Fingerprint of radix clematidis and application thereof in quality control of medicinal materials

Abstract

The present application relates to a kind of fingerprints of radix tetrapanacis and its application in medicinal material quality control, the radix tetrapanacis HPLC fingerprint spectrum established in the present application overcomes the shortcomings of single detection index in prior art, can not reflect the internal quality of medicinal materials, the provided radix tetrapanacis HPLC fingerprint spectrum establishment method is high in precision, good stability and repeatability, by comparing the presence or absence of common peaks in the obtained fingerprint spectrum, the quality of radix tetrapanacis can be effectively characterized, and it has important significance for ensuring clinical efficacy.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of traditional Chinese medicine, and particularly relates to a fingerprint spectrum of Physochlaina infundibularis Kuang and application thereof in quality control of medicinal materials. BACKGROUND

[0002] Physochlaina infundibularis Kuang is the dried root of Physochlaina infundibularis Kuang in Solanaceae, which is also known as Huashanrenshen, Reishen, Qinsen, Baomaorenshen, Zhixiancao, etc. and is mainly distributed in Shaanxi, Henan and Shanxi. Physochlaina infundibularis Kuang is listed as a national second-class protected plant and is recorded in the book Bencao Gangmu Shiyi written by Zhao Xue-min in the Qing Dynasty. Physochlaina infundibularis Kuang grows in mountain slopes, valleys or grasslands and has the effects of warming lung, expelling phlegm, relieving cough and asthma, and calming nerves. Physochlaina infundibularis Kuang is used for treating cold phlegm, cough and asthma, and restlessness and insomnia. Literature reports that Physochlaina infundibularis Kuang mainly contains alkaloids such as atropine, scopolamine and anisodine, coumarin compounds such as scopolin and anisatin, and amino acids and polysaccharides, and has the pharmacological effects of relieving cough, expelling phlegm, relieving asthma and inhibiting central nervous system. Scopolin is an important active ingredient in Physochlaina infundibularis Kuang and has the effects of anti-inflammatory analgesia, diuresis and asthma relief.

[0003] The 2020 edition of Chinese Pharmacopoeia adopts ultraviolet-visible spectrophotometry to determine the contents of scopolamine and scopolin in Physochlaina infundibularis Kuang. However, the ultraviolet-visible spectrophotometry is complex in operation and poor in accuracy and reproducibility. Meanwhile, scopolin is also contained in many Solanaceae plants such as Atropa belladonna, Lycium barbarum and other plants such as Tinospora crispa, and the specificity is insufficient. At present, there is still a lack of comprehensive quality detection method for Physochlaina infundibularis Kuang. Based on this, the present application establishes a detection method for HPLC fingerprint spectrum of Physochlaina infundibularis Kuang, which can control the quality of Physochlaina infundibularis Kuang as a whole. SUMMARY

[0004] The present application provides an HPLC fingerprint spectrum of Physochlaina infundibularis Kuang, which is basically consistent with the HPLC fingerprint spectrum of the reference sample when the HPLC chromatographic conditions are as follows. Figure 8

[0005] The HPLC chromatographic conditions are as follows:

[0006] The chromatographic column is Agilent 5TC-C 18 , with a specification of 250 mm x 4.6 mm and 5 μm.

[0007] The mobile phase is methanol as phase A and 0.1 wt% phosphoric acid aqueous solution as phase B.

[0008] ​Elution gradient: 0-5 min, 10%-13% A; 5-10 min, 13%-18% A; 10-33 min, 18%-26% A; 33-40 min, 26%-40% A; 40-45 min, 40%-10% A; Column temperature: 30℃; Injection volume: 10 μL; Flow rate: 1.0 mL·min -1 Another embodiment of the present application provides the HPLC fingerprint of the above-mentioned Radix Tetrapanacis, characterized in that the HPLC fingerprint of the Radix Tetrapanacis is basically consistent with Figure 8 wherein peak 5 is scopolamine, peak 8 is tetracene, peak 9 is scopolin, peak 11 is hyoscyamine, and peak 14 is scopoletin. With the retention time of peak 8 as a reference, the relative retention times of peaks 5, 8, 9, 11 and 14 in the HPLC chromatogram of 10 batches of Radix Tetrapanacis samples are shown in the following table:

[0009]

[0010]

[0011] Another embodiment of the present application provides the application of the HPLC fingerprint of the above-mentioned Radix Tetrapanacis in quality control and component analysis of Radix Tetrapanacis.

