precisely weighing salvianolic acid B, tanshinone IIA, saikoside a, schizandrin A and schizandrin B reference substances in a volumetric flask, and adding methanol into the volumetric flask to prepare mixed solution containing 80-120 mu g of salvianolic acid B, 10-30 mu g of tanshinone IIA, 380-containing saikoside a 420 mu g, 280-containing schizandrin A320 mu g, 480-containing schizandrin A530 mu g and 480-containing 530 mu g of schizandrin B per 1mL respectively; filtering the mixed solution to obtain a filtrate, namely a mixed reference solution; preferably, the mixed solution is filtered by a filter membrane; more preferably, precisely weighing salvianolic acid B, tanshinone IIA, saikosaponin a, schizandrin A, deoxyschizandrin and schizandrin B reference substances in a volumetric flask, and adding methanol to prepare a mixed solution containing 100 μ g of salvianolic acid B, 20 μ g of tanshinone IIA, 400 μ g of saikosaponin a, 300 μ g of schizandrin A, 500 μ g of deoxyschizandrin A and 500 μ g of schizandrin B in each 1 mL; filtering the mixed solution by using a 0.22 mu m microporous filter membrane, and taking a subsequent filtrate as a mixed reference solution.
Further, the test solution is prepared by the following steps: accurately weighing 0.2-0.6g of the content of the Wuling capsules, placing the content into a 50mL volumetric flask, and adding 30-50mL of methanol into the volumetric flask to obtain a methanol solution of the Wuling capsules; carrying out ultrasonic extraction on the methanol solution of the Wuling capsules, and then fixing the volume of methanol to obtain an extracting solution; filtering the extracting solution, and taking a subsequent filtrate as a test solution; preferably, the conditions of the ultrasonic extraction are: performing ultrasonic treatment for 20-40min under the ultrasonic condition with frequency of 50Hz and power of 200W; preferably, the extract is filtered with a filter membrane; more preferably, 0.4g of the content of the Wuling capsules is precisely weighed and placed in a 50mL volumetric flask, and then 40mL of methanol is added into the volumetric flask to obtain a methanol solution of the Wuling capsules; carrying out ultrasonic extraction on the methanol solution of the Wuling capsules, and then fixing the volume of methanol to obtain an extracting solution; ultrasonically extracting for 30min according to the ultrasonic condition with the frequency of 50Hz and the power of 200W, and fixing the volume with methanol to obtain an extracting solution; filtering the extractive solution with 0.22 μm filter membrane, and collecting the filtrate as sample solution.
Further, the first negative control solution, the second negative control solution, the third negative control solution and the fourth negative control solution were prepared by referring to the preparation method of wuling capsules in the "chinese pharmacopoeia (part one) of 2015 edition, respectively.
According to a second aspect of the application, a fingerprint characteristic spectrum of the Wuling preparation is provided, and the fingerprint characteristic spectrum of the Wuling preparation is established by adopting any one of the establishing methods.
According to the third aspect of the application, the application of the fingerprint characteristic spectrum of the Wuling preparation established by any one of the establishing methods in the quality detection of the Wuling capsules is provided.
According to a fourth aspect of the application, a quality detection method of a wuling preparation is provided, which comprises the following steps: establishing a fingerprint characteristic spectrum of the Wuling preparation according to any one establishing method of the fingerprint characteristic spectrum of the Wuling preparation; and detecting the quality of the Wuling preparation according to the fingerprint characteristic spectrum of the Wuling preparation.
Further, the detection comprises the step of comparing the fingerprint characteristic spectrum of the Wuling preparation of the sample to be detected with the fingerprint characteristic spectrum of the Wuling preparation of the standard sample; preferably, according to the comparison result, when the similarity is greater than or equal to 0.90, the quality of the sample to be detected is judged to be qualified.
