Method for identifying fritillaria producing area
By using ICP-MS technology and multivariate statistical analysis methods, a model for identifying the origin of Zhejiang Fritillaria was established, which solved the problem of distinguishing between Zhejiang Fritillaria and Hubei Fritillaria, and achieved efficient and accurate origin traceability and authenticity identification.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHOUSHAN INST FOR FOOD & DRUG CONTROL
- Filing Date
- 2026-02-06
- Publication Date
- 2026-06-12
Smart Images
Figure CN122192890A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of fritillaria origin identification technology, specifically relating to a method for identifying the origin of fritillaria. Background Technology
[0002] Fritillaria thunbergii, the dried bulb of the plant *Fritillaria thunbergii*, is a traditional Chinese medicine from Zhejiang Province, ranking first among the "Eight Treasures of Zhejiang." Its main production areas are Pan'an, Yinzhou, Jinyun, and Kaihua in Zhejiang Province, with cultivation now also occurring in Jiangsu and Anhui provinces. Its cultivation history dates back over 300 years. Fritillaria thunbergii possesses the effects of clearing heat, resolving phlegm, relieving cough, detoxifying, dispersing nodules, and reducing swelling. Clinically, it is mainly used to treat wind-heat cough, phlegm-fire cough, lung abscess, mastitis, scrofula, and carbuncles. Due to its wide clinical application, high market acceptance, and huge demand, other Fritillaria species are being used to confuse and impersonate Fritillaria thunbergii in the market, with Hubei Fritillaria being the most common. Hubei Fritillaria and Fritillaria thunbergii are similar in appearance, making traditional morphological, microscopic, and physicochemical identification difficult to effectively distinguish, and its market price is lower than that of Fritillaria thunbergii. Therefore, there is an urgent need to establish a rapid and sensitive method for tracing the origin of Fritillaria thunbergii to ensure the healthy development of the market for this medicinal material. Currently, research on tracing the origin and identifying the authenticity of Fritillaria thunbergii mainly focuses on the chromatographic and mass spectrometric analysis of organic components. However, the organic components in medicinal materials are prone to changes or loss during later processing and storage, which may affect the accuracy of traceability identification.
[0003] Plant growth, development, and the formation of secondary metabolites are significantly influenced by geographical factors. Inorganic elements, as essential components that medicinal plants cannot synthesize themselves, are mainly obtained from the environment, such as soil and water sources. The composition and content of inorganic elements vary characteristically across different regions, resulting in unique inorganic element "fingerprints" within plants growing in different locations. These fingerprints effectively reflect the regional characteristics of their growth. Given the high stability, strong exclusivity, and susceptibility to post-processing and storage effects of inorganic elements in traditional Chinese medicine (TCM), fingerprint analysis based on inorganic elements has shown significant advantages in the research of tracing the origin of TCM. With the widespread adoption of inductively coupled plasma mass spectrometry (ICP-MS), the application of multi-element determination combined with multivariate statistical analysis methods in tracing the origin and identifying authenticity of TCM is becoming increasingly widespread. However, research on the identification of origin and quality evaluation of Fritillaria cirrhosa based on inorganic element analysis has not yet been reported. Summary of the Invention
[0004] The purpose of this invention is to provide an origin discrimination model based on elemental fingerprint features, which provides a more accurate, faster, more sensitive and efficient identification method for tracing the origin and distinguishing the authenticity of Fritillaria thunbergii.
[0005] The technical solution adopted by the present invention to achieve the above objectives is as follows: A method for identifying the origin of Fritillaria cirrhosa includes: detecting the content of organic or inorganic components in Fritillaria cirrhosa, and identifying the origin of Fritillaria cirrhosa based on the detection results; the detection results include the score of Fritillaria cirrhosa from Zhejiang or Fritillaria cirrhosa from Hubei, the organic components include Fritillarin A or Fritillarin B; the inorganic components include at least one of Mg, Al, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Rb, Sr, Mo, Ag, Cd, Sn, Sb, Ba, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Hg, Tl, Pb and U.
[0006] Preferably, the inorganic component includes at least one of Mg, Zn, Rb, Mo, Ba, Eu, Tb, Nd, Pr, Ce, Gd, Al, La, Cd and Cu.
[0007] Preferably, the fraction of Fritillaria thunbergii is 6.299[Mg] + 0.946[Zn] + 1.189[Rb] − 0.345[Mo] - 1.772[Ba] + 1.103[Eu / Tb] − 0.313[Nd / Pr] − 1.136[Pr / Ce] + 0.677[Gd / Al] + 0.526[La / Gd] + 0.334[Cd / Cu] − 2.917.
[0008] Preferably, the fraction of Hubei fritillary bulb is: -15.598[Mg] - 2.193[Zn] - 2.673[Rb] + 0.752[Mo] + 4.221[Ba] - 2.923[Eu / Tb] + 0.983[Nd / Pr] + 3.011[Pr / Ce] - 1.634[Gd / Al] - 0.855[La / Gd] - 1.334[Cd / Cu] - 13.389.
[0009] Preferably, the results of the fritillary bulb origin identification are as follows: Fritillaria bulbs originate from Zhejiang Province, and Zhejiang Fritillaria bulbs have a higher score than Hubei Fritillaria bulbs; or, Fritillaria bulbs originate from Hubei Province, and Hubei Fritillaria bulbs have a higher score than Zhejiang Fritillaria bulbs.
[0010] Preferably, the results of the fritillary bulb origin identification are as follows: Fritillaria bulbs originate from Zhejiang Province; fritillary glycoside A is greater than fritillary glycoside B; or, The fritillaria bulbs are from Hubei province, and fritillarin B is greater than fritillarin A.
[0011] More preferably, fritin A or fritin B is obtained by separating fritin extract from fritillaria, which is obtained by extracting fritillaria with an extraction reagent.
[0012] More preferably, the extraction reagent includes chloroform and / or methanol.
[0013] More preferably, the extraction reagent includes 2-amino-4-hydroxybenzoic acid and / or 2-chloro-6-methoxyphenol. This invention also provides a method for extracting fritillaria extract from fritillaria. The fritillaria extract contains fritin A and / or fritin B. The content of fritin A and fritin B can assist in the identification of the origin of fritillaria. In the preparation of the fritillaria extract, concentrated ammonia is added first, followed by the extraction reagent. In addition to using chloroform and / or methanol as the extraction reagent, 2-amino-4-hydroxybenzoic acid and / or 2-chloro-6-methoxyphenol can also be added. Under the action of 2-amino-4-hydroxybenzoic acid and / or 2-chloro-6-methoxyphenol, the dissolution of components in fritillaria can be improved, and there is an excellent extraction effect on fritin A and fritin B. 2-amino-4-hydroxybenzoic acid and / or 2-chloro-6-methoxyphenol need to be used in reasonable amounts to be effective; too low or too low a dosage will not be effective.
