A method for quality analysis of ginseng medicinal materials

Abstract

The present application relates to the technical field of compound analysis, and in particular to a quality analysis method of ginseng medicinal materials, comprising the following steps: S100, preparation of a control solution: taking ginsenoside Rg1 control product, ginsenoside Re control product, ginsenoside Rf control product, ginsenoside Rb1 control product, ginsenoside Rc control product, ginsenoside Rb2 control product, ginsenoside Rd control product, 3-O-feruloylquinic acid control product, 4-coumaric acid control product, 8-O-4 / 8-O-4 dehydrogenated tri-ferulic acid control product, and adding methanol to prepare a mixed solution, and shaking to obtain the control solution. The present application synchronously monitors two components of saponins and phenolic acids under one detection method, monitors ten component contents of seven saponin components and three polyphenol components, obtains characteristic spectra of saponin components and polyphenol components, and the monitoring quality is more comprehensive and efficient.

Description

Technical Field

[0001] This invention relates to the field of compound analysis technology, and in particular to a method for quality analysis of ginseng medicinal materials. Background Technology

[0002] Modern medical research has proven that ginseng possesses a variety of biological and pharmacological properties, such as immune stimulation, anti-cancer, antiemetic, and antioxidant effects. These properties are closely related to the chemical components in ginseng, such as ginsenosides, ginseng polysaccharides, alkaloids, free amino acids, polyphenols, and volatile compounds such as limonene.

[0003] Current status of ginseng quality control research: The 2020 edition of the Chinese Pharmacopoeia specifies the content determination of three components, ginsenosides Rg1, Re, and Rb1, under the ginseng medicinal materials section. Some scholars have monitored the fingerprint spectrum of ginseng slices, monitoring nine common peaks and identifying five components: ginsenosides Rg1, Re, Rf, Rb1, and Rd. Some scholars have monitored nine components from wild ginseng: ginsenosides Rg1, Re, Rf, Rb1, Rc, F1, Rb2, Rb3, and Rd. Some scholars have monitored eleven components: ginsenosides Rb1, Rg1, Re, Rf, Rh1, Rc, Ro, Rb2, Rb3, Rd, and Rg3. Still others have established ginseng characteristic chromatogram monitoring, monitoring 11 characteristic peaks and the content of eight components: ginsenosides Rg1, Re, Rb1, Rf, Rc, Rb2, Rb3, and Rd. Some scholars analyzed the polyphenolic compounds in ginseng medicinal materials and detected thirteen components in sun-dried ginseng roots, including protocatechuic acid, p-hydroxybenzoic acid, syringic acid, gentianic acid, p-coumaric acid, ferulic acid, mesocoumaric acid, resveratrol, cinnamic acid, maltol, catechin, naringenin, and gentianin. Other scholars determined the content of seven components in wild ginseng grown under forest cover: fumaric acid, citric acid, maleic acid, succinic acid, tartaric acid, vanillic acid, and cinnamic acid.

[0004] However, these scholars' technical research only monitored or studied one category of components in ginseng, focusing on saponins or polyphenols, resulting in relatively singular and incomplete monitoring. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides the following technical solution:

[0006] A quality analysis method for ginseng medicinal materials includes the following steps:

[0007] S100. Preparation of reference solution: Take appropriate amounts of ginsenoside Rg1 reference, ginsenoside Re reference, ginsenoside Rf reference, ginsenoside Rb1 reference, ginsenoside Rc reference, ginsenoside Rb2 reference, ginsenoside Rd reference, 3-O-feruloylquinic acid reference, 4-coumaric acid reference, and 8-O-4 / 8-O-4 dehydrotriferulic acid reference, add methanol to prepare a mixed solution, shake well, and the solution is obtained.

[0008] S200. Preparation of the test solution: Weigh approximately 1-2 g of ginseng powder accurately, place it in a Soxhlet extractor, add chloroform and heat under reflux for 3 hours, discard the petroleum ether solution, evaporate the solvent from the residue, and transfer it along with the filter paper tube into a 100 ml conical flask. Accurately add 50 ml of water-saturated n-butanol, seal tightly, and let stand overnight. Sonicate for 6 hours, filter, discard the initial filtrate, accurately measure 25 ml of the subsequent filtrate, evaporate it to dryness in an evaporating dish, dissolve the residue in methanol and transfer it to a 5 ml volumetric flask, dilute with methanol to the mark, shake well, filter, and collect the subsequent filtrate to obtain the test solution.

