Extraction method and application of effective components of kadsura coccinea roots and method for identifying quality of kadsura coccinea roots

Abstract

The invention relates to the technical field of traditional Chinese medicinal material detection, in particular to a kadsura coccinea root effective component extraction method, application thereof and a kadsura coccinea root quality identification method. The invention provides an extraction method of effective components of kadsura coccinea roots. The extraction method comprises ultrasonic extraction or heating reflux-extraction combined extraction. The extraction method provided by the invention can be used for safely and effectively extracting the effective components of the kadsura coccinea roots. The invention also provides a method for identifying the quality of the kadsura coccinea roots. The method comprises the following steps: qualitatively identifying the effective components of the kadsura coccinea roots, detecting the relative retention time of an organic acid peak, detecting the content, detecting the moisture, detecting pesticide residues, and detecting heavy metals and harmful elements. The quality of the kadsura coccinea roots is controlled by performing multi-aspect detection on the effective components of the kadsura coccinea roots.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine testing technology, and in particular to a method for extracting the effective components of black tiger root and its application, as well as a method for quality identification of black tiger root. Background Technology

[0002] Black tiger root, a traditional medicinal plant, has significant application value in the field of traditional Chinese medicine. Its main bioactive components are lignans and triterpenoids, such as 12-acetoxy-black tiger acid and schisandrin a, which possess various pharmacological effects and have great potential in clinical applications. However, there are currently many problems to be solved regarding the quality control of black tiger root and the extraction technology of its effective components.

[0003] Regarding the quality control standard system, the current quality control standards for black tiger root medicinal materials are mainly concentrated at the local standard level, such as the "Guangdong Provincial Standard for Traditional Chinese Medicinal Materials" and the "Guangxi Yao Ethnic Minority Traditional Medicinal Materials Quality Standard." These standards mostly only include morphological identification, microscopic identification, and thin-layer chromatography, lacking key items such as content determination, and thus cannot comprehensively and accurately control the quality of the medicinal materials. At the same time, black tiger root medicinal materials from different producing areas are affected by factors such as the producing environment and planting conditions, resulting in certain differences in quality. However, systematic research on the differences in their chemical composition content is insufficient, and a quality grading standard based on producing area differences has not yet been formed. Furthermore, the lack of unified national or industry standards to regulate quality control leads to differences in the quality control of medicinal materials by different regions and enterprises, affecting the market circulation and quality stability of the medicinal materials.

[0004] From a quality control perspective, existing technologies primarily focus on morphological and microscopic identification, with limited attention paid to the determination of the content of active ingredients in medicinal materials. Even though some studies have used modern analytical techniques such as high-performance liquid chromatography (HPLC) to determine the content of certain components, providing new ideas and methods for quality control, these components cannot fully represent the overall quality of *Heterodon esculenta* root, nor can they comprehensively reflect its efficacy and safety. Commonly used thin-layer chromatography has limited specificity and sensitivity in identification, and is easily interfered with by other components, leading to inaccurate identification results. While modern analytical techniques such as HPLC offer high accuracy, they are complex to operate, costly, and have limited widespread adoption, making them difficult to apply extensively in grassroots testing units and enterprises. Furthermore, existing quality control technologies mainly focus on indicators such as the appearance and chemical composition of medicinal materials, with insufficient research into the correlation between these indicators and the actual efficacy of the medicinal materials. Moreover, the detection of safety indicators such as pesticide residues, heavy metal content, and microbial indicators is relatively limited, posing potential safety risks for medication use.

[0005] In terms of extracting active ingredients, there is currently a lack of efficient and stable extraction methods for active ingredients such as lignans and triterpenoids in black tiger root, which affects its further development and application in the pharmaceutical and related fields.

[0006] In summary, existing technologies for the quality identification and extraction of active ingredients from black tiger root medicinal materials suffer from incomplete standards, insufficient testing techniques, and extraction methods that require optimization. Therefore, there is an urgent need for a method that can accurately identify the quality of black tiger root and efficiently extract its active ingredients to meet market demands for quality control and utilization of active ingredients in black tiger root medicinal materials. Summary of the Invention

[0007] The purpose of this invention is to provide a method for extracting the effective components of black tiger root. This method can safely and effectively extract the effective components of black tiger root. By qualitatively identifying the effective components of black tiger root, detecting the relative retention time of organic acid peaks, detecting content, moisture content, pesticide residues, heavy metals, and harmful elements, the quality of tiger root medicinal materials can be controlled.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0009] This invention provides a method for extracting the effective components of black tiger root, the extraction method comprising ultrasonic extraction or a combination of heating reflux-extraction;

[0010] The ultrasonic extraction includes the following steps:

[0011] (1) Mix the black tiger root with extraction solvent 1 to obtain mixture 1; extract mixture 1 by ultrasound for 25-35 min to obtain an ultrasonic mixture;

[0012] (2) Cool the ultrasonic mixture, filter it, and take the filtrate to obtain the effective components of black tiger root;

[0013] In step (1), the extraction solvent 1 is a methanol solution and / or an ethanol solution; the mass fraction of the methanol solution is 90-100%; the mass fraction of the ethanol solution is 90-100%.

[0014] In step (1), the power of the ultrasound is 350-450W; the frequency of the ultrasound is 40-60kHz.

[0015] The combined heating reflux-extraction extraction includes the following steps:

[0016] 1) Mix black tiger root and extraction solvent 2 to obtain mixture 2; heat mixture 2 under reflux for 25-35 min to obtain a heated reflux mixture;

[0017] 2) Cool the heated reflux mixture, filter it to obtain a filtrate; dry the filtrate to obtain the primary extract;

[0018] 3) Mix the primary extract with water to obtain a primary extract solution; mix the primary extract solution with an extraction solvent and extract to obtain the effective components of black tiger root;

[0019] In step 1), the extraction solvent 2 is a methanol solution with a mass fraction of 40-60%;

[0020] In step 3), the extraction solvent is an ethyl acetate solution.

[0021] Preferably, in step (1), the mass-to-volume ratio of the black tiger root and the extraction solvent 1 is 1g: 20-30mL;

[0022] In step (2), the cooling temperature is 20-30°C.

[0023] Preferably, in step 1), the mass-to-volume ratio of the black tiger root and the extraction solvent 2 is 1g:20-30mL.

[0024] Preferably, in step 2), the drying is water bath drying; the drying temperature is 98–102°C.

[0025] Preferably, in step 3), the mass-to-volume ratio of the initial extract to water is 1g:18-22mL;

[0026] The volume ratio of the primary extract solution to the extraction solvent is 20:18 to 22;

[0027] The extraction is performed 1 to 3 times.

