CN115266997B - Construction method and application of puffball medicinal material and formula particle characteristic spectrum thereof - Google Patents
Construction method and application of puffball medicinal material and formula particle characteristic spectrum thereof Download PDFInfo
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Abstract
The invention discloses a construction method and application of a puffball medicinal material and formula particle characteristic spectrum thereof, wherein the construction method comprises the following steps: (1) Extracting Lasiosphaera Seu Calvatia medicinal material, standard decoction or granule thereof with solvent to obtain sample solution; (2) adding solvent into ergosterol to prepare reference substance solution; (3) Respectively injecting the sample solution and the reference substance solution into liquid chromatography to obtain characteristic patterns; and (4) judging the quality of the raw materials and distinguishing the counterfeit products. The method can be used for primarily distinguishing the pseudo large mouth gray ball, purple puffball and puffball, and provides a certain basis for identifying the authenticity and the primordial of puffball medicinal materials; and can comprehensively reflect the internal quality of puffball medicinal materials, standard decoction and prescription granules thereof.
Description
Technical Field
The invention relates to a construction method and application of a characteristic spectrum, in particular to a construction method and application of a characteristic spectrum of puffball medicinal materials and formula particles thereof.
Background
Puffball, alias ash bags, ma Fenbao, ma Pibo, etc., are dry fruit bodies of puffball Lasiosphaera fenzlii reich, puffball Calvatia gigantea (Batsch ex per.) Lloyd or puffball Calvatia lilacina (mont. Et Berk.) Lloyd, of the ash bag family fungi. Pungent taste, mild nature, lung meridian tropism, lung clearing, sore throat relieving, hemostasis and other effects, and can be used for treating wind-heat stagnation lung sore throat, hoarseness and cough; it is indicated for epistaxis and traumatic hemorrhage.
The puffball comprises the following main components: steroid (Jin Xiangqun, wang Lishu, cheng Dongyan, yi Xylon, lv Jingshan. Chemical composition study of Lasiosphaera seu Calvatia [ J ]. Chinese herbal medicine, 1998 (05): 298-300 ], terpene (Li Xiaoli. Ma Bohua chemical composition separation, identification and inhibition of cathepsin L study [ D ]. Xiangtan university, 2011 ]), small molecule nitrogen-containing compound (Zhang Jianmei, zhang Dancan. Chemical composition of Lasiosphaera Seu Calvatia and pharmacological composition development thereof [ J ]. Neuropharmacological report, 2020,10 (02): 11-14.), protein (Meng Jingjing, fan Yanhua, zhao Yunpeng, bai Junpeng, zhao Qingchun. HPLC method to determine white linear cyclic peptide content of Lasiosphaera's skin, 2013,16 (06): 833-835, amino acids (Zhang Jiangping, fan Xiaolong, wu Rui. Wild Lasiosphaera's disease nutritional composition analysis [ J ]. Shanxi agricultural science, 2013,41 (05): 456-457), polysaccharide and inorganic element, etc. Modern pharmacological action and clinical studies show that puffball has hemostatic effect (Yang Jianing, zhang Zhenling, chen Jingjing, zhang Ying, li Qingsong, lin Xiumin, dream. Comparative study of hemostatic action and adsorptive power of different parts of desquamation puffball [ J ]. Chinese journal of traditional Chinese medicine, 2020,38 (08): 157-161), anti-inflammatory (Zhang Sai, wang Dake, li Aixin, wang Shumin. Large Ma Bokang, analgesic, in vitro bacteriostasis studies [ J ]. Chinese edible fungi, 2014,33 (02): 50-52 ], bacteriostasis (bacteriostatic activity of extract of fruit bodies of Lasiosphaera Seu Calvatia in different maturity stages and analysis of their volatile oil components [ J ]. Protect, 2014,33 (03): 477-485), antioxidant (2014,33, 2014,33, 2014,33. The removal of oxygen radicals by the purple alopecia 2014,33. The soluble polysaccharides [ J ]. Anhui agriculture science, 2014,33 (08): 4053-4055 ], antitumor (Jingjing Meng, yanhua Fan, mingzhi Su, congqin Chen, tianshu Ren, jinhui ng, qinchun Zhao. WLIP 2014,33-2014,33 [ J ]. 2014,33, 2014,33) (521): 2014,33, 2014,33, and insecticidal activity studies [ 5255 ] of biological components [ J ]. 2014,33, 52.9655 ] of the extract of Lasiosphaera fruit bodies in different maturity stages.
The existing literature data about the quality control of puffball mainly concentrate on the content measurement of medicinal materials and decoction pieces, and most of researches take ergosterone and ergosterol as content measurement indexes, and a small amount of researches evaluate the puffball by measuring the content of polysaccharide, total triterpenes or white linear cyclic lipopeptid, and the like, so that the evaluation indexes are single, and the quality of puffball cannot be comprehensively reflected in whole. The characteristic spectrum and fingerprint research data of puffball medicinal materials are very few, and only one literature develops fingerprint research on the hemostatic active ingredients of puffball with desquamation. Gao Yunjia A high performance liquid fingerprint of hemostatic active ingredient of Lasiosphaera Seu Calvatia is established, 8 main chromatographic peaks are identified, fingerprint analysis is performed on 10 Lasiosphaera Seu Calvatia samples of different production places, and a certain basis is provided for quality and control of Lasiosphaera Seu Calvatia medicinal material. (Gao Yunjia) Lasiosphaera Seu Calvatia hemostatic active ingredient and its fingerprint (D) research of Shenyang pharmaceutical university, 2009). In the prior art, no research on fingerprint patterns or characteristic patterns in puffball (puffball) is seen.
As for research on Ma Boji original distinction, except traditional character identification, microscopic identification and thin-layer identification, research is carried out on content determination of ergosterone or ergosterol of different varieties and different producing places of puffball, and the results show that certain differences exist; (Congo and Kun, holly, guan Shengjiang, huang Huaipeng, wang Qiao, anfooli, chen Yingchun, wu Haiyan, zhu Shan. HPLC method measures ergosterol [ J ]. Chinese herbal medicine in different puffball medicinal materials of different producing places, 2016,47 (08): 1397-1400.) it is found by extracting volatile components in puffball medicinal materials and performing GC-MS analysis that the Pharmacopeia varieties Lasiosphaera seu calvatia, lasiosphaera Seu Calvatia, and Lasiosphaera Seu Calvatia contain ethyl hexadecanoate (also known as ethyl palmitate), while the non-genuine puffball (Lasiosphaera major static gray ball) does not contain such components. (Congo, guan Shengjiang, wang Qiao, anfoli, chen Yingchun, wu Haiyan. GC-MS analysis of volatile components of Lasiosphaera Seu Calvatia traditional Chinese medicine by different extraction methods [ J ]. Hebei medicine, 2016,38 (23): 3545-3547.) has been studied to compare types and contents of amino acids and inorganic elements of different Lasiosphaera Seu Calvatia, and Lasiosphaera Seu Calvatia varieties in pharmacopoeia can be classified into one type after the clustering analysis of amino acid contents, and are distinguished from other mixed pseudo products, and meanwhile, the contents of inorganic elements of Lasiosphaera of the three basic sources show a certain difference. (Zhang Qingkang, ding Yonghui.10 amino acid content determination and cluster analysis of puffball [ J ]. Chinese patent medicine, 1996 (08): 35-37.) (Yue Lidan, du Lijie, yang Jianing, zhang Zhenling. ICP-MS method determination and PCA analysis of different varieties of puffball inorganic element content study [ J ]. A traditional medicine, 2021,17 (04): 41-46.) in addition, the study finds that the Fourier transform infrared spectra of 3 varieties of medicinal materials under the pharmacopoeia item have obvious differences; the ITS sequence can be used for effectively distinguishing puffball pharmacopoeia variety medicinal materials from mixed and fake products (Bai Tuma Bo, lasiosphaera cephalotaxus, lasiosphaera anilox, lasiosphaera Seu Calvatia, lasiosphaera piricola, gray, lasiosphaera longifolia and Lasiosphaera longa). (Zhang Jiali, huang Yuhang, song Ming, ren Yangyang, zhang Mengting, liu Xia, sun Wei, chen Shilin) identifying puffball, a fungus medicinal material, and ITS mixed imitations [ J ]. World medicine, 2016,11 (05): 777-780+785. In summary, the research direction about Ma Boji original distinction and true-false identification is quite rich, but these technical means have certain limitations. At present, research on Ma Boji primary distinction by a characteristic spectrum or fingerprint spectrum technology is not seen.
