Method for constructing a characteristic map of Patrinia chinensis, a control map, and a method for distinguishing Patrinia chinensis from counterfeit products

Abstract

The present invention discloses a method for constructing a characteristic spectrum of Patrinia chinensis, a control spectrum, and a method for distinguishing Patrinia chinensis from a fake product. The construction method of the characteristic spectrum comprises the following steps: drawing a reference solution and a test solution respectively to carry out ultra-high performance liquid chromatography analysis, constructing a characteristic spectrum of a testing sample, wherein the characteristic spectrum includes at least 14 characteristic peaks, and there are 4 characteristic peaks in the characteristic peak corresponding to protocatechuic acid reference substance reference substance peak, dapoxetine reference substance reference substance peak, 3,4-O-dicaffeoylquinic acid reference substance reference substance peak, 4,5-O-dicaffeoylquinic acid reference substance reference substance peak; by the characteristic spectrum obtained, a qualitative analysis of the testing sample is carried out, while identifying Patrinia chinensis, Patrinia chinensis and Patrinia chinensis fake products. The method of the present invention is simple to operate, verified by methodology, and has good reproducibility and stability, low detection cost and high detection efficiency.

Description

Technical Field

[0001] The present invention relates to a method for constructing a drug characteristic map and an application thereof, and in particular to a method for constructing a Patrinia chinensis characteristic map, a control map, and a method for distinguishing Patrinia chinensis from counterfeit products. Background Art

[0002] Patrinia scabiosaefolia (Patrinia scabiosaefolia Fisch.) or Patrinia villosa Juss. (Patriniaceae family) is a dried herb. It has a pungent, bitter flavor and a slightly cold nature. It clears heat and detoxifies, eliminates carbuncles and pus, and relieves blood stasis and pain. It is used to treat appendicitis, dysentery, enteritis, hepatitis, postpartum abdominal pain due to blood stasis, carbuncles, furuncles, and other conditions (China Pharmacopoeia Commission. Pharmacopoeia of the People's Republic of China. Volume 1 [S]. Beijing: China Medical Science and Technology Press, 1977: 349-350).

[0003] Patrinia scabra is a traditional Chinese medicine, first recorded in Shennong's Herbal Classic. Studies have shown that the main chemical components of Patrinia scabra include triterpenoid saponins, cyclopentadiene ether terpenes, flavonoids, phenylpropanoids, volatile oils and organic acids (Chen Shuling, Han Liang. Modern research progress on Patrinia scabra [J]. Journal of Guangdong Pharmaceutical University, 2017, 33(06): 816-821.). Pharmacological studies have shown that Patrinia scabra has antiviral (Xiao Hehe, Liu Mingzhu, Yu Qing, et al. Antiviral effect of Patrinia scabra water extract on grouper iridovirus [J]. Journal of Guangxi Academy of Sciences, 2019, 35(03): 185-192.), anti-inflammatory (Sun Dajuan, Wei Xiunan, Cheng Yan, et al. Effect of Astragalus-Patrimonium herbal medicine pair on JAK1 / STAT6 / SOCS1 signaling pathway in HT-29 cell inflammation model [J]. Journal of Traditional Chinese Medicine, 2023, 64(03): 295-302.), anti-tumor (Zhang Pingping, Jia Ru, Cen Rong, et al. Research progress on the mechanism of action of flavonoids in Herba Patriniae on the occurrence and development of colorectal cancer [J]. Chinese Journal of Experimental Traditional Chinese Medicine, 2024, 30(04): 240-247.), liver protection (Li Aichen, Fang Lei. Protective effect of Herba Patriniae polysaccharides on liver tissue of rats with exercise fatigue [J]. Food Research and Development, 2016, 37(12): 174-177.) and other pharmacological activities.

[0004] Chinese herbal formula granules are made from single-ingredient Chinese herbal pieces through water extraction, concentration, drying, and granulation. They represent a new form of herbal medicine reform. Compared to traditional decoctions, they are more portable and simple to use, meeting the demands of modern, fast-paced work and life. In recent years, Chinese herbal formula granules have seen rapid development, with the release of national standards for formula granules for multiple Chinese herbal varieties. These standards have significantly contributed to the standardization of their production and the modernization of traditional Chinese medicine. Herba Patriniae Formula Granules are made from Herba Patriniae pieces through modern extraction, concentration, drying, and granulation processes. They retain the clinical efficacy of Herba Patriniae while ensuring portability.

[0005] Currently, there are few reports on the characteristic patterns of Patrinia chinensis and the identification of mixed and counterfeit products. Zhang Lei et al. studied the microscopic identification characteristics and HPLC fingerprint of Patrinia chinensis and its mixed and counterfeit products. The results showed that there were significant differences in the microscopic characteristics of the medicinal powder of Patrinia chinensis and its mixed and counterfeit products, as well as differences in the total saponin content. HPLC fingerprints of 26 batches of Patrinia chinensis and its mixed and counterfeit products were established, and 11 common peaks were calibrated. Through similarity analysis, cluster analysis and principal component analysis, Patrinia chinensis and its mixed and counterfeit products can be accurately distinguished (Zhang Lei, Huang Zilu, Lin Ziyi, et al. Study on the microscopic identification characteristics and HPLC fingerprint of Patrinia chinensis and its mixed and counterfeit products [J]. Chinese Herbal Medicine, 2024, 55(06): 2066-2076.). Yu Kunzi et al. established an identification method for Herba Patriniae and its counterfeits by using properties identification and microscopic identification, and conducted a comparative study on Herba Patriniae and its counterfeits on the market. They can distinguish each other through properties such as stem surface and cross-section characteristics, stem leaf morphology, inflorescence hair condition, odor, and microscopic characteristics such as stem cross section (Yu Kunzi, Han Shikai, Ma Siyu, et al. Pharmacognosy study on Herba Patriniae [J]. Journal of Pharmaceutical Analysis, 2024, 44(07): 1255-1266.). Wang Yinglan et al. used high performance liquid chromatography (HPLC) to study the fingerprint of Patrinia chinensis and obtained the HPLC fingerprint of Patrinia chinensis. At the same time, the key components of chlorogenic acid and rutin were determined, and 11 common peaks were calibrated as key indicators for the quality evaluation of Patrinia chinensis (Wang Yinglan, Liu Yuankun, Wan Haiqing, et al. Establishment and optimization of quality standards for Patrinia chinensis [J]. Clinical Rational Drug Use, 2024, 17(17): 154-157.). Liu Suxiang et al. established the HPLC fingerprint of Patrinia chinensis and calibrated 8 common peaks. Cluster analysis showed that the similarity between the fingerprints of Class I medicinal materials and the reference was 0.987-0.907, and the difference between the fingerprints of Class II medicinal materials and the reference was large. It can be used as an effective means to evaluate the quality of Patrinia chinensis from different origins and different varieties (Liu Suxiang, Liu Yi, Bai Xue, et al. Study on fingerprint of Patrinia chinensis [J]. Chinese Herbal Medicine, 2016, 47(12): 2074-2077.). According to existing literature, the quality control research of Patrinia chinensis is mainly concentrated on medicinal materials, and there are fewer research materials on the quality control of Patrinia chinensis water extract. Through patent retrieval, the patent No. CN111208253A adopts high performance liquid chromatography-mass spectrometry to detect Patrinia chinensis components, establishes Patrinia chinensis HPLC fingerprint, establishes Patrinia chinensis control fingerprint total pattern by system cluster analysis and similarity evaluation system, and judges the quality of medicinal materials. This invention patent does not carry out the different bases and authenticity identification research of Patrinia chinensis, and the quality evaluation research is not comprehensive enough, and can not effectively reflect the quality level of Patrinia chinensis. The characteristic spectrum method constructed by the patent of the present invention can identify the different bases and authenticity of Patrinia chinensis at the same time, can comprehensively control the quality of medicinal materials, and ensure the effectiveness and safety of product quality. Summary of the Invention

[0006] Purpose of the invention: The present invention aims to establish a characteristic spectrum of Patrinia chinensis with good reproducibility and stability, low detection cost and high detection efficiency, a method for constructing a reference spectrum, and a method for distinguishing Patrinia chinensis from counterfeits. The method can quickly and accurately identify Patrinia chinensis with yellow flowers, Patrinia chinensis with white flowers and Patrinia chinensis counterfeits, provide a theoretical basis for the quality control of Patrinia chinensis, and provide a more powerful guarantee for the industrialization of Patrinia chinensis formula granules.