[0028] This invention also provides the application of the effective components of black tiger root extracted by the extraction method described above in the quality identification of black tiger root.

[0029] This invention also provides a method for quality identification of black tiger root, the method including qualitative identification of the effective components of black tiger root, detection of the relative retention time of organic acid peaks, content detection, moisture detection, pesticide residue detection, heavy metal and harmful element detection;

[0030] The effective components of the black tiger root are those extracted using the extraction method described above.

[0031] Preferably, the qualitative identification method is thin-layer chromatography detection; the qualitative identification steps include: developing the active ingredients of black tiger root in a developing solvent, drying to obtain the analyte; spraying sulfuric acid ethanol solution onto the analyte, heating to develop color, and then examining it under ultraviolet light;

[0032] The developing solvent is petroleum ether-ethyl acetate-formic acid;

[0033] The volume ratio of petroleum ether-ethyl acetate-formic acid is 16-18:2-4:1;

[0034] The heating temperature is 100–110°C;

[0035] The heating time is 2 to 3 minutes.

[0036] Preferably, the organic acid includes meso-dihydroguaiac acid, open-ring schisandrin a, schisandrin a, and (+)-(8s,8′R)-3′,4,4′-trihydroxy-5,5′-dimethoxylignan.

[0037] Preferably, the components to be detected include open-ring schisandrin a and schisandrin a.

[0038] The beneficial effects of this invention are:

[0039] This invention provides a method for extracting the effective components of black tiger root and its application, as well as a method for quality identification of black tiger root. The extraction method of this invention can safely and effectively extract the effective components of black tiger root.

[0040] The quality of black tiger root medicinal materials is controlled through qualitative identification of its effective components, detection of the relative retention time of organic acid peaks, content determination, moisture content determination, pesticide residue detection, and detection of heavy metals and harmful elements. Thin-layer chromatography and characteristic chromatograms effectively distinguish black tiger root from its adulterants, such as black vine, preventing counterfeit products from entering the market and ensuring the authenticity and quality of the medicinal materials. Content determination methods accurately measure the content of key components in black tiger root, such as schisandrin a and schisandrin a, which helps ensure the efficacy of the medicinal materials and avoids adverse reactions or reduced clinical efficacy due to insufficient or excessive component content, thus ensuring the safety and effectiveness of medication. Moisture content, pesticide residue, heavy metal, and harmful element detection are then performed according to the Chinese Pharmacopoeia to ensure the safety of the medicinal materials. The establishment of this quality identification method provides a scientific basis for improving the quality standards of black tiger root medicinal materials, helps to fill the gaps in existing quality control standards, and provides strong support for the standardized production and quality supervision of medicinal materials.

[0041] The extraction method and quality identification method of the effective components of black tiger root of the present invention are of great practical significance for standardizing the black tiger root medicinal material market, improving its quality control level, and promoting its rational application in the field of traditional Chinese medicine. Attached Figure Description

[0042] Figure 1The results of thin-layer chromatography identification of 2 μl, 5 μl, and 10 μl of test solution in Example 1 are shown in the following chromatograms: lane 1 is 2 μl of test solution, lane 2 is 5 μl of test solution, and lane 3 is 10 μl of test solution.

[0043] Figure 2 The images shown are the thin-layer chromatographic results of the identification of 2 μl, 4 μl, and 5 μl of test solution, 1 μl, 2 μl, and 5 μl of open-ring schisandrin a reference solution, 1 μl, 2 μl, and 5 μl of schisandrin a reference solution, and 1 μl, 2 μl, and 5 μl of reference medicinal material solution in Example 1. Lane 1 is for 2 μl of test solution, lane 2 is for 4 μl of test solution, lane 3 is for 5 μl of test solution, and lane 4 is for 1 μl of open-ring schisandrin a reference solution. Schisandric acid a reference solution: Lane 5 contains 2 μl of open-ring schisandric acid a reference solution from Xinan; Lane 6 contains 5 μl of open-ring schisandric acid a reference solution from Xinan; Lane 7 contains 1 μl of Xinan schisandric acid a reference solution from Xinan; Lane 8 contains 2 μl of Xinan schisandric acid a reference solution from Xinan; Lane 9 contains 5 μl of Xinan schisandric acid a reference solution from Xinan; Lane 10 contains 1 μl of reference medicinal material solution; Lane 11 contains 2 μl of reference medicinal material solution; Lane 12 contains 5 μl of reference medicinal material solution.

[0044] Figure 3 The images show the results of thin-layer chromatography identification using different brands of thin-layer plates. From left to right, they are Qingdao Ocean, Merk, and Yantai Chemical. Lane 1 is the open-ring schisandrin a reference solution, lane 2 is the schisandrin a reference solution, lane 3 is the test solution, and lane 4 is the reference herb solution.

[0045] Figure 4 The results of thin-layer chromatography identification at different temperatures are shown in the following chromatograms from left to right: 4℃, 25℃, lane 1 is the reference solution of schistosic acid a, lane 2 is the reference solution of schistosic acid a, lane 3 is the test solution, and lane 4 is the reference medicinal material solution.

[0046] Figure 5 The results of thin-layer chromatography identification under different developing humidity are shown in the following chromatograms: from left to right, 50% and 78% are respectively. Lane 1 is the reference solution of schistosic acid a, lane 2 is the reference solution of schistosic acid a, lane 3 is the test solution, and lane 4 is the reference medicinal material solution.

[0047] Figure 6 The image shows the specific detection results. Lanes 1-5 are negative control solutions, lane 6 is the open-ring schisandrin a reference solution, lane 7 is the reference medicinal material solution, lane 8 is the schisandrin a reference solution, and lanes 9-10 are the test solution.