[0014] Preferably, the identification also includes PCA and / or OPLS-DA statistical analysis.
[0015] This invention aims to use ICP-MS technology to determine the content of 38 elements, including lead (Pb), cadmium (Cd), arsenic (As), selenium (Se), chromium (Cr), silver (Ag), thallium (Tl), molybdenum (Mo), barium (Ba), antimony (Sb), nickel (Ni), cobalt (Co), vanadium (V), tin (Sn), uranium (U), copper (Cu), zinc (Zn), rubidium (Rb), strontium (Sr), iron (Fe), manganese (Mn), aluminum (Al), magnesium (Mg), lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium (Nd), samarium (Sm), europium (Eu), gadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), thulium (Tm), ytterbium (Yb), lutetium (Lu), and mercury (Hg), in samples of Fritillaria thunbergii from Zhejiang and Hubei provinces. Combining principal component analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA), and Fisher linear discriminant analysis (LDA), a comprehensive discriminant analysis was conducted on two types of Fritillaria thunbergii. The aim was to construct an origin discrimination model based on elemental fingerprint characteristics, providing a more accurate, rapid, sensitive, and efficient identification method for tracing the origin and verifying the authenticity of Fritillaria thunbergii from Zhejiang.
[0016] This invention discloses a method for identifying the origin of fritillaria bulbs, comprising: S1, the content of Mg, Rb, Cd, Mo, La, Ce, Nd, Pr, Gd, Ba, Zn, Al, Tb, Eu, and Cu in the test fritillaria was determined. The elemental analysis was performed using ICP-MS, and the test fritillaria was prepared into ICP-MS samples by microwave digestion. S2, substitute the element content detected in S1 into the following two function models to calculate the score: The fraction of Fritillaria thunbergii is calculated as follows: 6.299[Mg] + 0.946[Zn] + 1.189[Rb] − 0.345[Mo] - 1.772[Ba] + 1.103[Eu / Tb] − 0.313[Nd / Pr] − 1.136[Pr / Ce] + 0.677[Gd / Al] + 0.526[La / Gd] + 0.334[Cd / Cu] − 2.917. The fraction of Fritillaria cirrhosa from Hubei Province is: -15.598[Mg] - 2.193[Zn] - 2.673[Rb] + 0.752[Mo] + 4.221[Ba] - 2.923[Eu / Tb] + 0.983[Nd / Pr] + 3.011[Pr / Ce] - 1.634[Gd / Al] - 0.855[La / Gd] - 1.334[Cd / Cu] - 13.389; S3, the identification results are as follows: The Fritillaria thunbergii to be tested was Zhejiang Fritillaria thunbergii, and its score was higher than that of Hubei Fritillaria thunbergii. The Fritillaria cirrhosa to be tested was from Hubei Province, and its score was higher than that of Fritillaria thunbergii from Zhejiang Province.
[0017] This invention discloses a method for identifying the origin of fritillaria bulbs, comprising: S1, the content of Mg, Al, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Rb, Sr, Mo, Ag, Cd, Sn, Sb, Ba, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Hg, Tl, Pb, and U in the tested fritillaria bulbs was determined. The elemental analysis was performed using ICP-MS, and the tested fritillaria bulbs were prepared for ICP-MS analysis by microwave digestion. S2, the elemental content was standardized and imported into SIMCA-p 14.1 software for OPLS-DA analysis.
[0018] A method for preparing fritillaria extract involves pulverizing fritillaria to obtain fritillaria powder, then treating it with concentrated ammonia for 0.5-3 hours, then adding an extraction solution, ultrasonicating for 20-120 minutes, filtering, taking the filtrate, evaporating it to dryness in a water bath, then adding chloroform and water for separation, taking the organic phase, filtering, and evaporating it to dryness in an evaporating dish to obtain fritillaria extract.
[0019] Preferably, the amount of fritillaria powder used is 10-30 wt% of concentrated ammonia.
[0020] More preferably, sodium lauryl tin can be added to the concentrated ammonia solution, wherein the content of sodium lauryl tin in the concentrated ammonia solution is 0.4 wt%. In this invention, sodium lauryl tin can also be added to the concentrated ammonia solution, and then further in an extraction reagent containing 2-amino-4-hydroxybenzoic acid and / or 2-chloro-6-methoxyphenol, which can further improve the dissolution of the components in fritillaria and also have a better extraction effect on fritillarin A and fritillarin B.
[0021] Preferably, the extraction solution is a mixture of chloroform and methanol, with a volume ratio of chloroform to methanol of 1-5:1-5.
[0022] Preferably, the amount of fritillaria powder used is 1-5 wt% of the extraction solution.
[0023] Preferably, the volume ratio of chloroform to water in the separation is 1-3:1-3.
[0024] Preferably, the ratio of the volume of water used in the separation to the mass of fritillaria powder used is 1-5 mL: 1 g.
[0025] Preferably, 2-amino-4-hydroxybenzoic acid may also be added to the extraction solution, and the content of 2-amino-4-hydroxybenzoic acid is 0.005-0.03 wt%.
[0026] Preferably, 2-chloro-6-methoxyphenol may also be added to the extraction solution, and the content of 2-chloro-6-methoxyphenol is 0.001-0.02 wt%.
[0027] This invention utilizes ICP-MS technology to perform multi-element analysis on 32 batches of Zhejiang and Hubei fritillaria samples, and combines multivariate statistical methods to screen characteristic elements, successfully establishing a model for identifying the origin of Zhejiang fritillaria. This invention provides a new method for identifying the origin and authenticity of Zhejiang fritillaria based on an inorganic element fingerprint-based origin tracing model. Combined with organic component analysis, a multi-dimensional quality identification and evaluation system for Zhejiang fritillaria is constructed. Therefore, it has the following beneficial effects: it can accurately, quickly, sensitively, and efficiently identify the origin of fritillaria. Thus, this invention is an origin discrimination model based on element fingerprint characteristics, providing a more accurate, rapid, sensitive, and efficient identification method for tracing the origin and identifying the authenticity of Zhejiang fritillaria. Attached Figure Description
[0028] Figure 1 PCA score chart for fritillaria from different origins.
[0029] Figure 2 OPLS-DA score chart for fritillaria from different origins.
[0030] Figure 3 This is a permutation test diagram for the OPLS-DA model of the sample.
[0031] Figure 4 This is a diagram of the OPLS-DA source tracing model.
[0032] Figure 5 This is a graph showing the total alkaloid content.
[0033] Figure 6 The recovery rate of friablel ethyl spiked solution is shown in the graph. Detailed Implementation
[0034] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0035] The concepts involved in this application will first be described with reference to the accompanying drawings. It should be noted that the following descriptions of various concepts are only for the purpose of making the content of this application easier to understand and do not constitute a limitation on the scope of protection of this application; furthermore, the embodiments and features in the embodiments of this application can be combined with each other unless otherwise specified. This application will now be described in detail with reference to the accompanying drawings and embodiments.