[0009] S300, Chromatographic conditions: C18 reversed-phase column; mobile phase A is acetonitrile and mobile phase B is a gradient eluent consisting of 0.2-1.0% phosphoric acid solution; detection wavelengths are 203 nm and 320 nm; column temperature is 33-37℃; flow rate is 0.8 ml / min-1.0 ml / min; determination time is 110 minutes.

[0010] S400. Determination: Accurately pipette the reference solution and the test solution into the high-performance liquid chromatograph, and determine the content of ten components by high-performance liquid chromatography. Determine the content of ten components by external standard method and obtain characteristic chromatograms. Analyze the obtained chromatograms and evaluate the quality of ginseng based on the analysis results.

[0011] As an improvement to the above technical solution, the gradient elution requirement is as follows:

[0012] From 0 to 35 min, mobile phase A was 19% and mobile phase B was 81%.

[0013] 35-55 min, mobile phase A is 19%-29%, mobile phase B is 81%-71%;

[0014] 55-70 min, mobile phase A is 29%, mobile phase B is 71%;

[0015] For 70-110 min, mobile phase A is 29%-38% and mobile phase B is 71%-62%.

[0016] As an improvement to the above technical solution, in step S400, the injection volume of the reference solution and the test solution injected into the high performance liquid chromatograph is precisely 10-20 μl.

[0017] As an improvement to the above technical solution, the content of seven saponin components, namely ginsenoside Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rb1, ginsenoside Rc, ginsenoside Rb2, and ginsenoside Rd, can be determined at a wavelength of 203nm.

[0018] As an improvement to the above technical solution, the contents of three phenolic acid components, namely 3-O-feruloylquinic acid, 4-coumaric acid, and 8-O-4 / 8-O-4 dehydrotriferulic acid, can be determined at a wavelength of 320 nm.

[0019] As an improvement to the above technical solution, based on the characteristic spectra of 15 batches of ginseng medicinal materials, the characteristic spectra at a wavelength of 203nm should have 12 characteristic peaks, of which peak 1 is ginsenoside Rg1; peak 2 is ginsenoside Re; peak 3 is ginsenoside Rf; peak 4 is ginsenoside Rb1 as a reference peak; peak 6 is ginsenoside Rc; peak 9 is ginsenoside Rb2; and peak 11 is ginsenoside Rd.

[0020] As an improvement to the above technical solution, the relative retention time and relative standard deviation of each characteristic peak are as follows:

[0021] Peak 1: Relative retention time 0.455±0.002, relative standard deviation 0.36%;

[0022] Peak 2: Relative retention time 0.490±0.003, relative standard deviation 0.40%;

[0023] Peak 3: Relative retention time 0.814 ± 0.002, relative standard deviation 0.12%;

[0024] Peak 4: Relative retention time 1.000±0.000, relative standard deviation 0.00%;

[0025] Peak 5: Relative retention time 1.051 ± 0.002, relative standard deviation 0.15%;

[0026] Peak 6: Relative retention time 1.063 ± 0.002, relative standard deviation 0.09%;

[0027] Peak 7: Relative retention time 1.089 ± 0.003, relative standard deviation 0.13%.