[0007] Technical solution: The present invention provides a method for constructing a characteristic map of Patrinia scabra, comprising the following steps:

[0008] (1) Take the powder of Patrinia herb, intermediate, decoction piece, standard decoction or its formula granules respectively, add water to dissolve, extract with n-butanol, evaporate the extract to dryness, and dissolve the residue in methanol-water solution to prepare the test solution;

[0009] (2) Protocatechuic acid, chondrosin, 3,4-O-dicaffeoylquinic acid, and 4,5-O-dicaffeoylquinic acid were taken separately and added with solvent to prepare reference substance solutions;

[0010] (3) The test solution and the reference solution were injected into the ultra-high performance liquid chromatography respectively to obtain a characteristic spectrum; the characteristic spectrum had 14 common peaks, protocatechuic acid was taken as the reference, and peak 1 was the chromatographic peak of protocatechuic acid; quinoline was taken as the reference, and peak 3 was the chromatographic peak of quinoline; 3,4-O-dicaffeoylquinic acid was taken as the reference, and peak 7 was the chromatographic peak of 3,4-O-dicaffeoylquinic acid; 4,5-O-dicaffeoylquinic acid was taken as the reference, and peak 10 was the chromatographic peak of 4,5-O-dicaffeoylquinic acid; the peak corresponding to the quinoline reference was the S peak, and the relative retention time of the remaining peaks to the S peak was calculated. The relative retention times should be within ±10% of the specified values. The specified values ​​of Peak 2, Peaks 4-6, Peak 8, Peak 9, and Peaks 11-14 are: 0.55, 1.05, 1.07, 1.17, 1.82, 1.94, 2.34, 2.51, 2.54, and 2.61, respectively.

[0011] The chromatographic conditions of the ultra-high performance liquid chromatography are as follows: GOLD aQ as a chromatographic column; methanol as mobile phase A, and 0.1% formic acid solution as mobile phase B; a flow rate of 0.23-0.27 ml / min; a column temperature of 25-30° C.; a detection wavelength of 254 nm; a gradient elution of 0-3 min, with a volume fraction of mobile phase A of 10% and a volume fraction of mobile phase B of 90%; from 3 to 22 min, the volume fraction of mobile phase A changes from 10 to 26%, and the volume fraction of mobile phase B changes from 90 to 74%; from 22 to 34 min, the volume fraction of mobile phase A changes from 26 to 36%, and the volume fraction of mobile phase B changes from 74 to 64%; from 34 to 55 min, the volume fraction of mobile phase A changes from 36 to 56%, and the volume fraction of mobile phase B changes from 64 to 44%; and from 55 to 58 min, the volume fraction of mobile phase A is 56%, and the volume fraction of mobile phase B is 44%.

[0012] The extraction times in step (1) are 1 to 3 times, and the extraction method is shaking.

[0013] Preferably, 0.2g to 1g of Patrinia scabra granules are taken, ground into powder, dissolved in water, extracted 1 to 3 times with n-butanol by shaking, the extract is evaporated to dryness, and the residue is dissolved in methanol and aqueous solution to serve as the test solution.

[0014] Preferably, 1 g to 3 g of Patrinia herba reference medicinal material is taken, water is added, the mixture is decocted on an electric stove, filtered, the filtrate is extracted 1 to 3 times by shaking with n-butanol or ethyl acetate, the extract is evaporated to dryness, and the residue is dissolved in methanol aqueous solution to serve as a reference medicinal material reference solution; separately, appropriate amounts of protocatechuic acid reference substance, chondramine glycoside reference substance, 3,4-O-dicaffeoylquinic acid reference substance, and 4,5-O-dicaffeoylquinic acid reference substance are taken, dissolved in methanol aqueous solution to serve as reference substance reference solution.

[0015] Preferably, 0.5 μl to 2 μl of the reference solution and the test solution are respectively aspirated.

[0016] The re-dissolution solvent is a methanol aqueous solution with a volume concentration of 10% to 70%.

[0017] Wherein, the concentration of the protocatechuic acid reference substance solution in step (1) is 15 to 40 μg / ml; the concentration of the glycoside reference substance solution is 100 to 200 μg / ml; the concentration of the 3,4-O-dicaffeoylquinic acid reference substance solution is 15 to 40 μg / ml; and the concentration of the 4,5-O-dicaffeoylquinic acid reference substance solution is 15 to 40 μg / ml.

[0018] The present invention also provides a method for constructing a reference map of Patrinia scabra, comprising the following steps:

[0019] (1) preparing multiple batches of Patrinia chinensis herb test solution, Patrinia chinensis intermediate test solution, Patrinia chinensis slice test solution, Patrinia chinensis standard decoction test solution, or Patrinia chinensis formula granule test solution;

[0020] (2) The test solution is tested according to the method for constructing the characteristic spectrum of Patrinia herba to obtain the characteristic spectrum of Patrinia herba raw materials, the characteristic spectrum of the intermediate, the characteristic spectrum of the decoction pieces, the characteristic spectrum of the standard decoction and the characteristic spectrum of the formula granules;

[0021] (3) The obtained characteristic spectrum is introduced into the Chinese medicine chromatographic fingerprint spectrum similarity evaluation system to establish a comparison spectrum of Patrinia herb materials, intermediates, decoction pieces, standard decoctions or their formula granules.

[0022] The present invention also provides a method for distinguishing Patrinia lutea from Patrinia whitei, comprising the following steps:

[0023] (1) Take the powder of Patrinia chrysanthemi or Patrinia dahurica, intermediate, decoction piece, standard decoction or its formula granules respectively, add water to dissolve, extract with n-butanol, evaporate the extract to dryness, and dissolve the residue with methanol-water solution to prepare the test solution;

[0024] (2) Protocatechuic acid, chondrosin, 3,4-O-dicaffeoylquinic acid, and 4,5-O-dicaffeoylquinic acid were taken separately and added with solvent to prepare reference substance solutions;

[0025] (3) The test sample solution and the reference sample solution were injected into the ultra-high performance liquid chromatography to obtain characteristic spectra; by analyzing the relative peak areas of peaks 8 and 11 relative to peak 3 in Patrinia chinensis, Patrinia chrysantha and Patrinia alba could be distinguished; when the relative peak area of ​​peak 11 was greater than 0.24 and the relative peak area of ​​peak 8 was greater than 0.30, the test sample was Patrinia chrysantha; when peak 11 was less than 0.24 and peak 8 was less than 0.30, the test sample was Patrinia alba.