[0036] Research basis: 1. Instruments, reagents, and samples 1.1 Instruments: Agilent 7850 inductively coupled plasma mass spectrometer (Agilent Technologies, USA); MARS 6 microwave digester (CEM, USA); Arium PRO UV ultrapure water system (Satorius, Germany); XSE205DU electronic balance (METTLER TOLEDO, Switzerland).
[0037] 1.2 Reagents: Standard solutions of 23 elements: Pb, Cd, As, Se, Cr, Ag, Tl, Mo, Ba, Sb, Ni, Co, V, Sn, U, Cu, Zn, Rb, Sr (all with a mass concentration of 10 µg·mL) -1 Fe, Mn, and Al (each with a mass concentration of 100 µg·mL) -1 ), Mg (mass concentration of 1000 µg·mL) -1 ); 14 rare earth standard solutions (each with a mass concentration of 100 µg·mL) -1The standard solutions for La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu were all purchased from Steel Research Institute Nake Testing Technology Co., Ltd. The Hg single-element standard solution (mass concentration 1000 µg·mL) was also included. -1 A mixed standard solution of germanium (Ge), indium (In), and bismuth (Bi) internal standards (each with a mass concentration of 100 µg·mL). -1 All were purchased from Beijing Manhag Biotechnology Co., Ltd.; gold (Au) single-element standard solution (mass concentration of 100 µg·mL) -1 The reagents were purchased from Inorganic, USA; nitric acid and hydrogen peroxide were of superior purity, and the water was ultrapure water.
[0038] 1.3 A total of 32 batches of medicinal materials (processed slices) of Zhejiang Fritillaria (numbers ZJ1-ZJ24) and Hubei Fritillaria (numbers HB1-HB8) were commercially available. Sample information is shown in Table 1. Among them, samples ZJ7 and ZJ12 are suspected to be Hubei Fritillaria masquerading as Zhejiang Fritillaria.
[0039] Table 1 Information on Fritillaria samples
[0040] Note: "*" indicates a suspicious sample. 2. Elemental determination methods and data processing 2.1 ICP-MS operating conditions: RF power 1550 W, plasma gas flow rate 15.0 L·min -1 The dilution gas flow rate is 0.15 L·min. -1 Atomizer flow rate: 0.95 L·min -1 Helium flow rate 5 mL·min -1 The temperature of the atomization chamber is 2.0℃.
[0041] 2.2 Preparation of Standard Solutions: Accurately measure each standard solution and dilute it with 10% nitric acid solution to prepare Pb, Cd, As, Se, Cr, Ag, Tl, Mo, Ba, Sb, Ni, Sr, Co, V, Sn, Rb, U, Cu, Zn, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu concentrations of 1, 2, 5, 10, 20, 50, and 100 ng·mL, respectively. -1 The mass concentrations of Fe, Mn, and Al were 10, 20, 50, 100, 200, 500, and 1000 ng·mL, respectively. -1 The Mg mass concentrations were 100, 200, 500, 1000, 2000, 5000, and 10000 ng·mL, respectively. -1A series of mixed standard working solutions were prepared. An appropriate amount of Hg single-element standard solution was accurately measured and diluted with 10% nitric acid solution to prepare mass concentrations of 0.2, 0.5, 1, 2, and 5 ng·mL. -1 Prepare fresh Hg elemental standard working solutions (freshly prepared before use); accurately measure an appropriate amount of Au single-element standard solution and dilute with water to prepare a mass concentration of 1 µg·mL. -1 The standard working solution. Separately, accurately measure appropriate amounts of 14 rare earth standard solutions and dilute them with 10% nitric acid solution to prepare a solution with a mass concentration of 2 μg·mL for each element. -1 Mixed standard stock solution I; accurately measure an appropriate amount of Hg single-element standard solution and dilute with 10% nitric acid solution to prepare a mass concentration of 200 ng·mL. -1 Standard stock solution II was used for recovery rate determination.
[0042] 2.3 Preparation of internal standard solution: Accurately measure the mixed standard solution of Ge, In, and Bi internal standards, and dilute with water to prepare a solution with a mass concentration of 0.5 µg·mL. -1 A mixed solution.
[0043] 2.4 Preparation of Test Sample Solutions: Accurately weigh 0.5 g of each of the Fritillaria cirrhosa samples listed in Table 1, place them in a microwave digestion vessel, add 5 mL of nitric acid and 0.5 mL of hydrogen peroxide, mix well, pre-digest at 80 ℃ for 30 min, cool, seal, and place in a microwave digester. Digest according to the digestion program in Table 2, with a power of 1.6 kW. After digestion, cool. Transfer the digestion solution to a 50 mL volumetric flask with water, add 200 µL of Au element standard working solution, dilute with water to the mark, and shake well to obtain the reagent blank solution (except for the absence of Au element, the other steps are the same as preparing the reagent blank solution).
[0044] Table 2 Microwave Digestion Procedure
[0045] 2.5 Determination Method The isotopes selected during the measurement were 24 Mg 27 Al、 51 V. 52 Cr 55 Mn, 56 Fe、 59 Co、 60 Ni、 63 Cu、 66 Zn, 75 As、 78 Se、 85 Rb、 88 Sr、 95 Mo、 107 Ag、 111 Cd,118 Sn、 123 Sb、 135 Ba、 139 La、 140 Ce、 141 Pr, 146 Nd, 147 Sm、 153 Eu、 157 Gd, 159 Tb, 163 Dy、 165 Ho、 166 Er、 169 Tm、 172 Yb、 175 Lu、 202 Hg, 205 Tl、 208 Pb, 238 U. Online internal standard calibration, combined with kinetic energy discrimination (KED) mode, read the instrument response value three times, calculate the content of each element in the sample according to the standard curve, and perform blank test under the same measurement conditions and subtract blank interference.
[0046] 2.6 Data Processing: SIMCA 14.1 software was used to perform PCA and OPLS-DA statistical analyses on the measured data, establishing geographical classification models for Zhejiang Fritillaria and Hubei Fritillaria, and screening characteristic elements that could distinguish the two species. SPSS 26.0 was used for Fisher LDA statistical analysis to establish discriminant function models for the two species, and cross-validation was performed.
[0047] 3. Results and Analysis 3.1 Methodological Examination 3.1.1 Linearity and Detection Limit Investigation: Take the series of standard solutions of each element under section 2.2, and inject them according to the working conditions under section 2.1 and the method under section 2.5 for determination. Plot the instrument response value of each element on the ordinate (Y) and the mass concentration on the abscissa (X, ng·mL). -1 A standard curve was plotted, and the regression equation and correlation coefficient (r) were obtained. For each element, r > 0.999, indicating a good linear relationship between the 38 elements and their response values within their respective concentration ranges. Eleven consecutive measurements of blank sample solutions were performed, and the detection limit was defined as the concentration of the analyte corresponding to three times the standard deviation (3SD) of the instrument response value. The results are shown in Table 3.