By applying the technical scheme of the invention, the method selects a mixed reference substance solution and a test substance solution as well as a negative reference solution without containing one of the medicinal materials aiming at 4 medicinal materials, namely the Chinese magnoliavine fruit, in the preparation of the Wuling capsule, so that the high performance liquid chromatography detection is carried out under the control of the mixed reference substance and each negative reference substance solution, thereby obtaining 16 characteristic peaks respectively belonging to the 3 medicinal materials, namely the Chinese thorowax root, the Chinese red sage root and the Chinese magnoliavine fruit, and taking the characteristic peaks as the fingerprint characteristic spectrum of the Wuling preparation. Compared with simple determination of the contents of several chemical components in the preparation, the method is favorable for comprehensively and effectively controlling the quality of the Wuling capsules.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 shows HPLC fingerprint of 10 batches of Wuling capsules according to the preferred embodiment of the present invention;
FIG. 2 shows a control fingerprint constructed according to the median method;
FIG. 3 shows chromatograms of 4 negative sample solutions;
fig. 4A and 4B show HPLC profiles of the mixed control and sample, respectively.
Detailed Description
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail with reference to examples.
As mentioned in the background art, although there are many reports on quality monitoring of traditional Chinese medicines by using a fingerprint spectrum method in the prior art, no corresponding fingerprint characteristic spectrum exists for a preparation of a wuling capsule, so that the application especially refers to a pharmaceutical preparation of the wuling capsule, searches and tries for establishment and application of a fingerprint spectrum of the pharmaceutical preparation, and finally provides a fingerprint spectrum establishment method suitable for the pharmaceutical preparation.
Specifically, the application refers to a preparation method for extracting Wuling capsules in 'Chinese pharmacopoeia' (department of China) of 2015 edition, and performs various exploration and optimization on chromatographic separation conditions:
(1) the samples are respectively treated by adopting methanol and acetonitrile, and the results show that the baseline is more stable and the number of peaks is more after the samples are treated by the methanol, so that the samples are treated by the methanol in the experiment.
(2) A reflux extraction method and an ultrasonic extraction method are adopted to process samples, and the difference of the two methods is not big through comparison, so that the simple and practical ultrasonic extraction method is selected as the extraction method.
(3) Ultrasonic extraction time (10min, 20min, 30min and 40min) is examined, and the result shows that the peak area reaches the maximum value at 30min and almost has no change after 30min, so that 30min is selected as the sample extraction time.
(4) The acetonitrile-water, the acetonitrile-0.1 percent phosphoric acid and the acetonitrile-0.25 percent phosphoric acid are respectively used for gradient investigation, and the result shows that the acetonitrile-0.1 percent phosphoric acid is used for gradient elution, the base line is stable, the separation degree of each peak is better, and therefore, the acetonitrile-0.1 percent phosphoric acid is used as a gradient elution mobile phase.
(5) When the sample solution was detected at different wavelengths (210nm, 250nm, 270nm, and 305nm), 210nm was used as the detection wavelength, since the number of peaks at 210nm was the largest and the amount of information was the largest.
(6) The flow rate, the column temperature and the like are also investigated, and finally the optimal flow rate is selected to be 0.8 mL/min; the optimum column temperature is 30 ℃.
By optimizing and adjusting the conditions, an HPLC fingerprint spectrum taking chemical components in the Chinese thorowax root, the lucid ganoderma, the red sage root and the Chinese magnoliavine fruit as main indexes is finally established, and the quality of the Wuling capsule is comprehensively controlled by attributing and identifying common peaks.
On the basis of the above research results, the applicant proposed the technical solution of the present application. In an exemplary embodiment of the present application, a method for establishing a fingerprint characteristic map of a wuling preparation is provided, which comprises the following steps: respectively preparing a mixed reference substance solution and a test substance solution of the Wuling preparation, wherein the Wuling preparation is a Wuling capsule; respectively preparing a first negative control solution, a second negative control solution, a third negative control solution and a fourth negative control solution; wherein, the first negative control solution is a Wuling capsule without bupleurum, and the second negative control solution is a Wuling capsule without lucid ganoderma; the third negative control solution is a Wuling capsule without salvia miltiorrhiza; the fourth negative control solution is a wuling capsule without schisandra chinensis; and (3) sucking the mixed reference substance solution, the test article solution, the first negative reference solution, the second negative reference solution, the third negative reference solution and the fourth negative reference solution, injecting into a high performance liquid chromatograph, and detecting according to the high performance liquid chromatography to obtain the fingerprint characteristic spectrum of the Wuling preparation.
