A method for detecting the quality of Saxifraga herbacea medicinal materials

Abstract

The invention discloses a method for detecting the quality of saxifrage medicinal materials, including identifying saxifrage medicinal materials by thin layer chromatography, including the following steps: A, taking saxifrage medicinal materials, adding 30% ethanol, ultrasonic treatment, filtering, evaporating the filtrate to dryness, suspending the residue in heated water, extracting with ethyl acetate for many times, merging the ethyl acetate extract, evaporating, dissolving the residue in methanol, and preparing a test solution; B, taking saxifrage control medicinal materials, adding 30% ethanol, ultrasonic treatment, filtering, evaporating the filtrate to dryness, suspending the residue in heated water, extracting with ethyl acetate for many times, merging the ethyl acetate extract, evaporating, dissolving the residue in methanol, and preparing a control medicinal material solution; C, taking the test solution and the control medicinal material solution, respectively, spotting on the same silica gel thin layer plate, using toluene ethyl acetate formic acid with a volume ratio of 5:5:1 as a developing agent, developing, taking out, drying, and inspecting. The present invention has the characteristics of high extraction rate and accurate identification.

Description

Technical Field

[0001] The invention relates to a traditional Chinese medicine analysis and detection, in particular to a method for detecting the quality of Saxifraga herbacea. Background Art

[0002] Saxifraga is the dried whole herb of Saxifraga of the Saxifragaceae family. There are about 370 species of Saxifraga plants in the world, and about 200 species in my country, which are widely distributed throughout the country, but the distribution of different species has obvious regional characteristics, and some species are still controversial in plant classification. According to literature records, there are about 36 species of plants in this genus (including sub-species levels), and the types and clinical applications of medicine in different regions and different ethnic groups are also quite different. Among them, Saxifraga, which is widely distributed in low-altitude areas, is the most widely used. It has the effects of anti-inflammatory and detoxification, and is used for acute otitis media, wind-heat cough, and externally for bullous tympanitis, urticaria itching, etc. The plants of this genus used in Tibetan medicine are mainly those distributed at high altitudes, such as S.umbellulata Hook.f.et Thoms., S.umbellullata Hook.f.et Thoms.var.pectinata (Marquand et Airy-Shaw) JTPan, S.tangutica Engl., etc., which are mainly used to treat liver and gallbladder diseases. Studies have shown that species of S.stolonifera distributed in low-altitude areas mainly contain phenolic acid components with bergenin as the characteristic component, while species distributed in high-altitude areas mostly do not contain bergenin, but contain characteristic diphenylheptanes, triterpenes and other components. This difference in components is closely related to its clinical application efficacy.

[0003] The original standards recorded under the name of "Saxifraga" include the "Pharmacopoeia of the People's Republic of China" (1977 edition) and various local standards, including the "Shanghai Traditional Chinese Medicine Standard" (1994 edition), the "Hubei Traditional Chinese Medicine Standard" (2009 edition), the "Guizhou Traditional Chinese Medicine and Ethnic Medicinal Materials Quality Standard" (2003 edition) and the "Hunan Traditional Chinese Medicine Standard" (2009 edition). Each standard stipulates [Properties], [Identification] (leaf surface observation, thin layer identification with bergenin or quercetin as the control), [Inspection] (water content, total ash content, acid-insoluble ash content), [Content measurement] (the limit of bergenin shall not be less than 0.08%), etc. At present, the standard only has thin layer identification of bergenin or quercetin as the control of Saxifraga, and there is no thin layer identification of Saxifraga control medicinal materials, which makes it easy to confuse the authenticity of Saxifraga and the overall quality cannot be well controlled. Summary of the invention

[0004] The purpose of the present invention is to provide a method for detecting the quality of Saxifraga herbacea, which has the characteristics of high extraction rate and accurate identification.

[0005] The technical solution of the present invention is a method for detecting the quality of Saxifraga herbaceous medicinal materials, which comprises using thin layer chromatography to identify Saxifraga herbaceous medicinal materials, and comprises the following steps:

[0006] A. Take Saxifraga herb, add 30% ethanol according to the material-liquid ratio of 1:40-50, ultrasonically treat, filter, evaporate the filtrate to dryness, suspend the residue in hot water, and extract with ethyl acetate for several times, combine the ethyl acetate extracts, evaporate to dryness, and dissolve the residue in methanol to prepare the test solution;

[0007] B. Take the reference medicinal material of Saxifraga sibiricum, add 30% ethanol according to the solid-liquid ratio of 1:40-50, ultrasonically treat, filter, evaporate the filtrate to dryness, suspend the residue in hot water, and extract with ethyl acetate for multiple times, combine the ethyl acetate extracts, evaporate to dryness, and dissolve the residue in methanol to prepare a reference medicinal material solution;

[0008] C. Take the test sample solution and the control medicinal material solution, spot them on the same silica gel thin layer plate respectively, use toluene-ethyl acetate-formic acid with a volume ratio of 5:5:1 as the developing solvent, develop, take out, dry, and examine under ultraviolet light.