[0028] Peak 8: Relative retention time 1.109 ± 0.002, relative standard deviation 0.15%;

[0029] Peak 9: Relative retention time 1.129 ± 0.001, relative standard deviation 0.09%;

[0030] Peak 10: Relative retention time 1.169 ± 0.003, relative standard deviation 0.15%;

[0031] Peak 11: Relative retention time 1.240 ± 0.002, relative standard deviation 0.11%;

[0032] Peak 12: Relative retention time 1.279 ± 0.003, relative standard deviation 0.14%;

[0033] The characteristic spectrum at 320 nm wavelength has five characteristic peaks, where peak 2 is 3-O-feruloylquinic acid; peak 3 is 4-coumaric acid as a reference peak; and peak 5 is 8-O-4 / 8-O-4 dehydrotriferulic acid. The relative retention time and relative standard deviation of each characteristic peak are as follows:

[0034] Peak 1: Relative retention time 0.655±0.000, relative standard deviation 0.03%;

[0035] Peak 2: Relative retention time 0.735±0.001, relative standard deviation 0.04%;

[0036] Peak 3: Relative retention time 7.211 ± 0.013, relative standard deviation 0.11%;

[0037] Peak 4: Relative retention time 7.524 ± 0.013, relative standard deviation 0.10%.

[0038] As an improvement to the above technical solution, the HPLC standard characteristic spectrum of ginseng at a wavelength of 203 nm is constructed, consisting of 12 characteristic peaks. Using a reference standard, peak 1 is ginsenoside Rg1; peak 2 is ginsenoside Re; peak 3 is ginsenoside Rf; peak 4 is ginsenoside Rb1; peak 6 is ginsenoside Rc; peak 9 is ginsenoside Rb2; and peak 11 is ginsenoside Rd. Ginsenoside Rb1 is used as a reference, peak 4 as a reference peak, and the corresponding peak of the reference peak is designated as the S peak. The relative retention times of each characteristic peak and the S peak are calculated. These relative retention times are within ±5% of the specified values, which are: peak 1: 0.46, peak 2: 0.49, peak 3: 0.81, peak 4: 1.00, peak 5: 1.05, and peak 6: 1. 06, Peak 7: 1.09, Peak 8: 1.11, Peak 9: 1.13, Peak 10: 1.17, Peak 11: 1.24, Peak 12: 1.28; The HPLC standard characteristic chromatogram of ginseng at 300 nm wavelength obtained by construction consists of 5 characteristic peaks. With reference standard, peak 2 is 3-O-feruloylquinic acid; peak 3 is 4-coumaric acid; peak 5 is 8-O-4 / 8-O-4 dehydrotriferulic acid. With 4-coumaric acid as reference, peak 3 is the reference peak, and peak 7 corresponding to the reference peak is the S peak. The relative retention time of each characteristic peak and the S peak is calculated. The relative retention time is within ±5% of the specified value. The specified value is peak 1: 0.66, peak 2: 0.74, peak 3: 1.00, peak 4: 7.21, peak 5: 7.52.

[0039] The beneficial effects of this invention are:

[0040] Simultaneous monitoring of two major components, saponins and phenolic acids, using a single detection method. This method monitors the content of ten components, including seven ginsenosides (Rg1, Re, Rf, Rb1, Rc, Rb2, and Rd) and three polyphenolic components (3-O-feruloylquinic acid, 4-coumaric acid, and 8-O-4 / 8-O-4 dehydrotriferulic acid). Characteristic chromatograms of saponin and polyphenolic components are obtained, resulting in more comprehensive and efficient monitoring. Attached Figure Description

[0041] Figure 1 A comparison chart showing the detection performance of the evaporative light scattering detector and the ultraviolet detector;

[0042] Figure 2 Chromatographic comparison of different mobile phase compositions applied to saponins at a wavelength of 203 nm;

[0043] Figure 3 Chromatographic comparison of different mobile phase compositions applied to phenolic acids at 320 nm wavelength;

[0044] Figure 4Chromatographic comparison of saponins at 203 nm wavelength, showing the application of different proportions of phosphoric acid in the mobile phase.

[0045] Figure 5 A comparative chromatogram of phenolic acids at 320 nm wavelength, showing the application of different proportions of phosphoric acid in the mobile phase;

[0046] Figure 6 Chromatographic comparison of the effects of different column temperatures on the preparation of saponins at a wavelength of 203 nm;

[0047] Figure 7 Chromatographic comparison at 320 nm wavelength: the effect of column temperature on the preparation of phenolic acids.