[0048] Table 3. Linear relationships and detection limits for 38 elements
[0049] Note: 1) The unit is μg·L -1 ; 2)The unit is ng·L -1 .
[0050] 3.1.2 Recovery and Stability Tests: Six portions of sample (ZJ21), each approximately 0.5 g, were accurately weighed. 0.25 mL each of the mixed standard solution of 23 elements (section 1.2), mixed standard stock solution I (section 2.2), and standard stock solution II (section 2.2) were accurately added to each. Test sample solutions were prepared according to the method in section 2.4. The samples were then injected and analyzed according to the working conditions in section 2.1 and the method in section 2.5. The instrument response values for each element were recorded, and the content was calculated. The average recovery rate of the 38 elements ranged from 81.73% to 114.85%, with an RSD of 0.57% to 6.86%, indicating good accuracy of the method. One portion of the sample solution was left at room temperature for 0, 2, 4, 6, 8, and 12 hours before being analyzed using the same method. The RSD values of the analysis results for each element were calculated, all ranging from 1.9% to 5.2%, indicating good stability of the test sample solution within 12 hours.
[0051] 3.1.3 Precision test: Take the spiked solution of ZJ21 and inject it continuously 6 times under the working conditions in section 2.1. The RSD of the instrument response value of each element was measured to be 0.32% to 4.02%, indicating that the instrument precision is good.
[0052] 3.2 Results and Analysis of Element Content in Samples: Test solutions were prepared from 32 batches of samples according to the method described in section 2.4. The samples were then analyzed under the working conditions described in section 2.1 and the method described in section 2.5, with three parallel replicates. Results showed that all elements except Sb, Tm, and Lu were detected in all samples. A total of 35 inorganic elements were included in the statistics. The average content and standard deviation of the inorganic elements are shown in Table 4. The highest content was found in the macroelement Mg, followed by the trace elements Fe, Al, Zn, Mn, Rb, Cu, and Ba. The average content of these elements in *Fritillaria thunbergii* from Zhejiang was generally higher than that in *Fritillaria hupehensis* from Hubei. The lowest content was mainly found in Ag, Hg, and rare earth elements. Among the rare earth elements, the contents of La, Ce, Pr, Eu, and Gd showed significant differences between the two types of *Fritillaria thunbergii*. Although rare earth elements have not long been considered essential elements for plant growth, they are easily transferred from the soil to the plant body and can serve as effective indicators for identifying plant origin. The difference test results showed that the content of inorganic elements in Fritillaria samples from different origins varied. Specifically, Mg, Zn, Rb, Mo, Cd, Ba, La, Ce, Pr, Nd, and Gd showed extremely significant differences (p < 0.01), while Al, Cu, Sm, Eu, Tb, and Pb showed significant differences (0.01 < p < 0.05). Furthermore, Pb, Cd, As, Hg, and Cu are key heavy metals and harmful elements controlled under the Chinese Pharmacopoeia. The results for both Fritillaria samples were below the consistency limits specified in the Pharmacopoeia for plant-based medicinal materials and processed products, indicating low levels of harmful element residues in both Fritillaria samples.
[0053] Table 4. Elemental content determination results of 32 batches of samples
[0054] Note: "*" indicates that the content of this element differed significantly between the two groups of Fritillaria. p <0.01), "**" indicates that there is a significant difference in the content of this element between the two groups of Fritillaria (0.01 < 0.01). p <0.05).
[0055] 3.3 PCA and OPLS-DA The contents of 35 elements measured in 30 batches of samples (excluding 2 suspicious batches) were standardized and then imported into SIMCA-p14.1 software for principal component analysis. The PCA score plot is shown below. Figure 1 The figure shows that the boundaries between fritillaria bulbs from different origins are not clear enough. Five batches of Zhejiang fritillaria bulb samples are clustered together with Hubei fritillaria bulbs and cannot be distinguished. However, overall, the two types of fritillaria bulbs still show a relatively obvious clustering trend. To achieve effective differentiation of fritillaria bulb origins, OPLS-DA modeling analysis was performed using this software to obtain OPLS-DA classification models for Zhejiang and Hubei fritillaria bulbs, see [link to model]. Figure 2 The data shows that the 32 batches of samples were distributed across different regions based on their origin, each clustering into a separate category, with clear differentiation among samples from different origins. The independent variables of the model... R 2 X The value is 0.685, and the dependent variable is... R 2 Y The cumulative predicted parameter is 0.858. Q 2 It is 0.700. R 2 and Q 2 All values > 0.5, indicating that the model is stable, reliable, and has good predictive ability. The established OPLS-DA model was subjected to 200 permutation tests to assess its interpretability. R 2 The model's predictive power is 0.386. Q 2 The value was -0.622, which is less than the original value, indicating that the model does not exhibit overfitting. (See...) Figure 3 Two batches of rejected suspicious samples, ZJ7 and ZJ12, were used as prediction groups and substituted into the model for verification. The results showed that ZJ7 and ZJ12 clustered in the Hubei Fritillaria section. Figure 4 The verification results are consistent with the expert evaluation results, indicating that the model is effective.
[0056] Building upon the effectiveness of the OPLS-DA model, the variable importance inprojection (VIP) value reflects the importance of a variable; a larger VIP value indicates a higher contribution of that element to the overall discriminant model.
[17] Based on the Kaiser principle, using VIP > 1 as the criterion, the distinguishing characteristic elements that can differentiate between the two types of fritillaria were selected. These elements are Mg, Rb, Cd, Mo, La, Ce, Nd, Pr, Gd, Ba, Zn, Al, Tb, Eu, and Cu, totaling 15 elements. Rare earth elements are the most numerous. The determination of these characteristic elements provides important support for the establishment of the discriminant function model and cross-validation in the next step.