[0012] Another embodiment of the present application provides the application of the HPLC fingerprint of the above-mentioned Radix Tetrapanacis in quality control of Radix Tetrapanacis, characterized in that the application comprises the following steps:

[0013] (1) crushing the Radix Tetrapanacis and extracting it by ultrasonic wave to prepare a test sample solution;

[0014] (2) taking the test sample solution obtained in step (1) and detecting it by HPLC to obtain the HPLC chromatogram of the test sample, wherein the chromatographic conditions are as follows:

[0015] The chromatographic column is Agilent 5TC-C 18 , with a size of 250 mm x 4.6 mm and a particle size of 5 μm;

[0016] The mobile phase is methanol as phase A and 0.1 wt% phosphoric acid aqueous solution as phase B;

[0017] Elution gradient: 0-5 min, 10%-13% A; 5-10 min, 13%-18% A; 10-33 min, 18%-26% A; 33-40 min, 26%-40% A; 40-45 min, 40%-10% A; Column temperature: 30℃; Injection volume: 10 μL; Flow rate: 1.0 mL·min -1; detection wavelength: 210 nm. (3) Comparing the HPLC chart of the test sample obtained in step (2) with the HPLC fingerprint chart of the medicinal material of Wushan- shen described in the present application, the medicinal material of Wushan-shen with the similarity of 0.90 or above (preferably the similarity of 0.95, 0.96 or above) is the qualified product.

[0018] The preparation method of the test sample solution in step (1) is as follows: the medicinal material of Wushan-shen is crushed, passed through a No. 3 sieve, 1.0 g is weighed, placed in a conical flask with a plug, 25 ml of 40% methanol by volume fraction is precisely added, tightly plugged, the weight is determined, ultrasonic treatment (power 300 W, frequency 40 kHz) is carried out for 1 hour, cooled (restored to room temperature), the weight is determined again, the lost weight is made up with 40% methanol by volume fraction, shaken uniformly, filtered with a 0.45 μm microporous organic filter membrane, and obtained.

[0019] Compared with the prior art, the present application has the following advantages: (1) the HPLC fingerprint chart of Wushan-shen established in the present application overcomes the shortcomings of single detection index and inability to reflect the internal quality of the medicinal material in the prior art, the HPLC fingerprint chart of Wushan-shen provided by the present application has high precision, good stability and repeatability, the presence or absence of common peaks in the obtained fingerprint chart can effectively characterize the quality of Wushan-shen, and has important significance for guaranteeing the clinical efficacy; (2) since there are many components in Wushan-shen, the solubility and separation degree of each component are different, multiple chromatographic conditions (extraction time, solvent, mobile phase, etc.) are investigated, and finally the chromatographic conditions of the present application are selected. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 chromatogram of Wushan-shen sample under different extraction time conditions;

[0021] Figure 2 chromatogram of Wushan-shen sample under different extraction solvent conditions;

[0022] Figure 3 chromatogram of Wushan-shen sample under different mobile phase conditions;

[0023] Figure 4 HPLC chromatogram of precision test;

[0024] Figure 5 HPLC chromatogram of repeatability test;

[0025] Figure 6 HPLC chromatogram of stability test;

[0026] Figure 7 HPLC fingerprint chart of 10 batches of medicinal material of Wushan-shen;

[0027] Figure 8 Wushan-shen control fingerprint chart measured in the present application;

[0028] Figure 9 The mixed reference solution chromatogram is prepared, wherein peak 5 represents scopolamine; peak 8 represents anisodamine; peak 9 represents scopolin; peak 11 represents hyoscyamine; and peak 14 represents scopoletin. DETAILED DESCRIPTION

[0029] In order to facilitate further understanding of the present application, the following examples are provided to make a more detailed description. However, these examples are only for better understanding of the application and are not intended to limit the scope or principles of the application, and the embodiments of the present application are not limited to the following.