[0048] Figure 8 Chromatographic comparison at 203 nm wavelength showing the effect of different flow rates on the preparation of saponins;

[0049] Figure 9 Chromatographic comparison at 320 nm wavelength showing the effect of different flow rates on the preparation of phenolic acids;

[0050] Figure 10 Chromatograms showing the effect of different injection volumes on saponins at a wavelength of 203 nm.

[0051] Figure 11 Chromatograms showing the effect of different injection volumes on phenolic acids at a wavelength of 320 nm.

[0052] Figure 12 Comparison of UV absorption spectra of saponins and phenolic acids;

[0053] Figure 13 The 3D chromatogram of the test solution;

[0054] Figure 14 Chromatograms comparing test solutions using different Soxhlet extraction solvents at a wavelength of 203 nm.

[0055] Figure 15 Chromatograms comparing test solutions using different Soxhlet extraction solvents at a wavelength of 320 nm.

[0056] Figure 16 The chromatograms of n-butanol used in the preparation of the test solution at a wavelength of 203 nm are shown.

[0057] Figure 17 The chromatograms of n-butanol used in the preparation of the test solution at a wavelength of 320 nm are shown.

[0058] Figure 18 Chromatographic comparison of test solutions prepared at different extraction times at a wavelength of 203 nm;

[0059] Figure 19 Chromatograms comparing test solutions prepared at different extraction times at a wavelength of 320 nm;

[0060] Figure 20 Chromatograms comparing different extraction methods of the test sample solution at a wavelength of 203 nm.

[0061] Figure 21 The chromatograms show the comparison of different extraction methods of the test sample solution at a wavelength of 320 nm.

[0062] Figure 22 The standard characteristic spectrum of ginseng at a wavelength of 203 nm;

[0063] Figure 23 Chromatographic comparison of multiple batches of ginseng medicinal materials at a wavelength of 203 nm;

[0064] Figure 24 The standard characteristic spectrum of ginseng at a wavelength of 320 nm;

[0065] Figure 25 This is a comparative chromatogram of multiple batches of ginseng medicinal materials at a wavelength of 320 nm. Detailed Implementation

[0066] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0067] See appendix Figure 1-25 As shown, a quality analysis method for ginseng medicinal materials includes the following steps:

[0068] S100, Preparation of the reference solution:

[0069] Take appropriate amounts of ginsenoside Rg1 reference standard, ginsenoside Re reference standard, ginsenoside Rf reference standard, ginsenoside Rb1 reference standard, ginsenoside Rc reference standard, ginsenoside Rb2 reference standard, ginsenoside Rd reference standard, 3-O-feruloylquinic acid reference standard, 4-coumaric acid reference standard, and 8-O-4 / 8-O-4 dehydrotriferulic acid reference standard, add methanol to prepare a mixed solution, shake well, and the solution is obtained.

[0070] The reference solutions contain saponins and phenolic acids, so they need to be prepared separately. This scheme provides specific preparation methods for saponin and phenolic acid reference solutions. It should be noted that these specific preparation methods are only for the purpose of explaining the preparation of reference solutions and should not be construed as limiting the preparation methods.

[0071] Specifically, in this scheme, the saponins are represented by the following components:

[0072] Ginsenoside Rg1 reference standard, ginsenoside Re reference standard, ginsenoside Rf reference standard, ginsenoside Rb1 reference standard, ginsenoside Rc reference standard, ginsenoside Rb2 reference standard, ginsenoside Rd;

[0073] The method for preparing the saponin reference standard is as follows:

[0074] S110. Take appropriate amounts of ginsenoside Rg1 reference standard, ginsenoside Re reference standard, ginsenoside Rf reference standard, ginsenoside Rb1 reference standard, ginsenoside Rc reference standard, ginsenoside Rb2 reference standard, and ginsenoside Rd reference standard, and accurately weigh them for later use.

[0075] S120 is prepared by adding methanol to a mixed solution containing 0.2 mg each of ginsenosides Rg1, Re, Rf, Rb1, Rc, Rb2, and Rd per ml. The solution is shaken well to obtain the ginsenoside reference solution.

