Quality detection method of cremastra applanata based on chemical fingerprint and mass difference component

Abstract

The present application relates to the technical field of traditional Chinese medicine detection, in particular to a quality detection method of Cymbidium lancifolium, which comprises a fingerprint of Cymbidium lancifolium medicinal materials, determination of the contents of two quality difference markers (4-methoxyphenanthrene-2,7-diol and Blestriarene C), and thin layer identification. The present application establishes the fingerprint of Cymbidium lancifolium medicinal materials and simultaneously determines the contents of 4-methoxyphenanthrene-2,7-diol and Blestriarene C in Cymbidium lancifolium by UHPLC, wherein the chromatographic column used is CORTECS T3, the column specification is 2.1 mm*100 mm*1.7 μm, the mobile phase is 0.1% formic acid aqueous solution (A)-methanol acetonitrile solution (B) for gradient elution, the flow rate is 0.3 mL*min ‑1 ; the column temperature is 40 DEG C; and the detection wavelength is 360 nm. The method of the present application has good accuracy, can comprehensively reflect the inherent chemical characteristics of Cymbidium lancifolium medicinal materials, can be used for identifying the authenticity of Cymbidium lancifolium traditional Chinese medicine, evaluating the quality, and ensuring the consistency and stability of Cymbidium lancifolium preparations.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of traditional Chinese medicine detection, in particular to a quality detection method and fingerprint spectrum of Cremastra appendiculata. BACKGROUND

[0002] Cremastra appendiculata Cremastra appendiculata (D. Don) Makino] is a rare medicinal plant of the genus Cremastra of the Orchidaceae family, is one of the authentic base origins of the traditional Chinese medicine Shancigu, is used as a medicine with dry pseudobulbs, is recorded in the 2020 edition of the Chinese Pharmacopoeia (Volume I), has the effects of clearing heat and resolving toxicity, reducing phlegm and resolving nodes, and is mainly used for treating carbuncle, pustule, toxic sore, scrofula, phlegm node and the like. The traditional Chinese medicine Shancigu is used for treating breast cancer, liver cancer, lung cancer, intestinal cancer and various tumor diseases in clinical practice, and various traditional Chinese medicine compound preparations (such as Cidan Capsules, Aiyu Capsules and Toujie Quweng Granules) taking Shancigu as the monarch drug have remarkable curative effects, which shows that Shancigu has good clinical application value. Although Shancigu is a multi-base medicinal material, in addition to Cremastra appendiculata Cremastra appendic-ulata (D. Don ) Makino, also includes the species of the genus Cremastra Pleione bulbocodioides ( Franch) Rolfe and Yunnan Cremastra appendiculata Pleione yunnanensis Rolfe two base origins, but in the modern pharmacological research on the anti-tumor effect of Shancigu, the research on Shancigu with the base origin of Cremastra appendiculata is the most.

[0003] In addition, due to the limitations of self-reproduction difficulty and artificial cultivation difficulty of Cremastra appendiculata, Cremastra appendiculata has been listed in the National Key Protected Wild Plant List and the Convention on International Trade in Endangered Species of Wild Fauna and Flora, and only “characteristics” and “microscopic identification” of Cremastra appendiculata are checked in the 2020 edition of the Chinese Pharmacopoeia (Volume I), lacking effective quality control standards. Therefore, it is of great significance to establish an effective quality detection method for the medicinal material of Cremastra appendiculata to ensure the quality consistency, effectiveness, germplasm resource collection and selection of artificial breeding of excellent varieties of the medicinal material.

[0004] The quality consistency of traditional Chinese medicine is the basis of the consistency of drug efficacy. The traditional Chinese medicine fingerprint spectrum, as a comprehensive and quantifiable chromatographic identification method, can reflect the types and quantities of chemical components contained in traditional Chinese medicine from a macroscopic and overall perspective, can reflect the overall changes of the internal quality of traditional Chinese medicine, is one of the effective methods for controlling the quality consistency of medicinal materials, and can fully integrate and correctly express complex chemical measurement data information by combining with the method of chemical metrology, can truly and vividly reflect the quality differences of traditional Chinese medicine, and reveal the hidden rules between complex compounds.

[0005] Therefore, it is necessary to develop a quantitative and qualitative quality detection method of Cremastra appendiculata based on chemical fingerprint spectrum combined with quality difference components, and it is necessary to effectively control the quality of the traditional Chinese medicinal material of Cremastra appendiculata. SUMMARY

[0006] In order to solve the above technical problems in the prior art, the present application provides a quality control mode for quantitative and qualitative determination of Duanlian based on chemical fingerprint combined with quality difference components, which is specifically as follows:

[0007] The fingerprint of Duanlian is specifically obtained by using a UHPLC method for sample determination, wherein a CORTECS T3 column is used, the column specification is 2.1 mm × 100 mm, 1.7 μm, a gradient elution is performed with 0.1% formic acid aqueous solution (A) - methanol acetonitrile solution (B) as the mobile phase, the flow rate is 0.3 mL·min -1 ; the column temperature is 40 ℃; and the detection wavelength is 360 nm.

[0008] The preparation methods of the test sample and the control sample are the same as above.

