CN115266997A - Construction method and application of puffball medicinal material and formula particle characteristic spectrum thereof - Google Patents

Abstract

The invention discloses a construction method and application of a puffball medicinal material and a formula particle characteristic spectrum thereof, wherein the construction method comprises the following steps: (1) Adding solvent into one of Lasiosphaera Seu Calvatia medicinal materials, standard decoction or its granule, and extracting to obtain test solution; (2) adding ergosterol into a solvent to prepare a reference substance solution; (3) Respectively injecting the test solution and the reference solution into a liquid chromatogram to obtain a characteristic spectrum; and (4) judging the quality of the raw materials and distinguishing the positive and the false products. The method can preliminarily distinguish counterfeit Lasiosphaera Seu Calvatia from Lasiosphaera Seu Calvatia, and provide certain basis for true and false identification and basic identification of Lasiosphaera Seu Calvatia medicinal material; and can comprehensively reflect the internal quality of puffball medicinal materials, standard decoction and formula granules thereof.

Description

Construction method and application of puffball medicinal material and formula particle characteristic spectrum thereof

Technical Field

The invention relates to a construction method and application of a characteristic map, in particular to a construction method and application of a puffball medicinal material and formula particle characteristic map thereof.

Background

Puffball, alias grey bag, horse dung bag, puffer, etc., is a dried fruit body of puffer Lasiosphaera fenzlii reich, puffer Calvatia gigantea (Batsch ex pers.), lloyd or purple-colored Calvatia lilacina (mont. Et berk.), lloyd. The medicine has pungent taste and mild nature, enters lung meridian, has the effects of clearing lung-heat, relieving sore throat, stopping bleeding and the like, and is used for treating wind-heat stagnation lung pharyngalgia, hoarseness and cough; it is used externally to treat epistaxis and traumatic hemorrhage.

The puffball comprises the following main components: steroids (Jin Xiangqun, wang Lishu, cheng Dongyan, yitong, lv Jingshan. Lasiobolus chemical composition research [ J ]. Chinese herbal medicine, 1998 (05): 298-300.), terpenes (Li Xiaoli. Puffball chemical composition separation, identification and research on cathepsin L inhibition [ D ]. Hunan Tan university, 2011.), small molecule nitrogen compounds (Zhang Jianmei, zhang Dancan. Puffball chemical composition and research on pharmacological effect [ J ]. Neuropharmacology news, 2020,10 (02): 11-14.), proteins (3535 zxft 35, fan Yanhua, zhao Yunpeng, bai Junpeng, zhao Qingchun. HPLC method determines white linear cyclic lipopeptide content in puffball [ J ]. Chinese pharmacist, 2013,16 (06): 833-835.), amino acids (Zhang Jiangping, 56 zxft 3456, 3838 zxft 5738. Puffball) and wild western 5749, western, 5749, etc. <xnotran> , (3456 zxft 3456, 3838 zxft 3838, 5749 zxft 5749, 6595 zxft 6595, 6898 zxft 6898, 3428 zxft 3428, 3476 zxft 3476 . [ J ]. , 3734 zxft 3734 (08): 157-161.), (, 3757 zxft 3757, 5852 zxft 5852, 3575 zxft 3575. [ J ]. , 3625 zxft 3625 (02): 50-52.), ( , . [ J ]. , 3826 zxft 3826 (03): 477-485.), (3828 zxft 3828, 3925 zxft 3925, 5483 zxft 5483. [ J ]. , 5678 zxft 5678 (08): 4053-4055.), (Jingjing Meng, yanhua Fan, mingzhi Su, congqin Chen, tianshu Ren, jinhui Wang, qingchun Zhao.WLIP derived from Lasiosphaera fenzlii Reich exhibits anti-tumor activity and induces cell cycle arrest through PPAR- γ associated pathways [ J ]. International Immunopharmacology, 7439 zxft 7439 (1).), (8624 zxft 8624, 9696 zxft 9696, 3235 zxft 3235. 3292 zxft 3292 [ J ]. , 3426 zxft 3426 (16): 9655-9656.) . </xnotran>

The existing literature data about puffball quality control mainly focuses on the content measurement of medicinal materials and decoction pieces, most researches take ergosterine and ergosterol as content measurement indexes, and a small amount of researches evaluate the contents of polysaccharides, total triterpenes or white linear cyclic lipopeptide and the like by measuring the contents of the polysaccharides, the total triterpenes or the white linear cyclic lipopeptide and the like, so that the evaluation indexes are single, and the quality of puffball cannot be integrally and comprehensively expressed. The research data on the characteristic spectrum and the fingerprint spectrum of puffball medicinal materials are very little, and only one document develops the fingerprint spectrum research on the desquamate puffball hemostatic active ingredients. Gao Yunjia establishes high performance liquid fingerprint of the hemostatic active component of peeled puffball, identifies 8 main chromatographic peaks, performs fingerprint analysis on 10 peeled puffball samples from different producing areas, and provides certain basis for quality control of peeled puffball medicinal materials. (Gao Yunjia research on desquamation puffball hemostatic active ingredient and its fingerprint spectrum [ D ]. Shenyang pharmaceutical university, 2009.). In the prior art, the research of fingerprint spectrum or characteristic spectrum in puffball (puffball) is not seen.