[0048] Figure 7 Thin-layer chromatographic results for the identification of 10 batches of black tiger root medicinal materials;

[0049] Figure 8 The characteristic chromatogram of the reference solution;

[0050] Figure 9 Characteristic chromatograms of test solution 1 and test solution 2;

[0051] Figure 10 Characteristic spectra of different extraction solvents;

[0052] Figure 11 Feature maps for different extraction times;

[0053] Figure 12 Characteristic spectra for different amounts of extraction solvent added;

[0054] Figure 13 Characteristic spectra for different extraction times;

[0055] Figure 14 Characteristic atlases of 15 batches of black old tree root medicinal materials;

[0056] Figure 15 The images show the chromatograms of the mixed reference solution and test solution 1. The top image shows the chromatogram of the mixed reference solution, and the bottom image shows the chromatogram of test solution 1. Detailed Implementation

[0057] This invention provides a method for extracting the effective components of black tiger root, the extraction method comprising ultrasonic extraction or a combination of heating reflux-extraction;

[0058] The ultrasonic extraction includes the following steps:

[0059] (1) Mix the black tiger root with extraction solvent 1 to obtain mixture 1; extract mixture 1 by ultrasound for 25-35 min to obtain an ultrasonic mixture;

[0060] (2) Cool the ultrasonic mixture, filter it, and take the filtrate to obtain the effective components of black tiger root;

[0061] The combined heating reflux-extraction extraction includes the following steps:

[0062] 1) Mix black tiger root and extraction solvent 2 to obtain mixture 2; heat mixture 2 under reflux for 25-35 min to obtain a heated reflux mixture;

[0063] 2) Cool the heated reflux mixture, filter it to obtain a filtrate; dry the filtrate to obtain the primary extract;

[0064] 3) Mix the primary extract with water to obtain a primary extract solution; mix the primary extract solution with an extraction solvent and extract to obtain the effective components of black tiger root;

[0065] In this invention, in step (1), before mixing the black tiger root and extraction solvent 1, the black tiger root needs to be crushed to a particle size of 40-60 mesh, preferably 45-55 mesh, and more preferably 50 mesh;

[0066] The extraction solvent 1 is a methanol solution and / or an ethanol solution; the mass fraction of the methanol solution is 90-100%, preferably 93-97%, more preferably 95%; the mass fraction of the ethanol solution is 90-100%, preferably 93-97%, more preferably 95%.

[0067] The extraction time is preferably 28-32 min, and more preferably 30 min;

[0068] The power of the ultrasound is 350-450W, preferably 380-420W, and more preferably 400W; the frequency of the ultrasound is 40-60kHz, preferably 45-55kHz, and more preferably 50kHz.

[0069] The mass-to-volume ratio of the black tiger root and extraction solvent 1 is 1g:20-30mL, preferably 1g:23-27mL, and more preferably 1g:25mL;

[0070] In this invention, in step (2), the cooling temperature is 20-30°C, preferably 23-27°C, and more preferably 25°C.

[0071] In this invention, in step 1), before mixing the black tiger root and the extraction solvent 2, the black tiger root needs to be crushed to a particle size of 40-60 mesh, preferably 45-55 mesh, and more preferably 50 mesh;

[0072] The extraction solvent 2 is a methanol solution with a mass fraction of 40-60%, preferably a methanol solution with a mass fraction of 45-55%, and more preferably a methanol solution with a mass fraction of 50%; the mass-volume ratio of the black tiger root and the extraction solvent 2 is 1g:20-30mL, preferably 1g:23-27mL, and more preferably 1g:25mL;

[0073] The heating and reflux treatment time is preferably 28 to 32 minutes, and more preferably 30 minutes.

[0074] In this invention, in step 2), the drying is water bath drying; the drying temperature is 98-102°C, preferably 99-101°C, and more preferably 100°C.

[0075] In this invention, in step 3), the extraction solvent is an ethyl acetate solution.

[0076] The mass-to-volume ratio of the primary extract to water is 1g:18-22mL, preferably 1g:19-21mL, and more preferably 1g:20mL;

[0077] The volume ratio of the primary extract solution to the extraction solvent is 20:18 to 22, preferably 20:19 to 21, and more preferably 20:20;

[0078] The extraction is performed 1 to 3 times, preferably 2 times.

[0079] This invention also provides the application of the effective components of black tiger root extracted by the extraction method described above in the quality identification of black tiger root.

[0080] This invention also provides a method for quality identification of black tiger root, the method including qualitative identification of the effective components of black tiger root, detection of the relative retention time of organic acid peaks, content detection, moisture detection, pesticide residue detection, heavy metal and harmful element detection;

[0081] The effective components of the black tiger root are those extracted by the extraction method described above.

[0082] In this invention, the method for moisture detection refers to Method II of General Chapter 0832, Part IV, Chinese Pharmacopoeia 2020 Edition;

[0083] The method for detecting pesticide residues refers to the fifth method in General Chapter 2341 of the First Supplement to the 2020 edition of the Chinese Pharmacopoeia, Part IV;

[0084] The method for detecting heavy metals and harmful elements refers to Inductively Coupled Plasma Mass Spectrometry (ICP-MS), General Chapter 2321, Part IV, Chinese Pharmacopoeia 2020 Edition.

[0085] In this invention, the qualitative identification method is thin-layer chromatography detection; the qualitative identification steps include: developing the effective components of black tiger root in a developing solvent, drying to obtain the analyte; spraying sulfuric acid ethanol solution onto the analyte, heating to develop color, and then examining it under ultraviolet light;

[0086] The developing agent is petroleum ether-ethyl acetate-formic acid, preferably the upper layer solution of petroleum ether-ethyl acetate-formic acid;

[0087] The volume ratio of petroleum ether-ethyl acetate-formic acid is 16-18:2-4:1, preferably 17:3:1;

[0088] The boiling range of the petroleum ether is 60–90°C;

[0089] The drying process refers to air drying.

[0090] The mass fraction of the sulfuric acid ethanol solution is 8-12%, preferably 9-11%, and more preferably 10%.

[0091] In the qualitative identification process, cyclic-open-ring schisandrin a and schisandrin a were used as reference standards.

[0092] Before the experiment, the reference medicinal material, reference standard, and test sample need to be prepared into a reference medicinal material solution, a reference standard solution, and a test sample solution, respectively.

[0093] The reference solution is prepared by dissolving the open-ring schisandrin a and schisandrin a in methanol solution to obtain a final concentration of 1 mg / mL, which is the reference solution.

[0094] The method for preparing the test sample solution is as follows: the test sample is evaporated and concentrated to 1 mL to obtain the test sample solution;

[0095] The preparation method of the reference medicinal material solution is as follows: Weigh 1.0g of black tiger root reference medicinal material, add 20mL of ethanol to dissolve it, sonicate it for 30min at a power of 400W and a frequency of 50KHz, cool it to 25℃, filter it, and evaporate and concentrate the filtrate to 1mL to obtain the reference medicinal material solution.

[0096] When developing, the amount of sample of the reference medicinal material solution is 1-5 μl, preferably 2.5 μl, the amount of sample of the reference standard solution is 1-5 μl, preferably 2.5 μl, and the amount of sample of the test solution is 2-10 μl, preferably 6 μl.