[0009] The aforementioned method for detecting the quality of Saxifraga herb comprises the following steps:

[0010] A. Take 0.5 g of Saxifraga herb, add 20 ml of 30% ethanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, suspend the residue in 10 ml of hot water, and extract with ethyl acetate 3 times, 10 ml each time, combine the ethyl acetate extracts, evaporate to dryness, and dissolve the residue in 1 ml of methanol to prepare the test solution;

[0011] B. Take 0.5 g of Saxifraga control medicinal material powder, add 20 ml of 30% ethanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, suspend the residue in 10 ml of hot water, and extract with ethyl acetate 3 times, 10 ml each time, combine the ethyl acetate extracts, evaporate to dryness, and dissolve the residue in 1 ml of methanol to prepare a control medicinal material solution;

[0012] C. Take the test sample solution and the control medicinal material solution, spot them on the same silica gel thin layer plate respectively, use toluene-ethyl acetate-formic acid with a volume ratio of 5:5:1 as the developing solvent, develop, take out, dry, and examine under ultraviolet light.

[0013] In the aforementioned method for detecting the quality of Saxifraga herbacea, in step C, in the chromatogram of the test solution, at positions corresponding to those in the chromatogram of the control medicinal material solution, both show spots of the same color.

[0014] In the aforementioned method for detecting the quality of Saxifraga herb, in step C, the amount of the test solution and the control medicinal material solution are both 10-15 μl.

[0015] In the aforementioned method for detecting the quality of Saxifraga herb, in step C, the wavelength of the ultraviolet lamp is selected to be 254 nm.

[0016] In the aforementioned method for detecting the quality of Saxifraga herbaceous medicinal materials, the detection method further comprises determining the content of bergenin in the Saxifraga herbaceous medicinal materials by liquid chromatography, comprising the following steps:

[0017] (1) Take bergenin reference substance and add 30% ethanol to prepare reference substance solution;

[0018] (2) Take Saxifraga herbacea, add 30% ethanol according to the solid-liquid ratio of 1:40-50, weigh the weight, ultrasonicate for 75 minutes, cool, weigh again, make up the lost weight with 30% ethanol, shake well, let stand, take the supernatant and filter it with a microporous membrane, take the filtrate, and obtain the test solution;

[0019] (3) The reference solution and the test solution were respectively aspirated and injected into a liquid chromatograph to measure the content of bergenin in the herb.

[0020] In the aforementioned method for detecting the quality of Saxifraga herbaceous medicinal materials, the detection method further comprises determining the content of bergenin in the Saxifraga herbaceous medicinal materials by liquid chromatography, comprising the following steps:

[0021] (1) Take a bergenin reference substance and add 30% ethanol to prepare a solution containing 110 μg of bergenin per 1 ml, which is the reference substance solution;

[0022] (2) Take 0.50 g of Saxifraga herb, add 20 ml of 30% ethanol, seal, weigh, ultrasonicate for 75 minutes, cool, weigh again, make up the lost weight with 30% ethanol, shake well, let stand, take the supernatant and filter with a 0.45 μm microporous membrane, take the filtrate, and obtain the test solution;

[0023] (3) Take the reference solution and the test solution respectively, inject them into the liquid chromatograph, and measure the content of bergenin in the Herba Saxifragae.

[0024] In the aforementioned method for detecting the quality of saxifrage herbs, the chromatographic conditions of the liquid chromatography method are as follows: using octadecylsilane bonded silica gel as a filler; using acetonitrile-0.4% formic acid solution with a volume ratio of 16:84 as an isocratic elution mobile phase; column temperature of 25°C; detection wavelength of 270nm; flow rate of 1.0ml·min -1 .

[0025] In the aforementioned method for detecting the quality of Saxifraga herb, in step (2), the power of ultrasonic treatment is 250 W and the frequency is 40 kHz.

[0026] In the aforementioned method for detecting the quality of Saxifraga herb, in step (3), 5 μl of each of the reference solution and the test solution is injected into a liquid chromatograph for determination.

[0027] Compared with the prior art, the present invention provides a quality detection method for Saxifragium aromaticum medicinal materials, which includes a thin layer identification method for Saxifragium aromaticum medicinal materials and a method for determining the content of bergenin in Saxifragium aromaticum medicinal materials. The thin layer identification method for Saxifragium aromaticum medicinal materials has the characteristics of high sensitivity, strong specificity and good separation effect, is simple and feasible, and has strong reproducibility, so that the authenticity of medicinal materials can be accurately identified, and Saxifragium aromaticum medicinal materials can be effectively distinguished from their counterfeits and confusions. The method for determining the content of bergenin in Saxifragium aromaticum medicinal materials can accurately and quickly determine the content of bergenin in Saxifragium aromaticum medicinal materials, and uses 30% ethanol as the extraction solvent, with a high extraction rate, providing an effective qualitative and quantitative analysis method for Saxifragium aromaticum medicinal materials, which is beneficial to the quality control of Saxifragium aromaticum medicinal materials. The quality detection method for Saxifragium aromaticum medicinal materials provided by the present invention can effectively control the overall quality of Saxifragium aromaticum medicinal materials and ensure the safety and effectiveness of Saxifragium aromaticum medicinal materials in clinical use.