[0076] In this scheme, the polyphenols are represented by the following components:

[0077] 3-O-feruloylquinic acid, 4-coumaric acid reference standards, 8-O-4 / 8-O-4 dehydrotriferulic acid;

[0078] The method for preparing the polyphenol reference standard is as follows:

[0079] S130. Take appropriate amounts of 3-O-feruloylquinic acid reference standard, 4-coumaric acid reference standard, and 8-O-4 / 8-O-4 dehydrotriferulic acid, and accurately weigh them for later use.

[0080] S140, add methanol to prepare a mixed solution containing about 1 μg of 3-O-feruloylquinic acid, about 2 μg of 4-coumaric acid and about 3 μg of 8-O-4 / 8-O-4 dehydrotriferulic acid per 1 ml, shake well to obtain the phenolic acid reference solution.

[0081] S200, Preparation of the test solution:

[0082] Accurately weigh approximately 1-2g of ginseng powder, place it in a Soxhlet extractor, add chloroform and heat under reflux for 3 hours. Discard the petroleum ether solution, evaporate the solvent from the residue, and transfer it along with the filter paper tube into a 100ml Erlenmeyer flask. Accurately add 50ml of water-saturated n-butanol, seal tightly, and let stand overnight. Sonicate for 6 hours, filter, discard the initial filtrate, accurately measure 25ml of the subsequent filtrate, evaporate it to dryness in an evaporating dish, dissolve the residue in methanol and transfer it to a 5ml volumetric flask, dilute with methanol to the mark, shake well, filter, and collect the subsequent filtrate to obtain the final product.

[0083] Specifically, in this scheme, the preparation method of the test solution is as follows:

[0084] Place 1g of accurately weighed ginseng powder in a Soxhlet extractor, add chloroform and heat under reflux for 3 hours. Discard the petroleum ether solution, evaporate the solvent from the residue, and transfer the residue along with the filter paper tube into a 100ml Erlenmeyer flask. Accurately add 50ml of water-saturated n-butanol, seal tightly, and let stand overnight. Sonicate for 6 hours, filter, discard the initial filtrate, accurately measure 25ml of the subsequent filtrate, evaporate to dryness in an evaporating dish, dissolve the residue in methanol and transfer to a 5ml volumetric flask, dilute with methanol to the mark, shake well, filter, and collect the subsequent filtrate to obtain the test solution.

[0085] The ultrasonic treatment conditions were: power 250W, frequency 50kHz, and time 6h.

[0086] The preparation of the reference solution and the test solution can be completed by following the above steps.

[0087] S300, Chromatographic conditions:

[0088] The chromatographic column was a C18 reversed-phase column; mobile phase A was acetonitrile and mobile phase B was a gradient elution solution consisting of 0.2%–1.0% phosphoric acid solution; the detection wavelengths were 203 nm and 320 nm, the column temperature was 33–37 °C, the flow rate was 0.8 ml / min–1.0 ml / min, and the measurement time was 110 minutes.

[0089] S400, Measurement:

[0090] Accurately pipette the reference solution and the test solution into the high-performance liquid chromatograph (HPLC), and determine the content of ten components by HPLC. Use the external standard method to determine the content of ten components and obtain characteristic chromatograms. Analyze the obtained chromatograms and evaluate the quality of ginseng based on the analysis results.

[0091] The injection volume of the reference solution and the test solution into the high-performance liquid chromatograph is 10–20 μl.

[0092] The reference solution and the test solution were injected into the liquid chromatograph, and the characteristic chromatograms were measured under chromatographic conditions. The characteristic chromatograms were then analyzed and compared.

[0093] The contents of seven saponin components—ginsenoside Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rb1, ginsenoside Rc, ginsenoside Rb2, and ginsenoside Rd—can be determined at a wavelength of 203 nm. The contents of three phenolic acids—3-O-feruloylquinic acid, 4-coumaric acid, and 8-O-4 / 8-O-4 dehydrotriferulic acid—can be determined at a wavelength of 320 nm.

[0094] The chromatographic conditions are as follows:

[0095] Octadecylsilane-bonded silica gel is used as a filler;

[0096] Acetonitrile was used as the mobile phase A.