Regarding the research of puffball primordial differentiation, except the traditional character identification, microscopic identification and thin-layer identification, the content of ergosterone or ergosterol is determined by the research on puffball of different varieties and different production areas, and the result shows that certain difference exists; (clever female, yunnan, guan Shengjiang, huang Huaipeng, wang Qiao, anfuli, chen Yingchun, wu Haiyan, zhu Shan. HPLC method for determining ergosterol [ J ] in different varieties of puffball medicinal materials in different producing areas, 2016,47 (08): 1397-1400.) from Lasiosphaera Seu Calvatia by extracting volatile components from Lasiosphaera Seu Calvatia, and performing GC-MS analysis, it has been found that Lasiosphaera Seu Calvatia, purple Lasiosphaera Seu Calvatia, and peeled Lasiosphaera Seu Calvatia all contain ethyl palmitate, but not Lasiosphaera Seu Calvatia (Dakou Silybum Gaertn.) contains no such components. (Smart Kun, guan Shengjiang, wang Qiao, anfuli, chen Yingchun, wu Haiyan. GC-MS analysis of volatile components in puffball traditional Chinese medicinal materials by different extraction methods [ J ]. Hebei medicine, 2016,38 (23): 3545-3547.) is researched to compare the types and contents of amino acids and inorganic elements of puffball of different varieties, and the puffball, purple puffball and peeled puffball varieties under the pharmacopoeia items can be classified into one type after the clustering analysis of the amino acid contents, and are different from other mixed fake products, and meanwhile, the contents of the inorganic elements of the puffball of the three primitive types show certain differences. (Zhang Qingkang, ding Yonghui. Determination of amino acid content and cluster analysis of 10 puffbas [ J ]. Chinese patent medicine, 1996 (08): 35-37.) (Yue Lidan, du Lijie, yang Jianing, zhang Zhenling. ICP-MS determination and PCA analysis for content research of inorganic elements of different varieties of puffbas [ J ]. Asia-Pacific traditional medicine, 2021,17 (04): 41-46.) in addition, research finds that Fourier transform infrared spectra of 3 varieties of medicinal materials under the pharmacopeia item have obvious differences; the ITS sequence can be used for effectively distinguishing medicinal materials of puffball pharmacopeia varieties from counterfeit products (white bald puffball, head bald puffball, reticulate puffball, small puffball, pear-shaped puffball, grey cocci with lead color, bark puffball, long root quiet grey cocci and bean package bacteria). (Zhang Jiali, huang Yuhang, song Ming, ren Yangyang, zhang Mengting, liu Xia, sun Wei, chen Shilin. Identifying the fungal drug puffba and ITS mixed counterfeit product [ J ]. World traditional Chinese medicine, 2016,11 (05): 777-780+ 785.). In summary, the research directions on the differentiation of puffball primordia and the identification of genuine and counterfeit products are very abundant, but these technical means have certain limitations. At present, no research about the differentiation of puffball primordia through a characteristic spectrum or a fingerprint spectrum technology is available.

Since ancient times, the medicinal variety of puffball has a rather confusing source, and the most commonly used counterfeit currently belongs to the large mouth resting grey ball bovistela sinesis Lloyd. At present, the 2020 edition of Chinese pharmacopoeia stipulates only 3 kinds of genuine medicinal materials (Lasiosphaera fenzlii Reich., lasiosphaera gigantea (Batsch ex Pers.) or Lasiosphaera lilacina (Mont. Et Berk.) Lloyd) of Bovidae fungi, and the medicinal basis of other varieties is lack of sufficient scientific proof.

The existing legal quality standard of puffball is recorded in the first part of 2020 edition of Chinese pharmacopoeia and mainly comprises characters, microscopic identification, thin layer identification, water, ash and extracts. At present, the research of the prior art about puffball quality research mainly focuses on content measurement, and the puffball medicinal materials are subjected to quality evaluation by measuring the content of ergosterone, ergosterol, polysaccharide, total triterpenes or white linear cyclic lipopeptide and the like. The quality of the puffball medicinal materials can be reflected to a certain extent by the detection items, but the evaluation indexes are single, the quality of the puffball cannot be integrally and comprehensively reflected, and the quality control effect is limited. The research data of the characteristic spectrum and the fingerprint spectrum of the puffball medicinal material are very little, only one document develops the fingerprint spectrum research on the hemostatic active ingredients of the desquamated puffball, but the process of the method is complex, and the puffball and other varieties of puffball are not compared.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to provide a puffball medicinal material and a method for constructing a characteristic spectrum of 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 simultaneously can provide a certain basis for distinguishing puffball authentic products and different basic raw medicinal materials; the invention also aims to provide application of the construction method of the characteristic map in quality evaluation and detection and distinguishing purple puffball, puffball and counterfeit big mouth static gray balls.

The technical scheme is as follows: the invention relates to a construction method of a puffball medicinal material and a formula particle characteristic spectrum thereof, which comprises the following steps:

(1) Taking one of puffball medicinal materials, standard decoctions or formula granules thereof, processing, and adding solvent for extraction to obtain a test solution;

(2) Taking ergosterol, and adding a solvent to prepare a reference substance solution;

(3) Respectively injecting the test solution and the reference solution into a liquid chromatogram to obtain a characteristic spectrum, wherein the liquid chromatogram conditions are as follows:

the stationary phase is a chromatographic column which takes octadecylsilane chemically bonded silica as a filler; the mobile phase A is acetonitrile, and the mobile phase B is phosphoric acid water solution with the phosphoric acid mass percent of 0.01-0.20%;

(4) Judging the quality of the raw materials and distinguishing the positive and the false products: if the characteristic spectrum obtains the following 6 characteristic peaks, the quality of the raw materials is qualified: peak 6 is the ergosterol peak, with peak 6 as the reference peak, the relative retention times of peak 1, peak 2, peak 3, peak 4 and peak 5 are respectively: 0.26-0.32, 0.27-0.33, 0.29-0.35, 0.32-0.40 and 0.80-0.98;

if the measured characteristic spectrum does not completely contain the 6 characteristic peaks, puffball is a fake product; and if the peak area of the characteristic peak 6 relative to the peak 5 in the characteristic map is more than 30, judging the purple puffball.

Preferably, when puffball is a medicinal material in the step (1), the specific steps are as follows: sieving puffball powder, placing in a conical flask, adding extraction solvent, treating with ultrasonic wave or heating or shaking for 15-60 min, cooling, adding corresponding solvent to the weight loss, shaking, filtering, and collecting the filtrate to obtain test solution;

when puffball is a standard decoction, the specific steps are as follows: taking puffball standard decoction powder, placing into a conical flask, adding an extraction solvent, wherein the mass of a test sample extracted by each 1ml of the solvent is 0.01-0.06g, carrying out ultrasonic treatment or heating or oscillation treatment for 15-60 min, cooling, supplementing the loss 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 a test sample solution;

when puffball is a formula particle, the specific steps are as follows: taking puffball formula particle powder, placing the puffball formula particle powder in a conical flask, adding an extraction solvent, wherein the mass of a test sample extracted by each 1ml of the solvent is 0.05-0.2g, carrying out ultrasonic treatment or heating or shaking treatment for 15-60 min, cooling, complementing the loss weight by the corresponding solvent, shaking up, centrifuging, taking supernatant, drying by distillation, dissolving residues by using methanol, transferring the residues to a measuring flask, adding the methanol to the scale, shaking up, filtering, and obtaining a subsequent filtrate, namely the test 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 standard decoction or formula particles, the extraction solvent is dichloromethane or methanol water solution containing 80-100% by volume of methanol or ethanol water solution containing 80-100% by volume of ethanol.