[0009] Further, different regions of Duanlian are taken for determination, the data is imported into a “Chinese medicine chromatographic fingerprint similarity evaluation system, 2012 version”, the time window width is 0.1 min, the median method is used to generate the superimposed fingerprint and the control fingerprint, 11 common peaks are calibrated, through comparison with the control sample and mass spectrum fragment information, 8 chemical components are identified, which are No. 1 peak Denneanoside B, No. 2 peak 7-hydroxy-4-methoxyphenanthrene-2,8-di-O-β-D-glucoside, No. 3 peak 8-hydroxy-4-methoxyphenanthrene-2,7-di-O-β-D-glucoside, No. 5 peak Ephemeranthoquinone B, No. 6 peak 4-methoxyphenanthrene-2,7-diol, No. 7 peak Blestriarene C, No. 8 peak 7-hydroxy-2,4-dimethoxyphenanthrene, and No. 11 peak 2,7,2'-trihydroxy-4,4',7'-trimethoxy-1,1'-biphenanthrene.

[0010] The finally obtained fingerprint is shown in Figure 2 .

[0011] The quality detection method of Duanlian is specifically a UHPLC method for simultaneously determining the contents of 4-methoxyphenanthrene-2,7-diol and Blestriarene C in Duanlian, wherein a CORTECS T3 column is used, the column specification is 2.1 mm × 100 mm, 1.7 μm, a gradient elution is performed with 0.1% formic acid aqueous solution (A) - methanol acetonitrile solution (B) as the mobile phase, the flow rate is 0.3 mL·min -1Column temperature: 40 ℃; detection wavelength: 360 nm.

[0012] Further, the UHPLC method, specific elution procedure as follows:

[0013]

[0014] Further, the test solution is prepared by the following method: the Cymbidium pulverized, through No. 3 sieve, to obtain Cymbidium powder; take 3 g of Cymbidium powder, accurately weighed, placed in a 100 mL conical flask with a plug, accurately added 25 mL of 50% ethanol, weighed, heated to reflux extraction for 1 h, cooled, weighed again, and the weight loss was made up with 50% ethanol, shaken, filtered, and the filtrate was taken and filtered through a 0.22 μm microporous filter membrane to obtain the test solution. The Cymbidium is the dry pseudobulb of Cymbidium appendiculatum (D. Don) Makino.

[0015] Further, the control solution is prepared by the following method: accurately weigh the 4-methoxyphenyl-2,7-diol, Blestriarene C control substance, and place them in 5 mL volumetric flasks, respectively, and add methanol to prepare a solution containing 2.01 mg of 4-methoxyphenyl-2,7-diol per 1 mL and a solution containing 1.72 mg of Blestriarene C per 1 mL, respectively; accurately measure the above two solutions and place them in 10 mL volumetric flasks, respectively, and dilute to the mark with methanol, shake well, and prepare a stock solution containing 0.201 mg of 4-methoxyphenyl-2,7-diol per 1 mL and a stock solution containing 0.172 mg of Blestriarene C per 1 mL; accurately measure the stock solutions 1 and 2 and place them in 10 mL volumetric flasks, and add methanol to make up the volume, to obtain a mixed control solution No. 1 containing 0.201 mg of 4-methoxyphenyl-2,7-diol per 1 mL and a mixed control solution No. 2 containing 0.172 mg of Blestriarene C per 1 mL; accurately measure 5 mL of mixed control solution No. 1 and place it in a 10 mL volumetric flask, add methanol to make up the volume, and mix well to obtain mixed control solution No. 2; and so on, to obtain a series of mixed control solutions with different concentrations, wherein the series of concentrations of 4-methoxyphenyl-2,7-diol is 0.126~16.08 μg / mL, and the series of concentrations of Blestriarene C is 0.161~20.64 μg / mL.

[0016] A quality detection method for Cymbidium, which adopts thin layer chromatography identification method, specifically, 8 μL of the test solution and the control solution are accurately measured and spotted on the same silica gel plate, toluene-acetone-glacial acetic acid (5:3:0.5) is used as the developing agent, pre-saturation is performed for 10 min, development is performed, the plate is taken out and dried, and the plate is observed under ultraviolet light (365 nm).

[0017] Further, the test solution of the sample is prepared by the following method: taking 3.0g of Cymbidium kanran powder, adding 50ml of ethanol into a conical flask with a stopper, refluxing for 1h, filtering through qualitative filter paper, evaporating the filtrate to dryness, dissolving the residue in 20ml of water, successively extracting with cyclohexane, dichloromethane and ethyl acetate, each reagent for three times, and each time with 20ml of extraction agent; combining the obtained same extraction layers, then evaporating the solvent with a rotary evaporator, and finally dissolving the residue in 2ml of methanol to obtain the test solution of the cyclohexane layer, the dichloromethane layer and the ethyl acetate layer.

[0018] Further, the control solution is a 4-methoxyphenanthrene-2,7-diol methanol solution with a concentration of 100.5ug / mL and a Blestriarene C methanol solution with a concentration of 86ug / mL.

[0019] Further, the silica gel plate is a thin layer chromatography silica gel GF254 plate.

[0020] A quality detection method of Cymbidium kanran, comprising the following three steps: (1) using the fingerprint and the construction method described above; (2) using the UHPLC method described above to simultaneously determine the content of 4-methoxyphenanthrene-2,7-diol and Blestriarene C in Cymbidium kanran; (3) using the thin layer chromatography identification method described above. The three methods are combined to simultaneously perform quality control, which can further enhance the quality control of Cymbidium kanran raw materials.

