Method for identifying schisandra chinensis and kadsura

Abstract

The invention discloses a method for identifying schisandra chinensis and kadsura longepedunculata. The method provided by the invention comprises the step of identifying whether a to-be-detected sample contains schisandra chinensis or not by detecting the content ratio of gomisin N to schisandrin B in the to-be-detected sample. The method provided by the invention can be used for rapidly identifying the schisandra chinensis and the kadsura longepedunculata, and a brand new identification and supervision means is provided for the counterfeit material feeding phenomenon of the schisandra chinensis in a prescription preparation.

Description

Technical Field

[0001] The present application relates to the field of analysis and detection, and in particular to a method for identifying Schisandra chinensis and Schisandra chinensis chinensis. Background Art

[0002] Schisandra chinensis and Schisandra chinensis are two varieties separately included in the 2020 edition of the Chinese Pharmacopoeia. They are derived from the dried mature fruits of Schisandra chinensis (Turcz.) Baill. and Schisandra phenanthera Rehd. et Wils., both of which are Magnoliaceae plants. The two have similar chemical compositions and mainly contain lignans, volatile oils, polysaccharides, triterpenes and organic acids. The functions and indications recorded in the pharmacopoeia are astringency, invigorating qi and promoting body fluid, and tonifying the kidney and calming the mind. For many years, the prescriptions for the two have been relatively chaotic; and the market prices of the two are very different. In the production process of Chinese patent medicines, there is a phenomenon of adulterating the raw materials with Schisandra chinensis instead of Schisandra chinensis, which is not conducive to the effectiveness of Schisandra chinensis prescription preparations.

[0003] There are a total of 107 Schisandrae Chinensis preparations included in the 2020 edition of the Chinese Pharmacopoeia, including 102 prescriptions containing northern Schisandrae Chinensis, 74 of which are extracted medicines, accounting for 72.5%, and 14 prescriptions containing southern Schisandrae Chinensis, 10 of which are extracted medicines. At present, there have been many studies on the identification of southern Schisandrae Chinensis and Schisandrae Chinensis. The researchers used the content ratio of anwuzhisu and schisandrae alcohol A to distinguish the adulteration of Fenghan Keke Tablets and Shihu Yeguang Pills, and the peak area ratio of the two was used for identification in Sishen Pills; in addition, there are also peak area ratios of schisandrae ester A / schisandrae alcohol A and schisandrae A / schisandrae B, and the false feeding of Jianganling Capsules was studied. At present, the existing research ideas are relatively fixed, and it is necessary to break through the existing chemical recognition mode and develop new and effective identification methods for southern Schisandrae Chinensis and Schisandrae Chinensis. Summary of the invention

[0004] The purpose of the present application is to provide a method for identifying Schisandra chinensis and Schisandra chinensis chinensis.

[0005] In order to solve the above technical problems, the first aspect of the present application provides a method for identifying Schisandra chinensis and Schisandra chinensis chinensis, the method comprising the steps of:

[0006] Whether the sample to be tested contains Schisandra chinensis is identified by detecting the content ratio of gomisin N to schisandrin B in the sample to be tested.

[0007] In some preferred embodiments, the method comprises the following steps:

[0008] S1, detecting the contents of gomisin N and schisandrin B in the sample to be tested;

[0009] S2, calculate the content ratio of gomisin N to schisandrin B;

[0010] S3, comparing the content ratio of gomisin N to schisandrae B with a preset value to identify whether the sample to be tested contains schisandrae.

[0011] In some preferred schemes, in step S3, the content ratio of gomisin N to schisandrin B is compared with a preset value. When the content ratio of gomisin N to schisandrin B is not higher than the preset value, the sample to be tested is Schisandra chinensis, otherwise it is not Schisandra chinensis.

[0012] In some preferred solutions, in step S3, the preset value is 0.21±5% to 0.28±5%.

[0013] In some preferred embodiments, in step S1, before detecting the sample to be tested, a sample pretreatment step is also included, and the sample pretreatment step includes: using a mixed solvent of water and ethanol to treat the sample to be tested to obtain an extract.