[0097] Using 0.5% phosphoric acid as mobile phase B;

[0098] Gradient elution was performed as specified, with a column temperature of 35°C and a flow rate of 1.0 ml per minute.

[0099] In this scheme, the gradient elution requirements are as follows:

[0100] From 0 to 35 min, mobile phase A was 19% and mobile phase B was 81%.

[0101] 35-55 min, mobile phase A is 19%-29%, mobile phase B is 81%-71%;

[0102] 55-70 min, mobile phase A is 29%, mobile phase B is 71%;

[0103] 70-110 min, mobile phase A is 29%-38%, mobile phase B is 71%-62%.

[0104] It should be noted that stage one is 0-35 min, with mobile phase A at 19% and mobile phase B at 81%.

[0105] At this stage, mobile phase A accounts for 19% and mobile phase B accounts for 81%. This fixed ratio allows the components of the sample to undergo preliminary distribution and movement in the chromatographic column under this specific flow environment, according to the interaction rules between their stationary and mobile phases, eluting those components that are relatively easy to separate at this ratio.

[0106] Phase 2 is 35-55 min, with mobile phase A at 19%-29% and mobile phase B at 81%-71%.

[0107] The proportion of mobile phase A gradually increases from 19% to 29%, while the proportion of mobile phase B gradually decreases from 81% to 71%. This gradual change in proportion alters the partition coefficients of the sample components between the stationary and mobile phases, allowing some components that were difficult to elute or not completely separated at previous proportions to be further separated in the column and eluted sequentially as the mobile phase proportions change.

[0108] Phase 3 is 55-70 min, with mobile phase A at 29% and mobile phase B at 71%.

[0109] Mobile phase A is maintained at a fixed ratio of 29%, and mobile phase B is maintained at a fixed ratio of 71%. After adjusting the ratios in the previous stages, this stage uses stable ratios to ensure that certain components can be stably eluted under these conditions, further refining the entire separation process and ensuring that each component has appropriate peak times and separation effects.

[0110] Stage four is 70-110 min, with mobile phase A at 29%-38% and mobile phase B at 71%-62%.

[0111] The proportion of mobile phase A increased from 29% to 38%, while the proportion of mobile phase B decreased from 71% to 62%. This gradual change in the mobile phase proportion helps to elute substances with polarity or other properties that differ from previously eluted components, making them more difficult to elute. This change in proportion promotes their elution from the chromatographic column, ultimately achieving a more complete separation of multiple components in the sample.

[0112] The mobile phase ratio variation setting is suitable for the analysis of samples with relatively complex components. By reasonably varying the mobile phase ratio, the resolution can be improved, ensuring that the chromatographic peaks of each component are well-shaped and completely separated, so as to facilitate accurate qualitative and quantitative analysis in the future.

[0113] This method simultaneously monitors two major components, saponins and phenolic acids, using a single detection method. It monitors the content of seven saponins (Rg1, Re, Rf, Rb1, Rc, Rb2, and Rd) and three polyphenols (3-O-feruloylquinic acid, 4-coumaric acid, and 8-O-4 / 8-O-4 dehydrotriferulic acid), totaling ten components. Characteristic spectra of saponins and polyphenols are obtained, making the monitoring more comprehensive and efficient.

[0114] The specific details will be explained based on the characteristic chromatograms of the 15 batches of ginseng medicinal materials obtained.

[0115] The characteristic spectrum at the 203nm wavelength should have 12 characteristic peaks, where peak 1 is ginsenoside Rg1; peak 2 is ginsenoside Re; peak 3 is ginsenoside Rf; peak 4 is ginsenoside Rb1 as a reference peak; peak 6 is ginsenoside Rc; peak 9 is ginsenoside Rb2; and peak 11 is ginsenoside Rd.

[0116] The relative retention times and relative standard deviations of each characteristic peak are as follows:

[0117] Peak 1: Relative retention time 0.455±0.002, relative standard deviation 0.36%;

[0118] Peak 2: Relative retention time 0.490±0.003, relative standard deviation 0.40%;

[0119] Peak 3: Relative retention time 0.814 ± 0.002, relative standard deviation 0.12%;

[0120] Peak 4: Relative retention time 1.000±0.000, relative standard deviation 0.00%;

[0121] Peak 5: Relative retention time 1.051 ± 0.002, relative standard deviation 0.15%;

[0122] Peak 6: Relative retention time 1.063 ± 0.002, relative standard deviation 0.09%;

[0123] Peak 7: Relative retention time 1.089 ± 0.003, relative standard deviation 0.13%.