Since ancient times, the sources of medicinal varieties of puffball have been confusing, and the most commonly used pseudo-products currently belong to the large mouth static ash ball Bovistella sinensis Lloyd. At present, the 2020 edition of Chinese pharmacopoeia prescribes that only 3 qualified medicinal materials (ash Bao Ke fungi, desquamation puffball Lasiosphaera fenzlii Reich., puffball Calvatia gigantea (Batsch ex Pers.) Lloyd or purple puffball Calvatia lilacina (Mont. Et Berk.) Lloyd) exist, and the medicinal basis of other varieties is not fully scientific proved.
The current legal quality standard of puffball is recorded in the part 2020 of Chinese pharmacopoeia and mainly consists of characters, microscopic identification, thin layer identification, moisture, ash and extract. The prior art on puffball quality research is mainly focused on content measurement, and puffball medicinal materials are subjected to quality evaluation by measuring the content of ergosterone, ergosterol, polysaccharide, total triterpenes or white linear cyclic lipopeptides and the like. The detection items can reflect the quality of puffball medicinal materials to a certain extent, but the evaluation index is single, the quality of puffball cannot be comprehensively reflected in whole, and the quality control effect is limited. With respect to characteristic patterns and fingerprint research data of puffball medicinal materials, only one literature develops fingerprint research on hemostatic active ingredients of puffball for desquamation, but the method is complex in process and does not compare puffball (puffball) and other puffball products.
Disclosure of Invention
The invention aims to: the invention aims to provide a construction method of a characteristic spectrum of puffball medicinal materials and formula particles thereof, which is helpful for providing a more comprehensive, simple, quick and effective quality control method of puffball medicinal materials, standard decoction and formula particles thereof, and can provide a certain basis for distinguishing Ma Bozhen counterfeit products and different basic raw medicinal materials; the invention further aims at providing application of the construction method of the characteristic spectrum in quality evaluation and detection and distinguishing purple puffball, puffball and pseudo puffball.
The technical scheme is as follows: the invention relates to a construction method of a characteristic spectrum of puffball medicinal materials and formula particles thereof, which comprises the following steps:
(1) Collecting one of Lasiosphaera Seu Calvatia, standard decoction or its granule, processing, and extracting with solvent to obtain sample solution;
(2) Adding solvent into ergosterol to obtain reference solution;
(3) Respectively injecting the sample solution and the reference substance solution into liquid chromatograph to obtain characteristic patterns, wherein the liquid chromatograph conditions are as follows:
the stationary phase is a chromatographic column taking octadecylsilane chemically bonded silica as a filler; the mobile phase A is acetonitrile, and the mobile phase B is phosphoric acid aqueous solution with the mass percent of phosphoric acid of 0.01-0.20%;
(4) Judging the quality of raw materials and distinguishing between false and positive products: if the characteristic spectrum obtains the following 6 characteristic peaks, the quality of the raw material is qualified: peak 6 is the peak of ergosterol, and relative retention times for peak 1, peak 2, peak 3, peak 4 and peak 5 are respectively: 0.26 to 0.32, 0.27 to 0.33, 0.29 to 0.35, 0.32 to 0.40 and 0.80 to 0.98;
if the measured characteristic patterns do not all contain the above 6 characteristic peaks, puffball is a pseudo product; if the peak area of the characteristic peak 6 relative to the peak 5 in the characteristic spectrum is greater than 30, the purple puffball is judged.
Preferably, in the step (1), when puffball is a medicinal material, the specific steps are as follows: sieving Lasiosphaera Seu Calvatia medicinal material powder, placing into a conical flask, adding extraction solvent, extracting with solvent with the mass of 0.03-0.15g per 1ml of solvent, performing ultrasonic treatment or heating or vibration treatment for 15-60 min, cooling, adding corresponding solvent to the weight of the filtrate, shaking, and filtering to obtain filtrate as sample solution;
when puffball is a standard decoction, the specific steps are as follows: placing Lasiosphaera Seu Calvatia standard decoction powder into a conical flask, adding extraction solvent, extracting with 0.01-0.06g of solvent per 1ml of solvent, performing ultrasonic treatment or heating or oscillating treatment for 15-60 min, cooling, adding corresponding solvent to the weight of the filtrate, shaking, centrifuging, collecting supernatant, evaporating, dissolving residue with methanol, transferring to a measuring flask, adding methanol to scale, shaking, filtering, and collecting filtrate to obtain sample solution;
When puffball is a formula particle, the specific steps are as follows: placing puffball formula particle powder into a conical flask, adding an extraction solvent, wherein the mass of a sample to be extracted per 1ml of solvent is 0.05-0.2g, carrying out ultrasonic treatment or heating or vibration treatment for 15-60 min, cooling, supplementing the reduced weight with the corresponding solvent, shaking up, centrifuging, taking supernatant, evaporating to dryness, dissolving residues with methanol, transferring to a measuring flask, adding methanol to scale, shaking up, filtering, and obtaining a subsequent filtrate, namely the sample solution.
Preferably, in the step (1), when puffball is a medicinal material, the extraction solvent is an aqueous methanol solution containing 80-100% by volume of methanol or an aqueous ethanol solution containing 80-100% by volume of ethanol; when puffball is a standard decoction or a formula particle, the extraction solvent is dichloromethane or a methanol water solution containing 80-100% of methanol by volume percent or an ethanol water solution containing 80-100% of ethanol by volume percent.
Preferably, the detecting instrument of the feature spectrogram in the step (3) is: ultra-high performance liquid chromatograph; the specification of the chromatographic column is as follows: the column length is 100-150 mm, and the grain diameter is 1.7-1.9 mu m; the conditions of the liquid chromatography further include: the flow rate is 0.25-0.35 ml/min; the column temperature is 20-30 ℃; the detection wavelength is 265-295 nm.
Preferably, the concentration of ergosterol in the control in step (2) is: 20-95 mug/ml; the dissolution solvent of the reference substance is methanol.