[0014] In some preferred embodiments, in the mixed solvent of water and ethanol, the volume proportion of ethanol is 60-90%, preferably 70%-80%, for example 75%.

[0015] In some preferred embodiments, the sample pretreatment step includes using a mixed solvent of water and ethanol to dissolve the sample to be tested, and heating and refluxing to obtain an extract.

[0016] In some preferred embodiments, liquid chromatography is used to detect the sample to be tested, and more preferably high performance liquid chromatography is used to detect the sample to be tested.

[0017] In some preferred embodiments, the chromatographic conditions of the liquid chromatography include:

[0018] Gradient elution was performed with acetonitrile as mobile phase A and water as mobile phase B.

[0019] In some preferred embodiments, the gradient elution procedure is shown in the following table:

[0020] Time (min) Mobile phase A (%) Mobile phase B (%) 0～6 5 95 6～7 5→45 95→55 7～25 45→50 55→50 25～35 50→70 50→30 35～45 70→90 30→10 .

[0021] In some preferred embodiments, the chromatographic conditions of the liquid chromatography further include: using a C18 chromatographic column for detection, preferably using a C18 chromatographic column whose filler is octadecylsilane bonded silica gel, such as an ACQUITY BEHC18 chromatographic column.

[0022] In some preferred embodiments, the chromatographic conditions of the liquid chromatography further include:

[0023] The detection wavelength is 280nm from 0 to 12 minutes and 220nm from 12 minutes to the end;

[0024] The column temperature is 30-40°C, for example 35°C;

[0025] The flow rate is 0.1-0.5 ml per minute, for example 0.3 ml.

[0026] In some preferred embodiments, in step S2, the content ratio of gomisin N and schisandrin B peaks is obtained by calculating the peak area ratio of gomisin N and schisandrin B.

[0027] In some preferred embodiments, the sample to be tested is a traditional Chinese medicine or a traditional Chinese medicine preparation.

[0028] In some preferred embodiments, the sample to be tested is a traditional Chinese medicine preparation, such as Schisandra chinensis granules, Shengmai Yin, Anshen capsules, Fenghan Keke granules or Zaoren Anshen capsules.

[0029] Based on the prior art, the present invention has at least the following advantages:

[0030] The present application provides a chemical evaluation method for identifying Schisandra chinensis and Schisandrae chinensis, and the evaluation method includes the peak area ratio or content ratio of gomisin N to schisandrin B. The present application firstly finds that there is a relatively fixed relationship between the peak area ratio or content ratio of gomisin N and schisandrin B in Schisandra chinensis, and the characteristic spectrum analysis by high performance liquid chromatography can be used for the rapid identification of Schisandra chinensis and Schisandrae chinensis, providing a new identification and supervision means for the phenomenon of counterfeit Schisandra chinensis in prescription preparations.

[0031] It should be understood that within the scope of this application, the above-mentioned technical features of this application and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] One or more embodiments are exemplarily described by the pictures in the corresponding drawings, and these exemplary descriptions do not constitute limitations on the embodiments.

[0033] Figure 1 It is a high performance liquid chromatogram of a Schisandra sample under HPLC-DAD mode according to an embodiment of the present invention (the peak positions of gomisin N and Schisandrin B are marked).

[0034] Figure 2 It is the characteristic spectrum of Schisandra chinensis Fructus (without gomisin N chromatographic peak) according to the embodiment of the present invention.

[0035] Figure 3 It is a chromatogram of gomisin N and schisandra chinensis B reference substances according to the examples of the present invention. DETAILED DESCRIPTION

[0036] After extensive and in-depth research, the inventors discovered for the first time that there is a relatively fixed relationship between the peak area ratio or content ratio of gomisin N and schisandrin B in Schisandra chinensis, and developed a characteristic spectrum analysis method based on high performance liquid chromatography. By analyzing the contents of gomisin N and schisandrin B in samples and calculating the peak area ratio or content ratio, Schisandra chinensis and its counterfeits in formula preparations can be quickly identified, providing a new identification and supervision means for the feeding phenomenon.