[0030] Example 1

[0031] 1 Instruments and reagents

[0032] 1.1 Instruments

[0033] Agilent 1260 high performance liquid chromatograph (Agilent, USA); SHIMADZU 2010A-HT high performance liquid chromatograph (Shimadzu, Japan); KQ5200DE digital ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); DFT-200A portable high-speed pulverizer (Wenling Lindai Machinery Co., Ltd.); BT25S electronic analytical balance (d = 0.01 mg), BS210S electronic analytical balance (d = 0.1 mg) (Beijing Sartorius Balance Co., Ltd.).

[0034] 1.2 Reagents

[0035] In this study, 10 batches of Huashan Shen medicinal materials from different origins were collected, which were identified by Professor Zhang Hong of Shaanxi Institute of Chinese Medicine as the dried roots of Physochlaina infundibularis Kuang of Solanaceae Physocarpus, and the sample information is shown in Table 1. The reference substances scopolamine hydrobromide (batch number: CHB210125), anisodamine hydrobromide (batch number: CHB210118), and scopoletin (batch number: CHB201202) were purchased from Chengdu Keluoma Biological Technology Co., Ltd. (purity ≥98%); scopolin (batch number: PS010518) was purchased from Chengdu Pusai Biological Technology Co., Ltd. (purity ≥98%); hyoscyamine (batch number: C12946842) was purchased from Shanghai Maikelin Biological Technology Co., Ltd. (purity ≥98%); methanol and acetonitrile were chromatographically pure (Thermo Fisher Scientific, USA); water was ultrapure water, and phosphoric acid was analytically pure.

[0036] Table 1 Sample source information of Huashan Shen

[0037]

[0038]

[0039] 2. Method and Result

[0040] 2.1 Preparation of solution

[0041] 2.1.1 Preparation of reference solution

[0042] Accurately weigh the reference substance of hyoscine hydrobromide, anisodamine hydrobromide, scopolin, hyoscyamine and scopoletin, respectively, dissolve in 10% methanol and dilute to volume to prepare a mixed reference solution with a mass concentration of 41, 43, 33, 34 and 24 μg·mL -1 , respectively.

[0043] 2.1.2 Preparation of test solution

[0044] Take about 1.0 g of Radix Tetrapanacis powder (pass through a No. 3 sieve), accurately weigh, place in a conical flask with a stopper, accurately add 40% methanol 25 mL, tightly stop, weigh, stand for 1 h, ultrasonic treatment (power 300 W, frequency 40 kHz) for 1 h, cool, weigh again, make up the weight loss with 40% methanol, shake well, filter, take the filtrate through a 0.45 μm microporous filter membrane, and obtain.

[0045] 2.2 Chromatographic conditions

[0046] Chromatographic column: Agilent 5TC-C 18 (250 mm x 4.6 mm, 5 μm); mobile phase: methanol (A)-0.1% phosphoric acid solution (B); elution gradient: 0-5 min, 10%-13% A; 5-10 min, 13%-18% A; 10-33 min, 18%-26% A; 33-40 min, 26%-40% A; 40-45 min, 40%-10% A; column temperature: 30°C; injection volume: 10 μL; flow rate: 1.0 mL·min -1 ; detection wavelength: 210 nm.

[0047] 2.3 Optimization of detection conditions

[0048] 2.3.1 Investigation of extraction time

[0049] The extraction conditions of ultrasonic extraction for 0.5 h, ultrasonic extraction for 1 h and ultrasonic extraction for 1 h after soaking for 1 h were investigated, respectively, and the obtained chromatograms are shown in Figure 1 . It was found that the extraction effects of the three were relatively close, and the total peak area of the method of soaking for 1 h and then ultrasonic treatment for 1 h was the largest, therefore, this method was selected to prepare the test solution.

[0050] 2.3.2 Investigation of extraction solvent

[0051] The same batch of Huashan-Shen medicinal materials was extracted with 40% methanol solution, 60% methanol solution, 80% methanol solution, methanol and water as the extraction solvent, respectively, and the obtained chromatograms are shown in Figure 2 . It was found that the number of peaks and the peak area were the largest when 40% methanol solution was used as the extraction solvent, so 40% methanol was finally selected as the extraction solvent.