The method selects 4 medicinal materials of radix bupleuri, lucid ganoderma, red sage root and schisandra chinensis in the Wuling capsule preparation, selects a mixed reference substance solution and a test sample solution, and also selects a negative reference substance without one of the medicinal materials, thereby carrying out high performance liquid chromatography detection under the reference of the mixed reference substance and each negative reference substance solution, obtaining 16 characteristic peaks respectively belonging to the 3 medicinal materials of radix bupleuri, red sage root and schisandra chinensis as fingerprint characteristic maps of the Wuling capsule preparation, and more comprehensively reflecting the distribution and proportion relation of various components in the Wuling capsule. Compared with simple determination of the contents of several chemical components in the preparation, the method is favorable for comprehensively and effectively controlling the quality of the Wuling capsules.
The column used in the high performance liquid chromatograph is preferably a C18 reverse phase column.
In the detection of the high performance liquid chromatography, gradient elution is carried out by adopting gradient eluent. As described above, by screening and optimizing the mobile phase, it was found that acetonitrile was used as the mobile phase A, 0.1% to 0.4% phosphoric acid solution was used as the mobile phase B, and the mixture of the two was used as the mobile phase to perform gradient elution with respect to the chromatographic column, whereby the baseline of the obtained chromatographic peak was stable and the degree of separation of each peak was relatively good. Preferably, the gradient elution time in the detection of the high performance liquid chromatography is less than or equal to 90 min.
In a more preferred embodiment, the elution gradient of the gradient eluent is performed by the following steps: 0-15min, 20-50% of mobile phase A and 80-50% of mobile phase B; 15-40min, 50-70% of mobile phase A and 50-30% of mobile phase B; 40-60min, 70-100% of mobile phase A and 30-0% of mobile phase B; 60-80min, 100-100% of mobile phase A and 0-0% of mobile phase B; 80-85min, 100-20% of mobile phase A and 0-80% of mobile phase B; 85-90min, 20-20% of mobile phase A and 80-80% of mobile phase B.
In the method for establishing the fingerprint characteristic spectrum, the flow velocity of gradient elution can be obtained by reasonably optimizing according to the shape and the separation degree of the peak. In a preferred embodiment of the present application, the flow rate of the gradient elution is 0.6ml/min to 1.4ml/min, at which the peak shape of each characteristic peak is good and the degree of separation is good.
In another preferred embodiment, the temperature of the chromatographic column is 25-35 ℃, more preferably 28-32 ℃, and the separation effect of each component in the temperature range is good.
In a preferred embodiment of the present application, the detection wavelength is 210nm, at which the number of peaks is the largest and the information content is the largest.
In the method for establishing the fingerprint characteristic spectrum, the preparation method of the mixed reference substance solution can refer to the known chemical components with determined content in the Wuling capsules, and has moderate retention time in HPLC separation and good separation degree of characteristic peaks, so that the known chemical components can be reasonably selected to be prepared according to the needs.
In a preferred embodiment, the preparation method of the mixed control solution is prepared by the following steps: precisely weighing salvianolic acid B, tanshinone IIA, saikoside a, schizandrin A and schizandrin B reference substances in a volumetric flask, and adding methanol into the volumetric flask to prepare mixed solution containing 80-120 mu g of salvianolic acid B, 10-30 mu g of tanshinone IIA, 380-containing saikoside a 420 mu g, 280-containing schizandrin A320 mu g, 480-containing schizandrin A530 mu g and 480-containing 530 mu g of schizandrin B per 1mL respectively; and filtering the mixed solution to obtain a filtrate, namely the mixed reference solution.
In a more preferred embodiment, precisely weighing salvianolic acid B, tanshinone IIA, saikosaponin a, schizandrin A and schizandrin B reference substances in a volumetric flask, adding methanol to prepare a mixed solution containing 100 μ g of salvianolic acid B, 20 μ g of tanshinone IIA, 400 μ g of saikosaponin a, 300 μ g of schizandrin A, 500 μ g of schizandrin A and 500 μ g of schizandrin B respectively per 1 mL; filtering the mixed solution by using a 0.22 mu m microporous filter membrane, and taking a subsequent filtrate as a mixed reference solution.