Preferably, the power of ultrasonic extraction in the step (1) is 250-600W, and the frequency is 35-45 kHz.
The construction method of the characteristic spectrum of the puffball medicinal materials and the formula particles thereof can be applied to quality evaluation and detection of the puffball medicinal materials, the standard decoction or the formula particles.
The construction method of the characteristic spectrum of puffball medicinal materials and the formula particles thereof can also be applied to distinguishing genuine puffball, puffball and pseudo puffball and majordomo dust balls.
The beneficial effects are that: compared with the prior art, the invention has the following remarkable advantages: (1) The invention establishes the characteristic map of puffball (puffball) medicinal materials, can distinguish the pseudo (puffball) medicinal materials from puffball (puffball) medicinal materials preliminarily, and provides a certain basis for the authenticity identification and basic source identification of puffball medicinal materials; (2) Because the formula particles lose the appearance forms of the original medicinal materials and decoction pieces, in order to ensure the consistency of the quality of the formula particles and the quality of the medicinal materials, the invention researches the characteristic components of puffball (puffball) medicinal materials, standard decoction and formula particles and uses the characteristic components as the determination basis of puffball (puffball) medicinal materials, standard decoction and formula particle characteristic patterns. The ergosterol reference substance is used as a reference substance, a characteristic map containing 6 characteristic peaks is established, and the internal quality of puffball (puffball) medicinal materials, standard decoction and formula particles thereof is comprehensively reflected; meanwhile, the characteristic spectrum researches of puffball (puffball) medicinal materials, standard decoction and formula particles have corresponding characteristic peaks, so that the consistency of the formula particles and the medicinal preparation extracts can be fully reflected, the basis is provided for tracing the characteristic components in puffball (puffball) formula particles and upstream raw materials, and the quality control in the industrialization process is also powerfully ensured; (3) The invention adopts ultra-high liquid chromatography to detect characteristic patterns; the chromatographic peak can be well separated, the chromatographic peak shape is good, the characteristic spectrum information is rich, the method is simple to operate, and the method has the advantages of high precision, good stability, good repeatability, high accuracy, high detection efficiency, low detection cost and the like through methodological verification, can effectively ensure the stability of the whole quality of puffball (puffball) medicinal materials, standard decoction and formula particles, and provides important guarantee for the production of the puffball (puffball) formula particles.
Drawings
FIG. 1 is a chromatogram of solvent extraction of Lasiosphaera Seu Calvatia (Lasiosphaera Seu Calvatia) medicinal material;
FIG. 2 is a chromatogram of solvent extraction of Lasiosphaera Seu Calvatia formula particles;
FIG. 3 is a graph of different wavelength detection chromatograms of Lasiosphaera Seu Calvatia (Lasiosphaera major) formula particles;
FIG. 4 is a chromatogram of different chromatographic column detection of Lasiosphaera Seu Calvatia (Lasiosphaera Seu Calvatia) formula particles;
FIG. 5 is a graph of different gradient detection chromatograms of Lasiosphaera Seu Calvatia (Lasiosphaera Seu Calvatia) formula particles;
FIG. 6 is a graph of different flow rate detection chromatograms of puffball (Lasiosphaera major) formula particles;
FIG. 7 is a graph of various column temperature detection chromatograms of Lasiosphaera Seu Calvatia (Lasiosphaera Seu Calvatia) formula particles;
FIG. 8 is a UPLC spectrum of a puffball (Lasiosphaera major) formula particle characteristic spectrum chromatographic peak assignment;
FIG. 9 is a UPLC spectrum of a puffball (Lasiosphaera major) formula particle feature spectrum integrity test;
FIG. 10 is a superimposed chart of UPLC spectra of 3 batches of Lasiosphaera Seu Calvatia (Lasiosphaera major) formula particles;
FIG. 11 is a superimposed chart of UPLC spectra of 15 batches of Lasiosphaera Seu Calvatia (Lasiosphaera seu Calvatia) medicinal material;
FIG. 12 is a UPLC control profile of Lasiosphaera Seu Calvatia formula particles;
FIG. 13 is a UPLC control characteristic map of Lasiosphaera Seu Calvatia (Lasiosphaera Seu Calvatia) medicinal material;
FIG. 14 is a characteristic map of various processes of puffball (Lasiosphaera seu Calvatia) mass production;
FIG. 15 is a characteristic map of various processes of puffball (Lasiosphaera seu Calvatia) mass production;
FIG. 16 is a graph showing the characteristics of various processes in the mass production of Lasiosphaera Seu Calvatia;
FIG. 17 is a diagram showing the superposition of the characteristic maps of pseudo-product (Dakou Jing Hui) and Lasiosphaera Seu Calvatia (Lasiosphaera Seu Calvatia) medicinal materials;
FIG. 18 is a UPLC control profile of Lasiosphaera Seu Calvatia (purple Lasiosphaera Seu Calvatia) medicinal material;
fig. 19 is a diagram showing the superposition of characteristic maps of puffball (purple puffball) medicinal materials and puffball (large puffball) medicinal materials.
Detailed Description
The technical scheme of the invention is further described below with reference to the accompanying drawings.
Example 1
Establishing a UPLC characteristic spectrum detection method of puffball formula particles:
1. liquid phase conditions
Chromatographic column: waters ACQUITYBEH C18，1.7μm，2.1mm×100mm。
Mobile phase a: acetonitrile
Mobile phase B:0.1% phosphoric acid aqueous solution (v/v)
Gradient elution procedure:
chromatographic parameters:
a detector: DAD,282nm
Flow rate: 0.3 ml/min
Column temperature: 25 DEG C
Sample injection amount: 3 μl of
2. Preparation of test solutions
Medicinal material test solution:
(1) Selection of extraction solvent
Because the index components of ergosterone and ergosterol in the puffball medicinal materials are fat-soluble components, the extraction effects of four different solvents, namely 100% methanol, 80% methanol aqueous solution, ethanol and 80% ethanol aqueous solution, on the puffball medicinal materials are examined, wherein the percentage is the volume percentage of the methanol or the ethanol.
The specific experimental method is as follows:
about 1.5g of Lasiosphaera Seu Calvatia medicinal material powder is taken, 5 groups are parallel, precisely weighed, placed in conical flasks with plugs, respectively and precisely added with 15ml of methanol, 80% aqueous methanol solution, ethanol and 80% aqueous ethanol solution, subjected to ultrasonic treatment (power 600W, frequency 40 kHz) for 45 minutes, cooled, shaken uniformly, filtered, and the subsequent filtrate is obtained. 3 μl of each sample solution was precisely sucked up, injected into a liquid chromatograph, measured according to the above chromatographic conditions, the chromatograms were recorded, and the peak areas/sample amounts of 6 chromatographic peaks were calculated, and the results are shown in Table 1.
TABLE 1 extraction solvent investigation results of characteristic patterns of Lasiosphaera Seu Calvatia (Lasiosphaera Seu Calvatia)
The comparison result of the chromatograms is shown in fig. 1.
The peak area data was analyzed and the characteristic peak area was lower when extracted with 80% aqueous methanol. When 100% methanol is used for extraction, the peak area of each characteristic peak is the highest, and can be used as a preferable condition parameter.