Preferably, the detecting instrument for the characteristic spectrum in the step (3) is: ultra-high performance liquid chromatography; 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 μm; the conditions of the liquid chromatography further comprise: the flow rate is 0.25 to 0.35ml/min; the column temperature is 20-30 ℃; the detection wavelength is 265-295 nm.

Preferably, the concentration of the control ergosterol in step (2) is: 20-95 mu g/ml; the dissolution solvent of the reference substance is methanol.

Preferably, the power of the 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 material and the formula granules thereof can be applied to the quality evaluation and detection of purple puffball and puffball medicinal materials, standard decoction or formula granules.

The construction method of the feature spectrum of the puffball medicinal material and the formula granules thereof can also be applied to distinguishing true purple puffball, puffball and fake large silent grey balls.

Has the advantages that: compared with the prior art, the invention has the following remarkable advantages: (1) The method establishes the characteristic spectrum of the puffball medicinal material, can preliminarily distinguish counterfeit (large static gray ball), puffball (purple puffball) medicinal material and puffball (puffball) medicinal material, and provides a certain basis for true and false identification and basic identification of the puffball medicinal material; (2) Because the formula particles lose the appearance 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 medicinal materials, standard decoctions and formula particles and takes the characteristics components as the basis for determining the characteristic spectrums of the puffball medicinal materials, the standard decoctions and the formula particles. The ergosterol reference substance is used as a reference substance, a characteristic map containing 6 characteristic peaks is established, and the internal quality of the Lasiosphaera Seu Calvatia medicinal material, the standard decoction and the formula granules thereof is comprehensively reflected; meanwhile, the characteristic spectrum research of puffball medicinal materials, standard decoction and formula particles has corresponding characteristic peaks, so that the consistency of the formula particles and medicinal preparation extracts can be more fully reflected, a basis is provided for tracing the source of characteristic components in the puffball formula particles and upstream raw materials, and a powerful guarantee is provided for quality control in an industrialization process; (3) The invention adopts the ultra-high liquid chromatography to detect the characteristic spectrum; the method has the advantages of good separation of chromatographic peaks, good chromatographic peak shape, rich characteristic spectrum information, simple operation, high precision, good stability, good repeatability, high accuracy, high detection efficiency, low detection cost and the like, is verified by methodology, can effectively ensure the stability of the overall quality of puffball medicinal materials, standard decoction and formula particles, and provides important guarantee for the production of standardized puffball formula particles.

Drawings

FIG. 1 is a chromatogram for examining the extraction solvent of Lasiosphaera Seu Calvatia;

FIG. 2 is a chromatogram for solvent investigation of puffball formulation;

FIG. 3 is a chromatogram of different wavelength measurements of puffball (Lasiosphaera Seu Calvatia) formulation particles;

FIG. 4 is a chromatogram obtained by detecting different chromatographic columns of puffball (Lasiosphaera Seu Calvatia) formula granules;

FIG. 5 is a chromatogram obtained by measuring different gradients of puffball formula particles;

FIG. 6 is a chromatogram of different flow rate measurements of puffball (Lasiosphaera Seu Calvatia) formulation granules;

FIG. 7 is a chromatogram obtained by measuring different column temperatures of Lasiosphaera Seu Calvatia (Lasiosphaera Seu Calvatia) formulation granules;

FIG. 8 is a UPLC spectrum for identifying the peaks of the characteristic chromatogram of puffball (puffball) formula granules;

FIG. 9 is a UPLC spectrum for integrity test of characteristic spectrum of puffball (big puffball) formula particles;

FIG. 10 is a UPLC chromatogram overlay of 3 puffball (big puffball) formula pellets;

FIG. 11 is a UPLC chromatogram superposition chart of 15 batches of Lasiosphaera Seu Calvatia (Lasiosphaera Seu Calvatia) medicinal materials;

FIG. 12 is a UPLC comparative profile for puffball (puffball) formula granules;

FIG. 13 is UPLC contrast characteristic spectrum of Lasiosphaera Seu Calvatia (Lasiosphaera Seu Calvatia) medicinal material;

FIG. 14 is a characteristic map of each process of puffball (puffball) mass production;

FIG. 15 is a characteristic map of each process of puffball (puffball) mass production;

FIG. 16 is a characteristic map of each process of puffball (big puffball) mass production;

FIG. 17 is an overlay of the characteristic spectra of counterfeit drug (Dakou static gray ball) and Lasiosphaera Seu Calvatia;

FIG. 18 is UPLC contrast characteristic spectrum of Lasiosphaera Seu Calvatia (purple Lasiosphaera Seu Calvatia) medicinal material;

FIG. 19 shows puffball (purple puffball) and puffball the characteristic map overlay of (Lasiosphaera Seu Calvatia) medicinal materials.

Detailed Description

The technical scheme of the invention is further explained by combining the attached drawings.

Example 1

The establishment of the UPLC characteristic spectrum detection method of puffball formula particles comprises the following steps:

1. liquid phase condition

A chromatographic column: waters ACQUITY

BEH C18，1.7μm，2.1mm×100mm。

Mobile phase A: acetonitrile

And (3) 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 introduction amount: 3 μ l

2. Preparation of test solution

Test solution of medicinal materials:

(1) Selection of extraction solvent

As the ergosterone and ergosterol which are index components in the puffball medicinal material are fat-soluble components, the extraction effect of four different solvents of 100 percent methanol, 80 percent methanol aqueous solution, ethanol and 80 percent ethanol aqueous solution on the puffball medicinal material is considered, and the percentage is the volume percentage of the methanol or the ethanol.

The specific experimental method is as follows:

taking about 1.5g of Lasiosphaera Seu Calvatia powder, paralleling to 5 groups, precisely weighing, placing into a conical flask with a plug, respectively and precisely adding 15ml of methanol, 80% methanol water solution, ethanol, and 80% ethanol water solution, respectively, performing ultrasonic treatment (power 600W, frequency 40 kHz) for 45 min, cooling, shaking up, filtering, and taking out the subsequent filtrate. Precisely absorbing 3 mu l of each test solution, injecting into a liquid chromatograph, measuring according to the chromatographic conditions, recording a chromatogram, and calculating peak areas/sample weighing of 6 chromatographic peaks, wherein the results are shown in table 1.

TABLE 1 Lasiosphaera Seu Calvatia (Lasiosphaera Seu Calvatia) medicinal material feature map extraction solvent investigation results

The chromatogram comparison results are shown in FIG. 1.

