Fingerprint detection method for distinguishing radix tetrastigme from producing areas of autonomous regions of Zhejiang, Fujian and Guangxi Zhuang

Abstract

The invention discloses a fingerprint detection method for distinguishing tetrastigma hemsleyanum Diels et Gilg from origin of Zhejiang, Fujian and Guangxi Zhuang autonomous districts, which comprises the following steps: (1) collecting tetrastigma hemsleyanum Diels et Gilg samples from origin of Zhejiang, Fujian and Guangxi Zhuang autonomous districts, and drying in the sun; (2) preparing a test sample solution for the samples, and determining the test sample solution by adopting a high performance liquid chromatography to respectively generate contrast fingerprints of radix tetrastigme in the production places of the autonomous regions of Zhejiang, Fujian and Guangxi Zhuang; (3) generating a chromatogram of an unknown tetrastigma hemsleyanum Diels et Gilg sample by adopting a method in the step (2), carrying out similarity calculation on the chromatogram and the fingerprints of tetrastigma hemsleyanum Diels et Gilg in the autonomous regions of Zhejiang, Fujian and Guangxi Zhuang by adopting traditional Chinese medicine chromatographic fingerprint similarity evaluation system software, and if the similarity is more than 0.8, considering that the tetrastigma hemsleyanum Diels et Gilg belongs to the corresponding origin; and evaluating the producing area of the tetrastigma hemsleyanum sample. Tetrastigma hemsleyanum Diels et Gilg in different producing areas such as Zhejiang, Fujian and Guangxi Zhuang autonomous regions can be effectively distinguished, and quality evaluation and clinical medication of the Tetrastigma hemsleyanum Diels et Gilg are facilitated.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicinal material origin identification, and particularly relates to a fingerprint detection method for distinguishing Tetrastigma obtetum from Zhejiang, Fujian and Guangxi Zhuang Autonomous Region. BACKGROUND

[0002] Tetrastigma obtetum is a plant of Vitaceae Tetrastigma hemsleyanum The fresh or dried tuber of Tetrastigma obtetum Diels et Gilg is one of the 'New Eight Flavors of Zhejiang', and has the effects of clearing heat and resolving toxins, relieving swelling and pain, resolving phlegm and resolving nodes, and is used for treating infantile convulsion with high fever, croup, carbuncle, phlegm node and snake bite. At present, the main producing areas of Tetrastigma obtetum are Zhejiang, Fujian and Guangxi Zhuang Autonomous Region, but researches show that there are certain differences in appearance and pharmacology and efficacy of Tetrastigma obtetum from different producing areas.

[0003] In recent years, Tetrastigma obtetum has been studied more, but most of the researches are focused on determination of some components and comparison of the determined components from different producing areas, but the different components that can be used to distinguish Tetrastigma obtetum from different producing areas are not pointed out; some researches establish fingerprint, but the fingerprint cannot distinguish the producing areas, and there is no simple and feasible method for tracing the producing areas of unknown samples.

[0004] Therefore, it is necessary to develop a method for distinguishing Tetrastigma obtetum from Zhejiang, Fujian and Guangxi Zhuang Autonomous Region, so as to facilitate the quality evaluation and clinical medication of Tetrastigma obtetum. SUMMARY

[0005] The technical problem solved by the present application is to overcome the technical defects of the background art, and to provide a fingerprint detection method for distinguishing Tetrastigma obtetum from Zhejiang, Fujian and Guangxi Zhuang Autonomous Region. Based on traditional Chinese medicine fingerprint, the present application collects representative samples of Tetrastigma obtetum with clear sources and establishes a simple and feasible fingerprint analysis method for tracing and distinguishing Tetrastigma obtetum from different producing areas, which can effectively distinguish Tetrastigma obtetum from Zhejiang, Fujian and Guangxi Zhuang Autonomous Region, and is beneficial to the quality evaluation and clinical medication of Tetrastigma obtetum.

[0006] The technical solution adopted by the present application to solve the above technical problem is as follows: A fingerprint detection method for distinguishing Tetrastigma obtetum from Zhejiang, Fujian and Guangxi Zhuang Autonomous Region, comprising the following steps: (1) collecting Tetrastigma obtetum samples from Zhejiang, Fujian and Guangxi Zhuang Autonomous Region with clear sources and drying them; (2) preparing test sample solutions from the above samples, and determining the test sample solutions by high performance liquid chromatography to generate control fingerprint chromatograms of Tetrastigma obtetum from Zhejiang, Fujian and Guangxi Zhuang Autonomous Region, respectively; (3) using the method of step (2) to generate the chromatogram of the unknown Radix Ampelopsis, using the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint to calculate the similarity between the chromatogram and the control fingerprint of Radix Ampelopsis from Zhejiang, Fujian and Guangxi Zhuang Autonomous Region, and if the similarity is above 0.8, it is considered to be Radix Ampelopsis from the corresponding producing area, so as to evaluate the producing area of the Radix Ampelopsis sample.