[0057] 3.4 Fisher LDA and Cross-Validation LDA is an important multivariate statistical analysis method. Its core lies in establishing a discriminant function. This invention uses this analysis method to establish discriminant function models for two types of Fritillaria thunbergii, which can quickly and effectively classify and predict suspected samples. After standardizing the content of 15 characteristic elements obtained from OPLS-DA analysis, the data was imported into SPSS 26.0 software as independent variables. ZJ7 and ZJ12 were listed as ungrouped, while the remaining Fritillaria thunbergii from Zhejiang were group 1, and Fritillaria thunbergii from Hubei were group 2. Collinearity diagnosis and correlation analysis were performed, and it was found that the variance inflation factor (VIF) values of rare earth elements were mostly >10, indicating that there was a high correlation between some elements. The data of highly correlated elements (Eu, Tb, Nd, Pr, Ce, Gd, Al, La, Cd, Cu) were processed by ratio analysis and standardized, then used as new independent variables. The data of Mg, Zn, Rb, Mo, Ba, Eu / Tb, Nd / Pr, Pr / Ce, Gd / Al, La / Gd, and Cd / Cu were then imported back into the software. The resulting VIF values were all ≤5, the canonical correlation was 0.956, and the significance was 0.00 < 0.05, indicating that the recombined variables significantly improved the model's interpretability and predictive ability. Table 5 shows the classification results of Fisher's linear discriminant function for elements from samples of different origins. The results show that the original grouping accuracy was 100.0%, and the ungrouped ZJ7 and ZJ12 were identified as Hubei Fritillaria, with a cross-validation accuracy of 100.0%. This indicates that the discriminant function established using the optimized feature elements has good classification performance. The two discriminant function models are as follows: Fritillaria =6.299[Mg]+0.946[Zn]+1.189[Rb]−0.345[Mo]-1.772[Ba]+1.103[Eu / Tb]−0. 313[Nd / Pr]−1.136[Pr / Ce]+0.677[Gd / Al]+0.526[La / Gd]+0.334[Cd / Cu]−2.917; Hubei Fritillaria = −15.598[Mg]−2.193[Zn]−2.673[Rb]+0.752[Mo]+4.221[Ba]−2.923[Eu / Tb]+0.983[Nd / Pr]+3.011[Pr / Ce]−1.634[Gd / Al]−0.855[La / Gd]−1.334[Cd / Cu]−13.389.
[0058] Table 5. Classification results of the discriminant function a,c
[0059] a. 100.0% of the original grouped cases were correctly classified. b. Cross-validation was performed only for the cases in the analysis. In cross-validation, each case was classified by functions derived from all other cases. c. 100.0% of the grouped cases that underwent cross-validation were correctly classified.
[0060] Example 1: A method for identifying the origin of fritillaria bulbs S1, the content of Mg, Rb, Cd, Mo, La, Ce, Nd, Pr, Gd, Ba, Zn, Al, Tb, Eu, and Cu in the test fritillaria was determined. The elemental analysis was performed using ICP-MS, and the test fritillaria was prepared into ICP-MS samples by microwave digestion.
[0061] S2, substitute the element content detected in S1 into the following two function models to calculate the score: The fraction of Fritillaria thunbergii is calculated as follows: 6.299[Mg] + 0.946[Zn] + 1.189[Rb] − 0.345[Mo] - 1.772[Ba] + 1.103[Eu / Tb] − 0.313[Nd / Pr] − 1.136[Pr / Ce] + 0.677[Gd / Al] + 0.526[La / Gd] + 0.334[Cd / Cu] − 2.917. The fraction of Fritillaria cirrhosa from Hubei Province is: -15.598[Mg] - 2.193[Zn] - 2.673[Rb] + 0.752[Mo] + 4.221[Ba] - 2.923[Eu / Tb] + 0.983[Nd / Pr] + 3.011[Pr / Ce] - 1.634[Gd / Al] - 0.855[La / Gd] - 1.334[Cd / Cu] - 13.389.
[0062] S3, the identification results are as follows: The Fritillaria thunbergii to be tested was Zhejiang Fritillaria thunbergii, and its score was higher than that of Hubei Fritillaria thunbergii. The Fritillaria cirrhosa to be tested was from Hubei Province, and its score was higher than that of Fritillaria thunbergii from Zhejiang Province.
[0063] Example 2: A method for identifying the origin of fritillaria bulbs S1, the content of Mg, Al, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Rb, Sr, Mo, Ag, Cd, Sn, Sb, Ba, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Hg, Tl, Pb, and U in the tested fritillaria bulbs was determined. The elemental analysis was performed using ICP-MS, and the tested fritillaria bulbs were prepared for ICP-MS analysis via microwave digestion.
[0064] S2, the elemental content was standardized and imported into SIMCA-p 14.1 software for OPLS-DA analysis.
[0065] Comparative Example 1: A method for identifying the origin of fritillaria bulbs S1, the content of Mg, Al, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Rb, Sr, Mo, Ag, Cd, Sn, Sb, Ba, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Hg, Tl, Pb, and U in the tested fritillaria bulbs was determined. The elemental analysis was performed using ICP-MS, and the tested fritillaria bulbs were prepared for ICP-MS analysis via microwave digestion.
[0066] S2. Cluster analysis was used to standardize the element content data using Z-scores, and a cluster dendrogram was drawn based on the inter-group linkage method and squared Euclidean distance.
[0067] In the clustering dendrogram, when the Euclidean distance is 25, 32 batches of samples were divided into two categories, but the two types of fritillaria samples had obvious overlap and failed to be effectively distinguished.
[0068] Comparative Example 2: A method for identifying the origin of fritillaria bulbs S1, the content of Mg, Al, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Rb, Sr, Mo, Ag, Cd, Sn, Sb, Ba, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Hg, Tl, Pb, and U in the tested fritillaria bulbs was determined. The elemental analysis was performed using ICP-MS, and the tested fritillaria bulbs were prepared for ICP-MS analysis via microwave digestion.
[0069] S2, the elemental content was standardized and imported into SIMCA-p 14.1 software for principal component analysis.
[0070] The results of principal component analysis are as follows Figure 1 As shown, samples of Fritillaria thunbergii from Zhejiang and Fritillaria thunbergii from Hubei were found to be clustered together, and the differentiation effect was still not ideal. The above analytical methods failed to clearly distinguish the origin of the two types of Fritillaria thunbergii.
[0071] This invention also employs OPLS-DA and Fisher LDA analysis methods. OPLS-DA focuses on the overall contribution of variables to classification and does not require variables to be independent, thus allowing for correlations between variables. Fisher discriminant analysis, on the other hand, requires variables to have a certain degree of independent explanatory power; high VIF values can affect the reliability of coefficient estimates. This invention first uses OPLS-DA for variable selection and discriminant analysis, which has high discriminant power, but the results are presented graphically, limiting their explanatory power. Furthermore, the feature variables selected by OPLS-DA are input into Fisher LDA. By reducing multicollinearity among variables, an intuitive, concise, and analytical discriminant function is obtained. The discriminant results for the two batches of counterfeit products in the dual model are consistent, verifying the effectiveness of this combined analysis framework. This strategy leverages the advantages of OPLS-DA in high-dimensional data while enhancing the interpretability and practicality of the model through Fisher LDA.