And (4) analyzing peak area data, and when the data are extracted by using 80% methanol aqueous solution, the peak area of the characteristic peak is lower. When 100% methanol is used for extraction, the peak area of each characteristic peak is the highest and can be used as an optimal condition parameter.

(2) Examination of extraction methods

100% methanol is selected as an extraction solvent, and the extraction effect of puffball medicinal materials by ultrasonic treatment (frequency 600W and power 40 kHz), heating reflux and shaking extraction for 45 minutes is examined. And analyzing the data of the number of extracted peaks and the peak area of each characteristic peak.

The specific experimental method is as follows:

taking about 1.5g of Lasiosphaera Seu Calvatia medicinal powder, paralleling 3 groups, precisely weighing, placing into a conical flask with a plug, precisely adding 15ml of methanol, respectively performing ultrasonic treatment (power 600W and frequency 40 kHz), heating, refluxing, shaking for 45 min, cooling, shaking uniformly, filtering, and taking the subsequent filtrate. Precisely absorbing 3 mu l of each test solution, injecting into a liquid chromatograph, measuring according to the chromatographic conditions, recording a chromatogram, and calculating peak areas/sample weighing of 6 chromatographic peaks, wherein the results are shown in a table 2.

TABLE 2 Lasiosphaera Seu Calvatia (Lasiosphaera Seu Calvatia) medicinal material feature map extraction mode investigation results

The results show that: the three extraction modes have no obvious difference in the number of extraction peaks, the ultrasonic treatment and the heating reflux mode have good extraction effects, and the ultrasonic treatment can be used as the most preferable extraction mode in consideration of the simplicity and convenience of operation.

(3) Selection of extraction time

100% methanol is selected as an extraction solvent, and the extraction effect of puffball medicinal materials is examined after ultrasonic treatment for 15 minutes, 30 minutes, 45 minutes and 60 minutes. And analyzing the data of the number of extracted peaks and the peak area of each characteristic peak.

The specific experimental method is as follows:

taking about 1.5g of Lasiosphaera Seu Calvatia medicinal powder, paralleling 4 groups, precisely weighing, placing into a conical flask with a plug, precisely adding 15ml of methanol, respectively performing ultrasonic treatment (power 600W, frequency 40 kHz) for 15 min, 30 min, 45 min and 60min, cooling, shaking up, filtering, and taking the subsequent filtrate. Precisely absorbing 3 μ l of each test solution, injecting into a liquid chromatograph, measuring according to the chromatographic conditions, recording the chromatogram, and calculating the peak area/sample weighing of 6 chromatographic peaks, wherein the results are shown in Table 3.

TABLE 3 Lasiosphaera Seu Calvatia (Lasiosphaera Seu Calvatia) medicinal material feature map extraction time investigation result

The results show that when the extraction time is 15 minutes, 30 minutes, 45 minutes and 60 minutes, the number of extraction peaks has no obvious difference, the extraction efficiency of each peak is not obviously increased after the heating and refluxing are carried out for 45 minutes, and the extraction time of 45 minutes can be used as an optimal 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 10ml, 15ml, 25ml and 50ml on puffball medicinal materials is examined. And analyzing the data of the number of extracted peaks and the peak area of each characteristic peak.

The specific experimental method is as follows:

taking about 1.5g of Lasiosphaera Seu Calvatia medicinal powder, paralleling 4 groups, precisely weighing, placing into a conical flask with a plug, precisely adding 10ml, 15ml, 25ml and 50ml of methanol respectively, performing ultrasonic treatment (power 600W and frequency 40 kHz) for 45 minutes, cooling, shaking up, filtering, and taking the subsequent filtrate. Precisely absorbing 3 μ l of each test solution, injecting into a liquid chromatograph, measuring according to the chromatographic conditions, recording the chromatogram, and calculating the peak area/sample weighing volume of 6 chromatographic peaks, the results are shown in Table 4.

TABLE 4 Lasiosphaera Seu Calvatia (Lasiosphaera Seu Calvatia) medicinal material feature map extraction mode investigation result

The results show that: the extraction efficiency is the highest when the volume of the extraction solvent is 15ml, and this is a preferred condition parameter.

Particle test solution:

(1) Selection of extraction solvent

The puffball formula particles are formed by water extraction and concentration of puffball, so that the water solubility is high, and the index components ergosterone and ergosterol in puffball medicinal materials are fat-soluble components. In summary, the extraction effect of four different solvents, namely 100% methanol, 80% methanol in water, ethanol and dichloromethane, on puffball formula particles was examined, wherein the percentage is methanol volume percentage.

The specific experimental method is as follows:

taking a proper amount of Lasiosphaera Seu Calvatia formula granules, grinding, taking about 5g, paralleling 5 groups, precisely weighing, placing into a conical flask with a plug, precisely adding 50ml of methanol, 80% methanol aqueous solution, ethanol and dichloromethane respectively, sealing the plug, weighing, respectively carrying out ultrasonic treatment (power 600W and frequency 40 kHz) for 30 minutes, cooling, weighing again, complementing the lost weight with corresponding solvents, shaking up, centrifuging, precisely weighing 25ml of supernate, evaporating to dryness, dissolving residues with methanol, transferring into a 5ml measuring flask, adding methanol to scale, shaking up, filtering, and taking the subsequent filtrate to obtain the Lasiosphaera Seu Calvatia powder. Precisely absorbing 3 μ l of each test solution, injecting into a liquid chromatograph, measuring according to the chromatographic conditions, recording the chromatogram, and calculating the peak area/sample weighing of 6 chromatographic peaks, the results are shown in Table 5.

TABLE 5 Lasiosphaera Seu Calvatia formula granule feature map extraction solvent investigation results

The chromatogram comparison results are shown in FIG. 2.

And (4) analyzing peak area data, and when dichloromethane and 80% methanol aqueous solution are adopted for extraction, the peak area of the characteristic peak is lower. When 100% methanol and ethanol are adopted for extraction, the peak area of each characteristic peak is higher.

When the liquid chromatogram is analyzed, the solvent effect is smaller when 100% methanol is adopted for extraction compared with ethanol.

Finally, when 100% methanol is used for extraction, the peak area of each characteristic peak is high, the peak shape of a chromatographic peak is good, and the solvent effect is small, so that the characteristic peaks can be used as optimal condition parameters.

(2) Examination of extraction methods

100% methanol is selected as an extraction solvent, and the extraction effect of puffball formula particles through ultrasonic treatment (frequency 600W and power 40 kHz), heating reflux and shaking extraction for 30 minutes is examined. And analyzing the data of the number of extracted peaks and the peak area of each characteristic peak.