[0021] Compared with the prior art, the technical effects of the present application are embodied in:

[0022] (1) The UHPLC method is used to simultaneously determine the content of 4-methoxyphenanthrene-2,7-diol and Blestriarene C in Cymbidium kanran, the chromatographic column used is CORTECS T3, the column specification is 2.1mm x 100mm, 1.7um, the mobile phase is 0.1% formic acid aqueous solution (A)-methanol acetonitrile solution (B) for gradient elution, the flow rate is 0.3mL·min -1 ; the column temperature is 40℃; and the detection wavelength is 360nm. According to the method of the present application, the content of 4-methoxyphenanthrene-2,7-diol and Blestriarene C in Cymbidium kanran can be simultaneously determined. The average sample addition recovery rates of 4-methoxyphenanthrene-2,7-diol and Blestriarene C are 106.32% and 98.59% respectively, and the RSDs are 1.73% and 1.2% respectively, indicating that the method of the present application has good accuracy.

[0023] (2) The Ducllanthus chinensis fingerprint spectrum is obtained by using the UHPLC method, which comprehensively reflects the internal chemical characteristics of the traditional Chinese medicinal material Ducllanthus chinensis, and can be used for identifying the authenticity of Ducllanthus chinensis, evaluating the quality, and ensuring the consistency and stability of Ducllanthus chinensis preparations.

[0024] (3) The present application optimizes the preparation of the test solution, the composition of the developing agent, and other aspects, and investigates the temperature, humidity, stability, and thin layer chromatography plate, and establishes a simple and feasible Ducllanthus chinensis thin layer chromatography identification method with obvious qualitative characteristics.

[0025] (4) The present application establishes an effective quality detection method for Ducllanthus chinensis. Fingerprint spectrum technology can comprehensively reflect the internal chemical characteristics of traditional Chinese medicinal materials, and is a powerful method for identifying the authenticity of traditional Chinese medicines, evaluating the quality, and ensuring the consistency and stability of preparations. Chemical pattern recognition technology is a comprehensive technology that reveals the internal rules of things by using computer processing based on the chemical composition information of substances. The effective information in the fingerprint spectrum is comprehensively analyzed, dimensionally reduced, and classified by using the chemical pattern recognition technology, so that the quality consistency of traditional Chinese medicinal materials can be better evaluated, and the quality difference components can be screened out. The present application evaluates the quality of 35 batches of Ducllanthus chinensis medicinal materials by using the ultra-high performance liquid chromatography fingerprint spectrum combined with the chemical pattern recognition method, and determines the content of two main difference markers, thereby providing a reference for the quality control of Ducllanthus chinensis medicinal materials. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 is the UHPLC superimposed fingerprint spectrum (S1-S35) of Ducllanthus chinensis.

[0027] Figure 2 is the control fingerprint spectrum of Ducllanthus chinensis.

[0028] Figure 3 is the UHPLC spectrum of the mixed control (A) and Ducllanthus chinensis S27 (B), wherein peak 6: 4-methoxyphenyl-2,7-diol; peak 7: Blestriarene C.

[0029] Figure 4 is the common peak identification spectrum information diagram of the UHPLC fingerprint spectrum of Ducllanthus chinensis.

[0030] Figure 5 is the clustering analysis result of 35 batches of Ducllanthus chinensis samples.

[0031] Figure 6 is the PCA score diagram of 35 batches of Ducllanthus chinensis medicinal materials from different producing areas.

[0032] Figure 7 is the score diagram of the orthogonal partial least squares discriminant analysis of 35 batches of Ducllanthus chinensis medicinal materials from different producing areas.

[0033] Figure 8 Figure 8 is an OPLS-DA permutation plot of 35 batches of different origin of Duanquanlan medicinal materials.

[0034] Figure 9 Figure 9 is a VIP value plot of orthogonal partial least squares discriminant analysis of 35 batches of different origin of Duanquanlan medicinal materials.

[0035] Figure 10 Figure 10 is a TLC identification method of Duanquanlan different extraction parts investigation results plot.

[0036] Figure 11 Figure 11 is a TLC identification method of Duanquanlan different developing agent investigation results plot.

[0037] Figure 12 Figure 12 is a TLC identification method of Duanquanlan different brand thin layer chromatography silica gel GF254 plate investigation results plot.

[0038] Figure 13 Figure 13 is a TLC identification method of Duanquanlan different developing humidity investigation results plot.

[0039] Figure 14 Figure 14 is a TLC identification method of Duanquanlan different developing temperature investigation results plot.

[0040] Figure 15 Figure 15 is a TLC identification method of Duanquanlan test solution stability investigation results plot. DETAILED DESCRIPTION

[0041] The technical solutions of the present application will be further limited by combining specific embodiments, but the scope of protection is not limited to the description.

[0042] EMBODIMENT

[0043] 1. Instruments and materials

[0044] 1.1 Instruments

[0045] Agilent 1290 type ultra-high performance liquid chromatography system (American Agilent Technology Co., Ltd.); FW100 type high-speed universal pulverizer (Tianjin Test Instrument Co., Ltd.); EL-104 type electronic analytical balance [Mettler-Toledo Instrument (Shanghai) Co., Ltd.]; Centrifuge type centrifuge (Beckman Coulter Company, USA); KQ-300DE type digital ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); ultrapure water machine (Sichuan Water Technology Development Co., Ltd.).

[0046] 1.2 Materials

[0047] 4-methoxyphenanthrene-2,7-diol reference substance (batch number wkq22060806, purity ≥ 97%); Blestriarene reference substance (batch number wkq22053109, purity ≥ 98%) were purchased from Sichuan Weikeqi Biological Technology Co., Ltd.; Acetonitrile, methanol were chromatographically pure, and other reagents were analytically pure, and water was self-made ultrapure water. The medicinal materials used in the experiment were identified by Professor Liu Chunhua, Associate Professor of Pharmacognosy Teaching and Research Section, School of Pharmacy, Guizhou Medical University, as the dry pseudobulb of the Orchidaceae Cremastra appendiculata (D. Don) Makino. See Table 1 for source.

