A method for identifying the characteristic spectrum of sinopodophyllum hexandrum and its counterfeit kadsura peltata and bucrania frutescens

Abstract

This invention discloses a method for identifying the characteristic chromatograms of Piper kadsura and its adulterants, Schisandra chinensis and Photinia serratifolia. The method includes preparing test and reference solutions, and using high-performance liquid chromatography (HPLC) under specific chromatographic conditions to obtain characteristic chromatograms. Piper kadsura exhibits eight characteristic peaks, Photinia serratifolia shows only four of these peaks, while Schisandra chinensis shows no obvious corresponding chromatographic peaks. By comparing the characteristic chromatograms, Piper kadsura can be quickly and accurately distinguished from its adulterants. This method is highly specific, stable, and reproducible, and is suitable for identifying the authenticity of Piper kadsura medicinal materials, processed slices, standard decoctions, and formulated granules.

Description

Technical Field

[0001] The present invention relates to a method for identifying Piper kadsura, in particular to a characteristic chromatogram method for identifying Piper kadsura and its counterfeits Kadsura heteroclita and Piper wallichii Background Art

[0002] Piper kadsura is the dried vine stem of Piper kadsura (Choisy) Ohwi of the Piperaceae family. It is harvested in summer and autumn, the roots and leaves are removed, and then dried in the sun. It has a pungent, bitter taste and is slightly warm in nature; it belongs to the Liver Meridian. It has the effects of dispelling wind-dampness, dredging collaterals, and stopping pain due to arthralgia, and is mostly used for wind-cold-damp arthralgia, pain in the joints, muscle spasms, and limited flexion and extension. There are many plants with the same name as Piper kadsura. At present, the original plants of Piper kadsura purchased and sold in various places are not only one kind, which is relatively chaotic, and there are also different medication habits in different regions. In Hainan, Guangdong and Guangxi regions, the vine stems of Kadsura heteroclita (Roxb.) Craib of the Magnoliaceae family are generally used as Piper kadsura for medicinal purposes, also known as Guangdong Piper kadsura and Guangxi Piper kadsura. In addition, Piper wallichii, also known as Piper wallichii and Small Wind Vine, of the Piperaceae family is used as Piper kadsura for medicinal purposes in many places. Piper wallichii is the dried vine stem of Piper wallichii (Miq.) Hand.-Mazz. of the genus Piper in the Piperaceae family, and is distributed in Hubei, Hunan, Guangdong, Guizhou, Sichuan, Yunnan and other places.

[0003] At present, for the identification of Piper kadsura, Kadsura heteroclita and Piper wallichii, traditional character, microscopic and thin-layer identification, and ultraviolet-visible spectroscopy are mostly used for identification. Qin Xiao et al. discussed the characteristics of the original plants, the characters, microscopy, thin-layer chromatography and ultraviolet spectra of the original medicinal materials and cut pieces of traditional Chinese medicine Piper kadsura and Kadsura heteroclita. Luo Heng et al. used RAPD technology for PCR amplification, and screened out 4 primers from 62 random primers that could accurately identify Piper kadsura and its substitutes (Piper wallichii and Piper puberulum), thus inferring that there are certain differences between Piper kadsura and Piper wallichii and Piper puberulum at the DNA molecular level. The above studies all have some defects in distinguishing Piper kadsura and its counterfeits: (1) With the development of society and the change of environment, the corresponding character identification indexes have not been established yet. Coupled with the requirement that the character identification requires the identifier to have rich practical experience and long-term knowledge accumulation, it causes certain difficulties in popularization; (2) Microscopic identification has low resolution and is relatively difficult to identify multi-origin and variety-confused medicinal materials; (3) DNA barcoding identification has insufficient sensitivity and poor operability. Summary of the Invention

[0004] Object of the Invention: The object of the present invention is to provide a characteristic chromatogram method for identifying Piper kadsura and its counterfeits Kadsura heteroclita and Piper wallichii with strong specificity, high stability and good repeatability, and solve the problem that the prior art lacks a high-performance liquid chromatography method for distinguishing Piper kadsura and its counterfeits.