The specific experimental method is as follows:

taking a proper amount of Lasiosphaera Seu Calvatia formula granules, grinding, taking about 5g, paralleling 3 groups, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of methanol, sealing the plug, weighing, respectively performing ultrasonic treatment (power 600W, frequency 40 kHz), heating reflux, shaking for extraction for 30 minutes, cooling, weighing again, supplementing the weight loss by methanol, shaking uniformly, centrifuging, precisely weighing 25ml of supernatant, evaporating to dryness, dissolving residues by methanol, transferring to a 5ml measuring flask, adding methanol to scale, shaking uniformly, filtering, and taking a subsequent filtrate to obtain the Lasiosphaera Seu Calvatia. Precisely absorbing 3 mu l of each test solution, injecting into a liquid chromatograph, measuring according to the chromatographic conditions, recording a chromatogram, and calculating peak areas/sample weighing of 6 chromatographic peaks, wherein the results are shown in Table 6.

TABLE 6 inspection results of characteristic spectrum extraction method of puffball (Lasiosphaera Seu Calvatia) formula particles

The results show that: the three extraction modes have no obvious difference in the number of extraction peaks, and the heating reflux extraction effect 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 the puffball formula particles is examined after heating and refluxing for 15 minutes, 30 minutes, 45 minutes and 60 minutes. And analyzing the data of the number of extracted peaks and the peak area of each characteristic peak.

The specific experimental method is as follows:

taking a proper amount of Lasiosphaera Seu Calvatia formula particles, grinding, taking about 5g, paralleling 4 groups, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of methanol, sealing the plug, weighing, respectively heating and refluxing for 15 min, 30 min, 45 min and 60min, cooling, weighing again, complementing the lost weight with methanol, shaking up, centrifuging, precisely weighing 25ml of supernate, drying by distillation, dissolving residues with methanol, transferring to a 5ml measuring flask, adding methanol to the scale, shaking up, filtering, and taking the subsequent filtrate. Precisely absorbing 3 mu l of each test solution, injecting into a liquid chromatograph, measuring according to the chromatographic conditions, recording a chromatogram, and calculating peak areas/sample weighing of 6 chromatographic peaks, wherein the results are shown in a table 7.

TABLE 7 puffball formula particle feature map extraction time study results

The results show that when the extraction time is 15 minutes, 30 minutes, 45 minutes and 60 minutes, the number of extraction peaks has no obvious difference, the extraction efficiency of each peak is not obviously increased after heating and refluxing for 30 minutes, and in order to ensure complete extraction, 45 minutes of extraction can be used as an optimal condition parameter.

(4) Investigation of extraction volume

100% methanol was selected as the extraction solvent, and the extraction effect of puffball formula particles of 25ml, 50ml and 100ml extraction volume was examined. And analyzing the data of the number of extracted peaks and the peak area of each characteristic peak.

The specific experimental method is as follows:

taking a proper amount of Lasiosphaera Seu Calvatia formula granules, grinding, taking about 5g, paralleling 3 groups, precisely weighing, placing into a conical flask with a plug, precisely adding 25ml, 50ml and 100ml of methanol, sealing the plug, weighing the weight, respectively heating and refluxing for 45 minutes, cooling, weighing the weight again, supplementing the weight loss by using methanol, shaking up, centrifuging, precisely weighing 15ml, 25ml and 50ml of supernate respectively, drying by distillation, dissolving residues by using methanol, transferring into a 5ml measuring flask, adding methanol to scale, shaking up, filtering, and taking the subsequent filtrate to obtain the Lasiosphaera Seu Calvatia. Precisely absorbing 3 μ l of each test solution, injecting into a liquid chromatograph, measuring according to the chromatographic conditions, recording the chromatogram, and calculating the peak area/sample weighing of 6 chromatographic peaks, wherein the results are shown in Table 8.

TABLE 8 Lasiosphaera Seu Calvatia formula granule feature map extraction mode investigation results

The results show that: after 50ml of extraction solvent is adopted, the extraction efficiency is not remarkably increased and can be taken as a preferable condition parameter.

3. Determination of chromatographic conditions

Preparation of a test solution:

taking a proper amount of puffball (big puffball) formula particles, grinding, taking about 5g, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of methanol, sealing the plug, weighing, heating and refluxing for 45 minutes, cooling, weighing again, supplementing the lost weight with methanol, shaking up, centrifuging, precisely measuring 25ml of supernatant, evaporating to dryness, dissolving the residue with methanol, transferring to a 5ml measuring flask, adding methanol to scale, shaking up, filtering, and taking the subsequent filtrate to obtain the medicine.

(1) Selection of detection wavelength

The chromatographic conditions were as follows:

a chromatographic column: waters ACQUITY

BEH 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 μ l

The results of detection at different wavelengths ranging from low to high wavelengths 3, 240nm, 282nm and 349nm, respectively, were examined and the resulting chromatogram was shown in FIG. 3.

As a result, the peak distribution was relatively uniform with a large amount of information at 282nm, and the peak distribution was found to be a preferable detection condition.

(2) Selection of different chromatography columns

The experiment inspects the influence of chromatographic columns of different manufacturers and fillers on the separation effect, and the information of the chromatographic columns is shown in the following table.

The results of the examination 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 preferred characteristic spectrum analysis chromatographic column.

(3) Selection of different gradients

The chromatographic conditions were as follows:

a chromatographic column: waters ACQUITY

BEH C18，1.7μm，2.1mm×100mm。

A mobile phase A: acetonitrile

And (3) 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 introduction amount: 3 μ l

The experiment investigated the influence 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 examination are shown in FIG. 5.

The result shows that when gradient 3 is adopted for elution, the chromatogram presents 6 chromatographic peaks with better peak shape and better separation degree, the tailing factor and the theoretical plate number of each characteristic peak are comprehensively considered, and when gradient 3 is adopted for elution, the separation effect is optimal and can be used as an optimal condition parameter.

(4) Selection of different flow rates

The chromatographic detection conditions were as follows:

and (3) chromatographic column: waters ACQUITY

BEH C18，1.7μm，2.1mm×100mm。

Mobile phase A: zxfoom

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 introduction amount: 3 μ l

The experiment examines the influence of three different flow phase flow rates of 0.25ml/min, 0.30ml/min and 0.35ml/min on the separation effect.

The results of the examination are shown in FIG. 6.