[0028] The quality detection method of the Herba Saxifragae provided by the present invention is helpful to make up for the deficiencies of the quality detection methods in the Pharmacopoeia of the People's Republic of China (1977 edition) and various local standards, such as the Shanghai Traditional Chinese Medicine Standard (1994 edition), the Hubei Traditional Chinese Medicine Standard (2009 edition), the Guizhou Traditional Chinese Medicine and National Medicinal Materials Quality Standard (2003 edition) and the Hunan Traditional Chinese Medicine Standard (2009 edition), and has strong practicality. The method of the present invention is of great significance for the further quality control of the Herba Saxifragae, and can provide a scientific basis for the formulation of the quality table of the Herba Saxifragae. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is the choice of developing agent solvent to examine the thin layer chromatogram;

[0030] Figure 2 It is the ratio of the developing agent to the solvent to examine the thin layer chromatogram;

[0031] Figure 3 It is the thin layer chromatogram of the test solution preparation method;

[0032] Figure 4 It is to examine the thin layer chromatogram by spotting the sample;

[0033] Figure 5 It is to examine the thin layer chromatogram under different wavelengths of ultraviolet light;

[0034] Figure 6 This is the thin layer chromatogram of the reference herb Saxifrage;

[0035] Figure 7This is the UV scan of Bergenin;

[0036] Figure 8 It is a liquid chromatogram with the mobile phase being methanol-pure water (30:70);

[0037] Fig. 9 is a liquid chromatogram with the mobile phase being methanol-0.4% formic acid water (25:75);

[0038] Fig.10 is a liquid chromatogram with the mobile phase being acetonitrile-0.4% formic acid water (16:84);

[0039] Fig.11 is a liquid chromatogram with the mobile phase being acetonitrile-0.4% formic acid water (12:88);

[0040] Fig.12 is a liquid chromatogram with the mobile phase being acetonitrile-0.4% formic acid water (16:84);

[0041] Fig.13 is a liquid chromatogram with the mobile phase being acetonitrile-0.4% formic acid water (20:80);

[0042] Fig.14 It is the liquid chromatogram of different flow rates;

[0043] Fig.15 It is the chromatogram of methanol extract with different concentrations;

[0044] Fig.16 It is the chromatogram of ethanol extracts with different concentrations;

[0045] Fig.17 It is the chromatogram of water extract;

[0046] Fig.18 The chromatographic results were investigated using different extraction solvent dosages. Figure 1 ;

[0047] Fig.19 The chromatographic results were investigated using different extraction solvent dosages. Figure 2 ;

[0048] Fig. 20 The chromatographic results of different ultrasonic extraction times were investigated. Figure 1 ;

[0049] Fig.21 The chromatographic results of different ultrasonic extraction times were investigated. Figure 2 ;

[0050] Fig. 22 is the standard curve of bergenin;

[0051] Fig.23 It is the result graph of precision experiment;

[0052] Fig.24 is the repeatability test chromatogram;

[0053] Fig.25 The sample recovery rate experiment chromatogram Figure 1 ;

[0054] Fig.26 The sample recovery rate experiment chromatogram Figure 2 ;

[0055] Fig. 27 The sample recovery rate experiment chromatogram Figure 3 . DETAILED DESCRIPTION

[0056] The present invention will be further described below in conjunction with the embodiments, but they are not intended to limit the present invention.

[0057] Example:

[0058] instrument:

[0059] Swiss CAMAG thin layer sampler (Auto sampler 4) and imager (Visualize 4); Shimadzu LC-20AT liquid chromatograph; chromatographic column: Waters sunfire (5μm, 4.6mm×250mm); AUW220D electronic balance: 1 / 10,000, Ohaus Corporation, USA; KQ250DB CNC ultrasonic cleaner: Gongyi Yuhua Instrument Co., Ltd.; KQ5200 water bath: Kunshan Ultrasonic Instrument Co., Ltd.; DHG-9140A electric constant temperature blast drying oven: Gongyi Yuhua Instrument Co., Ltd.; three-purpose ultraviolet analyzer: Shanghai Gucun Electro-Optical Instrument Factory.

[0060] Reagents and test drugs

[0061] 1. Reference

[0062] Bergenin and gallic acid were purchased from China Food and Drug Inspection Institute (Bergenin batch number: 110709-200304, purity 94.4%; Gallic acid batch number: 1102542-201452).

[0063] 2. Reagents

[0064] Silica gel GF for thin layer chromatography 254(Merck Chemical Technology Co., Ltd., batch number: HC30492153); methanol (analytical grade, Xilong Scientific Co., Ltd., batch number: 161004); toluene (analytical grade, Sinopharm Chemical Reagent Co., Ltd., batch number: 20151102); acetonitrile (chromatographic grade, USA, batch number: 810901 / 810905); chloroform (analytical grade, Sinopharm Chemical Reagent Co., Ltd., batch number: 20141206); ethyl acetate (analytical grade, Xilong Chemical Co., Ltd., batch number: 20140103); anhydrous ethanol (analytical grade, Tianjin Hengxing Chemical Preparation Manufacturing Co., Ltd., batch number: 20161125); formic acid (analytical grade, Xilong Chemical Co., Ltd., batch number: 20130143); acetone (analytical grade, Xilong Scientific Co., Ltd., batch number: 20142520); water was ultrapure water produced by Millipore pure water instrument; the rest of the reagents were of analytical grade.