[0005] Technical solution: The characteristic spectral method for identifying Piper kadsura and its adulterants Schisandra chinensis and Photinia serratifolia as described in this invention includes the following steps: (1) Prepare test solution and reference solution; the test sample is Piper kadsura, Schisandra chinensis, and Photinia serratifolia; (2) The test solution and the reference solution were respectively injected into the high performance liquid chromatograph and determined to obtain the characteristic chromatograms of Piper kadsura, Schisandra chinensis and Photinia serratifolia; (3) Compare the characteristic chromatogram of the sample to be tested with the control characteristic chromatograms of Piper kadsura, Schisandra chinensis and Photinia serratifolia; Piper kadsura has 8 characteristic peaks; Photinia serratifolia only has peaks 1, 5, 6 and 8 of the 8 characteristic peaks, and the other peaks are missing; Schisandra chinensis has basically no corresponding chromatographic peaks under the chromatogram conditions.

[0006] Preferably, the high-performance liquid chromatography (HPLC) conditions are as follows: the column is packed with octadecyl-bonded silica gel; acetonitrile is used as mobile phase A, and 0.18-0.22% phosphoric acid solution is used as mobile phase B for gradient elution; the column temperature is 25-35℃, the flow rate is 0.9-1.1 ml / min, and the detection wavelength is 230-260 nm. Mobile phase B is preferably 0.2% phosphoric acid solution.

[0007] Preferably, the gradient elution program is: 0 min-20 min-23 min-30 min-45 min, mobile phase A: 40%-41%-50%-53%-65%, mobile phase B: 60%-59%-50%-47%-35%.

[0008] Preferably, using peak 4 as a reference peak, the relative retention times of the other characteristic peaks and peak 4 are calculated as follows: peak 1: 0.72, peak 2: 0.82, peak 3: 0.96, peak 5: 1.38, peak 6: 1.44, peak 7: 1.69, peak 8: 1.83.

[0009] Preferably, among the eight characteristic peaks of the *Piper kadsura*, peak 4 is piperine and peak 8 is cypermethrin.

[0010] Preferably, the test solution is prepared by taking the test sample powder, adding an organic solvent for extraction, shaking well, filtering, and taking the supernatant to obtain the test solution.

[0011] Preferably, the organic solvent is 68-72% methanol; the extraction method is ultrasonic treatment. The organic solvent is preferably 70% methanol. Preferably, the reference solution is prepared by dissolving long pepper reference standard and cypermethrin reference standard in solvents to obtain long pepper reference solution and cypermethrin reference solution.

[0012] Preferably, the concentration of the long pepper reference solution is 3-7 μg / mL, the concentration of the purslane reference solution is 10-20 μg / mL, and the solvent is 68-72% methanol. More preferably, the concentration of the long pepper reference solution is 5 μg / mL, the concentration of the purslane reference solution is 15 μg / mL, and the solvent is 70% methanol.

[0013] The method described in this invention is applied to the identification of the authenticity of Piper kadsura medicinal materials, processed slices, standard decoctions, or formula granules.

[0014] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: This invention can distinguish between Piper kadsura and its adulterants, Schisandra chinensis and Photinia serratifolia, and can specifically evaluate the quality of Piper kadsura. The characteristic map construction method established by this invention has the advantages of strong specificity, high stability, and good repeatability, thus achieving the purpose of effectively identifying Piper kadsura and its adulterants, Schisandra chinensis and Photinia serratifolia. Attached Figure Description

[0015] Figure 1 Chromatograms of the standard decoction of Piper kadsura under different column temperatures.

[0016] Figure 2 Chromatograms of the standard decoction of Piper kadsura under different flow rates.

[0017] Figure 3 This is an overlay diagram of the standard decoction of Piper kadsura.

[0018] Figure 4 Comparative chromatograms of standard decoctions of Piper kadsura and its adulterants Schisandra chinensis and Photinia serratifolia.