[0037] the term

[0038] Unless otherwise indicated, the term "or" means and is used interchangeably with the term "and / or".

[0039] As used herein, including the appended claims, singular forms of words such as "a," "an," and "the" include their corresponding plural referents unless the context clearly dictates otherwise.

[0040] As used in the present invention, the CAS of "gomisin N" is 69176-52-9. The CAS of "schizandrin B" is 61281-37-6.

[0041] As used in the present invention, the term "Schisandra chinensis" refers to the dried mature fruit of Schisandrasphenanthera Rehd. et Wils., a plant of the Magnoliaceae family.

[0042] As used in the present invention, the term "Schisandra chinensis" or "Northern Schisandra" refers to the dried mature fruit of Schisandra chinensis (Turcz.) Baill. of the Magnoliaceae family. Herein, "Southern Schisandra" is different from "Schisandra chinensis".

[0043] As used in the present invention, the term "processed product" refers to a product obtained by processing Chinese medicine through a processing process. Processing processes include clean products; cut products; fried products; blanched products; calcined products; charcoal-making products; steamed products; stewed products; simmered products; boiled products; wine products; vinegar products; salt products; roasted products with ginger juice; roasted products with honey; roasted products with oil; water-fried products; frosted products; stewed products; extracted products; dry distilled products; baked products; fermented products; germinated products; replicated products; special preparation products, etc. In an embodiment of the present invention, the processed Schisandra chinensis product is a vinegar product of Schisandra chinensis, also known as "vinegar Schisandra chinensis". In an embodiment of the present invention, the processed Schisandra chinensis product is a vinegar product of Schisandra chinensis, also known as "vinegar Schisandra chinensis".

[0044] As used in the present invention, the term "raw product" refers to Chinese herbal medicine that has not been processed by any processing technology.

[0045] As used in the present invention, the term "vinegar schisandra chinensis" or "vinegar southern schisandra chinensis" refers to schisandra chinensis or southern schisandra chinensis that has been fumigated with vinegar. According to the theory of traditional Chinese medicine, vinegar schisandra chinensis and schisandra chinensis; vinegar southern schisandra chinensis and southern schisandra chinensis are different in chemical composition, efficacy, therapeutic diseases, contraindications and adverse reactions.

[0046] In an embodiment of the present invention, the content ratio of gomisin N to schisandrin B in Schisandra chinensis / vinegar Schisandra chinensis or its counterfeit is detected and then compared with a preset value range. If it falls within the range, it is considered that the sample batch to be tested belongs to Schisandra chinensis or its vinegar product. If it is not within the range, the sample is considered to be a counterfeit.

[0047] In one embodiment, the sample to be tested is detected by chromatography, and the content ratio of gomisin N to schisandrin B can also be replaced by a peak area ratio or a peak height ratio.

[0048] In one embodiment, the preset value ranges from 0.21±5% to 0.28±5%.

[0049] In one embodiment, liquid chromatography is used to detect the sample to be tested, and the peak area ratio of gomisin N to schisandrin B is 0.236, falling within the range of 0.21±5% to 0.28±5%, and the sample to be tested is Schisandra chinensis.

[0050] In one embodiment, liquid chromatography is used to detect the sample to be tested, and the peak area ratio of gomisin N to schisandrin B is 0.262, falling within the range of 0.21±5% to 0.28±5%, and the sample to be tested is vinegar schisandrae chinensis.

[0051] In one embodiment, liquid chromatography is used to detect the sample to be tested, and the peak area ratio of gomisin N to schisandrin B is 0, and the sample to be tested is Schisandra chinensis var.

[0052] In one embodiment, before using liquid chromatography to detect the sample, 75% ethanol (containing 75% ethanol aqueous solution by volume) is used to reflux extract the sample to obtain an extract. The alcohol reflux extraction method provides better stability.

[0053] Any method can be used to detect the content ratio of gomisin N and schisandrin B in the sample to be tested. The content ratio here also includes the use of any other ratio that directly or indirectly represents the content ratio. For example, when using chromatography to detect the sample, the ratio of peak area or peak height can be used. When the peak area ratio is used to indirectly represent the content ratio, its range is also within the range of the preset values ​​of the present invention, for example, within 0.21±5% to 0.28±5%.