[0026] The chromatographic conditions of the ultra-high performance liquid chromatography are as follows: GOLD aQ as a chromatographic column; methanol as mobile phase A, and 0.1% formic acid solution as mobile phase B; a flow rate of 0.23-0.27 ml / min; a column temperature of 25-30° C.; a detection wavelength of 254 nm; a gradient elution of 0-3 min, with a volume fraction of mobile phase A of 10% and a volume fraction of mobile phase B of 90%; from 3 to 22 min, the volume fraction of mobile phase A changes from 10 to 26%, and the volume fraction of mobile phase B changes from 90 to 74%; from 22 to 34 min, the volume fraction of mobile phase A changes from 26 to 36%, and the volume fraction of mobile phase B changes from 74 to 64%; from 34 to 55 min, the volume fraction of mobile phase A changes from 36 to 56%, and the volume fraction of mobile phase B changes from 64 to 44%; and from 55 to 58 min, the volume fraction of mobile phase A is 56%, and the volume fraction of mobile phase B is 44%.

[0027] The present invention also provides a method for distinguishing Patrinia lutea from counterfeit products, comprising the following steps:

[0028] (1) Take the powder of Patrinia chrysanthemi or counterfeit medicinal materials, intermediates, decoction pieces, standard decoctions or their formula granules, add water to dissolve them, extract them with n-butanol, evaporate the extract to dryness, and dissolve the residue with methanol-water solution to prepare the test solution;

[0029] (2) Protocatechuic acid, chondrosin, 3,4-O-dicaffeoylquinic acid, and 4,5-O-dicaffeoylquinic acid were taken separately and added with solvent to prepare reference substance solutions;

[0030] (3) The test sample solution and the reference sample solution were injected into the ultra-high performance liquid chromatography to obtain characteristic spectra; by analyzing the relative peak areas of peaks 8 and 11 relative to peak 3 in Patrinia herba, Patrinia herba and its counterfeits, Herba scutellariae and Herba cyperi, could be distinguished; when the relative peak area of ​​peak 11 was greater than 0.24 and the relative peak area of ​​peak 8 was greater than 0.30, the test sample was Patrinia chrysantha; when peaks 11 and 8 were not detected, the test sample was Herba scutellariae; when peak 3 was not detected, the test sample was Herba cyperi.

[0031] Among them, the preparation of the reference medicinal material reference solution: take 1g to 3g of the reference medicinal material Patrinia herba, place it in a stoppered conical flask, add water, decoct on an electric stove, filter, and extract the filtrate by shaking with n-butanol or ethyl acetate 1 to 3 times. The extract is evaporated to dryness, and the residue is dissolved in methanol and aqueous solution to serve as the reference medicinal material solution.

[0032] Preparation of reference substance solution: Take appropriate amount of protocatechuic acid reference substance, arachidylcholine reference substance, 3,4-O-dicaffeoylquinic acid reference substance, and 4,5-O-dicaffeoylquinic acid reference substance, accurately weigh them, and add 10% to 70% methanol to make a solution containing 15 to 40 μg of protocatechuic acid, 100 to 200 μg of arachidylcholine, 15 to 40 μg of 3,4-O-dicaffeoylquinic acid, and 15 to 40 μg of 4,5-O-dicaffeoylquinic acid per 1 ml as reference substance solution.

[0033] Preparation of the test solution of Patrinia scabra granules: Take about 0.3 g of Patrinia scabra granules powder, place it in a stoppered conical flask, add 20 ml of water to dissolve it, shake and extract with n-butanol twice, 20 ml each time, combine the n-butanol solutions, evaporate to dryness, add 2 ml of 50% methanol to dissolve the residue, and use it as the test solution.

[0034] Preparation of test sample solutions of standard decoctions of Patrinia scabra (Parinia chrysantha, Patrinia leucophylla) and Patrinia scabra counterfeits (Parinia ulmoides, Cyperus rotundus): Take about 0.2-1.0 g of standard decoction powder, place it in a stoppered conical flask, add water to dissolve it, shake and extract with n-butanol or ethyl acetate 1-3 times, evaporate the extract to dryness, and dissolve the residue in methanol-water solution to prepare the test sample solution.

[0035] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0036] (1) The method of the present invention is a simpler, faster, and more effective method for detecting the characteristic spectrum of Patrinia patriniae formula granules, which can simultaneously identify different origins and their counterfeits, providing a more powerful guarantee for the industrialization of Patrinia patriniae formula granules;

[0037] (2) The characteristic spectrum method established by the present invention can simultaneously distinguish different origins of Patrinia scabra and their counterfeits, providing a reliable analytical method for identifying different origins of Patrinia scabra and their counterfeits. The method is simple to operate and the results are relatively objective and accurate. After methodological verification, it has good reproducibility and stability, low detection cost, and high detection efficiency, providing an important guarantee for the standardization of the production of Patrinia scabra formula granules.

[0038] (3) The characteristic spectrum method established by the method of the present invention presents rich chromatographic peak information, including multiple chemical components, a large number of characteristic peaks, and 14 common characteristic peaks. Some characteristic peaks are identified, which is more than the number of characteristic peaks specified in the published standard of Patrinia scabra formula granules, and the evaluation is more comprehensive. In addition, the separation of each characteristic peak is good and the baseline is stable. At the same time, the use of control medicinal materials as reference materials can better reflect the consistency of the formula granules and the medicinal materials, and can more comprehensively control the quality of Patrinia scabra formula granules.

[0039] (4) The characteristic spectrum presents rich chromatographic peak information, including a variety of chemical components such as iridoids, phenolic acids, phenylpropanoids, and flavonoid glycosides, which can more comprehensively reflect the quality of Patrinia scabra.

[0040] (5) The present invention uses ultra-high performance liquid chromatography (UHPLC) and rationally optimizes the chromatographic conditions to establish a characteristic spectrum for Patrinia patriniae formula granules. This allows for identification of different bases and authenticity, providing an important guarantee for the quality control of Patrinia patriniae formula granules. The method is simple to operate, has high detection sensitivity, and reduces detection time and cost, providing a new analytical tool for quality control of Patrinia patriniae formula granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 UPLC chromatograms of Patrinia scabra granules at different detection wavelengths;

[0042] Figure 2 UPLC diagrams of different extraction methods and solvents;

[0043] Figure 3 UPLC diagrams for different extraction times;

[0044] Figure 4 The chromatograms of the reference substance, reference medicinal material, and Patrinia scabra granules are shown;

[0045] Figure 5 This is the characteristic spectrum of the Patrinia scabra granules, peak 1: protocatechuic acid; peak 3 (S): dandelion; peak 7: 3,4-O-dicaffeoylquinic acid; peak 10: 4,5-O-dicaffeoylquinic acid;

[0046] Figure 6 This is the characteristic spectrum of the intermediate of Patrinia scabra, Peak 1: protocatechuic acid; Peak 3 (S): dandelion; Peak 7: 3,4-O-dicaffeoylquinic acid; Peak 10: 4,5-O-dicaffeoylquinic acid;

[0047] Figure 7 This is the characteristic spectrum of the standard decoction of Patrinia chinensis, peak 1: protocatechuic acid; peak 3 (S): dandelion; peak 7: 3,4-O-dicaffeoylquinic acid; peak 10: 4,5-O-dicaffeoylquinic acid;