[0048] Table 1 Source of Cremastra appendiculata medicinal materials

[0049]

[0050] 2 Methods and results

[0051] 2.1 Preparation of solutions

[0052] 2.1.1 Test solution

[0053] The Cremastra appendiculata was crushed and passed through a No. 3 sieve. 3 g of Cremastra appendiculata powder was accurately weighed and placed in a 100 mL stoppered conical flask. 25 mL of 50% ethanol was accurately added, the mass was re-weighed, heated to reflux for 1 h, cooled, the mass was re-weighed, and the loss was made up with 50% ethanol. Shake well, filter, take the filtrate, and filter through a 0.22 μm microporous filter membrane to obtain the test solution.

[0054] 2.1.2 Reference solution

[0055] An appropriate amount of 4-methoxyphenanthrene-2,7-diol and Blestriarene C reference substances were accurately weighed and placed in 5 mL volumetric flasks, respectively. Methanol was added to prepare a solution containing 2.01 mg of 4-methoxyphenanthrene-2,7-diol per 1 mL and a solution containing 1.72 mg of Blestriarene C per 1 mL, respectively. An appropriate amount of each of the above two solutions was accurately measured and placed in a 10 mL volumetric flask, diluted to the mark with methanol, shaken well, and prepared into a mixed standard evaluation solution containing 4-methoxyphenanthrene-2,7-diol 0.201 mg and Blestriarene C 0.172 mg per 1 mL.

[0056] 2.2 Chromatographic conditions

[0057] The chromatographic column was CORTECS T3 (2.1 mm × 100 mm, 1.7 μm), and the mobile phase was 0.1% formic acid aqueous solution (A) - methanol acetonitrile solution (B) for gradient elution. The elution program is shown in Table 2. Flow rate: 0.3 mL·min -1Column temperature: 40 ℃; detection wavelength: 360 nm; injection volume: 2 μL.

[0058] Table 2 Gradient elution program

[0059]

[0060] 2.3 Fingerprint study

[0061] 2.3.1 Precision test

[0062] Take the Cymbidium test solution (S10), and continuously inject under the chromatographic conditions of item "2.2" for 6 times. Take No. 6 4-methoxyphenyl-2,7-diol chromatographic peak as the reference peak (S), and calculate the relative retention time RSD of each common peak <0.89%, and the relative peak area RSD <1.36%, which indicates that the instrument precision is good.

[0063] 2.3.2 Reproducibility test

[0064] Take the same batch of Cymbidium test solution (S10) and prepare 6 test solution samples in parallel according to the method of item "2.1.1". Inject and determine, take No. 6 4-methoxyphenyl-2,7-diol chromatographic peak as the reference peak (S), and calculate the relative retention time RSD of each common peak <1.26%, and the relative peak area RSD <2.10%, which indicates that the method has good reproducibility.

[0065] 2.3.3 Stability test

[0066] Take the Cymbidium test solution (S10), and inject and determine under the chromatographic conditions of item "2.2" at 0, 2, 4, 6, 8, 12, and 24 h after preparation, respectively. Take No. 6 4-methoxyphenyl-2,7-diol chromatographic peak as the reference peak (S), and calculate the relative retention time RSD of each common peak <1.59%, and the relative peak area RSD <1.76%, which indicates that the test solution has good stability within 24 h.

[0067] 2.3.4 Fingerprint establishment and similarity evaluation

[0068] Take 35 batches of Cymbidium sample solutions, and inject and determine under the chromatographic conditions of item "2.2". Record the chromatogram, import the data into "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version)", take the chromatogram of sample S1 as the reference chromatogram, the time window width is 0.1 min, and the median method is used to generate the superimposed fingerprint and the control fingerprint. 11 common peaks are identified, see Figure 1 , 2 . By comparing the chromatogram ( Figure 3 ) and the ultraviolet spectrum ( Figure 3), 6th peak was 4-methoxyphenanthrene-2,7-diol, 7th peak was Blestriarene C. Through consulting literature and comparing mass fragment information, 1st peak was identified as Denneanoside B, 2nd peak was 7-hydroxy-4-methoxyphenanthrene-2,8-di-O-β-D-glucoside, 3rd peak was 8-hydroxy-4-methoxyphenanthrene-2,7-di-O-β-D-glucoside, 5th peak was Ephemeranthoquinone B, 8th peak was 7-hydroxy-2,4-dimethoxyphenanthrene, 11th peak was 2,7,2'-trihydroxy-4,4',7'-trimethoxy-1,1'-biphenanthrene. Similarity evaluation results were shown in Table 3. The similarity of each sample from different producing areas with the control fingerprint was greater than 0.89 except S21, indicating that the established fingerprint method could be used for the identification and overall quality control of C. japonicum. Each sample of C. japonicum was basically similar to the control fingerprint, indicating that the types of chemical components contained in samples from different producing areas were consistent. With 6th peak (4-methoxyphenanthrene-2,7-diol) as the reference peak (S), the relative retention time RSD of each common peak was 0.06% ~ 0.27%, indicating that the peak time of common peaks in each batch was relatively stable, but the relative peak area RSD (41.13% ~ 74%) was quite different, indicating that there was certain difference in the content of components in different batches of C. japonicum.