[0097] The heating temperature is 100-110°C, preferably 103-107°C, and more preferably 105°C;

[0098] The heating time is 2 to 3 minutes, preferably 2.5 minutes;

[0099] The ultraviolet wavelength is 360–370 nm, preferably 363–367 nm, and more preferably 365 nm.

[0100] In this invention, the organic acids include meso dihydroguaiac acid, open-ring schisandrin a, schisandrin a, and (+)-(8s,8′R)-3′,4,4′-trihydroxy-5,5′-dimethoxyignan.

[0101] The reference solution used in the detection of the relative retention time of organic acid peaks is a methanol solution and includes the following components at final concentrations:

[0102] Mesodihydroguaiacol contains 30–50 μg / mL, preferably 35–45 μg / mL, and more preferably 40 μg / mL.

[0103] The concentration of the ring-opening schisandrin a is 30–50 μg / mL, preferably 35–45 μg / mL, and more preferably 40 μg / mL.

[0104] The concentration of schisandrin a in the new southern region is 30–50 μg / mL, preferably 35–45 μg / mL, and more preferably 40 μg / mL.

[0105] (+)-(8s,8′R)-3′,4,4′-trihydroxy-5,5′-dimethoxylignan 30-50 μg / mL, preferably 35-45 μg / mL, more preferably 40 μg / mL;

[0106] In this invention, the components for content detection include open-ring schisandrin a and schisandrin a.

[0107] In this invention, the standard for qualitative identification is: in the chromatogram of the test sample, two identical blue fluorescent spots appear at the corresponding positions as in the chromatogram of the reference medicinal material, and in the chromatogram of the reference substance, two identical blue fluorescent spots appear at the corresponding positions.

[0108] The standard for detecting the relative retention times of the organic acid peaks is as follows: the chromatogram of the test sample should show 8 characteristic peaks, and the retention times should correspond to 4 characteristic peaks in the chromatogram of the reference standard. Among them, peaks 3, 5, and 8 should correspond to the retention times of the (+)-(8s,8′R)-3′,4,4′-trihydroxy-5,5′-dimethoxylignan reference standard, meso dihydroguaiac acid reference standard, and neoschisandrin a reference standard, respectively. The peak corresponding to the reference peak of the open-ring schisandrin a is the S peak. Calculate the relative retention time of each characteristic peak and the S peak. The relative retention time of peaks 5, 6, and 8 should be within ±8% of the specified value. The relative retention time of peaks 1, 2, 3, and 4 should be within ±10% of the specified value. The specified values ​​are 0.14 (peak 1), 0.45 (peak 2), 0.52 (peak 3), 0.58 (peak 4), 0.63 (peak 5), 0.83 (peak 6), and 1.05 (peak 8).

[0109] The standard for content detection is as follows: the total amount of schistosic acid a and schistosic acid a in this product, calculated on a dried basis, shall not be less than 18.0402 mg / g.

[0110] The standard for moisture content testing is as follows: the moisture content shall not exceed 15% according to the moisture determination method (General Rule 0832, Method II).

[0111] The pesticide residue detection, heavy metal and harmful element detection comply with the provisions of the Chinese Pharmacopoeia.

[0112] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0113] In the embodiments of the present invention, the open-ring schisandrin a (124817-74-9, 99.78%) and schisandrin a (123929-80-6, 99.44%) were purchased from Shanghai Shidande Standard Technical Service Co., Ltd.; the batch numbers and places of origin of 15 batches of black tiger root medicinal materials are shown in Table 1.

[0114] Table 115: Batch Numbers and Origins of Black Tiger Root Medicinal Materials

[0115] Serial Number batch number Place of origin 1 HLH002 Lianshan, Guangdong 2 HLH003 Tianlin, Guangxi 3 HLH004 Shangsi, Guangxi 4 HLH005 Qingyuan, Guangdong 5 HLH006 Longzhou, Guangxi 6 HLH008 Yangshan, Guangdong 7 HLH009 Huaiji, Guangdong 8 HLH010 Renhua, Guangdong 9 HLH012 Lipu, Guangxi 10 HLH015 Yulin, Guangxi 11 HLH016 Meizhou, Guangdong 12 HLH017 Huizhou, Guangdong 13 HLH018 Shantou, Guangdong 14 HLH019 Yulin, Guangxi 15 YP202209 Yulin, Guangxi

[0116] Example 1 Thin-layer chromatography identification

[0117] Preparation of reference solution: The cyclic schisandrin a and schisandrin a were dissolved in methanol solution to prepare a solution with a final concentration of 1 mg / mL, which is the reference solution.

[0118] Preparation of test solution: (1) 1.0g of black tiger root was crushed to 50 mesh and then mixed with 20mL of 100% ethanol solution to obtain mixture 1; mixture 1 was ultrasonically treated for 30min at a power of 400W and a frequency of 50KHz to obtain ultrasonic mixture;

[0119] (2) Cool the ultrasonic mixture to 25°C, filter it, and take the filtrate to obtain the effective components of black tiger root;

[0120] (3) The effective components of black tiger root were evaporated and concentrated to 1 mL in a water bath at 100°C to obtain the test solution.

[0121] Preparation of the reference medicinal material solution: Weigh 1.0 g of black tiger reference medicinal material (China National Institutes for Food and Drug Control), add 20 mL of ethanol to dissolve it, cool to 25℃, filter, and evaporate and concentrate the filtrate to 1 mL in a water bath at 100℃ to obtain the reference medicinal material solution.

[0122] Thin-layer chromatography identification method: Take the test solution, the reference solution of schisandrin a, the reference solution of schisandrin a, and the reference medicinal material solution and spot them on the same silica gel G thin-layer plate. Use the upper layer of petroleum ether (60-90℃)-ethyl acetate-formic acid (volume ratio of 17:3:1) as the developing solvent. Develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ for 3 minutes to develop color, and examine under ultraviolet light (365nm).

[0123] Thin-layer chromatography (TLC) was used to detect 2 μl, 5 μl, and 10 μl of the test solution, respectively. The detection chromatograms are shown below. Figure 1 As shown;

[0124] Take 2 μl, 4 μl, and 5 μl of the test solution, 1 μl, 2 μl, and 5 μl of the open-ring schisandrin a reference solution, 1 μl, 2 μl, and 5 μl of the schisandrin a reference solution, and 1 μl, 2 μl, and 5 μl of the reference medicinal material solution, respectively, and perform thin-layer chromatography identification on the test solution, the open-ring schisandrin a reference solution, the schisandrin a reference solution, and the reference medicinal material solution. The results are as follows: Figure 2 As shown;

[0125] from Figures 1-2 As can be seen, when the sample volume of the test sample solution is 2-10 μl, the sample volume of the reference medicinal material solution is 1-5 μl, and the sample volume of the reference standard solution is 1-5 μl, the spots are clear after development, the separation is good, and there is no tailing. For ease of observation, the sample volume of the test sample solution is 2 μl, the sample volume of the reference medicinal material solution is 1 μl, and the sample volume of the reference standard solution is 2 μl.