[0124] Peak 8: Relative retention time 1.109 ± 0.002, relative standard deviation 0.15%;

[0125] Peak 9: Relative retention time 1.129 ± 0.001, relative standard deviation 0.09%;

[0126] Peak 10: Relative retention time 1.169 ± 0.003, relative standard deviation 0.15%;

[0127] Peak 11: Relative retention time 1.240 ± 0.002, relative standard deviation 0.11%;

[0128] Peak 12: Relative retention time 1.279 ± 0.003, relative standard deviation 0.14%;

[0129] The characteristic spectrum at 320 nm wavelength has five characteristic peaks, where peak 2 is 3-O-feruloylquinic acid; peak 3 is 4-coumaric acid as a reference peak; and peak 5 is 8-O-4 / 8-O-4 dehydrotriferulic acid. The relative retention time and relative standard deviation of each characteristic peak are as follows:

[0130] Peak 1: Relative retention time 0.655±0.000, relative standard deviation 0.03%;

[0131] Peak 2: Relative retention time 0.735±0.001, relative standard deviation 0.04%;

[0132] Peak 3: Relative retention time 7.211 ± 0.013, relative standard deviation 0.11%;

[0133] Peak 4: Relative retention time 7.524 ± 0.013, relative standard deviation 0.10%.

[0134] The constructed HPLC standard characteristic spectrum of ginseng at a wavelength of 203 nm consists of 12 characteristic peaks, with reference standards as the standard. Peak 1 is ginsenoside Rg1; peak 2 is ginsenoside Re; peak 3 is ginsenoside Rf; peak 4 is ginsenoside Rb1; peak 6 is ginsenoside Rc; peak 9 is ginsenoside Rb2; and peak 11 is ginsenoside Rd. Ginsenoside Rb1 is used as the reference, peak 4 is used as the reference peak, and the corresponding peak of the reference peak is used as the S peak. The relative retention time of each characteristic peak and the S peak is calculated. The relative retention time is within ±5% of the specified value. The specified values ​​are: peak 1: 0.46, peak 2: 0.49, peak 3: 0.81, peak 4: 1.00, peak 5: 1.05, peak 6: 1.06, peak 7: 0.49, peak 2: 0.49, peak 3: 0.81, peak 4: 1.00, peak 5: 1.05, peak 6: 1.06, peak 7: 0.49, peak 2: 0.49, peak 3: 0.81, peak 4: 0.81, peak 5: 1.0 ...5, peak 7: 0.81, peak 2: 0.49, peak 3: 0.81, peak 4: 0.81, peak 5: 0.81, peak 6: 0.81, peak 7: 0.81, peak 2: The HPLC standard characteristic chromatogram of ginseng at 300 nm wavelength obtained by constructing the standard consists of 5 characteristic peaks. Using the reference standard, peak 2 is 3-O-feruloylquinic acid; peak 3 is 4-coumaric acid; and peak 5 is 8-O-4 / 8-O-4 dehydrotriferulic acid. Using 4-coumaric acid as the reference, peak 3 is the reference peak, and peak 7 corresponding to the reference peak is the S peak. The relative retention times of each characteristic peak and the S peak are calculated. The relative retention times are within ±5% of the specified value, which is: peak 1: 0.66, peak 2: 0.74, peak 3: 1.00, peak 4: 7.21, peak 5: 7.52.

[0135] To facilitate understanding of the technical solution of this application, step S300 is further explained. The following provides a detailed comparison and explanation of the determination of characteristic spectra under chromatographic conditions, and presents the analytical comparison results of the characteristic spectra. The specific analysis is as follows:

[0136] See appendix Figure 1 As shown, the selection of testing instruments was analyzed in this scheme.