(2) Investigation of the extraction method
100% methanol is selected as an extraction solvent, and the extraction effect of ultrasonic treatment (frequency 600W, power 40 kHz), heating reflux and shaking extraction for 45 minutes on puffball medicinal materials is examined. And analyzing the number of extracted peaks and the peak area data of each characteristic peak.
The specific experimental method is as follows:
about 1.5g of Lasiosphaera Seu Calvatia medicinal material powder is taken, 3 groups are parallel, precisely weighed, placed in a conical bottle with a plug, precisely added with 15ml of methanol, respectively subjected to ultrasonic treatment (power 600W, frequency 40 kHz), heated reflux, shaken and extracted for 45 minutes, cooled, shaken uniformly, filtered, and the subsequent filtrate is taken to obtain the traditional Chinese medicine preparation. 3 μl of each sample solution was precisely aspirated, injected into a liquid chromatograph, measured according to the above chromatographic conditions, and the chromatograms were recorded, and the peak areas/sample amounts of 6 chromatographic peaks were calculated, and the results are shown in Table 2.
TABLE 2 extraction method of characteristic patterns of Lasiosphaera Seu Calvatia (Lasiosphaera Seu Calvatia) medicinal materials
The results show that: the number of the peaks extracted by the three modes is not obviously different, the ultrasonic treatment and the heating reflux mode have better extraction effects, and the ultrasonic treatment can be used as the most preferable extraction mode in consideration of the simplicity of operation.
(3) Selection of extraction time
100% methanol is selected as an extraction solvent, and the extraction effect of ultrasonic treatment on puffball medicinal materials for 15 minutes, 30 minutes, 45 minutes and 60 minutes is examined. And analyzing the number of extracted peaks and the peak area data of each characteristic peak.
The specific experimental method is as follows:
about 1.5g of Lasiosphaera Seu Calvatia medicinal material powder is taken, 4 groups are parallel, precisely weighed, placed in a conical bottle with a plug, precisely added with 15ml of methanol, respectively subjected to ultrasonic treatment (power 600W, frequency 40 kHz) for 15 minutes, 30 minutes, 45 minutes and 60 minutes, cooled, shaken uniformly, filtered, and the subsequent filtrate is taken, thus obtaining the preparation. 3 μl of each sample solution was precisely aspirated, injected into a liquid chromatograph, measured according to the above chromatographic conditions, and the chromatograms were recorded, and the peak areas/sample amounts of 6 chromatographic peaks were calculated, and the results are shown in Table 3.
TABLE 3 time of extraction of Lasiosphaera Seu Calvatia (Lasiosphaera Seu Calvatia) medicinal material characteristic spectrum
The results show that when the extraction time is 15 minutes, 30 minutes, 45 minutes and 60 minutes, the number of the extracted peaks is not obviously different, the extraction efficiency of each peak is not obviously increased after the heating reflux is carried out for 45 minutes, and the extraction time of 45 minutes can be used as a preferable condition parameter for ensuring the complete extraction.
(4) Investigation of extraction volume
100% methanol is selected as an extraction solvent, and the extraction effect of the puffball medicinal materials with extraction volumes of 10ml, 15ml, 25ml and 50ml is examined. And analyzing the number of extracted peaks and the peak area data of each characteristic peak.
The specific experimental method is as follows:
about 1.5g of Lasiosphaera Seu Calvatia medicinal powder is taken, 4 groups are parallel, precisely weighed, placed in conical flasks with plugs, respectively precisely added with 10ml of methanol, 15ml of methanol, 25ml of methanol and 50ml of methanol, subjected to ultrasonic treatment (power 600W, frequency 40 kHz) for 45 minutes, cooled, shaken uniformly, filtered, and the subsequent filtrate is taken to obtain the traditional Chinese medicine preparation. 3 μl of each sample solution was precisely aspirated, injected into a liquid chromatograph, measured according to the above chromatographic conditions, the chromatogram was recorded, and the peak area/sample weighing volume of 6 chromatographic peaks was calculated, and the results are shown in table 4.
TABLE 4 extraction method of characteristic patterns of Lasiosphaera Seu Calvatia (Lasiosphaera Seu Calvatia) medicinal materials
The results show that: when the extraction solvent volume is 15ml, the extraction efficiency is the highest, and the extraction solvent volume can be used as a preferable condition parameter.
Particulate test solution:
(1) Selection of extraction solvent
Because the puffball formula particles are prepared by concentrating Ma Boshui, the puffball has strong water solubility, and the index components of ergosterone and ergosterol in puffball medicinal materials are fat-soluble components. In summary, the extraction effect of four different solvents, namely 100% methanol, 80% aqueous methanol solution, ethanol and dichloromethane, on puffball formula particles was examined, wherein the percentage is the volume percentage of methanol.
The specific experimental method is as follows:
taking proper amount of puffball formula particles, grinding, taking about 5g, carrying out parallel 5 groups, precisely weighing, placing into a conical flask with a plug, precisely adding 50ml of methanol, 80% aqueous methanol solution, ethanol and dichloromethane, sealing, weighing, respectively carrying out ultrasonic treatment (power 600W and frequency 40 kHz) for 30 minutes, cooling, weighing again, supplementing the weight of loss with corresponding solvent, shaking uniformly, centrifuging, precisely measuring 25ml of supernatant, evaporating to dryness, dissolving residues with methanol, transferring into a 5ml measuring flask, adding methanol to scale, shaking uniformly, filtering, and taking subsequent filtrate. 3 μl of each sample solution was precisely aspirated, injected into a liquid chromatograph, measured according to the above chromatographic conditions, and the chromatograms were recorded, and the peak areas/sample amounts of 6 chromatographic peaks were calculated, and the results are shown in Table 5.
TABLE 5 extraction solvent investigation results of puffball (Lasiosphaera seu Calvatia) formula particle feature profile
The comparison result of the chromatograms is shown in fig. 2.
The peak area data was analyzed and the characteristic peak area was lower when extracted with methylene chloride, 80% aqueous methanol. When 100% methanol and ethanol are used for extraction, the peak area of each characteristic peak is higher.
When the liquid chromatogram is analyzed, compared with ethanol, the solvent effect is smaller when 100% methanol is adopted for extraction.
Finally, as the 100% methanol is extracted, each characteristic peak area is higher, the chromatographic peak shape is better, and the solvent effect is smaller, thus being used as a preferable condition parameter.
(2) Investigation of the extraction method
100% methanol is selected as an extraction solvent, and the extraction effect of ultrasonic treatment (frequency 600W, power 40 kHz), heating reflux and shaking extraction for 30 minutes on puffball formula particles is examined. And analyzing the number of extracted peaks and the peak area data of each characteristic peak.
The specific experimental method is as follows:
taking proper amount of puffball formula particles, grinding, taking about 5g, carrying out parallel 3 groups, precisely weighing, placing into a conical flask with a plug, precisely adding 50ml of methanol, sealing, weighing, respectively carrying out ultrasonic treatment (power 600W, frequency 40 kHz), heating reflux, shaking and extracting for 30 minutes, cooling, weighing again, supplementing the lost weight with methanol, shaking uniformly, centrifuging, precisely measuring 25ml of supernatant, evaporating to dryness, dissolving residues with methanol, transferring to a 5ml measuring flask, adding methanol to scale, shaking uniformly, filtering, and taking subsequent filtrate to obtain the puffball. 3 μl of each sample solution was precisely sucked up, injected into a liquid chromatograph, measured according to the above chromatographic conditions, and the chromatograms were recorded, and the peak areas/sample amounts of 6 chromatographic peaks were calculated, and the results are shown in Table 6.