[0054] In one embodiment, liquid chromatography is used to detect the sample to be tested. When the liquid chromatography is used to detect the sample to be tested, the chromatographic conditions (such as chromatographic column, column temperature, flow rate, mobile phase, elution procedure, etc.) are not particularly limited, as long as they can meet the requirements of the accuracy, precision, etc. in the detection standard. In one embodiment, the separation degree of each peak in the chromatogram is not less than 1.5.

[0055] In one embodiment, the sample to be tested is a Chinese medicine formula preparation such as a tablet, capsule, granule, pill or liquid preparation.

[0056] In one embodiment, the sample to be tested is selected from Wuzi Yanzong Tablets, Sishen Tablets, Ningshen Buxin Tablets, Anshen Buxin Pills, Ruyi Dingchuan Tablets, Funingkang Tablets, Gynecological Stopping Tablets, Gengnian An Tablets, Liver Protection Tablets, Bushen Yinao Tablets, Guben Kechuan Tablets, Shenqi Wuweizi Tablets, Shenqi Jiangtang Tablets, Shenrong Guben Tablets, Baizi Yangxin Tablets, Yangyin Jiangtang Tablets, Jiannao Anshen Tablets, Jianpi Shengxue Tablets, Yixin Ningshen Tablets, Yixin Shu Tablets, Yinao Tablets, Xiaokeping Tablets, Xiaokeling Tablets, Biyan Tablets, Fufang Chuanbei Jing Tablets, Fuheqing Tablets, Zhikebao Tablets, Shiwei Xiaoke Capsules, Jiuwei Gantai Capsules, San Bao capsule, Wuling capsule, Yuquan capsule, Shengmai capsule, Dengzhan Shengmai capsule, Anshen capsule, Fukezhile capsule, Suhuangzhike capsule, Gengnianan capsule, Hugan capsule, Zaoren Anshen capsule, Shenqi Schisandra capsule, Shenqijiangtang capsule, Chunxuean capsule, Fufang Yiganling capsule, Jiannao capsule, Yixinshu capsule, Dianiaole capsule, Shensongyangxin capsule, Yinhuangqingfei capsule, Hepatitis B nourishing yin and promoting blood circulation granules, Hepatitis B replenishing qi and relieving depression granules, Xiaoqinglong granules, Schisandra granules, Fenghan cough granules, Yuquan granules, Anshenbuxin granules, Cistanche Yishen granules, Qifeng Gubiao Granules, Hugan Granules, Liganlong Granules, Zaoren Anshen Granules, Shenqi Schisandra Granules, Yinshanlian Granules, Fuqi Zhihan Granules, Jianpi Shengxue Granules, Yixinshu Granules, Yishenling Granules, Huangqi Shengmai Granules, Longmu Zhuanggu Granules, Ermu Ningsou Pills, Qiwei Duqi Pills, Ginseng Yangrong Pills, Tianwang Buxin Pills, Tianwang Buxin Pills (Concentrated Pills), Wubi Yam Pills, Wuzi Yanzong Pills, Zhisou Huatan Pills, Fenghan Kettle Pills, Shihu Yeguang Pills, Sishen Pills, Quanlu Pills, Angong Jiangya Pills, Maiwei Dihuang Pills, Gengnian An Pills, Hugan Pills, Bushen Yinao Pills, Bushen Yijing Pills Pills, Jinhua Mingmu Pills, Shenjing Zhike Pills, Baizi Yangxin Pills, Compound Yigan Pills, Yangshen Baofei Pills, Jiannao Pills, Yixinshu Pills, Xiaoke Pills, Runfei Zhisou Pills, Qingshu Yiqi Pills, Qiangyang Baoshen Pills, Alopecia Pills, Juhong Huatan Pills, Compound Qingdai Pills, Tongmai Yangxin Pills, Peikun Pills, Twenty-seven Wei Dingkun Pills, Xiaoqinglong Mixture, Schisandra Syrup, Xinrong Oral Liquid, Shengmai Drink, Ruanmailing Oral Liquid, Shenbao Mixture, Shenbao Syrup, Guanxin Shengmai Oral Liquid, Shema Oral Liquid, Yiqi Yangxue Oral Liquid, Ciwujia Naoling Mixture, Juhong Tanke Liquid, Tongmai Yangxin Oral Liquid, etc. For example, Schisandra Granules, Shengmai Drink, Anshen Capsules, Fenghan Keke Granules, Zaoren Anshen Capsules.