[0048] Figure 8 This is the characteristic spectrum of Patrinia chinensis slices, Peak 1: protocatechuic acid; Peak 3 (S): dandelion; Peak 7: 3,4-O-dicaffeoylquinic acid; Peak 10: 4,5-O-dicaffeoylquinic acid;

[0049] Figure 9 This is the characteristic spectrum of Patrinia radix, peak 1: protocatechuic acid; peak 3 (S): dandelion; peak 7: 3,4-O-dicaffeoylquinic acid; peak 10: 4,5-O-dicaffeoylquinic acid;

[0050] Figure 10 This is a test on the specificity of Patrinia scabra granules;

[0051] Figure 11 This is a test on the integrity of Patrinia scabra granules;

[0052] Figure 12 To investigate the chromatographic column for the characteristic spectrum of Patrinia patriniae formula granules;

[0053] Figure 13 The column temperature survey of the characteristic spectrum of Patrinia patriniae granules;

[0054] Figure 14 It is an investigation on the flow rate of the characteristic spectrum of Patrinia patriniae formula granules;

[0055] Figure 15 This is a comparison of characteristic spectra of different standard decoctions of Patrinia chinensis.

[0056] Figure 16 This is a comparison of characteristic spectra of standard decoctions to distinguish between authentic and counterfeit Patriniae Herba. DETAILED DESCRIPTION

[0057] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0058] 1. Instruments and reagents

[0059] Agilent Technologies 1290 Infancy ultra-high performance liquid chromatograph; Thermo Vanquish ultra-high performance liquid chromatograph; KQ-250B ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); electronic analytical balance (Mettler-Toledo Instrument (Shanghai) Co., Ltd.); temperature-controlled water bath (Nantong Huatai Experimental Instrument Co., Ltd.); HY-4 oscillator (Jintan Kexing Instrument Factory); pure water system (Millipore); AS165W centrifuge (Azov (Shanghai) Trading Co., Ltd.); acetonitrile (chromatographic grade, Thermo Fisher); methanol (chromatographic grade, Thermo Fisher); formic acid (chromatographic grade, Aladdin); ultrapure water; all other reagents were of analytical grade.

[0060] Protocatechuic acid reference substance, arachidylcholine reference substance, and 4,5-O-dicaffeoylquinic acid reference substance were purchased from the China Food and Drug Administration with batch numbers 110809-201906, 111742-200501, and 111894-202205, respectively, with purities of 97.7%, 100%, and 94.9%, respectively. 3,4-O-dicaffeoylquinic acid reference substance was purchased from Chengdu Pusi Biotechnology Co., Ltd. with batch number PS001054 and a purity of 98.0%.

[0061] Patrinia scabra (Patrimonium wilfordii) was purchased from the China Food and Drug Administration with batch number 121271-201201.

[0062] Patrinia scabra (Patrimonium wilfordii) formula granules (batch numbers: KL1129, KL1139, KL1149) were provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0063] Patrinia scabra (Patrimonium wilfordii) standard decoction (batch numbers: DG2019, DG2020, DG2022, DG2023, DG2024, DG2027, DG2028, DG2029, DG3037, DG3039, DG3040, DG3043, DG3044, DG3045, DG3650, DG3651, DG3 652), the standard decoction of Patrinia jasminoides (Baihua Patrinia jasminoides) (batch numbers: DG6028, DG6029, DG6030), the standard decoction of the counterfeit Patrinia jasminoides Mutouhui (batch numbers: DG6047, DG6050, DG6052), and the standard decoction of the counterfeit Patrinia jasminoides (batch numbers: DG6053, DG6054, DG6055) were provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0064] Patrinia scabra (Patrimonium wilfordii) intermediates (batch numbers: ZJT1129, ZJT1139, ZJT1149), Patrinia scabra (Patrimonium wilfordii) slices (batch numbers: YP2019, YP2020, YP2022, YP2023, YP2024, YP2027, YP2028, YP2029, YP3037, YP3039, YP3040, YP3043, YP3044, YP3045, YP3650, Y P3651, YP3652), Patrinia chinensis (Patrimonium wilfordii) medicinal materials (batch numbers: YC2019, YC2020, YC2022, YC2023, YC2024, YC2027, YC2028, YC2029, YC3037, YC3039, YC3040, YC3043, YC3044, YC3045, YC3650, YC3651, YC3652) were provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0065] Example 1 Establishment of the characteristic spectrum method of Patrinia scabra formula granules

[0066] This example is used to confirm the optimal solution for establishing the characteristic spectrum of Patrinia scabra samples, specifically including comparison of chromatographic conditions, wavelength selection, and test solution preparation schemes, and select the optimal chromatographic conditions and test solution preparation scheme.

[0067] 1. Determination of detection wavelength

[0068] Take an appropriate amount of Patrinia scabra (Patrimonium wilfordii) granules (batch number: KL1129), grind them into powder, take 0.3g, place in a stoppered conical flask, add 20ml of water to dissolve, shake and extract with n-butanol twice, 20ml each time, combine the n-butanol solutions, evaporate to dryness, add 2ml of 50% methanol solution to dissolve the residue, as the test solution, accurately draw 1μl of each test solution, inject into the ultra-performance liquid chromatograph, and record the chromatograms at different wavelengths (235nm, 300nm, 254nm), as shown. Figure 1 The chromatographic conditions were as follows: GOLD aQ (Thermo, 2.1×150 mm, 1.9 μm) as the chromatographic column; methanol as the mobile phase A, 0.1% formic acid solution as the mobile phase B, gradient elution according to the requirements in Table 1; flow rate of 0.25 ml / min; column temperature of 25°C.

[0069] Table 1

[0070]

[0071] The results showed that the baseline of the Patrinia scabra granules was stable at a wavelength of 254 nm, the chromatographic peak information was rich and the response was good, so 254 nm was finally selected as the detection wavelength.

[0072] In summary, the chromatographic conditions were as follows: GOLD aQ (Thermo, 2.1×150 mm, 1.9 μm) was used as the chromatographic column; methanol was used as the mobile phase A, and 0.1% formic acid solution was used as the mobile phase B, with gradient elution as specified in Table 1; the flow rate was 0.25 ml per minute; the column temperature was 25°C; and the detection wavelength was 254 nm.

[0073] 2. Preparation of test solution

[0074] 2.1 Investigation of different extraction methods and solvents

[0075] Take an appropriate amount of Patrinia scabra (Patrimonium wilfordii) formula granules (batch number: KL1129), grind them into powder, take 0.3g, and make three parallel portions. Place them in stoppered conical flasks. Add 20ml of 50% methanol solution to one portion, ultrasonically treat (power 250W, frequency 40kHz) for 30 minutes, let cool, shake well, filter, and take the filtrate to obtain; add 20ml of water to the other two portions to dissolve them, shake and extract one portion twice with n-butanol, and shake and extract one portion twice with ethyl acetate, 20ml each time, combine n-butanol and ethyl acetate respectively, evaporate to dryness, add 2ml of 50% methanol solution to the residue to dissolve it, and use it as the test solution. Accurately draw 1μl of each test solution, inject it into the ultra-performance liquid chromatograph, measure it according to the above chromatographic conditions, and calculate the ratio of the characteristic peak area / sample weight. The results are shown in Table 2. Figure 2 .