TABLE 6 extraction of characteristic patterns of Lasiosphaera Seu Calvatia formula particles
The results show that: the number of the peaks extracted by the three modes is not obviously different, and the extraction effect of the heating reflux mode is far higher than that of ultrasonic treatment and shaking extraction, so that the method can be used as the most preferable extraction mode.
(3) Selection of extraction time
100% methanol is selected as an extraction solvent, and the extraction effect of heating reflux for 15 minutes, 30 minutes, 45 minutes and 60 minutes on puffball formula particles is examined. And analyzing the number of extracted peaks and the peak area data of each characteristic peak.
The specific experimental method is as follows:
taking proper amount of puffball formula particles, grinding, taking about 5g, precisely weighing in 4 groups in parallel, placing into a conical flask with a plug, precisely adding 50ml of methanol, sealing, weighing, respectively heating and refluxing for 15 minutes, 30 minutes, 45 minutes and 60 minutes, cooling, weighing again, supplementing the lost weight with methanol, shaking uniformly, centrifuging, precisely measuring 25ml of supernatant, evaporating to dryness, dissolving residues with methanol, transferring to a 5ml measuring flask, adding methanol to a scale, shaking uniformly, filtering, and taking subsequent filtrate to obtain the puffball. 3 μl of each sample solution was precisely aspirated, injected into a liquid chromatograph, measured according to the above chromatographic conditions, and the chromatograms were recorded, and the peak areas/sample amounts of 6 chromatographic peaks were calculated, and the results are shown in Table 7.
TABLE 7 time-of-extraction investigation of puffball (Lasiosphaera seu Calvatia) formula particle characteristics
The results show that when the extraction time is 15 minutes, 30 minutes, 45 minutes and 60 minutes, the number of the extracted peaks is not obviously different, the extraction efficiency of each peak is not obviously increased after heating and refluxing for 30 minutes, and 45 minutes can be used as a preferable condition parameter for ensuring the complete extraction.
(4) Investigation of extraction volume
100% methanol is selected as an extraction solvent, and the extraction effect of 25ml, 50ml and 100ml of extraction volume on puffball formula particles is examined. And analyzing the number of extracted peaks and the peak area data of each characteristic peak.
The specific experimental method is as follows:
taking proper amount of puffball formula particles, grinding, taking about 5g, carrying out parallel 3 groups, precisely weighing, placing into a conical flask with a plug, precisely adding 25ml, 50ml and 100ml of methanol, sealing, weighing, respectively heating and refluxing for 45 minutes, cooling, weighing again, supplementing the lost weight with methanol, shaking uniformly, centrifuging, precisely measuring 15ml, 25ml and 50ml of supernatant respectively, evaporating to dryness, dissolving residues with methanol, transferring to a 5ml measuring flask, adding methanol to a scale, shaking uniformly, filtering, and taking the subsequent filtrate to obtain the puffball. 3 μl of each sample solution was precisely sucked up, injected into a liquid chromatograph, measured according to the above chromatographic conditions, and the chromatograms were recorded, and the peak areas/sample amounts of 6 chromatographic peaks were calculated, and the results are shown in Table 8.
TABLE 8 extraction method of characteristic patterns of Lasiosphaera Seu Calvatia formula particles
The results show that: after the extraction solvent is 50ml, the extraction efficiency is not obviously increased, and the extraction solvent can be used as a preferable condition parameter.
3. Determination of chromatographic conditions
Preparation of test solution:
taking a proper amount of puffball (puffball) prescription particles, grinding, taking about 5g, precisely weighing, placing into a conical flask with a plug, precisely adding 50ml of methanol, sealing, weighing, heating and refluxing for 45 minutes, cooling, weighing again, supplementing the weight which is lost with methanol, shaking uniformly, centrifuging, precisely measuring 25ml of supernatant, evaporating to dryness, dissolving residues with methanol, transferring to a 5ml measuring flask, adding methanol to a scale, shaking uniformly, filtering, and taking subsequent filtrate.
(1) Selection of detection wavelength
The chromatographic conditions were as follows:
chromatographic column: waters ACQUITYBEH C18，1.7μm，2.1mm×100mm。
Mobile phase a: acetonitrile
Mobile phase B:0.1% phosphoric acid aqueous solution (v/v)
Gradient elution procedure:
chromatographic parameters:
a detector: DAD, full wavelength scan (200-400 nm)
Flow rate: 0.3 ml/min
Column temperature: 25 DEG C
Sample injection amount: 3 μl of
The results of detection at different wavelengths from low wavelength to high wavelength 3, 240nm, 282nm and 349nm respectively, were examined, and the obtained chromatograms are shown in FIG. 3.
As a result, the information content at 282nm was large, and the peak distribution was relatively uniform, which was considered as a preferable detection condition.
(2) Selection of different chromatographic columns
The experiment examines the influence of chromatographic columns of different factories and fillers on separation effect, and chromatographic column information is shown in the following table.
The results of the investigation are shown in FIG. 4.
The results show that the separation effect of each characteristic peak measured by 3 chromatographic columns is better. The chromatographic column 3 has moderate retention time and can be used as a preferable characteristic spectrum analysis chromatographic column.
(3) Selection of different gradients
The chromatographic conditions were as follows:
chromatographic column: waters ACQUITYBEH C18，1.7μm，2.1mm×100mm。
Mobile phase a: acetonitrile
Mobile phase B:0.1% phosphoric acid aqueous solution (v/v)
Chromatographic parameters:
a detector: DAD,282nm
Flow rate: 0.3 ml/min
Column temperature: 25 DEG C
Sample injection amount: 3 μl of
The experiment examined the effect of three different elution gradients on the separation effect.
Gradient 1:0/20/23 min, 5/95/95% acetonitrile;
gradient 2:0/10/23/33 min, 5/20/85/85% acetonitrile;
gradient 3:0/8/12/30 min, 55/91% acetonitrile;
the results of the investigation are shown in FIG. 5.
The result shows that when the gradient 3 is adopted for elution, the chromatogram shows 6 chromatographic peaks with better peak shape and better separation degree, the separation degree, tailing factor and theoretical plate number of each characteristic peak are comprehensively considered, and when the gradient 3 is adopted for elution, the separation effect is optimal and can be used as the preferable condition parameter.
(4) Selection of different flow rates
The chromatographic detection conditions were as follows:
chromatographic column: waters ACQUITYBEH C18，1.7μm，2.1mm×100mm。
Mobile phase a: acetonitrile
Mobile phase B:0.1% phosphoric acid aqueous solution (v/v)
Gradient elution procedure:
chromatographic parameters:
a detector: DAD,282nm
Column temperature: 25 DEG C
Sample injection amount: 3 μl of
The experiment examined the effect of three different flow phases, 0.25ml/min, 0.30ml/min and 0.35ml/min, on the separation effect.
The results of the investigation are shown in FIG. 6.
The result shows that the sample separation effect is good when the flow rate is 0.25 ml/min-0.35 ml/min, and the retention time is moderate under the condition of considering the flow rate of 0.30ml/min, so that the sample separation device can be used as a preferable condition parameter.