[0057] To make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the present application is further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods in the following examples that do not specify specific conditions are usually based on normal conditions or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts. The experimental materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified.

[0058] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the technical field to which the application belongs. It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments of the present application.

[0059] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the technical field to which the application belongs. It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments of the present application.

[0060] Example 1. Method development

[0061] (1) Sample pretreatment

[0062] Take about 0.25 g of Schisandra chinensis or vinegar Schisandra chinensis medicinal material powder (passed through No. 3 sieve), or an appropriate amount of prepared preparation containing about 0.25 g of Schisandra chinensis or vinegar Schisandra chinensis according to the prescription, accurately weigh, put in a stoppered conical flask, accurately add 25 ml of methanol, weigh the weight, ultrasonically treat (power 330 W, frequency 37 kHz) for 30 minutes, cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the filtrate.

[0063] (2) Instrument testing

[0064] Octadecylsilane bonded silica was used as filler, ACQUITY BEH C18 chromatographic column (column length: 15 cm, inner diameter: 2.1 mm, particle size: 1.7 μm); acetonitrile was used as mobile phase A, water was used as mobile phase B, and gradient elution was performed according to the provisions in Table 1; the detection wavelength was 280 nm from 0 to 12 minutes, and 220 nm from 12 minutes to the end; the column temperature was 35°C; and the flow rate was 0.3 ml per minute.

[0065] Table 1 Gradient elution program

[0066] Time (min) Mobile phase A (%) Mobile phase B (%) 0～6 5 95 6～7 5→45 95→55 7～25 45→50 55→50 25～35 50→70 50→30 35～45 70→90 30→10

[0067] Take an appropriate amount of gomisin N and schisandra chinensis B reference substances, weigh them accurately, add methanol to make a mixed solution containing 10μg of each per 1ml, and obtain a mixed reference substance solution. Then take about 0.25g of schisandra chinensis and southern schisandra chinensis powder (passed through No. 3 sieve), weigh them accurately, put them in a stoppered conical bottle, accurately add 25ml of methanol, weigh the weight, ultrasonically treat (power 330W, frequency 37kHz) for 30 minutes, cool, weigh the weight again, make up the lost weight with methanol, shake well, filter, and take the filtrate to obtain the sample solution. Accurately pipette 2μl of the reference substance solution and sample solution respectively, and inject them into the high performance liquid chromatograph for determination. Figure 1 The characteristic spectrum of Schisandra chinensis is shown. Figure 1 The characteristic spectrum of Schisandra chinensis (without the chromatographic peak of Gomisin N) is shown. Figure 3 Chromatograms of gomisin N and schisandrin B reference substances are shown.

[0068] After verification by gomisin N and schisandrin B reference substances, the chromatographic peaks corresponding to gomisin N and schisandrin B in 27 batches of Schisandrae Chinensis samples and 12 batches of vinegar Schisandrae Chinensis were determined, and the calculated gomisin N / schisandrin B peak area ratios were highly consistent, ranging from 0.21 to 0.28; the content ratio of the two calculated based on gomisin N and schisandrin B reference substances was also in the range of 0.21 to 0.28. The results are shown in Table 2.

[0069] Table 2 Peak area ratio and content ratio of gomisin N / schizandrin B in Schisandra samples

[0070]

[0071]

[0072] In summary, this study found that the ratio of gomisin N to schisandrin B in Schisandrae Chinensis samples was between 0.21 and 0.28, while the ratio of gomisin N to schisandrin B in Schisandrae Chinensis and its vinegar products was not within this range. Figure 2 As shown in the figure, the ratio of gomisin N to schisandrin B in Schisandrae Chinensis is 0. Therefore, the peak area ratio (0.21-0.28) or content ratio (0.21-0.28) of gomisin N to schisandrin B can be used as an effective reference for rapid identification of Schisandrae Chinensis and its vinegar products.