In the above method, the sample solution may be prepared from a plurality of preparation samples of different batches. In a preferred embodiment, the preparation is prepared by the following steps: accurately weighing 0.2-0.6g of the content of the Wuling capsules, placing the content into a 50mL volumetric flask, and adding 30-50mL of methanol into the volumetric flask to obtain a methanol solution of the Wuling capsules; carrying out ultrasonic extraction on the methanol solution of the Wuling capsules, and then fixing the volume of methanol to obtain an extracting solution; filtering the extractive solution, and collecting the filtrate as sample solution. Preferably, the conditions of the ultrasonic extraction are: performing ultrasonic treatment for 20-40min under the ultrasonic condition with the frequency of 50Hz and the power of 200W; preferably, the extract is filtered with a 0.22 μm filter;
in a more preferred embodiment, 0.4g of the content of the wuling capsules is precisely weighed and placed in a 50mL volumetric flask, and then 40mL of methanol is added to the volumetric flask to obtain a methanol solution of the wuling capsules; carrying out ultrasonic extraction on the methanol solution of the Wuling capsules, and then fixing the volume of methanol to obtain an extracting solution; ultrasonically extracting for 30min according to the ultrasonic condition with the frequency of 50Hz and the power of 200W, and fixing the volume with methanol to obtain an extracting solution; filtering the extractive solution with 0.22 μm filter membrane, and collecting the filtrate as sample solution.
As described above, in the fingerprint characteristic spectrum establishing method of the present application, besides the mixed reference substance solution, a plurality of negative reference solutions lacking one medicine are provided, and the obtaining process of each negative reference solution is completely the same as the preparation process of the wuling capsule, except that one medicine is absent in the formula. The first negative control solution, the second negative control solution, the third negative control solution and the fourth negative control solution are prepared according to the process of the extraction preparation method of the Wuling capsules in the' Chinese pharmacopoeia (one part) of 2015 edition.
Wherein, the process of the extraction preparation method of Wuling capsules in the' Chinese pharmacopoeia (one part) of 2015 edition is briefly described as follows:
prescription: 342g of bupleurum, 173g of ganoderma lucidum, 342g of salvia miltiorrhiza and 342g of schisandra chinensis.
The preparation method comprises the following steps: pulverizing bupleuri radix, sieving, and collecting 171g fine powder; and adding 75% ethanol into the coarse powder and the lucid ganoderma, performing reflux extraction twice for 1 hour each time, filtering, combining the filtrates, recovering the ethanol, and performing reduced pressure concentration to obtain a thick paste with the relative density of 1.36-1.38 (60-70 ℃). Adding ethanol into the schisandra chinensis and the salvia miltiorrhiza, performing reflux extraction for three times for 1 hour each, filtering, combining filtrates, recovering ethanol, pressing and concentrating to obtain thick paste with the relative density of 1.36-1.38 (60-70 ℃), combining the two pastes, simultaneously adding the fine powder of the radix bupleuri, uniformly mixing, drying, crushing, encapsulating, and preparing into 1000 granules.
In a second exemplary embodiment of the present application, a fingerprint characteristic spectrum of a wuling preparation is further provided, and the fingerprint characteristic spectrum of the wuling preparation is established by adopting any one of the methods. The fingerprint characteristic spectrum covers 16 characteristic peaks respectively belonging to 3 medicinal materials of radix bupleuri, salvia miltiorrhiza and schisandra chinensis, and more comprehensively reflects the distribution (retention time or appearance sequence) and proportion relation of various components in the Wuling capsule. Compared with simple determination of the contents of several chemical components in the preparation, the method is favorable for comprehensively and effectively controlling the quality of the Wuling capsules.
In a third exemplary embodiment of the present application, an application of the fingerprint characteristic spectrum of the wuling preparation established by any one of the above methods in quality detection of the wuling capsules is further provided.
In a fourth exemplary embodiment of the present application, a method for detecting the quality of a wuling preparation is further provided, which comprises the following steps: establishing a fingerprint characteristic spectrum of the Wuling preparation according to any one establishing method of the fingerprint characteristic spectrum of the Wuling preparation; and detecting the quality of the Wuling preparation according to the fingerprint characteristic spectrum of the Wuling preparation.