(5) Selection of different column temperatures
Chromatographic column: waters ACQUITYBEH C18，1.7μm，2.1mm×100mm。
Mobile phase a: acetonitrile
Mobile phase B:0.1% phosphoric acid aqueous solution (v/v)
Gradient elution procedure:
chromatographic parameters:
a detector: DAD,282nm
Flow rate: 0.3 ml/min
Sample injection amount: 3 μl of
The experiment examined the effect of three different temperatures at 20deg.C, 25deg.C and 30deg.C on the separation effect.
The results of the investigation are shown in FIG. 7.
The results show that the separation effect of the sample is better at the column temperature of 20-30 ℃, and the retention time is moderate when the column temperature is 25 ℃ is considered, so that the sample can be used as the preferable condition parameter.
Example 2
UPLC characteristic spectrum methodology verification of Lasiosphaera Seu Calvatia formula particles
(1) Chromatographic peak assignment
Preparation of test solution: preparing a test solution of puffball (puffball) formula particles according to the preferred experimental conditions
Preparation of reference solution: taking a proper amount of ergosterol reference substance, precisely weighing, adding methanol to prepare a solution containing 20 mug per 1 ml.
Preparation of negative control solution: a negative control solution of puffball (puffball) deficiency formula particles was prepared according to the experimental conditions set forth above.
The peaks of the characteristic spectrum of puffball (puffball) formula particles are positioned as shown in figure 8.
(2) Integrity inspection
Taking the sample, preparing a test sample solution according to the experimental conditions set forth above, measuring, maintaining the highest proportion of acetonitrile under the same chromatographic conditions, prolonging the elution time by one time, namely, recording a chromatogram, and comparing with a gradient 1 specified by a standard text, wherein the gradient 2 is shown in the table 9, and the result is shown in fig. 9.
TABLE 9 elution gradient Table for characteristic Profile integrity test of Lasiosphaera Seu Calvatia formula particles
The results show that: after the original gradient elution is finished, no obvious chromatographic peak flows out, which shows that the chromatographic condition basically meets the principle of maximum information content and has no interference to the analysis of the subsequent samples.
(3) Precision investigation
The sample is taken, a sample solution is prepared and measured according to the experimental conditions set forth above, the sample is continuously injected for 6 times, and the consistency of the characteristic peak relative retention time is examined, and the results are shown in Table 10.
TABLE 10 results of particle characterization by instrument (relative retention time) for Lasiosphaera Seu Calvatia (Lasiosphaera Seu Calvatia) formulations
The results show that: the relative retention time RSD of each characteristic peak is less than 0.4%, and the precision is good.
(4) Stability investigation
Sample solutions were prepared according to the experimental conditions set forth above, and measured at 0, 2, 4, 8, 10, 12, 14, 16, 18, and 24 hours, respectively, to examine the consistency of the characteristic peaks with respect to retention time, and the results are shown in table 11.
TABLE 11 results of stability test of particle characteristics of Lasiosphaera Seu Calvatia (Lasiosphaera Davida) formula (relative retention time)
The results show that: the relative retention time RSD of each characteristic peak of the test solution is less than 0.6%, and the test solution has good stability within 24 hours.
(5) Repeatability investigation
The sample solution was prepared and measured in parallel with 6 parts under the experimental conditions set forth above, and the consistency of the characteristic peaks with respect to retention time was examined, and the results are shown in Table 12.
TABLE 12 results of a particle characterization repeatability test of Lasiosphaera Seu Calvatia (Lasiosphaera Davida) (relative retention time)
The results show that: the RSD of each characteristic peak was less than 0.3% with good reproducibility.
(6) Intermediate precision investigation
The same batch of samples (batch number: 21090389) was taken by other analysts of the project group under the chromatograph of different manufacturers, and the test sample solutions were prepared and measured in parallel with 3 parts according to the experimental conditions set forth above. And (5) examining the consistency of characteristic peaks relative to retention time. The results are shown in Table 13.
TABLE 13 results of intermediate precision tests (relative retention time) of characteristic patterns of puffball (Lasiosphaera seu Calvatia) formula particles
The results show that: the characteristic spectrum of the puffball formula particle can be reproduced between two instruments of different brands, the relative retention time difference of each characteristic peak is smaller and is within the range of +/-10% of a specified value, the RSD is smaller than 3%, the separation degree and the peak type of each characteristic peak are better, and the intermediate precision is good.
Example 3
Puffball (puffball) prescription granule, detection of medicinal materials and establishment of control map
S11, taking a proper amount of puffball (puffball) formula particles, grinding, taking about 5g, precisely weighing, placing into a container with a plug, precisely adding 50ml of methanol, sealing, weighing, heating and refluxing for 45 minutes, cooling, weighing again, supplementing the weight of loss with methanol, shaking uniformly, centrifuging, precisely measuring 25ml of supernatant, evaporating to dryness, dissolving residues with methanol, transferring into a 5ml measuring flask, adding methanol to a scale, shaking uniformly, filtering, and taking subsequent filtrate.
S12, taking a proper amount of puffball (Lasiosphaera seu calvatia) medicinal material powder, taking about 1.5g, precisely weighing, placing into a container with a plug, precisely adding 15ml of methanol, sealing, weighing, performing ultrasonic treatment (power 600W, frequency 40 kHz) for 45 minutes, taking out, cooling, weighing again, supplementing the lost weight with methanol, shaking uniformly, filtering, and taking subsequent filtrate.
S13, taking a proper amount of ergosterol reference substance, and adding methanol to prepare a solution containing 20 mug or 95 mug of ergosterol per 1 ml.
S14, respectively precisely sucking 3 μl of a reference substance solution and puffball (puffball) formula particles or medicinal material test solution, and injecting into a liquid chromatograph under the following chromatographic conditions: chromatographic column, waters ACQUITYBEH C18 (1.7 μm,2.1 mm. Times.100 mm); acetonitrile is taken as a mobile phase A, 0.1% phosphoric acid aqueous solution is taken as a mobile phase B, and gradient elution is carried out according to the specifications in the following table; the flow rate is 0.3ml per minute; the column temperature is 25 ℃; the detection wavelength was 282nm.
(1) Preparing 3 batches of puffball (puffball) formula particles and 15 batches of puffball (puffball) medicinal material sample solution;
(2) According to the method for detecting the UPLC characteristic spectrum of puffball (puffball) formula particles, the test solution is respectively detected to obtain UPLC characteristic spectrum of 3 batches of puffball (puffball) formula particles and UPLC characteristic spectrum of 15 batches of puffball (puffball) medicinal materials;
(3) The obtained UPLC characteristic patterns of 3 batches of Lasiosphaera Seu Calvatia (Lasiosphaera seu Calvatia) formula particles and UPLC characteristic patterns of 15 batches of Lasiosphaera Seu Calvatia (Lasiosphaera Seu Calvatia) medicinal materials are respectively imported into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition) issued by the national formulary committee, and control patterns of Lasiosphaera Seu Calvatia (Lasiosphaera Seu Calvatia) formula particles and Lasiosphaera Seu Calvatia (Lasiosphaera Seu Calvatia) medicinal materials are established. See fig. 10 and 11.