[0007] Preferably, in step (1), the Radix Ampelopsis sample is identified as the plant Ampelopsis aconitifolia Batal. Tetrastigma hemsleyanum dried tubers of Diels and Gilg.

[0008] Preferably, in step (2), the preparation of the test solution uses the following conditions: the effects of extraction solvent (methanol, 50% methanol and n-butanol), extraction method (reflux and ultrasonic), extraction time (20, 30 and 40 minutes) and sample weight (0.3 g, 0.5 g and 0.7 g) on the method are investigated respectively, and finally determined as follows: about 0.5 g of sample powder is accurately weighed and placed in a conical flask with a plug, 25 mL of 50% methanol is accurately added, tightly sealed, the weight is determined, ultrasonic treatment (power 250 W, frequency 40 kHz) is performed for 30 minutes, then released, placed at room temperature, weighed again, the lost weight is made up with methanol, shaken uniformly, filtered, and the filtrate is obtained.

[0009] Preferably, in step (2), the high performance liquid chromatography uses the following conditions: octadecylsilane-bonded silica gel is used as the filler, acetonitrile is used as the mobile phase A, 0.1% phosphoric acid solution is used as the mobile phase B, gradient elution (0~5 min: 6% A; 5~22 min: 6%~15% A; 22~35 min: 15%~30% A; 35~50 min: 30%~100% A); flow rate: 1.0 mL•min -1 ; column temperature: 30 ℃; injection volume: 5 μL; detection wavelength: 270 nm.

[0010] Preferably, in step (2), the generation method of the control fingerprint includes the following steps: 15 batches of Radix Ampelopsis samples from Zhejiang are collected, and determined according to the preparation method of the test solution and the chromatographic conditions, the determination data is integrated, and the control fingerprint is generated by importing the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint; the control fingerprints of Radix Ampelopsis from Fujian and Guangxi Zhuang Autonomous Region are generated by the same method.

[0011] Compared with the prior art, the present application has the following beneficial effects: The present application adopts traditional Chinese medicine fingerprint whole evaluation technology, collects representative samples of Smilax riparia Wendel with clear sources, and establishes a simple and feasible fingerprint analysis method for tracing and distinguishing Smilax riparia Wendel samples from different producing areas, so that Smilax riparia Wendel from Zhejiang, Fujian, Guangxi Zhuang Autonomous Region and other producing areas can be effectively distinguished, and the quality evaluation and clinical medication of Smilax riparia Wendel are facilitated. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 A chromatogram of 15 batches of Smilax riparia Wendel samples from Zhejiang produced by the present application (Accucore C18 (4.6 mm x 150 mm, 2.6 μm) chromatographic column); Figure 2 A control fingerprint chromatogram of Smilax riparia Wendel produced in Zhejiang; Figure 3 A comparison chart of Smilax riparia Wendel fingerprint chromatograms from Zhejiang, Fujian and Guangxi Zhuang Autonomous Region (peaks I-VII are common peaks of Smilax riparia Wendel from Guangxi Zhuang Autonomous Region); Figure 4 A precision test chromatogram of the method of the present application; Figure 5 A repeatability test chromatogram of the method of the present application; Figure 6 A stability test chromatogram of the method of the present application; Figure 7 A comparison chromatogram of extraction solvents of the method of the present application; Figure 8 A comparison chromatogram of extraction methods of the method of the present application; Figure 9 A comparison chromatogram of extraction time of the method of the present application; Figure 10 A comparison chromatogram of sample weight of the method of the present application; Figure 11 A comparison chromatogram of chromatographic columns of the method of the present application; Figure 12 A comparison chromatogram of instruments of the method of the present application. DETAILED DESCRIPTION

[0013] In order to better understand the content of the present application, further description will be made in combination with specific examples and drawings. It should be understood that these examples are only used to further illustrate the present application, and are not used to limit the scope of the present application. In addition, it should be understood that after reading the content described in the present application, those skilled in the art can make some non-essential modifications or adjustments to the present application, which still belongs to the protection scope of the present application. Example 1