The result shows that the separation effect of the sample is better when the flow rate is 0.25 ml/min-0.35 ml/min per minute, and the retention time is moderate under the condition of considering the flow rate of 0.30ml/min, so that the sample can be used as the optimal condition parameter.

(5) Selection of different column temperatures

And (3) chromatographic column: waters ACQUITY

BEH C18，1.7μm，2.1mm×100mm。

A mobile phase A: acetonitrile

And (3) 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 introduction amount: 3 μ l

The experiment examines the influence of three different temperatures of 20 ℃, 25 ℃ and 30 ℃ on the separation effect.

The results of the examination are shown in FIG. 7.

The result shows that the separation effect of the sample is better when the column temperature is in the range of 20-30 ℃, and the retention time is moderate when the column temperature is 25 ℃, so that the separation effect can be taken as an optimal condition parameter.

Example 2

UPLC characteristic spectrum methodology verification of puffball formula particles

(1) Identification of chromatographic Peak

Preparation of a test solution: preparing puffball formula particle sample solution according to the optimized experimental conditions

Preparation of reference solutions: taking an appropriate amount of ergosterol reference substance, precisely weighing, and adding methanol to obtain a solution containing 20 μ g of ergosterol per 1 ml.

Preparation of negative control solution: negative control solution of formula granules lacking Lasiosphaera Seu Calvatia (Lasiosphaera Seu Calvatia) is prepared according to the above-defined experimental conditions.

The peak of the characteristic spectrum of the puffball formula particles is located, as shown in fig. 8.

(2) Integrity survey

Taking the product, preparing test solution according to the above-defined experimental conditions, measuring, keeping acetonitrile at the highest ratio under the same chromatographic conditions, prolonging elution time by one time, namely gradient 2 in table 9, recording chromatogram, and comparing with gradient 1 specified in standard text, wherein the result is shown in fig. 9.

TABLE 9 integrity test elution gradient chart for puffball formula granule feature profile

The results show that: no obvious chromatographic peak flows out after the original gradient elution is finished, which shows that the chromatographic condition basically meets the principle of maximum information content and has no interference on the analysis of subsequent samples.

(3) Precision survey

Taking the product, preparing a test solution according to the experiment conditions and determining, continuously injecting samples for 6 times, and inspecting the consistency of the characteristic peaks relative to the retention time, wherein the results are shown in a table 10.

TABLE 10 puffball formulation particle profile Instrument precision test results (relative retention time)

The results show that: the relative retention time RSD of each characteristic peak is less than 0.4 percent, and the precision is good.

(4) Stability survey

Taking the product, preparing a test solution according to the experimental conditions, respectively measuring at 0, 2, 4, 8, 10, 12, 14, 16, 18 and 24h, and examining the consistency of the characteristic peaks relative to the retention time, wherein the results are shown in Table 11.

TABLE 11 puffball formulations particles characteristic map stability test results (relative retention time)

The results show that: the RSD of the relative retention time of each characteristic peak of the test solution is less than 0.6 percent, and the test solution has good stability within 24 hours.

(5) Repeatability survey

Taking the product, preparing a test solution according to the experiment conditions and measuring, paralleling 6 parts, and inspecting the consistency of the characteristic peak relative to the retention time, wherein the results are shown in a table 12.

TABLE 12 puffball (puffball) recipe particle feature profile repeatability test results (relative retention time)

The results show that: the RSD of the relative retention time of each characteristic peak is less than 0.3 percent, and the repeatability is good.

(6) Intermediate precision investigation

Other analysts in the project group operate under different manufacturers' chromatographs, take samples in the same batch (batch number: 21090389), prepare test sample solutions according to the experiment conditions set up above, and determine the solutions in parallel by 3 parts. The consistency of the characteristic peaks with respect to retention time was examined. The results are shown in Table 13.

TABLE 13 Lasiosphaera Seu Calvatia recipe granule feature map intermediate precision test results (relative retention time)

The results show that: the characteristic spectrum of the puffball formula particles can be reproduced between two instruments of different brands, the relative retention time difference of each characteristic peak is small and is within +/-10% of a specified value, the RSD is less than 3%, and the separation degree and the peak type of each characteristic peak are good, and the intermediate precision is good.

Example 3

Lasiosphaera Seu Calvatia formula granule, detection of medicinal materials and establishment of reference map

S11, taking a proper amount of puffball (big puffball) formula particles, grinding, taking about 5g, precisely weighing, placing in a container with a plug, precisely adding 50ml of methanol, sealing the plug, weighing the weight, heating and refluxing for 45 minutes, cooling, weighing again, supplementing the weight loss by using methanol, shaking up, centrifuging, precisely weighing 25ml of supernate, drying by distillation, dissolving residues by using methanol, transferring to a 5ml measuring flask, adding methanol to scale, shaking up, filtering, and taking a subsequent filtrate to obtain the traditional Chinese medicine.

S12, taking a proper amount of puffball (puffball) medicinal material powder, taking about 1.5g, precisely weighing, placing in a container with a plug, precisely adding 15ml of methanol, sealing the plug, weighing, ultrasonically treating (with the power of 600W and the frequency of 40 kHz) for 45 minutes, taking out, cooling, weighing again, complementing the lost weight with methanol, shaking uniformly, filtering, and taking a subsequent filtrate.

S13, taking a proper amount of ergosterol reference substance, and adding methanol to prepare a solution containing 20 mu g or 95 mu g of ergosterol per 1ml, so as to obtain the ergosterol-containing composition.

S14, precisely absorbing 3 mu l of reference solution and puffball formula particle or medicinal material test solution respectively, and injecting into a liquid chromatograph under the chromatographic conditions: chromatography columns, waters ACQUITY

BEH C18 (1.7 μm,2.1 mm. Times.100 mm); acetonitrile is taken as a mobile phase A, 0.1 percent phosphoric acid aqueous solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; the flow rate was 0.3ml per minute; the column temperature was 25 ℃; the detection wavelength was 282nm.

(1) Preparing 3 batches of puffball formula particles and 15 batches of puffball medicinal material test solution;

(2) Respectively detecting the test solution according to the UPLC characteristic spectrum detection method of the puffball formula particles to obtain 3 batches of UPLC characteristic spectrums of the puffball formula particles and 15 batches of UPLC characteristic spectrums of puffball medicinal materials;

(3) Respectively introducing the obtained UPLC characteristic spectrums of 3 batches of puffball formula particles and 15 batches of puffball medicinal materials into a traditional Chinese medicine chromatography fingerprint similarity evaluation system (2012 edition) issued by the national pharmacopoeia committee to establish reference spectrums of the puffball formula particles and the puffball medicinal materials. See fig. 10, 11.