[0065] Medicinal Materials

[0066] There were a total of 38 batches of Saxifrage medicinal materials, including 19 batches (No. 1 to 19) provided by Guizhou Bailing Pharmaceutical Factory and 19 batches of self-sampled samples collected from Guizhou, Jiangxi, Anhui, Hubei, Chongqing, Hunan, Guangxi and Zhejiang, as shown in Table 1.

[0067] Table 1. Batch of Saxifraga herbacea medicinal materials

[0068]

[0069]

[0070] Regarding thin-layer identification, a comparative study on the components of Saxifraga stolonifera, which is mainly distributed in low-altitude areas, and three species of Saxifraga used in Tibetan medicine in high-altitude areas [S.umbellulata Hook.f.er Thoms.var.pectinata (Marquand et Airy-Shaw) JTPan, S.umbellulata Hook.f.er Thoms., and S.tangutica Engl.] were conducted. The results showed that the four species of Saxifraga had great differences in the types and compositions of components. Flavonoids were common components of the four species of Saxifraga. Bergenin and gallic acid were representative components of Saxifraga stolonifera compared with other plants of the same genus, and bergenin was the characteristic component of this species. Therefore, a thin-layer identification study was conducted using control medicinal materials, bergenin and gallic acid as controls, and various thin-layer identification methods were established.

[0071] A method for detecting the quality of Saxifraga herbaceous material comprises identifying the Saxifraga herbaceous material by thin layer chromatography, and comprises the following steps:

[0072] A. Take 0.5 g of Saxifraga herb, add 20 ml of 30% ethanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, suspend the residue in 10 ml of hot water (70-80°C), and extract with ethyl acetate 3 times, 10 ml each time, combine the ethyl acetate extracts, evaporate to dryness, and dissolve the residue in 1 ml of methanol to prepare the test solution;

[0073] B. Take 0.5 g of Saxifraga control medicinal material powder, add 20 ml of 30% ethanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, suspend the residue in 10 ml of hot water, and extract with ethyl acetate 3 times, 10 ml each time, combine the ethyl acetate extracts, evaporate to dryness, and dissolve the residue in 1 ml of methanol to prepare a control medicinal material solution;

[0074] C. According to the thin layer chromatography method (General Rule 0502), take 10-15 μl of the test solution and the reference drug solution, respectively, and spot them on the same silica gel GF 254 On the thin layer plate, use toluene-ethyl acetate-formic acid with a volume ratio of 5:5:1 as the developing agent, develop, take out, dry, and examine under an ultraviolet lamp with a wavelength of 254nm.

[0075] In the chromatogram of the test solution, at the corresponding position in the chromatogram of the control medicinal material solution, both show spots of the same color.

[0076] The identification of bergenin and gallic acid by thin layer chromatography includes the following steps:

[0077] (1) Take appropriate amounts of bergenin and gallic acid reference substances, add methanol to prepare reference substance solutions containing 110 μg of bergenin per 1 ml and 10 μg of gallic acid per 1 ml respectively;

[0078] (2) Take 0.5 g of Saxifraga herb powder, add 20 ml of 30% ethanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, add 10 ml of hot water to suspend, extract with ethyl acetate three times, 10 ml each time, combine the ethyl acetate extracts, evaporate to dryness, add 1 ml of methanol to dissolve the residue, and obtain the test solution;

[0079] (3) According to the thin layer chromatography method (General Method 0502), 5 to 10 μl of the test solution and the reference solution are respectively spotted on the same silica gel GF 254 On the thin layer plate, place it under ultraviolet light (254nm) for inspection; or spray sulfuric acid ethanol test solution for color development and inspect it under sunlight. 1. Selection of thin layer identification method:

[0080] 1.1 Selection of developing solvent

[0081] Chloroform-ethyl acetate-formic acid (volume ratio of 10:7:1), chloroform-acetone-formic acid (volume ratio of 7:5:1), toluene-ethyl acetate-formic acid (volume ratio of 5:5:1), ethyl acetate-methanol-formic acid (volume ratio of 40:1:0.05) were selected as the developing agent, the sample volume was 5 μl, developed, taken out, dried, and examined under ultraviolet light (254 nm); or sprayed with sulfuric acid ethanol test solution for color development, examined under sunlight, and saw Figure 1 .

[0082] The results showed that the development effect was best with toluene-ethyl acetate-formic acid (volume ratio of 5:5:1) as the developing agent, and it was best to inspect under ultraviolet light. Therefore, toluene-ethyl acetate-formic acid (volume ratio of 5:5:1) was selected as the developing agent and inspected under ultraviolet light.

[0083] 1.2 Selection of solvent ratio in the developing agent solvent system

[0084] After determining the toluene-ethyl acetate-formic acid developing solvent system, the ratio between the solvents was further optimized. Toluene-ethyl acetate-formic acid with a volume ratio of 5:9:1 and toluene-ethyl acetate-formic acid with a volume ratio of 5:5:1 were selected as developing solvents for measurement. The measurement results are as follows: Figure 2 The results show that toluene-ethyl acetate-formic acid (5:5:1) has the best development effect. Therefore, toluene-ethyl acetate-formic acid (5:5:1) is selected as the developing agent.