[0052] 2.3.3 Investigation of mobile phase

[0053] The same Huashan-Shen test solution was taken, and the elution systems of methanol-0.05% phosphoric acid aqueous solution, methanol-0.1% phosphoric acid aqueous solution, methanol-0.3% phosphoric acid aqueous solution and acetonitrile-0.3% phosphoric acid aqueous solution were investigated, respectively, and the obtained chromatograms are shown in Figure 3 . The experimental results showed that the separation effect, plate number and symmetry of the chromatographic peaks obtained by elution with methanol-0.1% phosphoric acid aqueous solution were better, the baseline was stable, and the peak time was also ideal, so methanol-0.1% phosphoric acid aqueous solution was finally used as the mobile phase.

[0054] 2.4 Establishment of HPLC fingerprint

[0055] 2.4.1 Precision test

[0056] The same batch of sample (S1) powder was taken, and the test solution was prepared according to the method under item “2.1.2”, and was continuously injected for 6 times according to the chromatographic conditions under item “2.2”, and the chromatograms were recorded. Taking peak No. 8 anisodine as the reference peak (S), the relative retention time and relative peak area RSD of each common peak were calculated. The results showed that the relative retention time RSD of each common peak was 0.27%-1.17%, as shown in Table 2. The relative peak area RSD was 1.41%-4.44%, as shown in Table 3. The overall appearance of the measured 6 fingerprint chromatograms had no obvious change, as shown in Figure 4 . The data file was imported into the “Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System” (2012 version) for similarity evaluation. The results showed that the similarity of each test fingerprint was greater than 0.990, the precision of the instrument was good, and met the requirements of the fingerprint research technology.

[0057] Table 2 Relative retention time of precision test

[0058]

[0059] Table 3 Relative peak area of precision test

[0060]

[0061]

[0062] 2.4.2 Reproducibility test

[0063] Take 6 portions of the same sample (S1) powder, respectively, accurately weigh, prepare the test solution according to the method under "2.1.2", and determine by sample injection under the chromatographic conditions under "2.2", and record the chromatogram. Take anhydrous anisodine as the reference peak (S), calculate the relative retention time and relative peak area RSD of each common peak. The results show that the relative retention time RSD of each common peak is 0.10% to 0.45%, as shown in Table 4. The relative peak area RSD is 0.70% to 4.74%, as shown in Table 5. The overall appearance of the six fingerprints measured has no obvious change, as shown in Figure 5 . Import the data file into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 version) for similarity evaluation. The results show that the similarity of each test sample fingerprint is greater than 0.990, the repeatability of the method is good, and it meets the requirements of fingerprint research technology.

[0064] Table 4 Relative retention time of repeatability test

[0065]

[0066] Table 5 Relative peak area of repeatability test

[0067]

[0068] 2.4.3 Stability test

[0069] Take the same sample (S1) powder, prepare the test solution according to the method under "2.1.2", and determine by sample injection at 0, 2, 4, 6, 8, 12, and 24 h, respectively, under the chromatographic conditions under "2.2", and record the chromatogram. Take anhydrous anisodine as the reference peak (S), calculate the relative retention time and relative peak area RSD of each common peak. The results show that the relative retention time RSD of each common peak is 0.24% to 1.76%, as shown in Table 6. The relative peak area RSD is 1.00% to 4.48%, as shown in Table 7. The overall appearance of the seven fingerprints measured has no obvious change, as shown in Figure 6 . Import the data file into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 version) for similarity evaluation. The results show that the similarity of each test sample fingerprint is greater than 0.970, the test sample solution has good stability within 24 h, and it meets the requirements of fingerprint research technology.

[0070] Table 6 Relative retention time of stability test

[0071]

[0072]

[0073] Table 7 Relative peak area of stability test

[0074]

[0075] 2.4.4 Establishment of fingerprint

[0076] Take 10 batches of sample powder, accurately weigh, prepare test solution according to the method under item “2.1.2”, then inject sample according to the chromatographic conditions under item “2.2” to determine, record chromatogram. Import the data into “Similarity Evaluation System of Chromatographic Fingerprint of Traditional Chinese Medicine” (2012 version) for analysis, take chromatogram of sample S1 as reference spectrum, time window width is 0.1, select average method, after multi-point correction, use Mark peak matching to generate HPLC fingerprint of 10 batches of Huashanpat sample (R Figure 7 ) and control fingerprint (R Figure 8 ), select 14 chromatographic peaks with obvious characteristics, good repeatability and stability as common peaks. Peak 8 is anisodamine with good stability, separation degree and symmetry, high chromatographic response value and moderate retention time, so it is selected as reference peak (S). The relative retention time and relative peak area of common peaks in 10 batches of Huashanpat are shown in Table 8 and Table 9.