Experimental results:
TABLE 14 measurement of samples of Lasiosphaera Seu Calvatia (Lasiosphaera major) formula particles (relative retention time)
TABLE 15 determination of samples of Lasiosphaera Seu Calvatia (Lasiosphaera Seu Calvatia)
The 3 batches of puffball (puffball) formula particles and the 15 batches of puffball (puffball) medicinal materials all show the same 6 common characteristic peaks, and standard research is carried out by taking peak 6 (ergosterol) as a reference peak. Finally, the characteristic spectrum standard of puffball (puffball) formula particles is specified as follows: the chromatogram of the test sample should show 6 characteristic peaks, wherein peak 6 should be consistent with the retention time of the reference peak of the ergosterol control. The peak corresponding to the ergosterol reference substance is an S peak, and the relative retention time of the peak 1, the peak 2, the peak 3, the peak 4, the peak 5 and the S peak is calculated, wherein the relative retention time is within +/-10% of a specified value, and the specified value is: 0.29 (Peak 1), 0.30 (Peak 2), 0.32 (Peak 3), 0.36 (Peak 4), 0.89 (Peak 5).
The established puffball (puffball) formula granule and medicinal material comparison map can be used for more accurately controlling the quality of puffball (puffball) formula granule, as shown in figures 12 and 13.
Example 4
Transfer of magnitudes of characteristic maps of samples from 3 batches of puffball (puffball) in each process
In this example, the magnitude transfer of characteristic patterns of medicinal materials, standard decoction and formula particles, which are samples of each process of puffball (puffball) 3 batches are discussed.
(1) The relative retention times of the samples of each process, peak 1, peak 2, peak 3, peak 4, peak 5 and S peak, were calculated for puffball (puffball) 3 batches using the corresponding peak of the ergosterol reference as the S peak, as shown in table 16.
(2) And respectively introducing 3 batches of pilot-scale puffball (Lasiosphaera seu calvatia) medicinal materials, corresponding standard decoction and traditional Chinese medicine formula particle chromatograms into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system 2012 edition, and comparing, wherein the overlapped chromatograms of the three batches of medicinal materials, the standard decoction and the traditional Chinese medicine formula particles are shown in fig. 14, 15 and 16.
Experimental results:
TABLE 16 Lasiosphaera Seu Calvatia (Lasiosphaera Seu Calvatia) 3 batch Mass production of sample characteristic peaks relative retention time for each process
From the graph, the patterns of the 3 batches of puffball (puffball) in each process sample (medicinal material and formula granule) have 6 characteristic peaks, the relative retention time of each characteristic peak is within +/-10% of a specified value, and the characteristic patterns are consistent with the standard decoction characteristic patterns, so that the basis of the puffball (puffball) mass production medicinal material and particles and puffball (puffball) standard decoction substances is basically consistent. The results show that the quantity value is stable in the puffball (puffball) production process, and the preparation process is reasonable.
Example 5
Ma Bozhen and identification of counterfeit medicinal materials
S11, taking a proper amount of pseudo product (large mouth static ash ball) powder, taking about 1.5g, precisely weighing, placing into a container with a plug, precisely adding 15ml of methanol, sealing, weighing, performing ultrasonic treatment (power 600W, frequency 40 kHz) for 45 minutes, taking out, cooling, weighing again, supplementing the lost weight with methanol, shaking uniformly, filtering, and taking subsequent filtrate.
S12, taking a proper amount of ergosterol reference substance, and adding methanol to prepare a solution containing 95 mug of ergosterol per 1 ml.
S13, respectively precisely sucking 3 mu l of a reference substance solution and 3 mu l of a pseudo product (large-mouth static ash ball) sample solution, and injecting into a liquid chromatograph under the chromatographic conditions that: chromatographic column, waters ACQUITYBEH C18 (1.7 μm,2.1 mm. Times.100 mm); acetonitrile is taken as a mobile phase A, 0.1% phosphoric acid aqueous solution is taken as a mobile phase B, and gradient elution is carried out according to the specifications in the following table; the flow rate is 0.3ml per minute; the column temperature is 25 ℃; the detection wavelength was 282nm.
(1) Preparing 1 batch of pseudo product (large mouth static gray ball) sample solution;
(2) Detecting the sample solution according to the method for detecting UPLC characteristic patterns of puffball (puffball) formula particles to obtain 1 UPLC characteristic pattern of a pseudo product (puffball static gray ball);
(3) The obtained UPLC characteristic patterns of 1 pseudo product (Dakou Jing Hui) and the UPLC characteristic patterns of puffball (Lasiosphaera seu Calvatia) are led into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system issued by the national formulary Committee for comparison. See fig. 17.
The results show that: from the number of characteristic peaks, the characteristic peaks of a genuine product (puffball) are stable, and 6 common peaks are presented; the number of characteristic peaks of the pseudo product (large mouth static gray ball) is 2 (the peak 1, the peak 2, the peak 3 and the peak 4 are deleted). Therefore, the pseudo product is larger in the difference between the characteristic peaks and the genuine product, and can be judged as the pseudo product when the number of the characteristic peaks is less than 6.
In summary, the existing data research shows that when the number of characteristic peaks is less than 6, the true products and the false products can be distinguished. Since there are few batches of counterfeit studies, this conclusion is only a preliminary study conclusion, and is to be further accumulated and perfected.
Example 6
Ma Bobu identification of the same-base (Lasiosphaera Seu Calvatia )
S11, taking a proper amount of puffball (Lasiosphaera Seu Calvatia) medicinal material powder, taking about 1.5g, precisely weighing, placing into a container with a plug, precisely adding 15ml of methanol, sealing, weighing, performing ultrasonic treatment (power 600W, frequency 40 kHz) for 45 minutes, taking out, cooling, weighing again, supplementing the lost weight with methanol, shaking uniformly, filtering, and taking subsequent filtrate.
S12, taking a proper amount of ergosterol reference substance, and adding methanol to prepare a solution containing 95 mug of ergosterol per 1 ml.
S13, respectively precisely sucking 3 μl of a reference substance solution and a puffball (purple puffball) medicinal material sample solution, and injecting into a liquid chromatograph under the chromatographic conditions that: chromatographic column, waters ACQUITYBEH C18 (1.7 μm,2.1 mm. Times.100 mm); acetonitrile is taken as a mobile phase A, 0.1% phosphoric acid aqueous solution is taken as a mobile phase B, and gradient elution is carried out according to the specifications in the following table; the flow rate is 0.3ml per minute; the column temperature is 25 ℃; the detection wavelength was 282nm.
(1) Preparing 2 batches of puffball (purple puffball) medicinal material sample solutions;
(2) Detecting the sample solution according to the method for detecting UPLC characteristic patterns of puffball (puffball) formula particles to obtain 2 UPLC characteristic patterns of puffball (puffball) medicinal materials;
(3) The obtained UPLC characteristic spectrum of 2 batches of Lasiosphaera Seu Calvatia (purple Lasiosphaera Seu Calvatia) medicinal materials is imported into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system issued by the national formulary Committee to establish a control spectrum of Lasiosphaera Seu Calvatia (purple Lasiosphaera Seu Calvatia) medicinal materials. See fig. 18.