[0126] Example 2

[0127] Thin-layer plate:

[0128] Take 2 μl of the test solution, 2 μl of the reference solution, and 1 μl of the reference medicinal material solution, respectively. Use the thin-layer chromatography identification method described in Example 1 to detect the test solution, the cyclic schisandrin a reference solution, the schisandrin a reference solution, and the reference medicinal material solution. The silica gel G thin-layer plates used in the detection process were from Qingdao Ocean, Mark, and Yantai Chemical, respectively. The results are as follows: Figure 3 As shown;

[0129] from Figure 3 As can be seen, thin-layer boards from different brands can all meet the identification requirements.

[0130] Temperature for identification:

[0131] Take 2 μl of the test solution, 2 μl of the reference solution, and 1 μl of the reference medicinal material solution, respectively. Use the thin-layer chromatography identification method described in Example 1 to detect the test solution, the cyclic-open-ring schisandrin a reference solution, the schisandrin a reference solution, and the reference medicinal material solution. During the detection process, the chromatography was performed at 4℃ and 25℃, respectively. The results are as follows: Figure 4 As shown;

[0132] from Figure 4 As can be seen, there are no significant differences in the chromatograms under different temperature conditions, and all of them can meet the identification requirements, indicating that the thin-layer chromatography identification method described in Example 1 has good durability under temperature conditions.

[0133] Humidity:

[0134] Take 2 μl of the test solution, 2 μl of the reference solution, and 1 μl of the reference medicinal material solution, respectively. Use the thin-layer chromatography identification method described in Example 1 to detect the test solution, the cyclic schistosic acid a reference solution, the schistosic acid a reference solution, and the reference medicinal material solution. During the detection process, the chromatography was carried out at 50% and 78% humidity, respectively. The results are as follows: Figure 5 As shown;

[0135] from Figure 5 As can be seen, the chromatograms under different humidity conditions are not significantly different and can all meet the identification requirements, indicating that the thin-layer chromatography identification method described in Example 1 has good durability under different humidity conditions.

[0136] Exclusivity:

[0137] Take 2 μl of the test solution, 2 μl of the reference solution, 2 μl of the negative control solution (ethanol solution), and 1 μl of the reference medicinal material solution, respectively. Use the thin-layer chromatography identification method described in Example 1 to detect the test solution, the cyclic schisandrin a reference solution, the schisandrin a reference solution, and the reference medicinal material solution. The results are as follows: Figure 6 As shown;

[0138] from Figure 6 As can be seen from the chromatogram, the test sample chromatogram shows two blue fluorescent spots of the same color at the corresponding positions as the reference medicinal material chromatogram, and the negative control chromatogram shows no spots corresponding to the reference medicinal material, reference material, and test sample. This indicates that the negative control solution does not interfere with the identification. The test sample chromatogram shows corresponding spots to the reference medicinal material and reference material, indicating that the thin-layer chromatography identification method described in Example 1 has good specificity.

[0139] Example 3 Sample Determination

[0140] The method described in Example 1 was used to identify 10 batches of black tiger root medicinal materials (Table 2), and the results are as follows: Figure 7 As shown.

[0141] Table 210: Sampling Information for Black Tiger Root Medicinal Materials

[0142] Serial Number batch number Sample quantity Serial Number batch number Sample quantity 1 HLH002 2μl 8 HLH010 2μl 2 HLH003 2μll 9 HLH012 2μl 3 HLH004 2μl 10 HLH015 2μl 4 HLH005 2μl 11 Open-ring schisandrin a 2μl 5 HLH006 2μl 12 Schisandra chinensis acid a 2μl 6 HLH008 2μl 13 Black Tiger Root Reference Material 1μl 7 HLH009 2μl / / /

[0143] from Figure 7 As can be seen from the chromatogram, the test sample shows a main spot with the corresponding fluorescent color at the corresponding position on the chromatogram of the reference medicinal material and the reference standard.

[0144] Example 4 Feature Map Detection

[0145] Reference solution: in methanol as solvent, comprising the following components at final concentrations: meso dihydroguaiac acid 40 μg / mL, schisandrin a 40 μg / mL, schisandrin a 40 μg / mL, (+)-(8s,8′R)-3′,4,4′-trihydroxy-5,5′-dimethoxyignan 40 μg / mL;

[0146] Preparation of test solution 1 (heated reflux extraction): 1) Crush 1.0 g of black tiger root to 50 mesh, then mix with 25 mL of 50% methanol solution to obtain mixture 2; heat mixture 2 under reflux for 30 min to obtain heated reflux mixture;

[0147] 2) Heat the reflux mixture and cool it for 25 minutes, then filter to obtain the filtrate; place the filtrate in a water bath at 100°C and dry it to obtain the initial extract;

[0148] 3) Mix the primary extract with water at a mass-volume ratio of 1g:20mL to obtain the primary extract solution; mix the primary extract solution with ethyl acetate solution at a volume ratio of 20:20 and extract twice, combine the extracts to obtain the effective components of black tiger root;

[0149] 4) Evaporate the active ingredients of black tiger root to dryness, dissolve in methanol solution, transfer to a 5 mL volumetric flask, and dilute to the mark to obtain test solution 1;

[0150] Preparation of test solution 2 (ultrasonic extraction): 1) 1.0 g of black tiger root was crushed to 50 mesh and then mixed with 25 mL of 50% methanol solution to obtain mixture 3; mixture 3 was ultrasonically treated at 400 W power and 50 kHz frequency for 30 min to obtain ultrasonic mixture;

[0151] 2) Cool the ultrasonic mixture to 25°C, filter it to obtain the filtrate; place the filtrate in a water bath at 100°C and dry it to obtain the initial extract;

[0152] 3) Mix the primary extract with water at a mass-volume ratio of 1g:20mL to obtain the primary extract solution; mix the primary extract solution with ethyl acetate solution at a volume ratio of 20:20 and extract twice, combine the extracts to obtain the effective components of black tiger root;

[0153] 4) Evaporate the active ingredients of black tiger root to dryness, dissolve in methanol solution, transfer to a 5 mL volumetric flask, and dilute to the mark to obtain test solution 2;

[0154] Characteristic chromatographic detection: Inject 10 μl of the reference solution, test solution 1, and test solution 2 into the liquid chromatograph for detection. The detection results are as follows: Figures 8-9 As shown;

[0155] The chromatographic conditions for the characteristic spectrum detection are as follows: Octadecylsilane-bonded silica gel is used as the packing material; the recommended column is CAPCELLPAKMGⅡC18 (5μm, 250×4.6mm); acetonitrile (A)-0.1% phosphoric acid solution (B) is used as the mobile phase, and elution is performed according to the gradient described in Table 3; the detection wavelength is 210nm; the theoretical plate number, calculated based on the open-ring schisandrin a, should not be less than 3000.