The experimental results are as follows:

TABLE 14 measurement results (relative Retention time) of puffball formula granules

TABLE 15 measurement results (relative Retention time) of puffball samples

The 3 batches of puffball formula particles and 15 batches of puffball medicinal materials all show the same 6 common characteristic peaks, and standard research is carried out by taking the peak 6 (ergosterol) as a reference peak. Finally, the characteristic spectrum standard of the puffball formula granules is defined as follows: the chromatogram of the test sample should show 6 characteristic peaks, wherein the peak 6 should be consistent with the retention time of the reference peak of the ergosterol control. The peak corresponding to the ergosterol control reference is the S peak, and the relative retention times of peak 1, peak 2, peak 3, peak 4, peak 5 and the S peak are calculated to be within ± 10% of the specified values: 0.29 (peak 1), 0.30 (peak 2), 0.32 (peak 3), 0.36 (peak 4), 0.89 (peak 5).

The established puffball formula particle and medicinal material contrast map can be used for more accurately controlling the quality of puffball formula particles, as shown in fig. 12 and 13.

Example 4

Puffball (big puffball) 3-batch production of quantity value transfer of sample characteristic spectrum of each process

This example discusses the magnitude transfer of characteristic patterns of puffball 3 batches of different process samples, i.e. herbs, standard decoctions and formula granules.

(1) The relative retention times of peak 1, peak 2, peak 3, peak 4, peak 5 and the S peak of the samples from each process of batch production of puffball (puffball) 3 were calculated using the peak corresponding to the ergosterol reference as the S peak, and are shown in Table 16.

(2) And (3) introducing chromatograms of puffball (puffball) medicinal materials produced in pilot production, corresponding standard decoction and traditional Chinese medicine formula granules into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system 2012 edition respectively, and comparing, wherein the superposed chromatograms of the three batches of medicinal materials, the standard decoction and the traditional Chinese medicine formula granules are shown in fig. 14, fig. 15 and fig. 16.

The experimental results are as follows:

TABLE 16 puffball (big puffball) 3 lots production of samples for each process run characteristic peak relative retention time

As can be seen from the above chart, the maps of the samples (medicinal materials and formula granules) of each process of the puffball (big puffball) 3 batches of mass production all have 6 characteristic peaks, the relative retention time of each characteristic peak is within the range of +/-10% of the specified value, and the characteristic peaks are consistent with the characteristic map of the standard decoction, which shows that the mass production medicinal materials and granules of the puffball (big puffball) are basically consistent with the material basis of the puffball (big puffball) standard decoction. The results show that the puffball (big puffball) has stable quantity value transmission in the production process and reasonable preparation process.

Example 5

Lasiosphaera Seu Calvatia identification of true and false medicinal materials

S11, taking a proper amount of counterfeit product (large-mouth static gray ball) powder, taking about 1.5g, precisely weighing, placing in a container with a plug, precisely adding 15ml of methanol, sealing the plug, weighing, carrying out ultrasonic treatment (power 600W and frequency 40 kHz) for 45 minutes, taking out, cooling, weighing again, complementing the lost weight with methanol, shaking up, filtering, and taking out a subsequent filtrate to obtain the finished product.

S12, taking a proper amount of ergosterol reference substances, and adding methanol to prepare a solution containing 95 micrograms of ergosterol per 1ml, so as to obtain the ergosterol-containing liquid.

S13, precisely absorbing 3 mu l of reference solution and fake product (large-opening static gray ball) test solution respectively, and injecting the solutions into a liquid chromatograph under the chromatographic conditions: chromatography columns, waters ACQUITY

BEH C18 (1.7 μm,2.1 mm. Times.100 mm); acetonitrile is taken as a mobile phase A, 0.1 percent phosphoric acid water solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; flow rate 0.3ml per minute; the column temperature was 25 ℃; the detection wavelength was 282nm.

(1) 1 batch of counterfeit product (large-mouth static gray ball) test solution is prepared;

(2) Detecting the test solution according to the method for detecting the UPLC characteristic spectrum of the puffball formula granules to obtain 1 UPLC characteristic spectrum of a fake product (big mouth static gray ball);

(3) The UPLC characteristic spectrum of 1 counterfeit product (Dakou static gray ball) and UPLC characteristic spectrum of Lasiosphaera Seu Calvatia (Lasiosphaera Seu Calvatia) are introduced into a similarity evaluation system of traditional Chinese medicine chromatogram fingerprint issued by the State Committee of pharmacopoeia for comparison. See fig. 17.

The results show that: from the number of the characteristic peaks, the characteristic peaks of the authentic product (Lasiosphaera Seu Calvatia) are relatively stable and present 6 common peaks; the number of characteristic peaks of the counterfeit product (Dakou static gray ball) is 2 (peak 1, peak 2, peak 3 and peak 4 are missing). Therefore, the difference of the characteristic peaks of the counterfeit product is larger than that of the genuine product, and when the number of the characteristic peaks is less than 6, the counterfeit product can be judged.

In summary, the existing data research shows that when the number of characteristic peaks is less than 6, genuine products and counterfeit products can be distinguished. Because of the small number of counterfeit research batches, this conclusion is only a preliminary conclusion to be accumulated and improved.

Example 6

Identification of different primordial (purple and big) medicinal materials of puffball

S11, taking a proper amount of puffball (purple puffball) medicinal material powder, taking about 1.5g, precisely weighing, placing in a container with a plug, precisely adding 15ml of methanol, sealing the plug, weighing, ultrasonically treating (power 600W, frequency 40 kHz) for 45 minutes, taking out, cooling, weighing again, complementing the lost weight with methanol, shaking uniformly, filtering, and taking a subsequent filtrate.

S12, taking a proper amount of ergosterol reference substances, adding methanol to obtain solution containing ergosterol 95 μ g per 1 ml.

S13, precisely absorbing 3 mu l of reference solution and puffball (purple puffball) medicinal material test solution respectively, and injecting into a liquid chromatograph under the chromatographic conditions that: chromatography columns, waters ACQUITY

BEH C18 (1.7 μm,2.1 mm. Times.100 mm); acetonitrile is taken as a mobile phase A, 0.1 percent phosphoric acid water solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; the flow rate was 0.3ml per minute; the column temperature was 25 ℃; the detection wavelength was 282nm.