[0069] Table 3 Similarity evaluation results of C. japonicum samples

[0070]

[0071] 2.5 Chemical pattern recognition

[0072] 2.5.1 Hierarchical cluster analysis (HCA)

[0073] SPSS 24.0 software was used to analyze 11 common peaks of 35 batches of C. japonicum with group-joining clustering method and square Euclidean distance as the distance between samples to construct a dendrogram, and the results were shown in Fig. 2. Figure 5 The samples were divided into different groups for evaluation. When the square Euclidean distance was 15, the samples of each batch of C. japonicum were mainly divided into two categories: S1 was a large category (I), and other samples were a large category (II). At the square Euclidean distance of 5, the samples of each batch of C. japonicum were divided into 5 small categories: S24 was a small category (A), S21 was a small category (B), S11 and S17 were a small category (C), and other samples were a small category (D).

[0074] 2.5.2 Principal Component Analysis (PCA)

[0075] The 11 common peaks of 35 batches of different origin of the D. candidum were imported into the SIMCA 14.0 software for principal component analysis. The scaling mode was Ctr (centering), and the eigenvalues and variance contribution rate are shown in Table 4. The results showed that there were 2 eigenvalues greater than 1, i.e. 2 principal components. The cumulative variance contribution rate of the first 2 principal components was 88.262%, indicating that the first 2 principal components could fully reflect the basic characteristics and main information of D. candidum and could be used as evaluation indicators of D. candidum. The coordinate system was established with 2 principal components, and projection was carried out to analyze the overall distribution of the samples and the contribution of each chromatographic peak to the sample distribution. Figure 6 The PCA scatter plot showed that the 35 batches of D. candidum samples could be divided into 5 categories, of which S1 was the first category, S24 was the second category, S21 was the third category; S11 and S17 were the fourth category, and the other samples were the fifth category. This result was basically consistent with the clustering analysis result, further verifying the effectiveness of CA. Table 5 is the principal component loading matrix, which mainly explains the correlation between the 11 original variables in the sample and the first and second principal components. Peak 5, peak 6 (4-methoxyphenanthrene-2,7-diol), peak 7 (Blestriarene C), and peak 8 showed obvious positive load values in the first principal component, indicating that the variance contribution rate of the first principal component was mainly affected by common peaks 5, 6, 7, and 8. Peak 5 showed obvious positive load values in the second principal component, indicating that the variance contribution rate of the second principal component was mainly affected by common peak 5. In summary, the above components had a major impact on the overall quality of D. candidum.

[0076] Table 4 Eigenvalues and contribution rates of principal components of D. candidum

[0077]

[0078] Table 5 Principal component matrix of D. candidum

[0079]

[0080] 2.5.3 Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA)

[0081] In order to better analyze the differences between the above different classification samples, supervised OPLS-DA was then used for modeling study. The 11 common peaks of 35 batches of different origin of the D. candidum were imported into the SIMCA 14.0 software for OPLS-DA analysis, and the score scatter plot is shown in Figure 7The results of the established model are as follows: R2X=0.799, R2Y=0.762, Q2=0.520, all greater than 0.5, indicating that the model is stable and has good prediction ability. To investigate whether the model is over-fitted, 200 times of permutation test were performed, and the results are shown in Table 2. Figure 8 As shown in Table 2, R2Y=0.496139, Q2Y=0.323559, indicating that the model is not over-fitted and can be used for discriminant analysis of 35 batches of samples of Cymbidium kanran. To further find the difference markers of 35 batches of Cymbidium kanran medicinal materials, the variable importance in projection (VIP) prediction values of the chemical components represented by the 11 common peaks in the OPLS-DA model were extracted, and the VIP values of each component are shown in Table 3. Figure 9 The VIP>1 is a meaningful variable, and the VIP values in descending order are peak 6 (4-methoxyphenanthrene-2,7-diol), peak 7 (Blestriarene C), peak 5, and peak 8. These components may be the main markers for the differences between different batches of samples, which is basically consistent with the importance weight variable found in the load matrix in PCA.

[0082] 2.4 Determination of the content of the components (determination of the content based on the chemical pattern recognition of the fingerprint)

[0083] Through the chemical pattern recognition of UHPLC fingerprint, it was found that the main components causing the differences between different batches of Cymbidium kanran samples were 4-methoxyphenanthrene-2,7-diol, Blestriarene C, peak 5, and peak 8. 4-methoxyphenanthrene-2,7-diol has good anti-tumor activity and antioxidant activity, and Blestriarene C has a significant inhibitory effect on the proliferation of human lung cancer A549 cells. Therefore, the contents of 4-methoxyphenanthrene-2,7-diol and Blestriarene C in Cymbidium kanran were determined simultaneously by UHPLC method, in order to provide a scientific basis for the quality control and evaluation of Cymbidium kanran.

[0084] 2.4.1 Investigation of linear relationship

[0085] Precisely take the 4-methoxyphenanthrene-2,7-diol and Blestriarene C reference substance solutions under item 2.1.2, and place them in a 10 mL volumetric flask. Add methanol to make up the volume, and obtain a mixed reference solution No. 1. Precisely take 5 mL of the mixed reference solution No. 1 into a 10 mL volumetric flask, add methanol to dilute and make up the volume, mix well, and obtain a mixed reference solution No. 2. In this way, a series of mixed reference solutions with different concentrations are prepared. Sample injection is performed under the chromatographic conditions in item 2.2, and the mass concentration is taken as the abscissa (X) and the peak area is taken as the ordinate (Y) to draw the standard curve, and the results are shown in Table 6.