[0156] Table 3 Gradient Elution

[0157] Time (min) Mobile phase A (%) Mobile phase B (%) 0～14 8→30 92→70 14～20 30→50 70→50 20～35 50→60 50→40 35～50 60→95 40→5 50～60 95 5 60～61 95→8 5→92 61～68 8 92

[0158] from Figures 8-9 As can be seen from the data, heating reflux extraction has a higher chromatographic response and better peak shape than ultrasonic extraction. The combined heating reflux-extraction method produces more complete chromatographic peaks, so the combined heating reflux-extraction method is selected.

[0159] Example 5

[0160] Refer to Example 4 for the reference solution;

[0161] The test solution was prepared according to the preparation of test solution 1 described in Example 4, wherein the extraction solvents were 30% methanol, 50% methanol, 100% methanol, 30% ethanol, 50% ethanol, and 100% ethanol, respectively, to obtain the test solution.

[0162] The test solution was subjected to characteristic spectrum detection according to the method described in Example 4, and the detection results are as follows: Figure 10 As shown;

[0163] from Figure 10 As can be seen from the data, chromatographic response is better with 50% methanol as solvent, characteristic peaks are more complete, peak shapes are better, and baselines are more stable. Therefore, 50% methanol solvent was selected for extraction.

[0164] Example 6

[0165] Refer to Example 4 for the reference solution;

[0166] The test solution was prepared according to the preparation of test solution 1 described in Example 4, wherein the extraction times were 15 min, 30 min, 45 min, and 60 min, respectively, to obtain the test solution;

[0167] The test solution was subjected to characteristic spectrum detection according to the method described in Example 4, and the detection results are as follows: Figure 11 As shown;

[0168] from Figure 11 It can be seen that the chromatographic response at an extraction time of 15 min is lower than that at 30, 45, and 60 min, while there is no significant difference in the chromatographic response at 30, 45, and 60 min. Therefore, a suitable extraction time of 30 min was selected.

[0169] Example 7

[0170] Refer to Example 4 for the reference solution;

[0171] The test solution was prepared according to the preparation of test solution 1 described in Example 4, wherein the amount of extraction solvent added was 15 mL, 25 mL, and 30 mL, respectively, to obtain the test solution.

[0172] The test solution was subjected to characteristic spectrum detection according to the method described in Example 4, and the detection results are as follows: Figure 12 As shown;

[0173] from Figure 12 As can be seen, the chromatographic response was low when extracted with 15 mL solvent, and there was no significant difference in the chromatographic response between 25 mL and 50 mL solvent extraction. The baselines were relatively stable. Considering the cost of reagents, 25 mL was selected as the appropriate solvent volume.

[0174] Example 8

[0175] Refer to Example 4 for the reference solution;

[0176] The test solution was prepared according to the preparation of test solution 1 in Example 4, wherein the extraction was performed 2 times and 3 times respectively to obtain the test solution;

[0177] The test solution was subjected to characteristic spectrum detection according to the method described in Example 4, and the detection results are as follows: Figure 13 As shown;

[0178] from Figure 13 As can be seen, there was no significant difference in the chromatographic response between different extraction times, and the baselines were all relatively stable. Therefore, the simpler method of extraction with ethyl acetate twice was selected.

[0179] Example 9

[0180] Refer to Example 4 for the reference solution;

[0181] The test solution was prepared by preparing 15 batches of black old tree root medicinal materials according to the preparation method of test solution 1 described in Example 4;

[0182] The test solution was subjected to characteristic spectrum detection according to the method described in Example 4, and the detection results are as follows: Figure 14 As shown in Table 4-5, the peak time of each characteristic peak was calculated. Using the S peak as a control, the relative retention time, relative peak area, and RSD value of the remaining characteristic peaks were calculated.

[0183] Table 415 shows the relative retention time of black tiger root medicinal materials.

[0184]

[0185]

[0186] Table 515 shows the relative peak area of ​​black tiger root medicinal materials.

[0187]

[0188]

[0189] The results showed that the relative retention time RSD of 15 batches of black tiger root medicinal materials was less than 3.0%, indicating that the elution time of each peak was consistent among batches; however, there were significant differences in the relative peak area among batches, so only the relative retention time was specified, while the relative peak area was not specified.

[0190] Based on the characteristic spectra of 15 batches of medicinal materials, 8 common peaks with good repeatability were selected as characteristic peaks. The relative retention time RSD of the 8 characteristic peaks of the 15 batches of black tiger root was less than 3.0%, indicating that the elution time of each characteristic peak was consistent among the batches. The relative peak area RSD of each characteristic peak was greater than 10%, indicating that there were significant differences in the relative peak area among the batches. Therefore, only the relative retention time was specified, while the relative peak area was not specified. The relative retention time of all samples was within ±8%, but considering the influence of different instruments and experimental conditions, the specified range of the relative retention time of each characteristic peak was specified as ±10%.

[0191] Based on the determination results of 15 batches of black tiger root, it is stipulated that: there should be a total of 8 characteristic peaks in the chromatogram of the test sample, and the retention times should correspond to the 4 characteristic peaks in the chromatogram of the reference standard. Among them, peaks 3, 5, and 8 should correspond to the retention times of the (+)-(8s,8′R)-3′,4,4′-trihydroxy-5,5′-dimethoxyignan reference standard, meso dihydroguaiac acid reference standard, and schisandrin a reference standard, respectively; and correspond to the retention times of the open-ring schisandrin a reference standard. The peak corresponding to each peak is called peak S. Calculate the relative retention times of peaks 1, 2, 3, 4, 5, 6, and 8 with peak S. The relative retention times of peaks 5, 6, and 8 should be within ±8% of the specified value, and the relative retention times of peaks 1, 2, 3, and 4 should be within ±10% of the specified value. The specified values ​​are: peak 1 (0.140), peak 2 (0.446), peak 3 (0.516), peak 4 (0.586), peak 5 (0.632), peak 6 (0.826), and peak 8 (1.045).