[0085] 1.3 Selection of test solution preparation method

[0086] Preparation method of test solution 1: Take 0.5 g of Saxifraga herb powder, add 20 ml of 30% ethanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, add 1 ml of methanol to dissolve, and obtain the solution.

[0087] Preparation method 2 of the test solution: Take 0.5 g of Saxifraga herb powder, add 20 ml of 30% ethanol, ultrasonically treat for 30 min, filter, spin-dry the filtrate, add 10 ml of hot water to suspend, extract with 10 ml of ethyl acetate three times respectively, separate the ethyl acetate extracts, combine the filtrates, evaporate to dryness, and dissolve the residue in 1 ml of methanol to obtain the solution.

[0088] 5 μl of the test solution and reference solution were respectively taken, spotted, developed with toluene-ethyl acetate-formic acid (volume ratio 5:5:1), and examined under ultraviolet light (254 nm).

[0089] Check the results in Figure 3 The results showed that the sample spots of the test solution preparation method 2 were clearer, so preparation method 2 was selected as the preparation method of the test solution.

[0090] 1.4 Sampling quantity inspection

[0091] Take the powder of Herba Saxifragae Herba and prepare the test solution according to the method for preparing the test solution. According to the above thin layer chromatography conditions, 15 μl, 10 μl and 5 μl of the sample were applied for development. Each application amount was repeated 3 times. The development results are shown in Figure 4 The results showed that the spotting volume of 10 to 15 μl was more appropriate.

[0092] 1.5 Check wavelength selection

[0093] Take powder of commercial medicinal materials of Saxifraga (samples 1 to 19) to prepare test solution, spot the sample, develop it, and inspect it under 254nm and 366nm ultraviolet light respectively. The inspection results are shown in Figure 5 The results show that the spots are clear when examined under 254nm UV light.

[0094] 1.6 Thin layer identification of reference medicinal materials

[0095] Take the reference medicinal materials of Saxifraga stolonifera (Table 1: No. 20, HRC-201607-7, collected from Fanjing Mountain, Tongren City, Guizhou Province, identified as Saxifraga stolonifera Meerb. by researcher Zhong Guoyue of Jiangxi University of Traditional Chinese Medicine, and the samples and wax leaf specimens are stored in the Research Center of Traditional Chinese Medicine Resources and Ethnic Medicine of Jiangxi University of Traditional Chinese Medicine), commercial medicinal materials of Saxifraga stolonifera (No. 1-6), self-collected counterpart medicinal materials (No. 21-28) and three species of Saxifraga plants produced in the plateau for Tibetan medicine, prepare the test solution, absorb each test solution and reference solution, spot and develop. The test results are shown in Figure 6 .

[0096] The results showed that the TLC of commercial and counterpart Saxifraga herb was consistent with that of the reference herb and reference substance; the TLC of three Saxifraga species from the plateau was significantly different from that of the reference herb and reference substance. Therefore, this TLC identification method is feasible.

[0097] Saxifraga resources are abundant and widely distributed. According to the investigation, there is no case of plateau Saxifraga mixed in the commercially available Saxifraga medicinal materials. The TLC identification of bergenin and gallic acid as the control is not very meaningful; Bergenin is one of the active ingredients in Saxifraga and has a high content, so it is suitable as a test index component; TLC identification of Saxifraga control medicinal materials is more meaningful in the authenticity identification of Saxifraga; flavonoids are common components of Saxifraga plants, and the characteristic of TLC identification of quercetin as the control in the original Guizhou standard is not strong. Therefore, the final standard only adds TLC identification of control medicinal materials, and cancels the TLC identification of bergenin and quercetin as the control in the original standard.

[0098] The original Guizhou and Hubei provincial standards stipulated that the limit of bergenin should not be less than 0.08%. The inventor's comparative study on the components of Saxifraga stolonifera and its three species of Saxifraga stolonifera showed that bergenin was a characteristic component compared with the other three species; at the same time, a large number of studies have reported that bergenin has antitussive and anti-inflammatory effects, which is consistent with the functions and indications of Saxifraga stolonifera, so it is reasonable to use bergenin as the index component of the test item. In the experiment, the HPLC determination method of bergenin content was investigated and the original determination method was optimized; the bergenin content of 19 batches of Saxifraga stolonifera materials was determined, and according to the determination results, the limit of bergenin content was revised to not less than 0.3%. This limit is significantly higher than the limit of the original standard (0.08%), because the original standard used methanol as the solvent for the preparation of the test solution, and its extraction rate was low. In this scheme, it was revised to use 30% ethanol as the solvent, and the extraction efficiency was significantly higher.

[0099] A method for detecting the quality of Saxifraga herbaceous material, further comprising using liquid chromatography to determine the content of bergenin in Saxifraga herbaceous material, and determining according to high performance liquid chromatography (General Rule 0512 of Part 4 of the 2020 edition of the Chinese Pharmacopoeia), comprising the following steps:

[0100] (1) Take a bergenin reference substance, accurately weigh it, and add 30% ethanol to make a solution containing 110 μg of bergenin per 1 ml, which is the reference solution;

[0101] (2) Take 0.50 g of Saxifraga herb powder (passed through a No. 4 sieve), place it in a stoppered conical flask, accurately add 20 ml of 30% ethanol, stopper it, weigh it, and treat it with ultrasound at a power of 250 W and a frequency of 40 kHz for 75 minutes. Let it cool, weigh it again, make up the lost weight with 30% ethanol, shake it well, let it stand, take the supernatant and filter it with a 0.45 μm microporous filter membrane, take the filtrate, and obtain the test solution;

[0102] (3) Take 5 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and measure the content of bergenin in the Herba Saxifragae.