[0014] A fingerprint detection method for distinguishing Smilax riparia Wendel from Zhejiang, Fujian and Guangxi Zhuang Autonomous Region, the steps are as follows: (1) Collecting the sample of Ampelopsis aconitifolia Batal. from Zhejiang, Fujian and Guangxi Zhuang Autonomous Region, and drying in the sun; the sample of Ampelopsis aconitifolia Batal. is identified as Ampelopsis aconitifolia Batal. of Vitaceae Tetrastigma hemsleyanum Diels et Gilg dried tuber; (2) Preparing the sample solution from the above sample, and determining the sample solution by high performance liquid chromatography to generate the control fingerprint of Ampelopsis aconitifolia Batal. from Zhejiang, Fujian and Guangxi Zhuang Autonomous Region, respectively; The preparation of the sample solution adopts the following conditions: the effects of extraction solvent (methanol, 50% methanol and n-butanol), extraction method (reflux and ultrasonic), extraction time (20, 30 and 40 minutes) and sample weight (0.3 g, 0.5 g and 0.7 g) on the method are investigated, and finally determined as follows: taking about 0.5 g of sample powder, accurately weighing, placing in a conical flask with a plug, accurately adding 25 mL of 50% methanol, tightly plugging, weighing, ultrasonic treating (power 250 W, frequency 40 kHz) for 30 minutes, releasing, placing at room temperature, weighing again, supplementing the lost weight with methanol, shaking uniformly, filtering, and taking the filtrate to obtain.

[0015] The high performance liquid chromatography adopts the following conditions: taking octadecylsilane bonded silica gel as the filler, acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution (0~5 min: 6% A; 5~22 min: 6%~15% A; 22~35 min: 15%~30% A; 35~50 min: 30%~100% A); flow rate: 1.0 mL•min -1 ; column temperature: 30 ℃; injection volume: 5 μL; detection wavelength: 270 nm.

[0016] The generation of the control fingerprint: collecting 15 batches of sample of Ampelopsis aconitifolia Batal. from Zhejiang with clear source, and determining according to the preparation method of the sample solution and the chromatographic conditions, and the chromatogram is shown in Figure 1 , integrating the determination data, importing into the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System, generating the control fingerprint, shown in Figure 2 , and the common fingerprint peak information is shown in Table 1; generating the fingerprint of Ampelopsis aconitifolia Batal. from Fujian, Guangxi Zhuang Autonomous Region and other places by the same method.

[0017] Table 1 Common peak attribution and compound information of Ampelopsis aconitifolia Batal. from Zhejiang No. t R (min)]]> ​ Compound name 1 6.06 5-hydroxytryptamine 2 7.36 Unknown 3 9.26 Unknown 4 10.45 3-mthoxy-4-hydrobenzoic acid 4-O-D-apifuranosyl(1→2)-O-D-glucopyranoside 5 16.34 Catechin 6 16.70 Unknown 7 19.00 Procyanidin B1 8 27.67 Kaempferol-3-(2'-d-apifuranosyl)-d-glucopyranoside-7-O-a-L-rhamnopyranoside 9 28.29 Unknown 10 30.06 Kaempferol-3-d-glucopyranoside-7-O-a-l-rhamnopyranoside (3) The chromatogram of the unknown Sanleyqing sample is generated by the method of step (2), and the similarity of the chromatogram to the fingerprint chromatograms of Sanleyqing from Zhejiang, Fujian and Guangxi Zhuang Autonomous Region is calculated by the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System software. If the similarity is greater than 0.8, it is considered to belong to Sanleyqing from the corresponding production area, thereby evaluating the production area of the Sanleyqing sample.

[0018] The comparison chart of Sanleyqing fingerprint chromatograms from different production areas such as Zhejiang, Fujian and Guangxi Zhuang Autonomous Region is shown in Figure 3 , and the different components are shown in Table 2, mainly showing differences in the types and contents of flavonoid glycosides; Sanleyqing from Guangxi Zhuang Autonomous Region mainly contains Kaempferol 3-apioside-7-rhamnosyl-(1->6)-galactoside, Astragalin, Rutin, Kaempferol 3-apioside-7-rhamnosyl-(1->6)-galactoside and Isoquercitrin, etc.; the contents of Kaempferol 3-apioside-7-rhamnosyl-(1->6)-galactoside and Kaempferol 3-apioside-7-rhamnosyl-(1->6)-galactoside in Sanleyqing from Zhejiang are higher, which are obviously different from those from other production areas, and can be used as characteristic components of Sanleyqing from Zhejiang; the contents of the above flavonoid glycosides in Fujian samples are smaller.