[0019] Figure 5 The characteristic chromatograms of Piper kadsura and its reference standard are shown, in which peak 4 is long pepperine and peak 8 is styracil. Detailed Implementation

[0020] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0021] Example 1: A method for identifying the characteristic chromatograms of Piper kadsura and its adulterants. 1. Instruments, reagents and samples The following equipment was used: Agilent Technologies 1260 high-performance liquid chromatograph; OpenLAB CDS 2.3 chromatography workstation; ThermoVanquish ultra-high-performance liquid chromatograph (Thermo Fisher Scientific); Chameleon 7.2 workstation (Thermo Fisher Scientific); KQ-250E ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); GKC114 temperature-controlled water bath (Nantong Huatai Experimental Instrument Co., Ltd.); HY-4 shaker (Jintan Kexing Instrument Factory); ME204E electronic analytical balance (Mettler-Toledo (Shanghai) Instrument Co., Ltd.); PO Lab plus-L pure water system (Satorius); TGL-16C centrifuge (Shanghai Anting Scientific Instrument Factory); METTLER TOLEDO XP6 ppm balance. Acetonitrile (chromatographic grade, Thermo Fisher Scientific); methanol (chromatographic grade, Thermo Fisher Scientific); phosphoric acid (chromatographic grade, Aladdin); water was ultrapure water; other reagents were analytical grade.

[0022] Wallwort alkaloid (batch number: 17094) was purchased from Shanghai Shidande Biotechnology Co., Ltd.; Piperine (batch number: Y03S11W123356) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. Sixteen batches of standard decoction of Piper kadsura (numbered S1~S16), three batches of standard decoction of Schisandra chinensis (numbered S17~S19), and three batches of standard decoction of Photinia serratifolia (numbered S20~S22) were all provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0023]

[0024] 2 Chromatographic conditions The column was packed with octadecylsilane-bonded silica gel (250 mm column length, 4.6 mm inner diameter, 5 μm particle size); acetonitrile was used as mobile phase A, and 0.2% phosphoric acid solution was used as mobile phase B, with gradient elution performed according to the specifications in the table below; the flow rate was 1.0 mL / min; the column temperature was 30 °C; and the detection wavelength was 240 nm. The theoretical plate number, calculated based on the alkaloid peak, should be no less than 5000.

[0025]

[0026] 3. Preparation of reference solution Take appropriate amounts of long pepper reference standard and cypermethrin reference standard, weigh them accurately, and add 70% methanol to prepare solutions containing 5 μg long pepper and 15 μg cypermethrin per ml, respectively, as reference solutions.

[0027] 4. Preparation of the test solution Take an appropriate amount of this product, grind it into a fine powder, take about 0.3g, weigh it accurately, place it in a stoppered conical flask, accurately add 15ml of 70% methanol, seal tightly, weigh it, sonicate (power 250W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 70% methanol, shake well, filter it, and take the filtrate to obtain the product.

[0028] 5. Robustness test of chromatographic conditions 5.1 Investigation of column temperature Take the S1 standard decoction of *Piper kadsura* (sea pine) as the test solution, and inject it into a ZORBAX SB-C18 (250×4.6mm, 5μm) column under the chromatographic conditions described above at different column temperatures (25℃, 30℃, 35℃). Record the chromatograms. See [link to chromatogram]. Figure 1 The results showed that the chromatographic peaks were well separated and the column exhibited good durability within a column temperature range of 25℃ to 35℃.

[0029] 5.2 Examination of Flow Velocity Take the S1 standard decoction of *Piper kadsura* (sea pine) as the test solution, and use a ZORBAX SB-C18 (250×4.6mm, 5μm) column. Under the chromatographic conditions described above, perform the determination at different flow rates of 0.9 ml / min, 1.0 ml / min, and 1.1 ml / min, and record the chromatograms. See [link to chromatogram]. Figure 2 The results showed that small flow rate variations within the range of 0.9 ml / min to 1.1 ml / min could meet the system applicability requirements.