[0076] Table 2 Comparison of extraction efficiency of different extraction methods and solvents (peak area / sample weight)

[0077]

[0078]

[0079] The results showed that the chromatographic peak information of the sample extracted by shaking with n-butanol was rich, the response was high and the separation was good, so n-butanol shaking extraction was finally selected.

[0080] 2.2 Investigation of different extraction times

[0081] Take an appropriate amount of Patrinia scabra (Patrimonium wilfordii) formula granules (batch number: KL1129), grind them into powder, take 0.3g, and place them in triplicate in a stoppered conical flask. Add 20ml of water to dissolve them. Shake and extract with n-butanol once, twice, and three times, 20ml each time. Combine the n-butanol solutions, evaporate to dryness, and dissolve the residue in 2ml of 50% methanol solution to prepare the test solution. Accurately pipette 1μl of each test solution and inject it into an ultra-high performance liquid chromatograph. Determine according to the above chromatographic conditions and calculate the characteristic peak area / sample weight. The results are shown in Table 3. Figure 3 .

[0082] Table 3 Comparison of extraction efficiency at different extraction times (peak area / sample weight)

[0083]

[0084] The results showed that the number of chromatographic peaks obtained with different extraction times was consistent. As the number of extractions increased, the response of some chromatographic peaks increased. The response of each chromatographic peak was good after two shaking extractions. In order to save extraction time and cost, the final extraction number was selected to be two.

[0085] 2.3 Determination of the test solution preparation method

[0086] According to the above research results, the preparation method of the test solution of the characteristic spectrum of Patrinia scabra granules is determined as follows:

[0087] Take an appropriate amount of Patrinia scabra granules, grind them into powder, take 0.3g, place it in a stoppered conical flask, add 20ml of water to dissolve it, shake and extract with n-butanol twice, 20ml each time, combine the n-butanol liquids, evaporate to dryness, add 2ml of 50% methanol to dissolve the residue, and use it as the test solution.

[0088] Example 2

[0089] Take 3 batches of Patriniae Herba (Patrimonium Chinense) formula granules, 17 batches of Patriniae Herba (Patrimonium Chinense) standard decoctions, 17 batches of Patriniae Herba (Patrimonium Chinense) decoction pieces, 17 batches of Patriniae Herba (Patrimonium Chinense) medicinal materials, 3 batches of Patriniae Herba (Patrimonium Chinense) intermediates, 3 batches of Baihua Patriniae standard decoctions, 3 batches of Mutouhui (Patrimonium Chinense counterfeit) standard decoctions, and 3 batches of Iris ternata (Patrimonium Chinense counterfeit) standard decoctions as Patriniae Herba samples, and measure them according to the following steps:

[0090] (1) Select Herba Patriniae and prepare the test solution; use Herba Patriniae as the reference medicinal material to prepare the reference medicinal material solution; use protocatechuic acid, chondramine, 3,4-O-dicaffeoylquinic acid and 4,5-O-dicaffeoylquinic acid as the reference medicinal material solutions, respectively.

[0091] Among them, the preparation of the reference medicinal material reference solution: take 2g of Patrinia scabra reference medicinal material, place it in a stoppered conical flask, add 50ml of water, boil on an electric stove for 30 minutes, cool, shake well, filter, and extract the filtrate by shaking with n-butanol twice, 50ml each time, combine the n-butanol liquid, evaporate to dryness, and add 1ml of 50% methanol to dissolve the residue as the reference medicinal material solution.

[0092] Preparation of reference substance solution: Take appropriate amount of protocatechuic acid reference substance, 3,4-O-dicaffeoylquinic acid reference substance, 4,5-O-dicaffeoylquinic acid reference substance and arachidonic acid reference substance, accurately weigh them, and add 50% methanol to make a mixed solution containing 30 μg of protocatechuic acid, 3,4-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid and 150 μg of arachidonic acid per 1 ml, which is used as the reference substance solution.

[0093] Preparation of the test solution of Patrinia scabra granules: Take an appropriate amount of Patrinia scabra granules, grind them into powder, take 0.3 g, place it in a stoppered conical flask, add 20 ml of water to dissolve it, shake and extract with n-butanol twice, 20 ml each time, combine the n-butanol solutions, evaporate to dryness, add 2 ml of 50% methanol to dissolve the residue, and use it as the test solution.

[0094] Preparation of test solutions of standard decoctions of Patrinia scabra (Parinia chrysantha, Patrinia leucophylla), Patrinia scabra counterfeits (Phellodendron chinense, Cyperus rotundus), and intermediates of Patrinia scabra (Parinia chrysantha): Take 0.3 g of the standard decoction and intermediate powder respectively, place them in a stoppered conical flask, add 20 ml of water to dissolve them, shake and extract with n-butanol twice, 20 ml each time, combine the n-butanol solutions, evaporate to dryness, and dissolve the residue in 2 ml of 50% methanol to prepare the test solution.

[0095] (2) Accurately pipette 1 μl of the reference substance solution, the reference medicinal material solution, and the test solution prepared in step (1), inject them into an ultra-high performance liquid chromatograph, perform liquid chromatography analysis, and obtain the corresponding ultra-high performance liquid chromatogram.

[0096] The chromatographic conditions were as follows: a GOLD aQ (Thermo, 2.1×150 mm, 1.9 μm) column; methanol as mobile phase A, 0.1% formic acid solution as mobile phase B, and gradient elution according to the requirements in Table 1; a flow rate of 0.25 ml / min; a column temperature of 25° C.; and a detection wavelength of 254 nm.

[0097] like Figure 4 As shown in the characteristic spectrum of Patrinia scabra granules, the test solution chromatogram shows 14 characteristic peaks, which correspond to the retention times of the 14 characteristic peaks in the chromatogram of the reference medicinal material. Peaks 1, 3, 7, and 10 should be consistent with the retention times of the protocatechuic acid reference, dapoxetine reference, 3,4-O-dicaffeoylquinic acid reference, and 4,5-O-dicaffeoylquinic acid reference, respectively. The peak corresponding to the dapoxetine reference is the S peak, and the relative retention times of the remaining peaks to the S peak are calculated. Their relative retention times should be within ±10% of the specified values, which are: 0.55 (peak 2), 1.05 (peak 4), 1.07 (peak 5), 1.17 (peak 6), 1.82 (peak 8), 1.94 (peak 9), 2.34 (peak 11), 2.51 (peak 12), 2.54 (peak 13), and 2.61 (peak 14).

[0098] The characteristic spectrum of Patrinia scabra granules was used to generate a control characteristic spectrum ( Figure 5 ).

[0099] According to the test method of Herba Patriniae Formula Granules, Herba Patriniae Intermediates, Herba Patriniae Formula Granules, Herba Patriniae Standard Decoction, Herba Patriniae Decoction Pieces, and Herba Patriniae Materials were tested. The test results of the test solution are shown in Tables 4 to 7 and Figures 6 to 9 .

[0100] Table 4 Patrinia scabra (Patrimonium wilfordii) formula granule sample measurement results (relative retention time)

[0101]

[0102]

[0103] Table 5 Patrinia chinensis (Patrimonium wilfordii) standard decoction sample determination results (relative retention time)

[0104]

[0105]

[0106]

[0107] Table 6 Patrinia chinensis (Patrimonium wilfordii) slice sample determination results (relative retention time)

[0108]

[0109]

[0110] Table 7 Patrinia chinensis (Patrimonium wilfordii) medicinal material sample determination results (relative retention time)

[0111]

[0112]

[0113]

[0114] From the above test results, it can be seen that from a qualitative analysis, the characteristic spectra of 3 batches of Herba Patriniae formula granules and 17 batches of Herba Patriniae standard decoctions, herbal slices, and medicinal materials meet the requirements of the characteristic spectra in step (2).