[0077] Table 8 Relative retention time of common peaks in 10 batches of Huashanpat spectrum

[0078]

[0079]

[0080] Table 9 Relative peak area of common peaks in 10 batches of Huashanpat spectrum

[0081]

[0082] 2.4.5 Identification of common peaks

[0083] Accurately take mixed reference solution under item “2.1.1” into a test tube, inject sample according to the chromatographic conditions under item “2.2” to determine, the results are shown in Figure 9 . After reference retention time positioning and chromatographic peak analysis, 5 peaks are identified, peak 5 is scopolamine, peak 8 is anisodamine, peak 9 is scopolin, peak 11 is hyoscyamine, and peak 14 is scopoletin.

[0084] 2.4.6 Similarity evaluation

[0085] The similarity evaluation was carried out by using the similarity evaluation system of chromatographic fingerprint of traditional Chinese medicines (2012 version) software, with the reference fingerprint R as the reference. The results showed that the similarity between the reference fingerprint and the 10 batches of Radix Lophatheri was 0.963-0.999, indicating that the consistency of the Radix Lophatheri samples was good, the quality was stable, and the established fingerprint could be used to evaluate the quality of Radix Lophatheri. The similarity results are shown in Table 10.

[0086] Table 10 Similarity evaluation results

[0087]

[0088]

Claims

1. The application of an HPLC fingerprint of Panax notoginseng in the quality control and component analysis of Panax notoginseng, wherein the HPLC fingerprint of Panax notoginseng has 14 characteristic fingerprint chromatographic peaks, wherein peak 5 is scopolamine, peak 8 is scopolamine, peak 9 is scopolamine glycoside, peak 11 is scopolamine, peak 14 is scopolamine lactone, and peak 8 is a reference peak; The HPLC chromatographic conditions are as follows: The chromatographic column was an Agilent 5 TC-C. 18 Specifications: 250 mm × 4.6 mm, 5μm; Mobile phase: Methanol as phase A, 0.1wt% phosphoric acid aqueous solution as phase B; Elution gradient: 0–5 min, 10%–13% A; 5–10 min, 13%–18% A; 10–33 min, 18%–26% A; 33–40 min, 26%–40% A; 40–45 min, 40%–10% A; Column temperature: 30 ℃; Injection volume: 10 μL; Flow rate: 1.0 mL·min -1 Detection wavelength: 210 nm; The extraction solvent for the Huashan ginseng medicinal material is selected from 40% methanol by volume.

2. The application according to claim 1, characterized in that... The application includes the following steps: (1) After pulverizing the Huashan ginseng medicinal material, ultrasonic extraction was performed to prepare a test solution; (2) Take the test solution obtained in step (1) and perform HPLC analysis to obtain the HPLC chromatogram of the test solution. The chromatographic conditions are as follows: The chromatographic column was an Agilent 5 TC-C. 18 Specifications: 250 mm × 4.6 mm, 5μm; Mobile phase: Methanol as phase A, 0.1wt% phosphoric acid aqueous solution as phase B; Elution gradient: 0–5 min, 10%–13% A; 5–10 min, 13%–18% A; 10–33 min, 18%–26% A; 33–40 min, 26%–40% A; 40–45 min, 40%–10% A; Column temperature: 30 ℃; Injection volume: 10 μL; Flow rate: 1.0 mL·min -1 Detection wavelength: 210 nm; (3) Compare the HPLC chromatogram of the test sample obtained in step (2) with the HPLC fingerprint chromatogram of the Huashan ginseng medicinal material according to claim 1. The Huashan ginseng medicinal material with a similarity of 0.90 or higher is a qualified product.

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