[0086] Table 6 Linear relationship investigation results of three components in the Cymbidium kanran

[0087]

[0088] 2.4.2 Precision test

[0089] Take the mixed reference standard solution under item "2.1.2", dilute 40 times with methanol, and determine by continuous injection for 6 times under the chromatographic conditions of item "2.2", and calculate the RSD of the peak area of 4-methoxyphenanthrene-2, 7-diol and Blestriarene C to be 0.31% and 0.93% respectively, indicating that the instrument precision is good.

[0090] 2.4.3 Reproducibility test

[0091] Take an appropriate amount of the same batch of Cymbidium kanran test sample, and prepare 6 test sample solutions in parallel according to the method of item "2.2", and determine by injection under the chromatographic conditions of item "2.2", and calculate the RSD of the peak area of 4-methoxyphenanthrene-2, 7-diol and Blestriarene C to be 0.62% and 1.25% respectively, indicating that the method has good reproducibility.

[0092] 2.4.4 Stability test

[0093] Take the Cymbidium kanran test sample solution, and determine by injection under the chromatographic conditions of item "2.2" at 0, 2, 4, 8, 12 and 24 h after preparation, and calculate the RSD of the peak area of 4-methoxyphenanthrene-2, 7-diol and Blestriarene C to be 0.39% and 0.49% respectively, indicating that the test sample solution has good stability within 24 h.

[0094] 2.4.5 Spiked recovery test

[0095] Take 6 portions of Cymbidium kanran sample (S27) with known content of two components, each 0.5 g, accurately weigh, add each reference solution according to the content of the sample to the reference amount 1:1, prepare the test sample solution according to the method of item "2.1.1", and determine by injection under the chromatographic conditions of item "2.2", and calculate the average spiked recovery of 4-methoxyphenanthrene-2, 7-diol and Blestriarene C to be 106.32% and 98.59% respectively, and the RSD is 1.73% and 1.2% respectively, indicating that the method has good accuracy.

[0096] 2.4.6 Sample content determination

[0097] The content of 4-methoxyphenanthrene-2,7-diol and Blestriarene C in 35 batches of D. candidum was determined by the method described in item 2.2. The results are shown in Table 7. The content of 4-methoxyphenanthrene-2,7-diol and Blestriarene C in 35 batches of D. candidum was 0.7329-69.4868 µg / g and 2.3272-81.6388 µg / g, respectively, indicating that the content of 4-methoxyphenanthrene-2,7-diol and Blestriarene C in D. candidum from different habitats varied greatly.

[0098] Table 7. Determination results of the content of two components in D. candidum (µg / g)

[0099]

[0100] 3 DISCUSSION

[0101] 3.1 Investigation of the preparation of the test solution

[0102] In order to make the fingerprint as much as possible to reflect the chemical composition of D. candidum, and maximize the content of the main components in D. candidum, the extraction method was optimized. The effects of different extraction methods (ultrasonic and reflux), solvents (methanol, 70% methanol, 50% methanol, ethanol, 70% ethanol, 50% ethanol) and extraction time (30, 60, 90, 120 min) on the chromatogram were investigated. The results showed that the extraction efficiency of heating reflux was higher than that of ultrasonic extraction, and 50% ethanol as the extraction solvent could extract relatively more comprehensive components, and the extraction time had no obvious change on the number of chromatographic peaks and the size of the main chromatographic peak area. Therefore, 50% ethanol was selected as the extraction solvent, and heating reflux extraction for 60 min was selected as the final extraction method.

[0103] 3.2 Selection of chromatographic conditions

[0104] In the process of optimizing the chromatographic conditions, two kinds of chromatographic columns, Agilent Eclipse Plus C18 RRHD (2.1 mm x 100 mm, 1.8 um) and CORTECS T3 (2.1 mm x 100 mm, 1.7 um), were mainly investigated. Finally, the separation effect of CORTECS T3 C18 was better. Gradient elution was used, and the mobile phase was investigated, including water-acetonitrile, 0.1% formic acid water-acetonitrile, water-methanol, 0.1% formic acid water-methanol, 0.1% formic acid water-acetonitrile methanol. The baseline stability and separation effect were used as evaluation indexes, and finally 0.1% formic acid water-acetonitrile methanol was selected as the mobile phase. In addition, the flow rate of the mobile phase, column temperature and wavelength were also investigated, and finally the flow rate was 0.3 mL / min, the column temperature was 30℃, and the detection wavelength was 260 nm.

[0105] 3.3 Establishment and analysis of the fingerprint

[0106] The similarity of the different origin of the Cymbidium lancifolium medicine was higher, and the similarity value was more than 0.89. The RSD value of the relative retention time in the fingerprint spectrum was not much different, but the RSD value of the relative peak area was quite different, which might be related to the wild environment, growth period and other factors. Through PCA analysis, the results showed that 35 batches of Cymbidium lancifolium medicine samples could be divided into 5 categories, in which S1 (Anshun, Guizhou) was the first category, S24 (Xingyi, Guizhou) was the second category, S21 (Renhuai, Guizhou) was the third category; S11 (Enshi, Hubei) and S17 (Qinglong, Guizhou) were the fourth category, and other samples were the fifth category. The results were basically consistent with the clustering analysis results. In general, the four batches of Cymbidium lancifolium medicine in Guizhou (S1 Anshun, Guizhou, S17 Qinglong, Guizhou, S21 Renhuai, Guizhou, S24 Xingyi, Guizhou) were clustered into one category. It was speculated that the quality of these batches of Cymbidium lancifolium medicine might be greatly affected by the origin of the medicine.