[0192] Example 10 Content Detection

[0193] Mixed reference solution: in methanol as solvent, comprising the following components at final concentrations: 0.20 mg / mL of open-ring schisandrin a and 0.20 mg / mL of schisandrin a;

[0194] Preparation of test solution 1: 1) Crush 1.0g of black tiger root to 50 mesh, then mix with 25mL of 100% ethanol solution to obtain mixture 1 and weigh it; sonicate mixture 1 at 400W power and 50KHz frequency for 30min to obtain ultrasonic mixture;

[0195] (2) Cool the ultrasonic mixture to 25°C, filter it, and take the filtrate to obtain the effective components of black tiger root;

[0196] (3) Make up the weight loss with methanol, shake well, filter through a 0.22 μm filter membrane to obtain test solution 1;

[0197] Preparation of test solution 2: 1) Crush 1.0g of black tiger root to 50 mesh, then mix with 25mL of 50% methanol solution to obtain mixture 2 and weigh it; heat mixture 2 under reflux for 30min to obtain reflux mixture;

[0198] 2) Heat the reflux mixture and cool it for 25 minutes, then filter to obtain the filtrate; place the filtrate in a water bath at 100°C and dry it to obtain the initial extract;

[0199] 3) Mix the primary extract with water at a mass-volume ratio of 1g:20mL to obtain the primary extract solution; mix the primary extract solution with ethyl acetate solution at a volume ratio of 20:20 and extract twice, combine the extracts to obtain the effective components of black tiger root;

[0200] 4) Make up the weight loss with methanol, shake well, filter through a 0.22μm filter membrane to obtain test solution 2;

[0201] Content determination: Accurately pipette 10 μl each of test solution 1, test solution 2, and mixed reference solution into the liquid chromatograph, obtain the chromatograms, and measure the peak areas. The results are as follows: Figure 15 As shown in Table 7, the contents of open-ring schisandrin a and schisandrin a were calculated based on the peak area.

[0202] The chromatographic conditions are as follows: octadecylsilane-bonded silica gel is used as the packing material; the recommended column is a CAPCELL PAKMGⅡ C18 column (4.6 × 250 mm, 5 μm); the mobile phase is gradient elution with acetonitrile (A) - 0.1% phosphoric acid (B) (as shown in Table 6); the detection wavelength is 210 nm; the column temperature is 35 ℃; the flow rate is 1.0 mL / min; the injection volume is 10 μl; the run time is 25 min; and the theoretical plate number, calculated based on the open-ring schisandrin a, should not be less than 3000.

[0203] Table 6 Gradient Elution Table

[0204] Time (min) Mobile phase A (%) Mobile phase B (%) 0～5 70→95 30→5 5～18 95 5 18～19 95→70 5→30 19～25 70 30

[0205] Table 7 Content of open-ring schisandrin a and schisandrin a

[0206] Test sample solution Open-ring schisandrin a (mg / g) Schisandra chinensis acid a (mg / g) 1 14.7100 7.6481 2 14.7162 7.6580

[0207] The results showed that there was no significant difference in the content of schisandrin a and schisandrin a in the sample between the extraction methods of heating reflux and ultrasonic treatment. Due to the simplicity of ultrasonic treatment, ultrasonic treatment for 30 min was chosen as the extraction method.

[0208] Example 11

[0209] Refer to Example 10 for the mixed reference solution;

[0210] The test solution was prepared according to the preparation of test solution 1 described in Example 10, wherein the extraction solvents were 30% methanol, 50% methanol, 100% methanol, 30% ethanol, 50% ethanol, and 100% ethanol, respectively, to obtain the test solution;

[0211] The contents of schisandrin a and schisandrin a in the test solution were determined according to the method described in Example 10. The test results are shown in Table 8.

[0212] Table 8. Content of open-ring schisandrin a and schisandrin a

[0213] Extraction solvent Open-ring schisandrin a (mg / g) Schisandra chinensis acid a (mg / g) 30% methanol Not detected Not detected 50% methanol Not detected Not detected 100% methanol 14.7100 7.6481 30% ethanol Not detected Not detected 50% ethanol 5.0289 Not detected 100% ethanol 14.6047 7.5924

[0214] As can be seen from Table 8, methanol and ethanol solutions yield higher content, with the content increasing progressively with increasing alcohol content. There is no significant difference in content between the two extraction solvents, methanol and ethanol, with methanol extraction yielding slightly higher content than ethanol. Considering all factors, 100% methanol solution was ultimately chosen as the extraction solvent because methanol solution yields higher content.

[0215] Example 12

[0216] Refer to Example 10 for the mixed reference solution;

[0217] The test solution was prepared according to the preparation of test solution 1 described in Example 10, wherein the ultrasonic time was 15 min, 30 min, 45 min, and 60 min, respectively, to obtain the test solution;

[0218] The contents of schisandrin a and schisandrin a in the test solution were determined according to the method described in Example 10. The test results are shown in Table 9.

[0219] Table 9. Content of open-ring schisandrin a and schisandrin a from Xinan.

[0220] Ultrasound time (min) Open-ring schisandrin a (mg / g) Schisandra chinensis acid a (mg / g) 15 13.4798 7.4062 30 14.7100 7.6481 45 14.6321 7.6161 60 14.9730 7.9756

[0221] As can be seen from Table 9, the content increases gradually with the extension of ultrasonic time; there is no significant difference in content between extraction times of 30 min, 45 min, and 60 min. Taking all factors into consideration, a moderate extraction time of 30 min is selected as the ultrasonic time.

[0222] Example 13

[0223] Refer to Example 10 for the mixed reference solution;

[0224] The test solution was prepared according to the preparation of test solution 1 described in Example 10, wherein the amount of methanol solution added was 15 mL, 25 mL, and 50 mL, respectively, to obtain the test solution.

[0225] The contents of cyclic schisandrin a and schisandrin a were determined in the test solution according to the method described in Example 10. The test results are shown in Table 10.

[0226] Table 10 Content of open-ring schisandrin a and schisandrin a

[0227] Solvent volume (mL) Open-ring schisandrin a (mg / g) Schisandra chinensis acid a (mg / g) 15 13.9484 7.3575 25 14.7100 7.6481 50 14.8573 8.0541

[0228] As can be seen from Table 10, the content is relatively low when the solvent volume is 15 mL, and there is no significant difference in content between 25 mL and 50 mL. In order to save reagents, considering all factors, a moderate solvent volume of 25 mL is selected as the extraction solvent volume for the test sample.