[0103] During the liquid chromatography treatment, octadecylsilane bonded silica gel was used as the filler; acetonitrile-0.4% formic acid solution with a volume ratio of 16:84 was used as the mobile phase for isocratic elution; the column temperature was 25°C; the detection wavelength was 270nm; and the flow rate was 1.0ml·min -1 The number of theoretical plates calculated based on the bergenin peak should be no less than 3000.

[0104] According to the standard, this product contains bergenin (C 14 H 16 O9) shall not be less than 0.30%.

[0105] 2. Selection of the method for determining the content of bergenin:

[0106] 2.1 Selection of detection wavelength

[0107] The UV absorption characteristics of bergenin are as follows Figure 7 Based on the existing data, the detection wavelength was finally determined to be 270nm.

[0108] 2.2 Selection of mobile phase solvent in mobile phase system

[0109] Using methanol or acetonitrile as the organic phase and pure water or 0.4% formic acid as the aqueous phase, the peak shape of bergenin in the Saxifraga herbaceous material samples under different chromatographic conditions was tested. The chromatogram test results of the Saxifraga herbaceous material samples are shown in Figure 8 to Figure 10 The results showed that when acetonitrile and formic acid aqueous solution were used as organic phase, the baseline drift of the sample chromatogram was small and the chromatographic peak shape of bergenin was better. Based on this, acetonitrile and formic acid aqueous solution were selected as mobile phase solvents.

[0110] 2.3 Selection of mobile phase solvent ratio

[0111] The mobile phases were tested with different ratios of acetonitrile-0.4% formic acid water, and the peak shapes of bergenin in the chromatograms were compared. The test results are shown in Figure 11 to Figure 13 The results showed that the baseline drift of the sample chromatogram with acetonitrile-0.4% formic acid water (16:84) was small, and the chromatographic peak shape of bergenin was better, so the mobile phase solvent ratio was determined to be acetonitrile-0.4% formic acid water (16:84).

[0112] 2.4 Flow rate selection

[0113] Acetonitrile-0.4% formic acid water (16:84) was used as the mobile phase, and the peak shapes of bergenin at different flow rates were compared. The test results are shown in Fig.14 The results showed that the flow rate was 1.0 ml min -1 When the sample chromatogram baseline drift is small, the bergenin chromatographic peak shape is better and the peak time is short, so the flow rate is selected as 1.0 ml min -1 .

[0114] 2.5 Selection of test solution preparation method

[0115] Different extraction solvents (methanol and ethanol at different concentrations), solvent dosage and extraction time were investigated respectively.

[0116] 2.5.1 Investigation of extraction solvent

[0117] Take 9 portions of the same batch of Saxifraga herb powder, 0.5g each, accurately weigh, accurately add 25ml of ethanol, methanol and water of different concentrations, seal, weigh, ultrasonically extract for 1 hour, cool, weigh again, make up the lost weight with ethanol, methanol and water of corresponding concentrations, shake well, let stand, take the supernatant and filter with 0.45μm microporous filter membrane, take the filtrate as the test solution. Accurately take 5μl of each of the above 9 test solutions, inject into liquid chromatograph, and measure. The results are shown in Table 2. Figures 15 to 17 . Calculate the size of the specific peak area (the ratio of the peak area to the injection volume is used as an experimental investigation indicator. The larger the ratio, the more complete the extraction of the component).

[0118] The results show that under the same conditions, the extraction rate of bergenin with 100% methanol as the extraction solvent is the lowest; the extraction rates of pure water, 30% ethanol and 30% methanol for bergenin in medicinal materials are similar, and the extraction rates are higher than those of other polar solvents, indicating that the high-polarity solvents can extract bergenin from medicinal materials more completely. However, considering that the pure water extraction solution has more high-polarity impurities and is not easy to filter, the 30% methanol extraction solution was also found to be difficult to filter during the experiment, so 30% ethanol was finally selected as the extraction solvent for preparing the test solution.

[0119] Table 2 Extraction results of different extraction solvents

[0120]

[0121]

[0122] 2.5.2 Investigation of the amount of extraction solvent

[0123] Take 7 portions of Saxifraga herb powder from the same batch, 0.5g each, and place in stoppered conical flasks. Accurately add 30% ethanol 5ml, 10ml, 15ml, 20ml, 25ml, 30ml, 40ml to each portion, stopper tightly, weigh, and ultrasonically treat (power 250W, frequency 40kHz) for 30 minutes. Let cool, weigh again, make up the lost weight with 30% ethanol, shake well, let stand, take the supernatant and filter through a 0.45μm filter membrane, and take the filtrate as the test solution. Accurately pipette 5μl of each of the above test solutions, inject into the liquid chromatograph, and measure. Calculate the size of the specific peak area (the ratio of peak area to injection volume is used as an experimental investigation indicator. The larger the ratio, the more complete the extraction of the component). The measurement results are shown in Table 3. Fig.18 , 19 It can be seen that when the solid-liquid ratio is 1:40, the extraction rate is the highest, so the solid-liquid ratio is selected to be 1:40, that is, when 0.50g of Saxifraga is taken, the amount of ethanol added is 20ml.