[0019] Table 2 Key difference components of Sanleyqing from different production areas No. tR / min formula m / z Messured ions Mass error / ppm Fragment ions Tentative Identification Origin 8 14.20 C 32 H 38 O 19 ]]> 725.1971 [M-H]- -5.8 579.1362,430.0902,283.0244 Kaempferol-3-(2'-d-apifuranosyl)-d-glucopyranoside-7-O-a-L-rhamnopyranoside Zhejiang 10 16.11 [CAT 27 H 30 O 15 ]]> 593.1542 [M-H]- -6.0 430.0909,283.0250 Kaempferol-3-d-glucopyranoside-7-O-a-l-rhamnopyranoside Zhejiang Ⅲ 16.53 C 27 H 30 O 16 ]]> 609.1462 [M-H]- -1.0 607.1321 Rutin Guangxi Zhuang Autonomous Region Ⅳ 16.67 C 21 H 20 O 12 ]]> 463.0880 [M-H]- -0.7 300.1002, 271.3786 Isoquercitrin Guangxi Zhuang Autonomous Region Ⅵ 17.96 C 27 H 30 O 15 ]]> 593.1515 [M-H]- -1.4 285.0412 Kaempferol-3-O-rutinoside Guangxi Zhuang Autonomous Region Ⅶ 18.42 C 21 H 20 O 11 ]]> 447.0924 [M-H]- 0.8 284.0305,255.0233 Astrin Guangxi Zhuang Autonomous Region The methodological validation of precision, repeatability, stability and durability of the above step (2) method is as follows: I. Precision test According to the above test sample solution preparation method and chromatographic conditions, prepare 1 part of test sample solution, continuously sample 6 times, record the chromatogram, and evaluate the similarity according to the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System provided by the National Pharmacopoeia Committee. The results show that the similarity is greater than 0.99, which meets the technical requirements of the fingerprint chromatogram, and the precision of the method is good, see Figure 4 .

[0020] II. Repeatability test According to the above test sample solution preparation method and chromatographic conditions, prepare 6 parts of test sample solution, and perform determination, record the chromatogram, and evaluate the similarity according to the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System, respectively. The results show that the similarity is greater than 0.99, and the method has good repeatability, see Figure 5 .

[0021] III. Stability test According to the above test solution preparation method and chromatographic conditions, the test solution of each dosage form was prepared, respectively injected into the liquid chromatograph at different time points within 26 hours, the chromatogram was recorded, and the similarity was evaluated according to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint provided by the State Pharmacopoeia Commission. The results showed that the similarity was greater than 0.98, and each test solution was stable within 26 hours. See Figure 6 .

[0022] Four, durability experiment (1) Extraction solvent investigation According to the above test solution preparation method and chromatographic conditions, the test solution of each dosage form was prepared, respectively using methanol, 50% methanol and n-butanol as extraction solvent, recording the chromatogram, and evaluating the similarity according to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint provided by the State Pharmacopoeia Commission. The results showed that the similarity was greater than 0.95, but the front peak of n-butanol was obviously less, and the chromatographic peak number of 50% methanol extraction was more and the extraction rate was high, so 50% methanol was selected as the extraction solvent. See Figure 7 .

[0023] (2) Extraction method investigation According to the above test solution preparation method and chromatographic conditions, the test solution of each dosage form was prepared, respectively using reflux for 30 minutes and ultrasonic for 30 minutes as two extraction methods, recording the chromatogram, and evaluating the similarity according to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint provided by the State Pharmacopoeia Commission. The results showed that the similarity was greater than 0.99, and considering the convenient operation, ultrasonic was selected as the extraction method. See Figure 8 .

[0024] (3) Extraction time investigation According to the above test solution preparation method and chromatographic conditions, the test solution of each dosage form was prepared, respectively using ultrasonic extraction for 20, 30 and 40 minutes, recording the chromatogram, and evaluating the similarity according to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint provided by the State Pharmacopoeia Commission. The results showed that the similarity was greater than 0.99, and considering the convenient operation, ultrasonic for 30 minutes was selected as the extraction method. See Figure 9 .

[0025] (4) Investigation of sample weight According to the above test solution preparation method and chromatographic conditions, the test solution of each dosage form was prepared, respectively taking 0.3g, 0.5g and 0.7g, recording the chromatogram, and evaluating the similarity according to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint provided by the State Pharmacopoeia Commission. The results showed that the similarity was greater than 0.95, and considering the convenient operation, 0.5g was selected as the sample weight. See Figure 10 .

[0026] (5) Chromatographic column investigation Prepare test sample solution according to the above preparation method and chromatographic conditions, respectively using Chromcore core C18 chromatographic column (4.6 mm x 250 mm, 5 μm), Ultimate plus C18 chromatographic column (4.6 mm x 250 mm, 5 μm) and Accucore C18 (4.6 mm x 150 mm, 2.6 μm), record the chromatogram, and evaluate the similarity according to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint provided by the State Pharmacopoeia Commission. The results show that the similarity is greater than 0.92 and the separation is good, and the durability is good, see Figure 11 .