Specifically, when the quality is detected, comparing the fingerprint characteristic spectrum of the Wuling preparation of the sample to be detected with the fingerprint characteristic spectrum of the Wuling preparation of the standard sample; preferably, according to the comparison result, when the similarity is greater than or equal to 0.90, the quality of the sample to be detected is judged to be qualified.
The following examples are presented to further illustrate the benefits of the present application. It should be noted that, in the following examples, with reference to the 2015 edition of chinese pharmacopoeia (a) and related documents, HPLC fingerprints using chemical components in bupleurum, ganoderma lucidum, salvia miltiorrhiza and schisandra chinensis as main indicators are established, and the quality of the wuling capsules is comprehensively controlled by attribution and identification of common peaks.
1. Material
1.1 apparatus Agilent1260 infinityII high performance liquid chromatograph (Agilent, USA); an AG285 electronic balance (mertler-toledo instruments shanghai ltd); KQ-500DE model digital control ultrasonic cleaner (Kunshan ultrasonic instruments Co., Ltd.);
1.2 reagent and reagent reference: salvianolic acid B (batch No. 111562-; wuling capsules (Qinghua de ren xi' an happy pharmaceutical Co., Ltd.); chromatographic acetonitrile (Fisher corporation, usa); phosphoric acid (chromatographically pure) Tianjin, Daloco chemical reagent works; methanol (analytical grade) Tianjin, Kemiou Chemicals, Inc.; the water is the Wahaha purified water.
2. Method and results
2.1 chromatographic conditions
(1) A chromatographic column: agilent ZORBAX SB-C18 column (250 mm. times.4.6 mm, 5 μm);
(2) a mobile phase;
acetonitrile (a) -0.1% phosphoric acid (B); gradient elution procedure: (0-15min, 80-50% B, 15-40min, 50-30% B, 40-60min, 30-0% B, 60-80min, 0-0% B, 80-85min, 0-80% B, 85-90min, 80-80% B);
(3) volumetric flow (i.e., flow rate): 0.8 mL/min;
(4) detection wavelength: 210 nm;
(5) column temperature: 30 ℃;
(6) sample introduction amount: 10 μ L.
2.2 preparation of the solution
2.2.1 mixing the reference solutions to obtain a proper amount of salvianolic acid B, tanshinone IIA, saikosaponin a, schizandrin A and schizandrin B, precisely weighing, placing in a volumetric flask, adding methanol to obtain a mixed reference solution containing 100 μ g of salvianolic acid B, 20 μ g of tanshinone IIA, 400 μ g of saikosaponin a, 300 μ g of schizandrin A, 500 μ g of schizandrin A and 500 μ g of schizandrin B in each 1mL, filtering with a 0.22 μm microporous membrane, and collecting the subsequent filtrate.
2.2.2 sample solution 0.4g of the content of the Wuling capsule is taken, precisely weighed, placed in a 50mL volumetric flask, added with about 40mL of methanol, subjected to ultrasonic treatment (frequency 50Hz and power 200W) for 30min, added with methanol to scale after the temperature is reduced to room temperature, shaken up, filtered by a 0.22 mu m filter membrane, and the subsequent filtrate is taken, thus obtaining the Wuling capsule. The 10 batches of wuling capsules were produced by Xian Happy pharmaceutical Co., Ltd, Qinghua de people, and the batch numbers are shown in Table 1.
TABLE 1 sample information
2.2.3 the negative sample solution refers to the preparation method of the Wuling capsule, and negative samples of the medicinal materials of bupleurum, lucid ganoderma, salvia miltiorrhiza and schisandra chinensis are respectively prepared, and the negative sample solution is prepared according to the method under the item 2.2.2.
2.3 methodological investigation
2.3.1 precision investigation
Preparing a sample solution by using a sample with the batch number of 200503 according to the method under the item '2.2.2', continuously injecting 6 needles under the chromatographic condition of the item '2.1', and taking the schizandrol A as a reference peak, wherein the measurement result of each chromatographic condition is as follows: the relative peak area RSD of each common peak is measured to be less than 3 percent, and the relative retention time RSD is measured to be less than 1 percent, which indicates that the precision of the instrument is good.