[0072] The elemental distribution of Fritillaria thunbergii from Zhejiang and Hubei provinces differs significantly. Zhejiang Province, located at the eastern end of the South China Fold System, is characterized by volcanic and metamorphic rocks. Hubei Province, spanning the Qinling Fold System and the Yangtze Platform, comprises sedimentary, metamorphic, and igneous rocks. This geological background leads to regional differences in the distribution of various mineral elements, with some trace elements exhibiting enrichment or symbiotic characteristics. For example, Pr, Ce, and Nd, all belonging to the light rare earth elements (LREEs), share similar atomic radii and chemical properties due to the lanthanide contraction effect, often undergoing synergistic migration during magmatic differentiation and sedimentary diagenesis. The LREE content in Zhejiang Fritillaria thunbergii is significantly higher than that in Hubei Fritillaria thunbergii, while the heavy rare earth element (HREE) content is also slightly higher. The LREE / HREE ratio reflects the degree of fractionation in the geological environment. For instance, the La / Gd ratio in Hubei Fritillaria thunbergii is generally lower than that in Zhejiang Fritillaria thunbergii, possibly because La migrates more easily than Gd in the weathering crust of Hubei carbonate rocks, a process that differs in the volcanic rock areas of Zhejiang. To effectively reduce the impact of multicollinearity among variables on the model, this invention employs ratio and standardization processing to optimize the VIF value to 1.8–5.4, significantly improving the reliability and stability of the model.
[0073] Comparative Example 3: A method for identifying the origin of fritillaria bulbs S1, Fritillary A and Fritillary B in the target Fritillaria were detected by liquid phase analysis.
[0074] The amounts of fritillary acetyl and fritillary acetyl in the tested fritillaria were compared by analysis.
[0075] The Fritillaria thunbergii tested was from Zhejiang, and fritillary glycoside B was higher than fritillary glycoside A; the Fritillaria thunbergii tested was from Hubei, and fritillary glycoside A was higher than fritillary glycoside B.
[0076] The characteristic components of Fritillaria species are steroidal alkaloids. In Fritillaria thunbergii (Zhejiang) and Fritillaria hubei, the highest content of alkaloids is fritillary alkaloid A and fritillary alkaloid B. The research progress on the chemical components and pharmacological activities of alkaloids in Fritillaria species reveals significant differences in the content of these two alkaloids between the two species; fritillary alkaloid A is higher in Zhejiang Fritillaria than in Hubei Fritillaria, while the opposite is true for Hubei Fritillaria. The alkaloid content in ZJ7 and ZJ12 was determined, and the results showed that fritillary alkaloid B was higher in ZJ7 and ZJ12 than in fritillary alkaloid A, consistent with the characteristics of Hubei Fritillaria. However, given that the organic components of traditional Chinese medicine are easily affected by processing and storage conditions, relying solely on the quantitative analysis of one or two organic components for tracing the origin has certain limitations, but it can still provide a preliminary judgment.
[0077] Example 3: A method for preparing a fritillaria extract Preparation of Fritillaria extract: Fritillaria was pulverized to obtain Fritillaria powder, which was then treated with concentrated ammonia for 1 hour. The extraction solution was then added, and the mixture was sonicated for 45 minutes. The mixture was filtered, and the filtrate was evaporated to dryness in a water bath. Trichloromethane and water were then added for separation. The organic phase was collected, filtered, and evaporated to dryness in an evaporating dish to obtain the Fritillaria extract. The amount of Fritillaria powder used was 20 wt% of concentrated ammonia. The extraction solution was a mixture of trichloromethane and methanol, with a volume ratio of 4:1. The amount of Fritillaria powder used was 2.5 wt% of the extraction solution. The volume ratio of trichloromethane to water in the separation was 3:1, and the ratio of water volume to Fritillaria powder mass was 5 mL: 1 g.
[0078] Example 4: A method for preparing a fritillaria extract Preparation of Fritillaria extract: Fritillaria was pulverized to obtain Fritillaria powder, then treated with concentrated ammonia for 1 hour, followed by the addition of extraction solution, ultrasonic treatment for 45 minutes, filtration, collection of filtrate, evaporation to dryness in a water bath, separation of chloroform and water, collection of organic phase, filtration, and evaporation to dryness in an evaporating dish to obtain Fritillaria extract. The amount of Fritillaria powder used was 20 wt% of concentrated ammonia. The extraction solution was a mixture of chloroform, 2-amino-4-hydroxybenzoic acid, 2-chloro-6-methoxyphenol, and methanol, with a volume ratio of chloroform to methanol of 4:1. The content of 2-amino-4-hydroxybenzoic acid in the extraction solution was 0.025 wt%, the content of 2-chloro-6-methoxyphenol in the extraction solution was 0.018 wt%, the amount of Fritillaria powder used was 2.5 wt% of the extraction solution, the volume ratio of chloroform to water in the separation was 3:1, and the relationship between the volume of water used and the mass of Fritillaria powder used in the separation was 5 mL: 1 g.
[0079] Example 5: A method for preparing a fritillaria extract Preparation of Fritillaria extract: Fritillaria was pulverized to obtain Fritillaria powder, then treated with concentrated ammonia for 1 hour, followed by the addition of extraction solution, ultrasonic treatment for 45 minutes, filtration, collection of filtrate, evaporation to dryness in a water bath, separation of chloroform and water, collection of organic phase, filtration, and evaporation to dryness in an evaporating dish to obtain Fritillaria extract. The amount of Fritillaria powder used was 20 wt% of concentrated ammonia. The extraction solution was a mixture of chloroform, 2-amino-4-hydroxybenzoic acid, 2-chloro-6-methoxyphenol, and methanol, with a volume ratio of chloroform to methanol of 4:1. The content of 2-amino-4-hydroxybenzoic acid in the extraction solution was 0.009 wt%, the content of 2-chloro-6-methoxyphenol in the extraction solution was 0.018 wt%, the amount of Fritillaria powder used was 2.5 wt% of the extraction solution, the volume ratio of chloroform to water in the separation was 3:1, and the relationship between the volume of water used and the mass of Fritillaria powder used in the separation was 5 mL: 1 g.
[0080] Example 6: A method for preparing a fritillaria extract Preparation of Fritillaria extract: Fritillaria was pulverized to obtain Fritillaria powder, then treated with concentrated ammonia for 1 hour, followed by the addition of extraction solution, ultrasonic treatment for 45 minutes, filtration, collection of filtrate, evaporation to dryness in a water bath, separation of chloroform and water, collection of organic phase, filtration, and evaporation to dryness in an evaporating dish to obtain Fritillaria extract. The amount of Fritillaria powder used was 20 wt% of concentrated ammonia. The extraction solution was a mixture of chloroform, 2-amino-4-hydroxybenzoic acid, 2-chloro-6-methoxyphenol, and methanol, with a volume ratio of chloroform to methanol of 4:1. The content of 2-amino-4-hydroxybenzoic acid in the extraction solution was 0.025 wt%, the content of 2-chloro-6-methoxyphenol in the extraction solution was 0.002 wt%, the amount of Fritillaria powder used was 2.5 wt% of the extraction solution, the volume ratio of chloroform to water in the separation was 3:1, and the relationship between the volume of water used and the mass of Fritillaria powder used in the separation was 5 mL: 1 g.