[0124] Table 3 Investigation of extraction solvent dosage

[0125]

[0126]

[0127] 2.6.3 Ultrasonic time investigation

[0128] Take 7 portions of Saxifraga herb powder from the same batch, 0.5 g each, put them in a stoppered conical flask, accurately add 20 ml of 30% ethanol, seal it, weigh it, and treat it with ultrasound (power 250 W, frequency 40 kHz) for 15 minutes, 30 minutes, 45 minutes, 60 minutes, 75 minutes, 90 minutes, and 105 minutes, respectively. Let it cool, weigh it again, make up the lost weight with 30% ethanol, shake it well, let it stand, take the supernatant and filter it with a 0.45 μm filter membrane, and take the filtrate as the test solution. Accurately pipette 5 μl of each of the above test solutions, inject it into the liquid chromatograph, and measure it. Calculate the size of the specific peak area (the ratio of the peak area to the sampling amount is used as an experimental investigation indicator. The larger the ratio, the more complete the extraction of the component). The measurement results are shown in Table 4. Fig. 20 , 21 .

[0129] The results showed that the content of bergenin no longer increased significantly after 75 minutes of ultrasonic extraction and tended to be stable. Therefore, the ultrasonic extraction time was selected to be 75 minutes.

[0130] Table 4 Results of different ultrasonic extraction times

[0131]

[0132]

[0133] According to the above test results, the preparation method of the test solution was finally determined as follows: take 0.50 g of the powder of the product (passed through a No. 4 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 20 ml of 30% ethanol, seal it, weigh it, ultrasonically treat it (power 250 W, frequency 40 kHz) for 75 minutes, cool it, weigh it again, make up the lost weight with 30% ethanol, shake it well, let it stand, take the supernatant and filter it with a 0.45 μm microporous filter membrane, and take the filtrate to obtain it.

[0134] 3. Methodological Validation

[0135] 3.1 Preparation of standard curve

[0136] Take 10 mg of bergenin, weigh accurately, place in a 10 ml volumetric flask, and dilute to the mark with 30% ethanol to obtain a bergenin reference solution. Under the above chromatographic conditions, inject 2, 4, 6, 8, 10, and 12 μl (n=3) respectively to measure the bergenin peak area. The results are shown in Table 5. Draw a standard curve (see Table 5) with the peak area integral value as the ordinate and the amount of the reference substance (μg) as the abscissa. Fig. 22 ), and the linear regression equation of bergenin was calculated to be y=1218741.65x-7055.93 (r=1), with a good linear relationship in the range of 0.2077μg-1.2461μg.

[0137] Table 5 Preparation of standard curve

[0138]

[0139]

[0140] 3.2 Precision test

[0141] Take the same bergenin reference solution and inject it into the liquid chromatograph. Repeat the injection 5 times, 5 μl each time, and measure the results. See Table 6. Fig.23 The results showed that the RSD value was less than 3%, and the instrument precision was good.

[0142] Table 6 Precision test results

[0143]

[0144] 3.3 Repeatability test

[0145] Take 6 portions of Saxifraga powder, 0.5 g each, weigh accurately, prepare according to the preparation method of the test solution, inject into the liquid chromatograph, inject 5 μl, measure the bergenin peak area, substitute into the standard curve equation, and calculate the bergenin content. The results are shown in Table 7. Fig.24 The results showed that the average content of the samples was 0.34% and the RSD value was 0.8%, indicating that the method had good reproducibility.

[0146] Table 7 Repeatability test results

[0147]

[0148]

[0149] 3.4 Stability test

[0150] Take the same test solution and inject it once every 2 hours for determination. The results are shown in Table 8.

[0151] The results showed that the tested samples were stable within 24 hours and had good stability.

[0152] Table 8 Stability Study

[0153]

[0154] 3.5 Sample recovery test

[0155] Take 6 portions of the same batch of saxifrage powder with known bergenin content, namely samples 1-6, about 0.25g each, add bergenin reference substance at a ratio of 1:1 to the bergenin content in the medicinal material, and prepare the test solution according to the test solution preparation method. Inject and measure respectively, and calculate the recovery rate according to the following formula:

[0156]

[0157] The results showed that the average recovery rate was 97.42 and the RSD value was 1.65%, indicating that the method had good accuracy. Each sample was injected three times and the average value was taken. The results are shown in Table 9. Figures 25-27 .

[0158] Table 9 Sample recovery test results (n = 3)

[0159]

[0160] 3.6 Sample content determination and content limitation:

[0161] 38 batches of Saxifraga samples (19 batches of commercial medicinal materials and 19 batches of counterpart medicinal materials) were taken to prepare the test solution. Under the above chromatographic conditions, 5 μl of the reference solution and the test solution were accurately pipetted and injected into the liquid chromatograph for determination. The results are shown in Table 10.