2.3.2 sample with the batch number of 200503 for stability investigation, preparing a test solution according to the method under the item '2.2.2', injecting samples under the chromatographic condition of the item '2.1' for 0, 2, 4, 8, 12 and 24h, taking the schizandrol A as a reference peak, measuring that the relative peak area RSD of each common peak is less than 5 percent, and the relative retention time RSD is less than 2 percent, and showing that the stability of the test solution is good within 24 h.
2.3.3 for repeatability tests, a sample with the batch number of 200503 is used, 6 test sample solutions are prepared in parallel according to the method under the item of 2.2.2, sample introduction is carried out under the chromatographic condition of the item of 2.1, schizandrol A is taken as a reference peak, the relative peak area RSD of each common peak is measured to be less than 5%, and the relative retention time RSD is measured to be less than 2%, so that the method has good reproducibility.
2.4 fingerprint map establishment
2.4.1 common Peak calibration
Selecting a schizandrol A peak as a reference peak (S), using the ratio of retention time of each chromatographic peak in a chromatogram to retention time of the reference peak (S) as relative retention time, and calibrating a common peak through the relative retention time values, wherein 16 peaks are shared by 10 batches of samples and are calibrated as common peaks, and the common peak area accounts for more than 75% of the total peak area, and the result is shown in figure 1.
2.4.2 evaluation of similarity
The common peaks are analyzed by a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012 edition), and a control fingerprint is established by a median method, which is shown in figure 2.
The similarity between the 10 batches of sample fingerprints and the comparison fingerprint is shown in Table 2, and is respectively 0.998, 0.999, 0.998, 0.997, 0.995, 0.999, 0.962, 0.997 and 0.986, which are all more than 0.960.
TABLE 2 analysis results of similarity
2.4.3 specialization examination
Taking 4 negative samples and samples with the batch number of 200503, preparing the samples into solutions according to the method under the item of 2.2.2, respectively absorbing the 4 negative samples and the sample solutions, injecting samples under the chromatographic condition of the item of 2.1, wherein the chromatogram is shown in figure 3, S1: a salvia miltiorrhiza negative sample; s2: a schisandra chinensis negative sample; s3: a ganoderma lucidum negative sample; s4: a Bupleurum negative sample; s5: wuling capsule.
As can be seen from the figure, the common peaks were assigned as follows by comparison with the negative samples: common peaks 1, 11, 13, and 16 belong to Salvia miltiorrhiza, common peak 2 belongs to bupleuri radix, and common peaks 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, and 15 belong to Schisandra chinensis.
2.4.4 consensus Peak assignments
Respectively injecting sample under the condition of '2.1' chromatography by using mixed reference substance solution and sample solution, and identifying the chromatographic peak of the reference substance shown in figure 4A and the common peak in HPLC chromatogram of the sample shown in figure 4B, wherein the peak 1 is salvianolic acid B, the peak 2 is saikosaponin a, the peak 3 is schizandrol A, the peak 12 is deoxyschizandrin, the peak 13 is tanshinone IIA, and the peak 15 is schisandrin B.
From the above description, it can be seen that the 16 common peaks provided by the method of the present application are respectively attributed to 3 herbs including bupleurum, salvia miltiorrhiza and schisandra chinensis, and the distribution and proportion relationship of various components in the wuling capsule are relatively comprehensively reflected (since the main components contained in ganoderma lucidum are polysaccharide, terpene and sterol compounds, the types of triterpene and sterol compounds are complex and low, and the absorption in the ultraviolet light (200nm-400nm) range is very weak, the spectrum of the present application cannot reflect the distribution and proportion relationship of ganoderma lucidum components in the wuling capsule).