[0073] Example 2: Stability Verification

[0074] Considering that Schisandra chinensis is used as fine powder or extracted in traditional Chinese medicine formula preparations, in order to investigate the stability of the peak area ratio or content ratio of gomisin N to schisandrin B, water reflux extraction and 75% ethanol reflux, which are the most widely used in actual production, were selected to investigate the stability of the ratio. The results showed that the peak area ratio or content ratio of gomisin N to schisandrin B was still stable under heating reflux, and it met the basic conditions as a judgment standard. The results are shown in Table 3.

[0075] Table 3 Peak area ratios of gomisin N and schisandrin B in Schisandrae Chinensis and its vinegar products in different extraction solvents

[0076]

[0077] Example 3: Sample testing

[0078] Based on the relationship between the peak area ratio or content ratio of gomisin N and schisandrin B, five varieties of Schisandra Chinese herbal preparations, including Schisandra Granules, Shengmai Drink, Anshen Capsules, Fenghan Keshen Granules, and Zaoren Anshen Capsules produced by different manufacturers, were tested. The actual detected peak area ratio or content ratio of gomisin N and schisandrin B was in the range of 0.21-0.28, which can be used as a basis for the judgment of actual samples of Schisandra raw materials and preparations.

[0079] Table 4 Peak area ratio of gomisin N to schisandrin B in Schisandrae Chinensis Chinese medicine formula

[0080]

[0081] Those skilled in the art will appreciate that the above-mentioned embodiments are specific examples for implementing the present application, and in actual applications, various changes may be made thereto in form and detail without departing from the spirit and scope of the present application.

Claims

1. A method for identifying Schisandra chinensis and Schisandra chinensis, characterized in that, The method comprises the steps of: Whether the sample to be tested contains Schisandra chinensis is identified by detecting the content ratio of gomisin N to Schisandrin B in the sample to be tested.

2. The method according to claim 1, characterized in that The method steps are: S1, detecting the contents of gomisin N and schisandrin B in the sample to be tested; S2, calculate the content ratio of gomisin N to schisandrin B; S3, comparing the content ratio of gomisin N to schisandrae B with a preset value to identify whether the sample to be tested contains schisandrae.

3. The method according to claim 2, characterized in that In step S3, the content ratio of gomisin N to schisandrin B is compared with a preset value. When the content ratio of gomisin N to schisandrin B is not higher than the preset value, the sample to be tested is Schisandra chinensis, otherwise it is not Schisandra chinensis.

4. The method according to claim 3, characterized in that In step S3, in step S3, the preset value is 0.21±5% to 0.28±5%.

5. The method according to claim 3, characterized in that: In step S1, before detecting the sample to be tested, a sample pretreatment step is also included, and the sample pretreatment step includes: using a mixed solvent of water and ethanol to treat the sample to be tested to obtain an extract.

6. The method according to claim 5, characterized in that The sample pretreatment step includes using a mixed solvent of water and ethanol to dissolve the sample to be tested, and heating it under reflux to obtain an extract.

7. The method according to claim 2, characterized in that In step S2, liquid chromatography is used to detect the sample to be tested, and the chromatographic conditions of the liquid chromatography include: using acetonitrile as mobile phase A and water as mobile phase B for gradient elution.

8. The method according to claim 7, characterized in that The procedure of the gradient elution is shown in the following table: 。 9. The method according to claim 7, characterized in that: The chromatographic conditions of the liquid chromatography also include: The detection wavelength is 280nm from 0 to 12 minutes and 220nm from 12 minutes to the end; Column temperature is 30-40°C; The flow rate is 0.1-0.5 ml per minute.

10. The method according to any one of claims 1 to 9, characterized in that The sample to be tested is a traditional Chinese medicine or a traditional Chinese medicine preparation.

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