[0030] 6. Establishment of Feature Maps Sixteen batches of standard decoction of Piper kadsura, three batches of standard decoction of Schisandra chinensis, and three batches of standard decoction of Photinia serratifolia were taken, with 0.3g of each sample. The characteristic chromatograms of the samples were determined according to the above preparation method and chromatographic conditions. The characteristic chromatograms of the 16 batches of Piper kadsura standard decoction samples were overlaid using the "Similarity Evaluation System for Chromatographic Characteristic Chromatography of Traditional Chinese Medicine (Version 2012.0)" issued by the National Pharmacopoeia Commission. Figure 3 Comparative charts of standard decoctions of Piper kadsura, Schisandra chinensis, and Photinia serratifolia are shown below. Figure 4 See the identification diagram for the standard decoction of *Hypericum perforatum*. Figure 5 .

[0031] 7. Identification Study of Standard Decoctions of Piper kadsura and its Adulterants: The characteristic chromatograms of *Piper kadsura* and its adulterants, *Schisandra chinensis* and *Photinia serratifolia*, differ significantly from those of the standard decoction. *Piper kadsura* has eight characteristic peaks, while *Photinia serratifolia* has peaks 1, 5, 6, and 8, but the others are absent. *Schisandra chinensis* is from a different family, and under the characteristic chromatogram conditions of *Piper kadsura*, it shows almost no corresponding chromatographic peaks.

Claims

1. A method for identifying the characteristic chromatograms of Piper kadsura and its adulterants, Schisandra chinensis and Photinia serratifolia, characterized in that, Includes the following steps: (1) Prepare test solution and reference solution; the test sample is Piper kadsura, Schisandra chinensis, and Photinia serratifolia; (2) The test solution and the reference solution were respectively injected into the high performance liquid chromatograph and determined to obtain the characteristic chromatograms of Piper kadsura, Schisandra chinensis and Photinia serratifolia; (3) Compare the characteristic chromatogram of the sample to be tested with the control characteristic chromatograms of Piper kadsura, Schisandra chinensis and Photinia serratifolia; Piper kadsura has 8 characteristic peaks; Photinia serratifolia only has peaks 1, 5, 6 and 8 of the 8 characteristic peaks, and the other peaks are missing; Schisandra chinensis has basically no corresponding chromatographic peaks under the chromatogram conditions.

2. The method according to claim 1, characterized in that, The conditions for the high performance liquid chromatography are as follows: the column is packed with octadecyl bonded silica gel; acetonitrile is used as mobile phase A and 0.18-0.22% phosphoric acid solution is used as mobile phase B for gradient elution; the column temperature is 25-35℃, the flow rate is 0.9-1.1 ml / min, and the detection wavelength is 230-260 nm.

3. The method according to claim 4, characterized in that, The gradient elution program is as follows: 0 min-20 min-23 min-30 min-45 min, mobile phase A: 40%-41%-50%-53%-65%, mobile phase B: 60%-59%-50%-47%-35%.

4. The method according to claim 1, characterized in that, Using peak 4 as a reference peak, the relative retention times of the other characteristic peaks and peak 4 were calculated as follows: peak 1: 0.72, peak 2: 0.82, peak 3: 0.96, peak 5: 1.38, peak 6: 1.44, peak 7: 1.69, peak 8: 1.

83.

5. The method according to claim 1, characterized in that, Of the eight characteristic peaks of the *Piper kadsura* species, peak 4 is piperine and peak 8 is cypermethrin.

6. The method according to claim 1, characterized in that, The test solution is prepared by taking the test sample powder, adding an organic solvent for extraction, shaking well, filtering, and taking the supernatant to obtain the test solution.

7. The method according to claim 6, characterized in that, The organic solvent is 68-72% methanol; the extraction method is ultrasonic treatment.

8. The method according to claim 1, characterized in that, The reference solutions are prepared by dissolving long pepper and cypermethrin reference standards in solvents to obtain long pepper reference solutions and cypermethrin reference solutions.

9. The method according to claim 8, characterized in that, The concentration of the long pepper reference solution is 3~7 μg / mL, the concentration of the wallwort alkaloid reference solution is 10~20 μg / mL, and the solvent is 68~72% methanol.

10. The application of the method of claim 1 in identifying the authenticity of Piper kadsura medicinal materials, processed slices, standard decoctions, or formula granules.

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