Therefore, the fingerprint characteristic spectrum established by the method through comparison and analysis of various components in 3 medicinal materials including the radix bupleuri, the salvia miltiorrhiza and the schisandra chinensis in the Wuling capsules has the advantages of simplicity and convenience in operation and large information amount compared with the method for directly measuring the contents of various chemical components in a preparation, and can comprehensively control the quality of the Wuling capsules.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method for establishing fingerprint characteristic spectrum of a Wuling preparation is characterized by comprising the following steps:
respectively preparing a mixed reference substance solution and a test substance solution of a Wuling preparation, wherein the Wuling preparation is a Wuling capsule;
respectively preparing a first negative control solution, a second negative control solution, a third negative control solution and a fourth negative control solution; wherein, the first negative control solution is the Wuling capsule without bupleurum, and the second negative control solution is the Wuling capsule without lucid ganoderma; the third negative control solution is a Wuling capsule without salvia miltiorrhiza; the fourth negative control solution is a Wuling capsule without schisandra chinensis;
and (3) sucking the mixed reference substance solution, the test article solution, the first negative reference solution, the second negative reference solution, the third negative reference solution and the fourth negative reference solution, injecting into a high performance liquid chromatograph, and detecting according to the high performance liquid chromatography to obtain the fingerprint characteristic spectrum of the Wuling preparation.
2. The establishing method according to claim 1, wherein the chromatographic column used in the high performance liquid chromatography is a C18 reversed phase chromatographic column;
preferably, in the detection by the high performance liquid chromatography, acetonitrile is used as a mobile phase A, and phosphoric acid with the volume percentage content of 0.1-0.4% is used as a mobile phase B for gradient elution;
preferably, the gradient elution time in the high performance liquid chromatography detection is less than or equal to 90 min;
preferably, the flow of gradient elution is as follows:
0-15min, 20-50% of mobile phase A and 80-50% of mobile phase B;
15-40min, 50-70% of mobile phase A and 50-30% of mobile phase B;
40-60min, 70-100% of mobile phase A and 30-0% of mobile phase B;
60-80min, 100-100% of mobile phase A and 0-0% of mobile phase B;
80-85min, 100-20% of mobile phase A and 0-80% of mobile phase B;
85-90min, 20-20% of mobile phase A and 80-80% of mobile phase B.
3. The method of establishing according to claim 2, wherein the flow rate of the gradient elution is between 0.6ml/min and 1.4 ml/min;
preferably, in the detection of the high performance liquid chromatography, the column temperature of a chromatographic column is 25-35 ℃, and more preferably 28-32 ℃;
preferably, in the detection by the high performance liquid chromatography, the detection wavelength is 210 nm.
4. The building method according to any one of claims 1 to 3, wherein the mixed control solution is prepared by the following steps:
precisely weighing salvianolic acid B, tanshinone IIA, saikosaponin a, schizandrol A, schizandrin A and schizandrin B reference substances in a volumetric flask,
adding methanol into the volumetric flask to prepare a mixed solution containing 80-120 mu g of salvianolic acid B, 10-30 mu g of tanshinone IIA, 420 mu g of saikosaponin a 380-;
filtering the mixed solution to obtain filtrate, namely the mixed reference solution;
preferably, the mixed solution is filtered by a filter membrane;
more preferably, precisely weighing salvianolic acid B, tanshinone IIA, saikosaponin a, schizandrin A, deoxyschizandrin and schizandrin B reference substances in a volumetric flask, and adding methanol to prepare a mixed solution containing 100 μ g of salvianolic acid B, 20 μ g of tanshinone IIA, 400 μ g of saikosaponin a, 300 μ g of schizandrin A, 500 μ g of deoxyschizandrin A and 500 μ g of schizandrin B in each 1 mL;
and filtering the mixed solution by adopting a 0.22-micron microporous filter membrane, and taking a subsequent filtrate as the mixed reference solution.
5. The establishing method according to any one of claims 1 to 3, wherein the test solution is prepared by:
accurately weighing 0.2-0.6g of the content of the Wuling capsules, placing the content into a 50mL volumetric flask, and adding 30-50mL of methanol into the volumetric flask to obtain a methanol solution of the Wuling capsules;
carrying out ultrasonic extraction on the methanol solution of the Wuling capsules, and then fixing the volume of methanol to obtain an extracting solution;
filtering the extracting solution, and taking a subsequent filtrate as the test solution;
preferably, the ultrasonic extraction conditions are: performing ultrasonic treatment for 20-40min under the ultrasonic condition with frequency of 50Hz and power of 200W;
preferably, the extract is filtered with a filter membrane;
more preferably, 0.4g of the content of the Wuling capsules is precisely weighed and placed in a 50mL volumetric flask, and then 40mL of methanol is added into the volumetric flask to obtain a methanol solution of the Wuling capsules;
carrying out ultrasonic extraction on the methanol solution of the Wuling capsules, and then fixing the volume of methanol to obtain an extracting solution;
ultrasonically extracting for 30min according to the ultrasonic condition with the frequency of 50Hz and the power of 200W, and fixing the volume with methanol to obtain the extracting solution;
and filtering the extracting solution by adopting a 0.22-micron filter membrane, and taking a subsequent filtrate as the test solution.