[0115] Example 3 Characteristic Spectrum Methodology Study

[0116] 1. Specificity inspection

[0117] The excipients added to the Herba Patriniae formula granules are maltodextrin, silicon dioxide and magnesium stearate. This experiment investigates the effect of negative samples lacking Herba Patriniae on Herba Patriniae formula granules. Take the negative sample lacking Herba Patriniae and prepare a negative sample solution according to the test sample preparation method. Inject 1μl of the sample according to the above chromatographic conditions. The results are shown in Figure 2. Figure 10 .

[0118] The results showed that the solvents and excipients had no interference in the determination of the characteristic spectrum of Patrinia patriniae formula granules, and the method was specific for the determination of Patrinia patriniae formula granules.

[0119] 2. Overall investigation

[0120] Take an appropriate amount of Patrinia scabra (Patrimonium wilfordii) formula granules (batch number: KL1129), grind them into powder, take 0.3g, place in a stoppered conical flask, add 20ml of water to dissolve, shake and extract with n-butanol twice, 20ml each time, combine the n-butanol solution, evaporate to dryness, add 2ml of 50% methanol to dissolve the residue, and use it as the test solution. Refer to the chromatographic conditions of Example 2, only the elution gradient is changed. Under the same chromatographic conditions, keep the elution gradient when the methanol ratio is the highest, and double the elution time. The results are shown in Table 2. Figure 11 .

[0121] The results showed that no obvious chromatographic peaks were eluted after the original gradient elution, indicating that the chromatographic conditions basically met the principle of maximum information content.

[0122] 3. Precision inspection

[0123] Take Patrinia herba (Patrimonium wilfordii) formula granules (batch number: KL1129) and prepare a test solution according to the test solution preparation method in Example 2. The sample was injected 6 times continuously, 1 μl each time, and the retention time of the characteristic peak was recorded. The relative retention time was calculated according to the method of Example 2. The results are shown in Table 8.

[0124] Table 8 Precision test results (relative retention time)

[0125]

[0126]

[0127] The results showed that the RSDs of the relative retention times of the characteristic peaks were less than 1%, indicating good precision.

[0128] 4. Intermediate precision inspection

[0129] Take the formula granules of Patrinia scabra (Patrimonium wilfordii) (batch number: KL1129) and prepare 3 samples by two experimenters A and B according to the test solution preparation method determined in Example 2. According to the chromatographic conditions of Example 2, 1 μl was injected at different times and on different instruments, and the retention time of the characteristic peak was recorded. The relative retention time was calculated. The results are shown in Table 9.

[0130] Table 9 Intermediate precision investigation (relative retention time)

[0131]

[0132]

[0133] The results showed that the RSD of the relative retention time of each characteristic peak was less than 3%, and the intermediate precision was good.

[0134] 5. Stability inspection

[0135] Take Patrinia herba (Patrimonium wilfordii) formula granules (batch number: KL1129) and prepare a test solution according to the test solution preparation method determined in Example 2. 1 μl was injected at 0, 2, 4, 8, 12, 18, and 24 h, respectively. The detection was performed according to the chromatographic conditions of Example 2, and the retention time of the characteristic peak was recorded. The relative retention time was calculated. The results are shown in Table 10.

[0136] Table 10 Stability study (relative retention time)

[0137]

[0138]

[0139] The results showed that the RSDs of the relative retention times of the characteristic peaks were all less than 1%, and the test solution had good stability within 24 hours.

[0140] 6. Repeatability inspection

[0141] Take Patrinia herba (Patrimonium wilfordii) formula granules (batch number: KL1129) in 6 parallel groups, prepare test solutions according to the test solution preparation method of Example 2, inject 1 μl of each solution, and detect according to the chromatographic conditions of Example 2. Record the retention time of its characteristic peak and calculate the relative retention time. The results are shown in Table 11.

[0142] Table 11 Repeatability test (relative retention time)

[0143]

[0144]

[0145] The results showed that the RSD of the relative retention time of each characteristic peak was less than 1%, and the repeatability of the method was good.

[0146] 7. Durability inspection

[0147] (1) Chromatographic column inspection

[0148] Take Patrinia scabra (Patrimonium wilfordii) formula granules (batch number: KL1129), prepare the test solution according to the preparation method of the test solution in Example 2, and use GOLD aQ (Thermo, 2.1×150mm, 1.9μm); SB-C18 (Agilent, 2.1×150mm, 1.8μm); Syncronis C18 (Thermo, 2.1mm×100mm, 1.7μm) three different chromatographic columns, other chromatographic conditions are the same as in Example 2, and the separation effect is investigated. The results are shown in Figure 12 , Table 12.

[0149] Table 12 Column investigation (relative retention time)

[0150]

[0151]

[0152] The results showed that the Patrinia herba sample was well separated on a GOLD aQ (Thermo, 2.1 × 150 mm, 1.9 μm) column, while some peaks overlapped on SB-C18 (Agilent, 2.1 × 150 mm, 1.8 μm) and Syncronis C18 (Thermo, 2.1 mm × 100 mm, 1.7 μm) columns. A GOLD aQ (Thermo, 2.1 × 150 mm, 1.9 μm) column was used in subsequent studies.

[0153] (2) Column temperature investigation

[0154] Take Patrinia chinensis (Patrimonium wilfordii) formula granules (batch number: KL1129), prepare the test solution according to the preparation method of the test solution in Example 2, use GOLD aQ (Thermo, 2.1×150mm, 1.9μm) chromatographic column, and investigate the sample separation effect when the column temperature is 20℃, 25℃, and 30℃ respectively. Other chromatographic conditions are the same as in Example 2. The results are shown in Figure 13 , Table 13.

[0155] Table 13 Column temperature investigation (relative retention time)

[0156]

[0157]

[0158] The results showed that at a column temperature of 20°C, peaks 4 and 5 formed an envelope peak. Within the temperature range of 25°C to 30°C, the relative retention times of the characteristic peaks remained within the specified range. Small column temperature fluctuations met the system's robustness requirements. A column temperature of 25°C was selected for subsequent studies.

[0159] (3) Flow rate investigation

[0160] Take Patrinia scabra (Patrimonium wilfordii) formula granules (batch number: KL1129), prepare the test solution according to the preparation method of the test solution in Example 2, use GOLD aQ (Thermo, 2.1×150mm, 1.9μm) chromatographic column, and examine the sample separation effect at flow rates of 0.23ml / min, 0.25ml / min, and 0.27ml / min, respectively. Other chromatographic conditions are the same as in Example 2. The results are shown in Figure 14 , Table 14.

[0161] Table 14 Flow rate investigation (relative retention time)

[0162]

[0163] The results showed that within the flow rate range of 0.23 to 0.27 ml / min, the relative retention times of the characteristic peaks were within the specified range, and small flow rate fluctuations met the system's robustness requirements. A flow rate of 0.25 ml / min was selected for subsequent studies.

[0164] Example 4 Identification study of different origins of Patrinia scabra

[0165] 1. Preparation of test solution

[0166] Take 0.3 g of the powder of the standard decoction of Patrinia chrysanthemi and the standard decoction of Patrinia dahurica, place it in a stoppered conical flask, add 20 ml of water to dissolve it, shake and extract it with n-butanol twice, 20 ml each time, combine the n-butanol solutions, evaporate to dryness, and add 2 ml of 50% methanol solution to dissolve the residue as the test solution.