[0027] (6) Instrument investigation Prepare test sample solution according to the above preparation method and chromatographic conditions, respectively using Shimadzu LC-20AT, Agilent 1200 and Thermo U3000 RSLC, record the chromatogram, and evaluate the similarity according to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint provided by the State Pharmacopoeia Commission. The results show that the similarity is greater than 0.90 and the separation is good, and the durability is good, see Figure 12 .

[0028] The above description is not a limitation of the present application, and the present application is not limited to the above examples. Changes, modifications, additions or substitutions made by those skilled in the art within the spirit and scope of the present application should also be within the protection scope of the present application.

Claims

1. A fingerprint detection method for distinguishing the origin of Radix Trifolii in Zhejiang, Fujian and Guangxi Zhuang Autonomous Region, characterized by, It comprises the following steps: (1) Collecting the samples of Tetrastigma hemsleyanum from Zhejiang, Fujian and Guangxi Zhuang Autonomous Region, and drying them; (2) Preparing the sample solution of the above samples, and determining the sample solution by high performance liquid chromatography to generate the control fingerprint of Tetrastigma hemsleyanum from Zhejiang, Fujian and Guangxi Zhuang Autonomous Region, respectively; (3) Generating the chromatogram of the unknown sample of Tetrastigma hemsleyanum by the method of step (2), and calculating the similarity between the chromatogram and the fingerprint of Tetrastigma hemsleyanum from Zhejiang, Fujian and Guangxi Zhuang Autonomous Region by the similarity evaluation system for chromatographic fingerprint of traditional Chinese medicine software, and regarding the similarity above 0.8 as belonging to the corresponding Tetrastigma hemsleyanum from the corresponding region, so as to evaluate the origin of the sample of Tetrastigma hemsleyanum.

2. The fingerprint detection method for distinguishing the producing areas of Zhejiang, Fujian and Guangxi Zhuang Autonomous Region of Radix Trifolii according to claim 1, characterized in that, In step (1), the sample of Radix Ampelopsis was identified as a plant of the grape family, Ampelopsis aconitifolia Batal. Tetrastigma hemsleyanum Dried tubers of Diels et Gilg.

3. The method for distinguishing the fingerprints of Radix Trifolii according to claim 1, wherein, In step (2), the preparation method of the sample solution comprises the following steps: taking about 0.5 g of the sample powder, accurately weighing, placing in a conical flask with a plug, accurately adding 25 mL of 50% methanol, tightly sealing, weighing, ultrasonic treating for 30 minutes, releasing, placing at room temperature, re-weighing, supplementing the weight loss with methanol, shaking, filtering, and taking the filtrate to obtain the sample solution.

4. The fingerprint spectrum detection method for distinguishing the producing areas of Zhejiang, Fujian and Guangxi Zhuang Autonomous Region of Radix Trifolii according to claim 3, characterized in that, The power of the ultrasonic treatment is 250 W, and the frequency is 40 kHz.

5. The fingerprint detection method for distinguishing the producing areas of Zhejiang, Fujian and Guangxi Zhuang Autonomous Region of Radix Trifolii according to claim 1, characterized in that, In step (2), the high performance liquid chromatography adopts the following conditions: octadecylsilane bonded silica gel as the filler, acetonitrile as the mobile phase A, 0.1% phosphoric acid solution as the mobile phase B, gradient elution; flow rate: 1.0 mL•min -1 ; column temperature: 30 ℃; injection volume: 5 μL; detection wavelength is 270 nm.

6. The fingerprint detection method for distinguishing Tetrastigma hemsleyanum from Zhejiang, Fujian and Guangxi Zhuang Autonomous Region according to claim 5, wherein the gradient elution conditions are as follows: 0~5 min: 6% A; 5~22 min: 6%~15% A; 22~35 min: 15%~30% A; 35~50 min: 30%~100% A.

7. The method for distinguishing the fingerprints of Radix Trifolii according to claim 1, wherein, In step (2), the generation method of the control fingerprint comprises the following steps: collecting 15 batches of samples of Tetrastigma hemsleyanum from Zhejiang with clear sources, determining according to the above sample solution preparation method and chromatographic conditions, integrating the determination data, importing the similarity evaluation system for chromatographic fingerprint of traditional Chinese medicine, and generating the control fingerprint; and generating the control fingerprints of Tetrastigma hemsleyanum from Fujian and Guangxi Zhuang Autonomous Region by the same method.

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