6. The establishing method according to any one of claims 1 to 3, wherein the first negative control solution, the second negative control solution, the third negative control solution and the fourth negative control solution are prepared by referring to the preparation method of Wuling capsules in the national pharmacopoeia (part of the 2015 edition), respectively.
7. A fingerprint characteristic spectrum of a Wuling preparation, which is characterized in that the fingerprint characteristic spectrum of the Wuling preparation is established by the establishing method of any one of claims 1 to 6.
8. The application of fingerprint characteristic spectrum of the Wuling preparation established by the establishing method of any one of claims 1 to 6 in the quality detection of the Wuling capsule.
9. A quality detection method of a Wuling preparation is characterized by comprising the following steps:
establishing a fingerprint characteristic spectrum of a Wuling preparation according to the establishing method of the fingerprint characteristic spectrum of the Wuling preparation as claimed in any one of claims 1 to 6;
and detecting the quality of the Wuling preparation according to the fingerprint characteristic spectrum of the Wuling preparation.
10. The quality detection method according to claim 9, wherein the detection comprises comparing the fingerprint feature of the wuling preparation of the sample to be detected with the fingerprint feature of the wuling preparation of the standard sample;
preferably, according to the comparison result, when the similarity is greater than or equal to 0.90, the quality of the sample to be detected is judged to be qualified.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115267007A (en) * | 2022-08-11 | 2022-11-01 | 清华德人西安幸福制药有限公司 | Method for establishing fingerprint of antipyretic syrup for treating infantile common cold and fingerprint thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103808842A (en) * | 2014-01-17 | 2014-05-21 | 黑龙江葵花药业股份有限公司 | Quality detection method for liver protection dropping pill of traditional Chinese medicine preparation |
| CN104950069A (en) * | 2015-06-23 | 2015-09-30 | 南京海昌中药集团有限公司 | Method for detecting quality of depression-relieving heart-comforting tablet |
| CN111735900A (en) * | 2020-07-01 | 2020-10-02 | 清华德人西安幸福制药有限公司 | Method for detecting effective components in Wuling capsules |
| CN111796047A (en) * | 2020-07-13 | 2020-10-20 | 清华德人西安幸福制药有限公司 | Method for establishing fingerprint characteristic spectrum of Reyanning preparation and application thereof |
-
2020
- 2020-11-10 CN CN202011248575.7A patent/CN112345679B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103808842A (en) * | 2014-01-17 | 2014-05-21 | 黑龙江葵花药业股份有限公司 | Quality detection method for liver protection dropping pill of traditional Chinese medicine preparation |
| CN104950069A (en) * | 2015-06-23 | 2015-09-30 | 南京海昌中药集团有限公司 | Method for detecting quality of depression-relieving heart-comforting tablet |
| CN111735900A (en) * | 2020-07-01 | 2020-10-02 | 清华德人西安幸福制药有限公司 | Method for detecting effective components in Wuling capsules |
| CN111796047A (en) * | 2020-07-13 | 2020-10-20 | 清华德人西安幸福制药有限公司 | Method for establishing fingerprint characteristic spectrum of Reyanning preparation and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 杨琳等: "基于HPLC指纹图谱结合化学模式识别的护肝片质量控制研究", 《中草药》 * |
| 靳晓峰等: "一测多评法同时测定解郁胶囊中9种成分", 《中成药》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115267007A (en) * | 2022-08-11 | 2022-11-01 | 清华德人西安幸福制药有限公司 | Method for establishing fingerprint of antipyretic syrup for treating infantile common cold and fingerprint thereof |
| CN115267007B (en) * | 2022-08-11 | 2024-02-23 | 清华德人西安幸福制药有限公司 | Method for establishing fingerprint of syrup for treating common cold and fever in children and fingerprint thereof |
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