(1) Preparing 2 batches of puffball (purple puffball) medicinal material test solution;

(2) Detecting the test solution according to the method for detecting the UPLC characteristic spectrum of the puffball (Lasiosphaera Seu Calvatia) formula granules to obtain 2 UPLC characteristic spectrums of the puffball (purple puffball) medicinal material;

(3) The contrast map of the puffball (purple puffball) medicinal material is established by leading the obtained UPLC characteristic map of 2 batches of puffball (purple puffball) medicinal material into a traditional Chinese medicine chromatography fingerprint similarity evaluation system issued by the State pharmacopoeia Committee. See fig. 18.

(4) And comparing UPLC reference spectra of the puffball (purple puffball) medicinal material and the puffball (big puffball) medicinal material, and calculating relative peak areas of the other 5 characteristic peaks by taking the peak 5 as a reference to distinguish the puffball (purple puffball) medicinal material and the puffball (big puffball) medicinal material. See table 17, fig. 19.

The experimental results are as follows:

TABLE 17 measurement results (relative peak area) of puffball batches and 15 puffball batches

The results show that: the purple Lasiosphaera Seu Calvatia medicinal material and Lasiosphaera Seu Calvatia medicinal material have slight difference in characteristic spectrum. Both of them present 6 common peaks, and the peak area of the purple Lasiosphaera Seu Calvatia medicinal material peak 6 relative to the peak 5 is much higher than that of the Lasiosphaera Seu Calvatia medicinal material from the relative peak area of the characteristic peak.

In summary, when the peak areas of the characteristic peak 6 relative to the peak 5 are all larger than 30, it is judged that the purple puffball is obtained.

Claims (10)

1. A construction method of a puffball medicinal material and a formula particle characteristic spectrum thereof is characterized by comprising the following steps:

(1) Taking one of puffball medicinal materials, standard decoctions or formula granules thereof, processing, and adding solvent for extraction to obtain a test solution;

(2) Adding solvent into ergosterol to obtain reference solution;

(3) Respectively injecting the test solution and the reference solution into a liquid chromatogram to obtain a characteristic spectrum, wherein the liquid chromatogram conditions are as follows:

the stationary phase is a chromatographic column which takes octadecylsilane chemically bonded silica as a filler; the mobile phase A is acetonitrile, and the mobile phase B is phosphoric acid water solution with the phosphoric acid mass percent of 0.01-0.20%;

(4) Judging the quality of the raw materials and distinguishing the positive and the false products: if the characteristic spectrum obtains the following 6 characteristic peaks, the quality of the raw materials is qualified: peak 6 is the ergosterol peak, with peak 6 as the reference peak, the relative retention times of 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 spectrum does not completely contain the 6 characteristic peaks, puffball is a fake product; and if the peak area of the characteristic peak 6 relative to the peak 5 in the characteristic map is more than 30, judging the purple puffball.

2. The method for constructing the feature spectrum of puffball medicinal materials and the formula granules thereof according to claim 1, wherein when puffball is a medicinal material in the step (1), the specific steps are as follows: sieving puffball powder, placing in a conical flask, adding extraction solvent, performing ultrasonic treatment or heating or shaking treatment for 15-60min, cooling, adding corresponding solvent to the weight loss, shaking, filtering, and collecting filtrate to obtain test solution;

when puffball is a standard decoction, the specific steps are as follows: taking puffball standard decoction powder, placing into a conical flask, adding extraction solvent, wherein the mass of a test sample extracted by every 1ml of solvent is 0.01-0.06g, carrying out ultrasonic treatment or heating or oscillation treatment for 15-60min, cooling, supplementing the loss weight with corresponding solvent, shaking up, centrifuging, taking supernatant, evaporating to dryness, dissolving residues with methanol, transferring into a measuring flask, adding methanol to scale, shaking up, filtering, and obtaining a subsequent filtrate, namely a test sample solution;

when puffball is a formula particle, the specific steps are as follows: taking puffball formula particle powder, placing the puffball formula particle powder in a conical flask, adding an extraction solvent, wherein the mass of a test sample extracted by each 1ml of the solvent is 0.05-0.2g, carrying out ultrasonic treatment or heating or shaking treatment for 15-60min, cooling, complementing the loss weight by using a corresponding solvent, shaking up, centrifuging, taking supernatant, drying by distillation, dissolving residues by using methanol, transferring the residues into a measuring flask, adding the methanol to the scale, shaking up, filtering, and obtaining a subsequent filtrate, namely a test sample solution.

3. The method for constructing the puffball medicinal material and the formula particle characteristic spectrum according to claim 1, wherein in the step (1), when puffball is the medicinal material, the extraction solvent is a methanol aqueous solution containing 80 to 100% by volume of methanol or an ethanol aqueous solution containing 80 to 100% by volume of ethanol; when puffball is standard decoction or formula granules, the extraction solvent is dichloromethane or aqueous methanol solution containing 80 to 100 percent by volume of methanol or aqueous ethanol solution containing 80 to 100 percent by volume of ethanol.

4. The method for constructing puffball medicinal materials and formula granules feature spectrum according to claim 1, wherein the detecting instrument of the feature spectrum in the step (3) is: an ultra-high performance liquid chromatograph.

5. The method for constructing puffball herb and its prescription granule feature map as claimed in claim 1, wherein the specification of the chromatographic column in step (3) is: the column length is 100 to 150mm, and the particle size is 1.7 to 1.9 mu m.

6. The method for constructing the characteristic spectrum of puffball and the formulation thereof as claimed in claim 1, wherein the conditions of the liquid chromatography in step (3) further comprise: the flow rate is 0.25 to 0.35ml/min; the column temperature is 20 to 30 ℃; the detection wavelength is 265 to 295nm.

7. The method for constructing the characteristic spectrum of puffball and the formula granules thereof according to claim 1, wherein the concentrations of the ergosterol as the reference substance in the step (2) are as follows: 20 to 95 mu g/ml; the dissolution solvent of the reference substance is methanol.

8. The method for constructing the feature spectrum of puffball and the formula granules thereof according to claim 2, wherein the method comprises the following steps: the power of ultrasonic extraction is 250 to 600W, and the frequency is 35 to 45kHz.

9. The use of the method for constructing the characteristic spectrum of puffball and its formula granules according to any one of claims 1 to 8 in the quality evaluation and detection of purple puffball and puffball herbs, standard decoctions or formula granules.

10. The use of the puffball herbs and their formula granules as defined in any one of claims 1 to 8 in the construction of characteristic maps for distinguishing true purple puffball, puffball and counterfeit big mouth static gray balls.

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