[0167] 2. Sample measurement and data analysis

[0168] 17 batches of standard decoction of Patrinia cava and 3 batches of standard decoction of Patrinia dahurica were taken respectively, and sample test solutions were made according to the above-mentioned test solution preparation method. 1 μl was accurately pipetted and injected into an ultra-performance liquid chromatograph. According to the chromatographic conditions of Example 2, the characteristic spectra of the standard decoction of Patrinia cava and Patrinia dahurica were respectively determined. The "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" recommended by the State Pharmacopoeia Commission was used to generate a control characteristic spectrum, and then the control characteristic spectra of the two were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" for comparative analysis.

[0169] The results are as follows Figure 15 As shown, some characteristic peaks in the standard decoction of Patrinia cava were not detected in Patrinia dahurica, and only 7 common characteristic peaks in Patrinia cava were presented, among which peaks 5, 8 and 12 only existed in some batches of Patrinia dahurica; while peaks 9, 11, 13 and 14 were not detected in the standard decoction of Patrinia dahurica.

[0170] The differences between the quasi-decoctions of Patrinia dahurica and Patrinia leucoderma were further analyzed by relative peak areas. Peak 3 was used as the reference peak to compare the relative peak area differences of the characteristic peaks in the standard decoctions of the two. The results are shown in Tables 15 and 16.

[0171] Table 15 Relative peak area ranges of the standard decoction samples of Patrinia chrysanthemi and Patrinia dahurica

[0172]

[0173]

[0174] Table 16 Relative peak areas of the standard decoction samples of Patrinia chrysanthemi and Patrinia dahurica

[0175]

[0176]

[0177] The results showed that peak 11 was not detected in the standard decoction of Patrinia dahurica. According to the relative peak area range, the minimum value was taken as the lower limit of the relative peak area by 40%, and the relative peak area of ​​peak 11 was not less than 0.24. Peak 8 was relatively small in the standard decoction of Patrinia dahurica. According to the relative peak area range, the minimum value was taken as the lower limit of the relative peak area by 40%, and the relative peak area of ​​peak 8 was not less than 0.30. This can effectively distinguish the Patrinia dahurica samples with different origins.

[0178] Example 5 Study on the identification of authentic Patrinia scabra

[0179] 1. Preparation of test solution

[0180] Take 0.3 g of the powder of this product, place it in a stoppered conical flask, add 20 ml of water to dissolve it, shake and extract it twice with 20 ml of n-butanol each time, combine the n-butanol solutions, evaporate to dryness, and add 2 ml of 50% methanol to dissolve the residue as the test solution.

[0181] 2. Sample measurement and data analysis

[0182] 17 batches of standard decoction of Patrinia chinensis, 3 batches of standard decoction of Mutouhui (counterfeit Patrinia chinensis), and 3 batches of standard decoction of Cyperus rotundus (counterfeit Patrinia chinensis) were taken respectively, and sample test solutions were made according to the above-mentioned test solution preparation method. 1 μl was accurately pipetted and injected into an ultra performance liquid chromatograph. According to the chromatographic conditions of Example 2, the characteristic spectra of Patrinia chinensis and its counterfeit standard decoction were respectively determined. The “Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)” recommended by the State Pharmacopoeia Commission was used to generate a control characteristic spectrum, and then the control characteristic spectrum of Patrinia chinensis and its counterfeit was imported into the “Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)” for comparative analysis.

[0183] (1) Patrinia scabra and Mutouhui

[0184] The results are as follows Figure 15 As shown, the number of characteristic peaks differed between the standard decoction of Mutouhui and the standard decoction of Patriniae. The standard decoction of Patriniae has 14 common characteristic peaks, while the standard decoction of Mutouhui only exhibits 8 of the common characteristic peaks of the standard decoction of Patriniae. Peak 12 is present only in some batches of Mutouhui. In addition, Peak A is a new characteristic peak of Mutouhui. This peak has poor resolution in Patriniae and low response in most batches, and was not identified as a characteristic peak of Patriniae. Therefore, the established characteristic spectrum can effectively distinguish Patriniae and Mutouhui standard decoctions.

[0185] (2) Patrinia chinensis and Cyperus rotundus

[0186] The results are as follows Figure 16 As shown, the number of characteristic peaks in the standard decoction of Herba Patriniae differs from that in the standard decoction of Herba Patriniae. The standard decoction of Herba Patriniae has 14 common characteristic peaks, while the standard decoction of Herba Patriniae only exhibits 7 of the common characteristic peaks found in the standard decoction of Herba Patriniae. Furthermore, peaks B through E are newly added to the common characteristic peaks of Herba Patriniae and are absent in Herba Patriniae. Therefore, the presence or absence of peaks B through E can effectively distinguish the standard decoctions of Herba Patriniae and Herba Patriniae.

[0187] The differences between Patrinia chinensis and the standard decoction of Mutouhui were further analyzed by relative peak area. Peak 3 was used as the reference peak to compare the relative peak area differences of each characteristic peak in Patrinia chinensis and the standard decoction of Mutouhui. The results are shown in Tables 17 and 18.

[0188] Table 17 Relative peak area ranges of Herba Patriniae and Herba Mutouhui standard decoction samples

[0189]

[0190]

[0191] Table 18 Relative peak areas of Herba Patriniae and Herba Mutouhui standard decoction samples

[0192]

[0193]

[0194] The results showed that peak 11 in the standard decoction of Patrinia salviae miltiorrhizae was not detected in the standard decoctions of Herba Epimedii and Herba Cynanchifoliae. According to the relative peak area range, the minimum value was taken as 40% lower as the lower limit of the relative peak area, and the relative peak area of ​​peak 11 was not less than 0.24. Peak 8 was smaller in the standard decoction of Herba Patriniae miltiorrhizae. According to the relative peak area range, the minimum value was taken as 40% lower as the lower limit of the relative peak area, and the relative peak area of ​​peak 8 was not less than 0.30. At the same time, neither peak 11 nor peak 8 was detected as Herba Cynanchifoliae, and Herba Cynanchifoliae was not detected in peak 3. This can effectively distinguish the fake Patrinia salviae miltiorrhizae Herba from Herba Cynanchifoliae.