(4) Comparing UPLC control maps of Lasiosphaera Seu Calvatia (purple Lasiosphaera Seu Calvatia) and Lasiosphaera Seu Calvatia (Lasiosphaera Seu Calvatia) and calculating relative peak areas of the other 5 characteristic peaks by taking peak 5 as reference, and distinguishing Lasiosphaera Seu Calvatia (purple Lasiosphaera Seu Calvatia) and Lasiosphaera Seu Calvatia (Lasiosphaera Seu Calvatia). See table 17, fig. 19.
Experimental results:
TABLE 17 measurement results (relative Peak area) of Lasiosphaera Seu Calvatia (purple Lasiosphaera Seu Calvatia) medicinal material and samples of Lasiosphaera Seu Calvatia (big Lasiosphaera Seu Calvatia) medicinal material of 15 batches
The results show that: the purple puffball medicinal materials are slightly different from the puffball medicinal materials in characteristic patterns. Both show 6 common peaks, and the peak area of the peak 6 of the purple puffball medicinal material relative to the peak 5 is far higher than that of the puffball medicinal material from the aspect of the characteristic peak relative peak area.
In summary, when the peak areas of the characteristic peaks 6 and 5 are both greater than 30, it is determined that puffball is purple.
Claims (6)
1. A quality evaluation and detection method for puffball medicinal materials, standard decoction or formula particles is characterized by comprising the following steps:
(1) Collecting one of Lasiosphaera Seu Calvatia, standard decoction or its granule, processing, and extracting with solvent to obtain sample solution; when puffball is a medicinal material, the extraction solvent is aqueous solution containing 80-100% of methanol by volume or aqueous solution containing 80-100% of ethanol by volume; when puffball is a standard decoction or a formula particle, the extraction solvent is dichloromethane or a methanol water solution containing 80-100% of methanol by volume percent or an ethanol water solution containing 80-100% of ethanol by volume percent;
(2) Adding solvent into ergosterol to obtain reference solution;
(3) Respectively injecting the sample solution and the reference substance solution into liquid chromatograph to obtain characteristic patterns, wherein the liquid chromatograph conditions are as follows:
the stationary phase is a chromatographic column taking octadecylsilane chemically bonded silica as a filler; the mobile phase A is acetonitrile, and the mobile phase B is phosphoric acid aqueous solution with the mass percent of phosphoric acid of 0.01-0.20%;
the detection instrument of the characteristic spectrogram comprises: the specification of the ultra-high performance liquid chromatograph and the chromatographic column is as follows: the column length is 100-150 mm, and the grain diameter is 1.7-1.9 mu m;
the gradient elution procedure was as follows:
(4) Judging the quality of raw materials and distinguishing between false and positive products: if the characteristic spectrum obtains the following 6 characteristic peaks, the quality of the raw material is qualified: peak 6 is the peak of ergosterol, and relative retention times for peak 1, peak 2, peak 3, peak 4 and peak 5 are respectively: 0.28 to 0.29, 0.29 to 0.31, 0.31 to 0.33, 0.35 to 0.37 and 0.88 to 0.90;
if the measured characteristic patterns do not all contain the above 6 characteristic peaks, puffball is a pseudo product; if the peak area of the characteristic peak 6 relative to the peak 5 in the characteristic spectrum is greater than 30, the purple puffball is judged.
2. The method for evaluating and detecting the quality of puffball medicinal materials, standard decoction or formula particles according to claim 1, wherein in the step (1), when puffball is medicinal material, the specific steps are as follows: sieving Lasiosphaera Seu Calvatia medicinal material powder, placing into a conical flask, adding extraction solvent, extracting with solvent with the mass of 0.03-0.15g per 1ml of solvent, performing ultrasonic treatment or heating or vibration treatment for 15-60 min, cooling, adding corresponding solvent to the weight of the filtrate, shaking, and filtering to obtain filtrate as sample solution;
When puffball is a standard decoction, the specific steps are as follows: placing Lasiosphaera Seu Calvatia standard decoction powder into a conical flask, adding extraction solvent, extracting with 0.01-0.06g of solvent per 1ml of solvent, performing ultrasonic treatment or heating or oscillating treatment for 15-60 min, cooling, adding corresponding solvent to the weight of the filtrate, shaking, centrifuging, collecting supernatant, evaporating, dissolving residue with methanol, transferring to a measuring flask, adding methanol to scale, shaking, filtering, and collecting filtrate to obtain sample solution;
when puffball is a formula particle, the specific steps are as follows: placing puffball formula particle powder into a conical flask, adding an extraction solvent, wherein the mass of a sample to be extracted per 1ml of solvent is 0.05-0.2g, carrying out ultrasonic treatment or heating or vibration treatment for 15-60 min, cooling, supplementing the reduced weight with the corresponding solvent, shaking up, centrifuging, taking supernatant, evaporating to dryness, dissolving residues with methanol, transferring to a measuring flask, adding methanol to scale, shaking up, filtering, and obtaining a subsequent filtrate, namely the sample solution.
3. The method for evaluating and detecting the quality of puffball, standard decoction or formulated particles according to claim 1, wherein the conditions of the liquid chromatography in step (3) further comprise: the flow rate is 0.25-0.35 ml/min; the column temperature is 20-30 ℃; the detection wavelength is 265-295 nm.
4. The method for evaluating and detecting the quality of puffball, standard decoction or formulation particles according to claim 1, wherein the concentration of ergosterol in the control solution in step (2) is: 20-95 mug/ml; the dissolution solvent of the reference substance solution is methanol.
5. The method for evaluating and detecting the quality of puffball medicinal materials, standard decoction or formula particles according to claim 2, which is characterized in that: the power of ultrasonic extraction is 250-600W, and the frequency is 35-45 kHz.
6. Use of the quality evaluation and detection method of puffball medicinal materials, standard decoction or formulation particles according to any one of claims 1-5 for distinguishing true purple puffball, puffball and pseudo puffball.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105985402A (en) * | 2015-02-01 | 2016-10-05 | 复旦大学 | Sterol lactam compound puffball lactam and use thereof in preparation of anticomplement drugs |
| CN105985403A (en) * | 2015-02-01 | 2016-10-05 | 复旦大学 | Fortisterol and use thereof in preparation of anticomplement drugs |
-
2022
- 2022-08-03 CN CN202210925290.5A patent/CN115266997B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105985402A (en) * | 2015-02-01 | 2016-10-05 | 复旦大学 | Sterol lactam compound puffball lactam and use thereof in preparation of anticomplement drugs |
| CN105985403A (en) * | 2015-02-01 | 2016-10-05 | 复旦大学 | Fortisterol and use thereof in preparation of anticomplement drugs |
Non-Patent Citations (4)
| Title |
|---|
| 周贤 等.星裂硬皮马勃不同部位麦角甾醇含量的高效液相色谱法测定.时珍国医国药 .2014,25(5),1062-1063. * |
| 杨秀璐 等.HPLC 双波长法测定小柄马勃子实体、 液体发酵菌丝中麦角甾醇和麦角甾酮含量.中国食用菌 .2018,37(5),57-61. * |
| 王大可 等.药用真菌星裂硬皮马勃的生药学研究.中国食用菌 .2015,34(6),50-53. * |
| 相聪坤 等.HPLC法测定不同产地不同品种马勃药材中麦角甾醇.中草药.2016,47(8),1397-1400. * |
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