[0229] Example 14

[0230] The mixed reference solution is shown in Example 10;

[0231] The test solution was prepared by preparing 15 batches of black old tree root medicinal materials according to the preparation method of test solution 1 described in Example 10;

[0232] The contents of schisandrin a and schisandrin a in the test solution were determined according to the method described in Example 10. The test results are shown in Table 11.

[0233] Table 11 Content of open-ring schisandrin a and schisandrin a

[0234]

[0235]

[0236] Based on the content data of 15 batches of black tiger root medicinal materials, the average content of the 15 batches of cyclic schisandrin a was 22.3942 mg / g; the average total content of cyclic schisandrin a and schisandrin a was 42.3638 mg / g; according to the average plus or minus 2 times the SD, it is tentatively determined that the total content of cyclic schisandrin a and schisandrin a in this product, calculated on a dried basis, should not be less than 18.0402 mg / g.

[0237] Example 15 Moisture Detection

[0238] The moisture content of 15 batches of black tiger root was determined according to Method 2 of General Chapter 0832 in Part IV of the 2020 edition of the Chinese Pharmacopoeia. The test results are shown in Table 12.

[0239] Table 1215 shows the moisture content test results of black tiger root medicinal materials.

[0240]

[0241]

[0242] The results show that the moisture content should not exceed 15%.

[0243] Example 16 Detection of Heavy Metals and Harmful Elements

[0244] The contents of lead, cadmium, arsenic, mercury and copper in 15 batches of black tiger root were determined by inductively coupled plasma mass spectrometry according to the General Chapter 2321 of Part IV of the 2020 edition of the Chinese Pharmacopoeia. The test results are shown in Table 13.

[0245] Table 1315 shows the test results of heavy metals and harmful elements in black tiger root medicinal materials.

[0246]

[0247]

[0248] As shown in Table 13, the levels of heavy metals and harmful elements meet the requirements.

[0249] Example 17 Pesticide Residue Detection

[0250] The residues of 33 prohibited pesticides, including methamidophos, methyl parathion, parathion, monocrotophos, phosphamidon, total hexachlorocyclohexane, and total DDT, were determined in 15 batches of black tiger root medicinal materials according to Method 5 of Part IV General Chapter 2341 of the 2020 edition of the Chinese Pharmacopoeia. The test results are shown in Table 14.

[0251] Table 14 shows the pesticide residue test results for batch 15 of black tiger root medicinal materials.

[0252]

[0253]

[0254] As can be seen from Table 14, the residue levels of the 33 banned pesticides meet the regulations.

[0255] As can be seen from the above embodiments, the present invention provides a method for extracting the effective components of black tiger root and its application, as well as a method for quality identification of black tiger root. The extraction method of the present invention can safely and effectively extract the effective components of black tiger root. By qualitatively identifying the effective components of black tiger root, detecting the relative retention time of organic acid peaks, detecting content, detecting moisture, detecting pesticide residues, detecting heavy metals and harmful elements, the quality of tiger root medicinal materials can be controlled.

[0256] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for extracting the effective components from the root of *Hemiberlesia lataniae*, characterized in that, The extraction method includes ultrasonic extraction or a combined heating reflux-extraction extraction; The ultrasonic extraction includes the following steps: (1) Mix the black tiger root with extraction solvent 1 to obtain mixture 1; extract mixture 1 by ultrasound for 25-35 min to obtain an ultrasonic mixture; (2) Cool the ultrasonic mixture, filter it, and take the filtrate to obtain the effective components of black tiger root; In step (1), the extraction solvent 1 is a methanol solution and / or an ethanol solution; the mass fraction of the methanol solution is 90-100%; the mass fraction of the ethanol solution is 90-100%. In step (1), the power of the ultrasound is 350-450W; the frequency of the ultrasound is 40-60kHz. The combined heating reflux-extraction extraction includes the following steps: 1) Mix black tiger root and extraction solvent 2 to obtain mixture 2; heat mixture 2 under reflux for 25-35 min to obtain a heated reflux mixture; 2) Cool the heated reflux mixture, filter it to obtain a filtrate; dry the filtrate to obtain the primary extract; 3) Mix the primary extract with water to obtain a primary extract solution; mix the primary extract solution with an extraction solvent and extract to obtain the effective components of black tiger root; In step 1), the extraction solvent 2 is a methanol solution with a mass fraction of 40-60%; In step 3), the extraction solvent is an ethyl acetate solution.

2. The extraction method according to claim 1, characterized in that, In step (1), the mass-to-volume ratio of the black tiger root and extraction solvent 1 is 1g: 20-30mL; In step (2), the cooling temperature is 20-30°C.

3. The extraction method according to claim 2, characterized in that, In step 1), the mass-to-volume ratio of the black tiger root and the extraction solvent 2 is 1g: 20-30mL.

4. The extraction method according to claim 3, characterized in that, In step 2), the drying is water bath drying; the drying temperature is 98-102℃.

5. The extraction method according to claim 4, characterized in that, In step 3), the mass-to-volume ratio of the initial extract to water is 1g:18-22mL; The volume ratio of the primary extract solution to the extraction solvent is 20:18 to 22; The extraction is performed 1 to 3 times.

6. The application of the effective components of black tiger root extracted by the extraction method according to any one of claims 1 to 5 in the quality identification of black tiger root.

7. A method for quality identification of black tiger root, characterized in that, The method includes qualitative identification of the effective components of black tiger root, detection of the relative retention time of organic acid peaks, content detection, moisture detection, pesticide residue detection, heavy metal and harmful element detection; The effective components of the black tiger root are those extracted by the extraction method described in any one of claims 1 to 5.

8. The method according to claim 7, characterized in that, The qualitative identification method is thin-layer chromatography detection; the qualitative identification steps include: developing the effective components of black tiger root in a developing solvent, drying to obtain the analyte; spraying sulfuric acid ethanol solution onto the analyte, heating to develop color, and then examining it under ultraviolet light; The developing solvent is petroleum ether-ethyl acetate-formic acid; The volume ratio of petroleum ether-ethyl acetate-formic acid is 16-18:2-4:1; The heating temperature is 100–110°C; The heating time is 2 to 3 minutes.

9. The method according to claim 8, characterized in that, The organic acids include meso-dihydroguaiac acid, open-ring schisandrin a, schisandrin a, and (+)-(8s,8′R)-3′,4,4′-trihydroxy-5,5′-dimethoxylignan.

10. The method according to claim 9, characterized in that, The components detected in the content test include open-ring schisandrin a and schisandrin a.

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