[0107] In addition, the harvesting period of S1 (Anshun, Guizhou), S17 (Qinglong, Guizhou), and S24 (Xingyi, Guizhou) was winter, while the harvesting period of S21 (Renhuai, Guizhou) was autumn. According to the similarity evaluation results, the similarity value of S21 (Renhuai, Guizhou) with the control fingerprint spectrum was 0.739, and the similarity values of the other three batches of Cymbidium lancifolium were more than 0.9. It was speculated that the difference in the quality of these batches of traditional Chinese medicine might be related to the origin and the harvesting period of the medicine. The three batches of Cymbidium lancifolium medicine outside Guizhou Province (S22 Shiquan, Shaanxi, S23 Leshan, Sichuan, S25 Yunnan, West of Yunnan) could not be separated from the other 27 batches of Cymbidium lancifolium medicine in Guizhou Province, indicating that the quality of traditional Chinese medicine might be affected not only by the origin of the medicine, but also by the growth period, harvesting time, processing method of the medicine in the production area. Using the loading plot to analyze the difference of the classification components, combined with OPLS-DA analysis, 4 strong characteristic peaks were found, which were peak 6 (4-methoxyphenyl-2, 7-diol), peak 7 (Blestriarene C), peak 5, and peak 8.

[0108] 3.4 Content determination analysis

[0109] Four compounds were screened by the fingerprint of C. japonica and chemical pattern recognition technology, including 4-methoxy-2,7-dihydrophenanthrene, Blestriarene C, Peak 5 and Peak 8. Among them, 4-methoxy-2,7-dihydrophenanthrene and Blestriarene C had good anti-tumor activity. And the previous experimental study of the research group showed that the above two compounds had a significant inhibitory effect on the proliferation of human breast cancer MCF-7 cells. Therefore, it is speculated that 4-methoxy-2,7-dihydrophenanthrene and Blestriarene C are both mass difference components and active ingredients, so the UHPLC method is used to determine the content of 4-methoxy-2,7-dihydrophenanthrene and Blestriarene C in C. japonica at the same time. The results show that among 35 batches of C. japonica, the total content (A) of 4-methoxy-2,7-dihydrophenanthrene and Blestriarene C in the sample (S17) of C. japonica collected in January 2021 in Qinglong, Guizhou is higher. There is no significant difference in A among the other three batches, and the average is about 5 times that of S17. Among the six batches of C. japonica in Guizhou Zhenning, the A of C. japonica collected in January 2022 (S27) is the highest, which is quite different from the other five batches. It is speculated that there is a large difference in A among C. japonica of the same origin collected at different times, which may be affected by many factors such as collection time and processing method. And among C. japonica of different origins collected at the same time (December 2021), there is a large difference in A, among which the A of C. japonica in Anshun, Guizhou (S1) is the highest. It is speculated that there is a large difference in A among C. japonica of different origins collected at the same time, which is mainly due to the influence of the production area. And the content of the two index components is low, which may be due to the low content of the medicinal material itself, the change of related components after the processing of the medicinal material such as decoction, which further leads to the low content. Through the analysis of the fingerprint and the content of two components of 35 batches of C. japonica, this study can provide a scientific basis for the quality control and evaluation of C. japonica.

[0110] Qualitative identification of C. japonica based on TLC

[0111] 1. Materials and methods

[0112] 1.1 Materials

[0113] 1.1.1 Instruments

[0114] FW100 type high-speed universal pulverizer (Tianjin Test Instrument Co., Ltd.); EL-104 type electronic analytical balance [Mettler Toledo Instrument (Shanghai) Co., Ltd.]; KQ-300DE type digital ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.).

[0115] 1.1.2 Reagents

[0116] 4-methoxyphenanthrene-2, 7-diol control (batch number wkq22060806, purity ≥97%); blestriarene control (batch number wkq22053109, purity ≥98%) were purchased from Sichuan Weikeqi Biological Technology Co., Ltd.; thin layer chromatography silica gel GF254 plate (Qingdao Haoyang Chemical Factory Branch); petroleum ether (analytical pure, Tianjin Cheryou Chemical Reagent Co., Ltd.); acetone (analytical pure, Sichuan Xilong Science Co., Ltd.); methanol (analytical pure, National Pharmaceutical Group Chemical Reagent Co., Ltd.); ethyl acetate (analytical pure, Sichuan Xilong Science Co., Ltd.); chloroform, dichloromethane, glacial acetic acid, hydrochloric acid, concentrated sulfuric acid (all purchased from Chongqing Chuandong Chemical Co., Ltd.); cyclohexane; experimental water is self-made ultrapure water.

[0117] 1.1.2 Instruments

[0118] Thin layer digital imaging system (model: LINOMAT5, manufacturer: Switzerland CAMAG), thin layer chromatography semi-automatic electric sampler (REPORSTAR3); silica gel GF254 thin layer plate (Qingdao Haoyang Chemical Co., Ltd., Yantai Jiangyou Silica Gel Development Co., Ltd.); silica gel HSGF254 thin layer plate (Yantai Jiangyou Silica Gel Development Co., Ltd.); constant temperature water bath.

[0119] 1.2 Methods

[0120] 1.2.1 Investigation of thin layer conditions

[0121] The pre-experiment was to investigate the crude extract of Cymbidium goeringii, the development of different developing systems: chloroform:methanol (13:1), xylene-chloroform-methanol-glacial acetic acid (5:12:2:0.5), toluene-acetone-glacial acetic acid (5:3:0.5), etc., and the development of different sample application methods: strip and dot, and finally establish the thin layer chromatography method.