Claims

1. A method for constructing a characteristic map of Patrinia scabra, characterized in that: The following steps are involved: (1) Take the powder of Patrinia herb, intermediate, decoction piece, standard decoction or its formula granules respectively, add water to dissolve, extract with n-butanol, evaporate the extract to dryness, and dissolve the residue in methanol-water solution to prepare the test solution; (2) Take protocatechuic acid, choline, 3,4-O-dicaffeoylquinic acid, and 4,5-O-dicaffeoylquinic acid respectively, and add solvent to prepare reference substance solution; (3) The test solution and the reference solution were injected into an ultra-high performance liquid chromatography (UPLC) to obtain characteristic spectra. The chromatographic conditions of the ultra-high performance liquid chromatography (UPLC) were as follows: GOLD aQ as the chromatographic column; methanol as the mobile phase A and 0.1% formic acid solution as the mobile phase B; the flow rate was 0.23-0.27 ml / min; the column temperature was 25-30 °C; the detection wavelength was 254 nm; the gradient elution was 0-3 min, the volume fraction of the mobile phase A was 10%, and the volume fraction of the mobile phase B was 90%; from 3 to 22 min, the volume fraction of the mobile phase A changed from 10 to 26%, and the volume fraction of the mobile phase B changed from 90 to 74%; from 22 to 34 min, the volume fraction of the mobile phase A changed from 26 to 36%, and the volume fraction of the mobile phase B changed from 74 to 64%; from 34 to 55 min, the volume fraction of the mobile phase A changed from 36 to 56%, and the volume fraction of the mobile phase B changed from 64 to 44%; ~58min, the volume fraction of mobile phase A is 56%, and the volume fraction of mobile phase B is 44%; the characteristic spectrum has 14 common peaks, taking protocatechuic acid as the reference substance, peak 1 is the chromatographic peak of protocatechuic acid; taking cinnamomea as the reference substance, peak 3 is the chromatographic peak of cinnamomea; taking 3,4-O-dicaffeoylquinic acid as the reference substance, peak 7 is the chromatographic peak of 3,4-O-dicaffeoylquinic acid; taking 4,5-O-dicaffeoylquinic acid as the reference substance, peak 10 is the chromatographic peak of 4,5-O-dicaffeoylquinic acid; the peak corresponding to the cinnamomea reference substance is the S peak, and the relative retention times of the remaining peaks and the S peak are calculated; their relative retention times should be within ±10% of the specified values, and the specified values ​​of peak 2, peaks 4-6, peak 8, peak 9, and peaks 11-14 are: 0.55、1.05、1.07、1.17、1.82、1.94、2.34、2.51、2.54、2.61。 2. The construction method according to claim 1, characterized in that: The number of extractions in step (1) is 1 to 3 times, and the extraction method is shaking.

3. The construction method according to claim 1, characterized in that: In step (1), the concentration of the protocatechuic acid reference substance solution is 15-40 μg / ml; the concentration of the glycoside reference substance solution is 100-200 μg / ml; the concentration of the 3,4-O-dicaffeoylquinic acid reference substance solution is 15-40 μg / ml; and the concentration of the 4,5-O-dicaffeoylquinic acid reference substance solution is 15-40 μg / ml.

4. A method for constructing a reference map of Patrinia chinensis, characterized in that: The following steps are involved: (1) Prepare multiple batches of Patrinia chinensis herb test solution, Patrinia chinensis intermediate test solution, Patrinia chinensis slice test solution, Patrinia chinensis standard decoction test solution, or Patrinia chinensis formula granule test solution; (2) The method for constructing a characteristic spectrum of Patrinia herba according to claim 1 is used to detect the test solution respectively to obtain a characteristic spectrum of Patrinia herba raw materials, a characteristic spectrum of intermediates, a characteristic spectrum of decoction pieces, a characteristic spectrum of standard decoctions, and a characteristic spectrum of formula granules; (3) The obtained characteristic spectrum is introduced into the Chinese medicine chromatographic fingerprint similarity evaluation system to establish a comparison spectrum of Patrinia herb materials, intermediates, decoction pieces, standard decoctions or their formula granules.

5. A method for distinguishing Patrinia chrysantha from Patrinia leucoderma, characterized in that: The following steps are involved: (1) Take the powder of Patrinia chrysanthemi or Patrinia dahurica, intermediate, slice, standard decoction or its formula granules respectively, add water to dissolve, extract with n-butanol, evaporate the extract to dryness, and dissolve the residue with methanol-water solution to prepare the test solution; (2) Take protocatechuic acid, choline, 3,4-O-dicaffeoylquinic acid, and 4,5-O-dicaffeoylquinic acid respectively, and add solvent to prepare reference substance solution; (3) The test sample solution and the reference sample solution were injected into the ultra-high performance liquid chromatography to obtain characteristic spectra; by analyzing the relative peak areas of peaks 8 and 11 relative to peak 3 in Patrinia chinensis, Patrinia chrysantha and Patrinia leucophylla could be distinguished; when the relative peak area of ​​peak 11 was greater than 0.24 and the relative peak area of ​​peak 8 was greater than 0.30, the test sample was Patrinia chrysantha; when peak 11 was less than 0.24 and peak 8 was less than 0.30, the test sample was Patrinia leucophylla; the chromatographic conditions of the ultra-high performance liquid chromatography were as follows: GOLD aQ was used as the chromatographic column; methanol was used as the mobile phase A, and 0.1% formic acid solution was used as the mobile phase B; the flow rate was 0.23-0.27 ml / min; the column temperature was 25-30°C; the detection wavelength was 254 nm; gradient elution was performed from 0 to 3 min, with the volume fraction of mobile phase A being 10% and the volume fraction of mobile phase B being 90%; from 3 to 22 min, the volume fraction of mobile phase A changed from 10 to 26%, and the volume fraction of mobile phase B changed from 90 to 74%; from 22 to 34 min, the volume fraction of mobile phase A changed from 26 to 36%, and the volume fraction of mobile phase B changed from 74 to 64%; from 34 to 55 min, the volume fraction of mobile phase A changed from 36 to 56%, and the volume fraction of mobile phase B changed from 64 to 44%; from 55 to 58 min, the volume fraction of mobile phase A was 56%, and the volume fraction of mobile phase B was 44%.

6. A method for distinguishing Patrinia chrysantha from counterfeits, characterized in that: The following steps are involved: (1) Take the powder of Patrinia chrysanthemi or counterfeit medicinal materials, intermediates, decoction pieces, standard decoction or its formula granules, add water to dissolve, extract with n-butanol, evaporate the extract to dryness, and dissolve the residue in methanol-water solution to prepare the test solution; (2) Take protocatechuic acid, choline, 3,4-O-dicaffeoylquinic acid, and 4,5-O-dicaffeoylquinic acid respectively, and add solvent to prepare reference substance solution; (3) The test solution and the reference solution were injected into an ultra-high performance liquid chromatography (UPLC) to obtain characteristic spectra. The chromatographic conditions of the ultra-high performance liquid chromatography (UPLC) were as follows: GOLD aQ as the chromatographic column; methanol as the mobile phase A and 0.1% formic acid solution as the mobile phase B; the flow rate was 0.23-0.27 ml / min; the column temperature was 25-30 °C; the detection wavelength was 254 nm; the gradient elution was 0-3 min, the volume fraction of the mobile phase A was 10%, and the volume fraction of the mobile phase B was 90%; from 3 to 22 min, the volume fraction of the mobile phase A changed from 10 to 26%, and the volume fraction of the mobile phase B changed from 90 to 74%; from 22 to 34 min, the volume fraction of the mobile phase A changed from 26 to 36%, and the volume fraction of the mobile phase B changed from 74 to 64%; From 34 to 55 min, the volume fraction of mobile phase A changed from 36 to 56%, and the volume fraction of mobile phase B changed from 64 to 44; from 55 to 58 min, the volume fraction of mobile phase A was 56%, and the volume fraction of mobile phase B was 44%; by analyzing the relative peak areas of peaks 8 and 11 relative to peak 3 in Patrinia herb, Patrinia herb and its counterfeits, Mutouhui and Ichthyophthirius, could be distinguished; when the relative peak area of ​​peak 11 was greater than 0.24 and the relative peak area of ​​peak 8 was greater than 0.30, the test sample was Patrinia chrysantha; when peaks 11 and 8 were not detected, the test sample was Mutouhui; when peak 3 was not detected, the test sample was Ichthyophthirius.

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