[0081] Example 7: A method for preparing a fritillaria extract Preparation of Fritillaria extract: Fritillaria was pulverized to obtain Fritillaria powder, then treated with concentrated ammonia for 1 hour, followed by the addition of extraction solution, ultrasonic treatment for 45 minutes, filtration, collection of filtrate, evaporation to dryness in a water bath, separation of chloroform and water, collection of organic phase, filtration, and evaporation to dryness in an evaporating dish to obtain Fritillaria extract. The amount of Fritillaria powder used was 20 wt% of concentrated ammonia. The extraction solution was a mixture of chloroform, 2-amino-4-hydroxybenzoic acid, 2-chloro-6-methoxyphenol, and methanol, with a volume ratio of chloroform to methanol of 4:1. The content of 2-amino-4-hydroxybenzoic acid in the extraction solution was 0.01 wt%, the content of 2-chloro-6-methoxyphenol in the extraction solution was 0.005 wt%, the amount of Fritillaria powder used was 2.5 wt% of the extraction solution, the volume ratio of chloroform to water in the separation was 3:1, and the relationship between the volume of water used and the mass of Fritillaria powder used in the separation was 5 mL: 1 g.
[0082] Example 8: A method for preparing a fritillaria extract Preparation of Fritillaria extract: Fritillaria was pulverized to obtain Fritillaria powder, then concentrated ammonia was added and treated for 1 hour, then extraction solution was added, ultrasonic treatment was performed for 45 minutes, filtered, the filtrate was collected, evaporated to dryness in a water bath, then chloroform and water were added for separation, the organic phase was collected, filtered, and evaporated to dryness in an evaporating dish to obtain Fritillaria extract. The concentrated ammonia solution also contains sodium laurylate, with a sodium laurylate content of 0.4 wt%. The amount of fritillaria powder used is 20 wt% of the concentrated ammonia solution. The extraction solution is a mixture of chloroform, 2-amino-4-hydroxybenzoic acid, 2-chloro-6-methoxyphenol, and methanol, with a volume ratio of chloroform to methanol of 4:1. The content of 2-amino-4-hydroxybenzoic acid in the extraction solution is 0.025 wt%, the content of 2-chloro-6-methoxyphenol in the extraction solution is 0.018 wt%, and the amount of fritillaria powder used is 2.5 wt% of the extraction solution. The volume ratio of chloroform to water in the separation is 3:1, and the relationship between the volume of water used and the mass of fritillaria powder used in the separation is 5 mL: 1 g.
[0083] Example 9: A method for preparing a fritillaria extract Preparation of Fritillaria extract: Fritillaria was pulverized to obtain Fritillaria powder, then concentrated ammonia was added and treated for 1 hour, then extraction solution was added, ultrasonic treatment was performed for 45 minutes, filtered, the filtrate was collected, evaporated to dryness in a water bath, then chloroform and water were added for separation, the organic phase was collected, filtered, and evaporated to dryness in an evaporating dish to obtain Fritillaria extract. The concentrated ammonia solution also contains sodium laurylate, with a sodium laurylate content of 0.05 wt%. The amount of fritillaria powder used is 20 wt% of the concentrated ammonia solution. The extraction solution is a mixture of chloroform, 2-amino-4-hydroxybenzoic acid, 2-chloro-6-methoxyphenol, and methanol, with a volume ratio of chloroform to methanol of 4:1. The content of 2-amino-4-hydroxybenzoic acid in the extraction solution is 0.025 wt%, the content of 2-chloro-6-methoxyphenol in the extraction solution is 0.018 wt%, and the amount of fritillaria powder used is 2.5 wt% of the extraction solution. The volume ratio of chloroform to water in the separation is 3:1, and the relationship between the volume of water used and the mass of fritillaria powder used in the separation is 5 mL: 1 g.
[0084] Comparative Example 4: A method for preparing a fritillaria extract Preparation of Fritillaria extract: Fritillaria was pulverized to obtain Fritillaria powder, then treated with concentrated ammonia for 1 hour, followed by the addition of extraction solution, ultrasonic treatment for 45 minutes, filtration, collection of filtrate, evaporation to dryness in a water bath, separation of chloroform and water, collection of organic phase, filtration, and evaporation to dryness in an evaporating dish to obtain Fritillaria extract. The amount of Fritillaria powder used was 20 wt% of concentrated ammonia. The extraction solution was a mixture of chloroform, 2-amino-4-hydroxybenzoic acid, 2-chloro-6-methoxyphenol, and methanol, with a volume ratio of chloroform to methanol of 4:1. The content of 2-amino-4-hydroxybenzoic acid in the extraction solution was 0.025 wt%, the content of 2-chloro-6-methoxyphenol in the extraction solution was 0.0005 wt%, and the amount of Fritillaria powder used was 2.5 wt% of the extraction solution. The volume ratio of chloroform to water in the separation was 3:1, and the relationship between the volume of water used and the mass of Fritillaria powder used in the separation was 5 mL: 1 g.
[0085] Comparative Example 5: A method for preparing a fritillaria extract Preparation of Fritillaria extract: Fritillaria was pulverized to obtain Fritillaria powder, then treated with concentrated ammonia for 1 hour, followed by the addition of extraction solution, ultrasonic treatment for 45 minutes, filtration, collection of filtrate, evaporation to dryness in a water bath, separation of chloroform and water, collection of organic phase, filtration, and evaporation to dryness in an evaporating dish to obtain Fritillaria extract. The amount of Fritillaria powder used was 20 wt% of concentrated ammonia. The extraction solution was a mixture of chloroform, 2-amino-4-hydroxybenzoic acid, 2-chloro-6-methoxyphenol, and methanol, with a volume ratio of chloroform to methanol of 4:1. The content of 2-amino-4-hydroxybenzoic acid in the extraction solution was 0.002 wt%, the content of 2-chloro-6-methoxyphenol in the extraction solution was 0.018 wt%, the amount of Fritillaria powder used was 2.5 wt% of the extraction solution, the volume ratio of chloroform to water in the separation was 3:1, and the relationship between the volume of water used and the mass of Fritillaria powder used in the separation was 5 mL: 1 g.