[0162] Table 10 Determination results of bergenin content in 38 batches of Saxifraga samples

[0163]

[0164]

[0165] The results showed that the content of bergenin in 38 batches of Saxifraga samples was between 0.15% and 0.65%, with an average content of 0.43%. The average value was 0.34% when the average was reduced by 20%, and 10 batches of 38 samples failed (the qualified rate was 71%). Based on this, it was determined that the content of bergenin should not be less than 0.30% (the qualified rate of 38 batches of samples was 86%).

Claims

1. A method for detecting the quality of Saxifraga herbacea, characterized in that: The method comprises the following steps: A. Take Saxifraga herb, add 30% ethanol according to the material-liquid ratio of 1:40-50, ultrasonically treat, filter, evaporate the filtrate to dryness, suspend the residue in hot water, and extract with ethyl acetate for several times, combine the ethyl acetate extracts, evaporate to dryness, and dissolve the residue in methanol to prepare the test solution; B. Take the reference medicinal material of Saxifraga sibiricum, add 30% ethanol according to the solid-liquid ratio of 1:40-50, ultrasonically treat, filter, evaporate the filtrate to dryness, suspend the residue in hot water, and extract with ethyl acetate for multiple times, combine the ethyl acetate extracts, evaporate to dryness, and dissolve the residue in methanol to prepare a reference medicinal material solution; C. Take the test sample solution and the control medicinal material solution, spot them on the same silica gel thin layer plate, use toluene-ethyl acetate-formic acid with a volume ratio of 5:5:1 as the developing solvent, develop, take out, dry, and inspect; The detection method also includes using liquid chromatography to determine the content of bergenin in Saxifraga herb, comprising the following steps: (1) Take bergenin reference substance and add 30% ethanol to prepare reference substance solution; (2) Take Saxifraga herbacea, add 30% ethanol according to the material-liquid ratio of 1:40-50, weigh the weight, ultrasonicate, cool, weigh again, make up the lost weight with 30% ethanol, shake well, let stand, take the supernatant and filter it with a microporous filter membrane, take the filtrate, and obtain the test solution; (3) respectively taking the reference solution and the test solution, injecting them into a liquid chromatograph, and measuring the content of bergenin in the Saxifraga herb; The chromatographic conditions of the liquid chromatography method are: using octadecylsilane bonded silica gel as a filler; using acetonitrile-0.4% formic acid solution with a volume ratio of 16:84 as an isocratic elution mobile phase; column temperature of 25°C; detection wavelength of 270nm; flow rate of 1.0ml·min -1 .

2. The method for detecting the quality of Saxifraga herb according to claim 1, characterized in that: The following steps are involved: A. Take 0.5 g of Saxifraga herb, add 20 ml of 30% ethanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, suspend the residue in 10 ml of hot water, and extract with ethyl acetate 3 times, 10 ml each time, combine the ethyl acetate extracts, evaporate to dryness, and dissolve the residue in 1 ml of methanol to prepare the test solution; B. Take 0.5 g of Saxifraga control medicinal material powder, add 20 ml of 30% ethanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, suspend the residue in 10 ml of hot water, and extract with ethyl acetate 3 times, 10 ml each time, combine the ethyl acetate extracts, evaporate to dryness, and dissolve the residue in 1 ml of methanol to prepare a control medicinal material solution; C. Take the test sample solution and the control medicinal material solution, spot them on the same silica gel thin layer plate respectively, use toluene-ethyl acetate-formic acid with a volume ratio of 5:5:1 as the developing solvent, develop, take out, dry, and examine under ultraviolet light.

3. A method for detecting the quality of Saxifraga herbacea according to claim 1 or 2, characterized in that: In step C, in the chromatogram of the test solution, at the corresponding position in the chromatogram of the control medicinal material solution, both show spots of the same color.

4. A method for detecting the quality of Saxifraga herbacea according to claim 1 or 2, characterized in that: In step C, the amount of the test solution and the control medicinal material solution is 10-15 μl.

5. A method for detecting the quality of Saxifraga herbacea according to claim 1 or 2, characterized in that: In step C, the wavelength of the ultraviolet lamp is selected to be 254nm.

6. The method for detecting the quality of Saxifraga herbacea according to claim 1, characterized in that: The liquid chromatography method is used to determine the content of bergenin in Saxifraga herb, which specifically includes the following steps: (1) Take a bergenin reference substance and add 30% ethanol to prepare a solution containing 110 μg of bergenin per 1 ml, which is the reference substance solution; (2) Take 0.50 g of Saxifraga herb, add 20 ml of 30% ethanol, seal, weigh, ultrasonicate for 75 minutes, cool, weigh again, make up the lost weight with 30% ethanol, shake well, let stand, take the supernatant and filter with a 0.45 μm microporous membrane, take the filtrate, and obtain the test solution; (3) Take the reference solution and the test solution respectively, inject them into the liquid chromatograph, and measure the content of bergenin in the Herba Saxifragae.

7. A method for detecting the quality of Saxifraga herbacea according to claim 1 or 6, characterized in that: In step (2), the power of ultrasonic treatment is 250W and the frequency is 40kHz.

8. A method for detecting the quality of Saxifraga herbacea according to claim 1 or 6, characterized in that: In step (3), 5 μl of each of the reference solution and the test solution is injected into a liquid chromatograph for determination.

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