[0122] 1.2.2 Preparation of medicinal material test solution

[0123] Take 3.0 g of the powder into a conical flask with a stopper, add 25 ml of 50 ethanol, reflux extract for 1 h, filter through qualitative filter paper, evaporate the filtrate to dryness, and dissolve the residue in 20 mL of water. Extract with cyclohexane, dichloromethane and ethyl acetate in turn, and each reagent is extracted three times with a volume of 20 mL. Combine the obtained same extraction layers, then evaporate the solvent with a rotary evaporator, and then dissolve with 2 mL of methanol to obtain the cyclohexane layer, dichloromethane layer, and ethyl acetate layer test solution.

[0124] 1.2.3 Preparation of control solution

[0125] Take 4-methoxyphenanthrene-2,7-diol control 2.01 mg, constant volume to 1 ml volumetric flask, dilution 10 times, configuration into the concentration of 100.5 ug / mL control solution. Blestriarene C control 3.44 mg, constant volume to 2 ml volumetric flask, dilution 10 times, configuration into the concentration of 86 ug / mL control solution.

[0126] 1.2.4 Investigation of different extraction parts

[0127] The specific results are Figure 10 .

[0128] 1.2.5 Investigation of different developing agents

[0129] The effects of three developing agents, chloroform:methanol (13:1), xylene-chloroform-methanol-glacial acetic acid (5:12:2:0.5) and toluene-acetone-glacial acetic acid (5:3:0.5), were investigated respectively, and the results are shown in Figure 11 .

[0130] From Figure 11 the three different developing agents, it can be seen that in toluene-acetone-glacial acetic acid (5:3:0.5) developing agent, the separation effect of the main fluorescent spots presented in the chromatogram of the three batches of test samples is better, and the spot coloration is clear.

[0131] 2.3.4 Establishment of thin layer identification method

[0132] According to the test of thin layer chromatography (general rule 0502), 8 μL of test sample solution and control solution were precisely measured and spotted on the same silica gel plate, toluene-acetone-glacial acetic acid (5:3:0.5) was used as developing agent, pre-saturation was performed for 10 min, development was carried out, and then the plate was taken out, dried, and observed under ultraviolet light (365 nm).

[0133] 2.3.5 Investigation of different brands of thin layer chromatography silica gel GF254 plates

[0134] According to the established thin layer chromatography identification method, the development effects of Qingdao Marine silica gel GF254 plate, Yantai Yellow Sea silica gel GF254 plate and Yantai Yellow Sea silica gel HSGF254 plate were investigated respectively, and the results are shown in Figure 12 . From the results of the three brands of silica gel G plates, it can be seen that the separation degree of Qingdao Marine silica gel G plate is the best, so Qingdao Marine silica gel G plate is selected.

[0135] 2.3.6 Investigation of different developing humidities

[0136] The relative humidity of the developing environment was adjusted to 32%, 47% and 72% respectively by different concentrations of sulfuric acid, and the effects of development under different relative humidities were investigated at 25℃, and the results are shown in Figure 13 . Figure 13The results showed that the chromatogram bands were clear and the Rf values had certain differences under different humidity, but the medicinal materials could still be well identified, indicating that the humidity endurance was good.

[0137] 2.3.7 Investigation of different development temperatures

[0138] The development was carried out at 35℃, 25℃, 15℃ and 58% relative humidity, respectively, to investigate the influence of different temperatures on development, and the results are shown in Figure 14 .

[0139] From Figure 14 The results showed that the chromatogram bands were clear and the Rf values had certain differences under different humidity, but the medicinal materials could still be well identified, indicating that the humidity endurance was good.

[0140] 2.3.8 Investigation of stability of test solution

[0141] The test solution of the same batch of product was prepared according to the proposed method 3 days ago, 2 days ago and on the same day, and was spotted on the same silica gel G plate, developed and colored, as shown in Figure 15 .

[0142] From Figure 15 The results showed that the chromatogram bands were clear and the Rf values had certain differences under different humidity, but the medicinal materials could still be well identified, indicating that the humidity endurance was good.

[0143] In this study, the test solution preparation and developing agent composition were optimized, and the temperature, humidity, stability, thin layer chromatography plate and other factors affecting the thin layer chromatography were investigated, and a simple and feasible method for identifying Cymbidium lancifolium was established.

[0144] Finally, it should be pointed out that the above examples are only more representative examples of the present application. Obviously, the technical solutions of the present application are not limited to the above examples, and there can be many variations. All variations that can be directly derived or inferred by those skilled in the art from the disclosed content should be considered as the protection scope of the present application.

Claims

1. A method for detecting a plant of the genus Haberlea, characterized in that, The thin layer chromatography identification method is specifically the test of general test for thin layer chromatography 0502. 8 μL of the test solution and the control solution are precisely taken and spotted on the same silica gel plate. Toluene: acetone: glacial acetic acid = 5:3:0.5 is used as a developing agent, is pre-saturated for 10 min, is developed, is taken out, is dried, and is observed under ultraviolet light 365 nm. The test solution is prepared by the following method: 3.0 g of the Cymbidium kanran powder is taken into a conical flask with a plug, 25 ml of 50% ethanol is added, and reflux extraction is performed for 1 h. The solution is filtered through a qualitative filter paper, the filtrate is evaporated to dryness, and the residue is dissolved in 20 mL of water. The solution is extracted with cyclohexane, dichloromethane and ethyl acetate in turn, and each reagent is extracted three times with a volume of 20 mL. The obtained same extraction layers are combined, the solvent is evaporated by a rotary evaporator, 2 mL of methanol is added for dissolution, and the cyclohexane layer, the dichloromethane layer and the ethyl acetate layer test solution are obtained. The control substance is 4-methoxyphenyl-2,7-diol and Blestriarene C.

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