[0086] The total alkaloid content in the Fritillaria cirrhosa extracts prepared in Examples 3-9 and Comparative Examples 4-5 was tested according to the method for determining the content of Fritillaria cirrhosa in the Chinese Pharmacopoeia. The results of the total alkaloids obtained are as follows: Figure 5 As shown, S3 is Example 3, S4 is Example 4, S5 is Example 5, S6 is Example 6, S7 is Example 7, S8 is Example 8, S9 is Example 9, D4 is Comparative Example 4, and D5 is Comparative Example 5. This invention involves first mixing concentrated ammonia with fritillaria powder, then adding an extraction reagent for extraction, and finally preparing a fritillaria extract by separation. The fritillaria extract contains total alkaloids. The extraction reagent includes at least chloroform and methanol. In this invention, the extraction reagent may also include 2-amino-4-hydroxybenzoic acid and 2- Chloro-6-methoxyphenol, 2-amino-4-hydroxybenzoic acid, and 2-chloro-6-methoxyphenol, when used in appropriate amounts, can increase the total alkaloid content in Fritillaria cirrhosa extract. However, if the amount of 2-amino-4-hydroxybenzoic acid or 2-chloro-6-methoxyphenol used is too low, and the amount of the other component used is appropriate, the total alkaloid content in Fritillaria cirrhosa extract cannot be significantly increased. Only when 2-amino-4-hydroxybenzoic acid and 2-chloro-6-methoxyphenol are used together can the total alkaloid content in Fritillaria cirrhosa extract be increased. In the preparation of Fritillaria extract, sodium lauryl laurate can be added to concentrated ammonia solution. Under the pretreatment of concentrated ammonia solution containing sodium lauryl laurate, and further extraction treatment using an extraction reagent containing 2-amino-4-hydroxybenzoic acid and 2-chloro-6-methoxyphenol, the total alkaloid content of the prepared Fritillaria extract is higher. This indicates that in addition to using 2-amino-4-hydroxybenzoic acid and 2-chloro-6-methoxyphenol, the further use of sodium lauryl laurate has a better effect.
[0087] In preparing Fritillaria extract using the methods of Examples 3-9 and Comparative Examples 4-5, the effectiveness of the method for Fritillaria extraction was tested using the standard addition method, with fritillary ethyl as a representative. The mass of the fritillary ethyl standard solution used was the same as that used for Fritillaria powder. The spiked recovery rate of fritillary ethyl was as follows: Figure 6 As shown, S3 is Example 3, S4 is Example 4, S5 is Example 5, S6 is Example 6, S7 is Example 7, S8 is Example 8, S9 is Example 9, D4 is Comparative Example 4, and D5 is Comparative Example 5. In this invention, concentrated ammonia water is first mixed with fritillaria powder, then an extraction reagent is added for extraction, and finally, fritillaria extract is obtained by separation. The fritillaria extract contains total alkaloids. The extraction reagent includes at least chloroform and methanol. In this invention, 2-amino-4-hydroxybenzoic acid and 2-chloro-6-methoxyphenol may also be added to the extraction reagent. 2-Amino-4-hydroxybenzoic acid and 2-chloro-6-methoxyphenol, when used in appropriate amounts, showed good spiked recoveries of fritillary ethylsin from fritillaria, indicating a good extraction effect. However, if the amount of 2-amino-4-hydroxybenzoic acid or 2-chloro-6-methoxyphenol used was too low, and the amount of the other component was appropriate, the extraction effect of fritillary ethylsin from fritillaria could not be significantly improved. Only when 2-amino-4-hydroxybenzoic acid and 2-chloro-6-methoxyphenol were used together could the extraction effect of fritillary ethylsin from fritillaria be improved. In the preparation of Fritillaria extract, sodium lauryl ester can be added to concentrated ammonia solution. Under the pretreatment of concentrated ammonia solution containing sodium lauryl ester, and further extraction treatment using an extraction reagent containing 2-amino-4-hydroxybenzoic acid and 2-chloro-6-methoxyphenol, the extraction effect of fritin ethyl in Fritillaria is better. This indicates that in addition to using 2-amino-4-hydroxybenzoic acid and 2-chloro-6-methoxyphenol, the further use of sodium lauryl ester has a better effect.
[0088] The embodiments and / or implementation methods described above are merely preferred embodiments and / or implementation methods for implementing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any way. Any person skilled in the art can make some modifications or alterations to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as the technology or embodiments that are substantially the same as the present invention.
[0089] This document uses specific examples to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. The above descriptions are only preferred embodiments of this application. It should be noted that due to the limitations of written expression, while there are objectively infinite specific structures, those skilled in the art can make several improvements, modifications, or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner. These improvements, modifications, changes, or combinations, or the direct application of the inventive concept and technical solution to other situations without modification, should all be considered within the scope of protection of this application.
Claims
1. A method for identifying the place of origin of Fritillaria cirrhosa, comprising: The content of organic or inorganic components in fritillaria is detected, and the origin of fritillaria is identified based on the test results. The test results include the score of Zhejiang fritillaria or Hubei fritillaria. The organic components include fritillarin A or fritillarin B. The inorganic components include at least one of Mg, Al, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Rb, Sr, Mo, Ag, Cd, Sn, Sb, Ba, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Hg, Tl, Pb, and U.
2. The identification method according to claim 1, characterized in that, The inorganic components include at least one of Mg, Zn, Rb, Mo, Ba, Eu, Tb, Nd, Pr, Ce, Gd, Al, La, Cd, and Cu.
3. The identification method according to claim 1, characterized in that, The fraction of Fritillaria thunbergii is 6.299[Mg] + 0.946[Zn] + 1.189[Rb] - 0.345[Mo] - 1.772[Ba] + 1.103[Eu / Tb] - 0.313[Nd / Pr] - 1.136[Pr / Ce] + 0.677[Gd / Al] + 0.526[La / Gd] + 0.334[Cd / Cu] - 2.
917.
4. The identification method according to claim 1, characterized in that, The fraction of Fritillaria cirrhosa from Hubei Province is: -15.598[Mg] - 2.193[Zn] - 2.673[Rb] + 0.752[Mo] + 4.221[Ba] - 2.923[Eu / Tb] + 0.983[Nd / Pr] + 3.011[Pr / Ce] - 1.634[Gd / Al] - 0.855[La / Gd] - 1.334[Cd / Cu] - 13.
389.
5. The identification method according to claim 1, characterized in that, The results of the fritillary bulb origin identification are as follows: Fritillaria bulbs originate from Zhejiang Province, and Zhejiang Fritillaria bulbs have a higher weighting than Hubei Fritillaria bulbs; or, Fritillaria bulbs originate from Hubei Province, and Hubei Fritillaria bulbs have a higher score than Zhejiang Fritillaria bulbs.
6. The identification method according to claim 1, characterized in that, The results of the fritillary bulb origin identification are as follows: Fritillaria bulbs originate from Zhejiang Province; fritillary glycoside A is greater than fritillary glycoside B; or, The fritillaria bulbs are from Hubei province, and fritillarin B is greater than fritillarin A.
7. The identification method according to claim 6, characterized in that, Fritillin A or Fritillin B is obtained by separating Fritillin extract, which is obtained by extracting Fritillin with an extraction reagent.
8. The identification method according to claim 7, characterized in that, Extraction reagents include chloroform and / or methanol.
9. The identification method according to claim 7, characterized in that, The extraction reagents include 2-amino-4-hydroxybenzoic acid and / or 2-chloro-6-methoxyphenol.
10. The identification method according to claim 1, characterized in that, The identification also includes PCA and / or OPLS-DA statistical analysis.