[0137] In this scheme, an evaporative light scattering detector (EVS) and an ultraviolet detector were not used because the EVS detector is only suitable for monitoring saponin components. See the attached figure for comparison.

[0138] See appendix Figure 2-3 As shown, the selection of mobile phase A was analyzed in this scheme.

[0139] In this scheme, acetonitrile is used as mobile phase A. The following comparison method is adopted:

[0140] Control group:

[0141] Control group 1: Acetonitrile (A) - 0.5% formic acid solution (B);

[0142] Control group 2: Acetonitrile (A) - 0.5% glacial acetic acid solution (B);

[0143] Control group 3: Acetonitrile (A) - Water (B); and

[0144] Group used: Acetonitrile (A) - 0.5% phosphoric acid (B).

[0145] The experimental comparison showed that when using control group 1 and control group 2 as mobile phases, the baseline fluctuation of saponin components at 203nm was large, so control group 1 and control group 2 are not applicable.

[0146] When using control group three as the mobile phase, the peak shape of some saponin components at 203 nm was abnormal, and polyphenol components at 320 nm could not be detected; therefore, control group three is not applicable. In conclusion, using the controlled group as the mobile phase yielded the best results.

[0147] See appendix Figure 4-5 As shown, the selection of mobile phase B was analyzed in this scheme.

[0148] In this scheme, mobile phase B uses 0.5% phosphoric acid, and its value was obtained through experimental comparison.

[0149] Experimental comparisons show that, compared to 0.1%-1.0% phosphoric acid solutions, 0.2%-1.0% phosphoric acid is superior in terms of peak separation at 203 nm, while there is no significant difference at 320 nm. Therefore, 0.5% phosphoric acid was selected as mobile phase B.

[0150] See appendix Figure 6-7 As shown, the selection of column temperature was analyzed in this scheme.

[0151] In this scheme, the column temperature is 35℃, and its value was obtained through experimental comparison.

[0152] Experimental comparisons show that, comparing column temperatures of 25℃-40℃, peak separation at 203nm is better at a column temperature of 35℃, while there is no significant difference at 320nm. Therefore, a column temperature of 35℃ was selected.

[0153] See appendix Figure 8-9 As shown, the selection of flow velocity was analyzed in this scheme.

[0154] In this protocol, the flow rate is 1.0 ml per minute, and this value was obtained through comparison.

[0155] Experimental comparisons showed that, comparing flow rates of 0.8 ml / min, 1.0 ml / min, and 1.2 ml / min, the separation of chromatographic peaks at 203 nm was optimal at a flow rate of 0.8-1.0 ml / min, while there was no significant difference at 320 nm. Therefore, a flow rate of 1.0 ml / min was selected.

[0156] See appendix Figure 10-11As shown, the precise amounts of the reference solution and the test solution described in this protocol were analyzed.

[0157] In this procedure, 10 μl of each of the reference solution and the test solution is precisely pipetted, and their values ​​are obtained by comparison.

[0158] Experimental comparisons show that, when comparing injection volumes of 10-50 μl, the peak shapes are better with 10-20 μl, while the peak shapes at 50 μl are abnormal and unsuitable. Compared to using 20 μl, using 10 μl yields essentially the same results, but requires less of both the reference solution and the test solution, resulting in higher efficiency.

[0159] See appendix Figure 12 As shown in a, 12b, 12c, 12d, 12e, 12f, 12h, 12i, and 12j, the optimal absorption wavelengths for saponins and phenolic acids were analyzed in this scheme.

[0160] Through comparative experiments, it was found that the maximum absorption wavelength of saponin components is 190nm, and 203nm was selected for baseline stability; the maximum absorption wavelength of polyphenol components is around 320nm, and 320nm was selected.

[0161] See appendix Figure 13 As shown in the attached figure, the main chromatographic peaks are concentrated in the 200-210 nm range, and the peaks show a decreasing trend. There are also relatively typical chromatographic peaks in the 300-360 nm range. Considering the consistency with the content determination, the wavelengths are determined to be 200 nm (characteristic spectrum of saponin components) and 320 nm (characteristic spectrum of polyphenol components).

[0162] See appendix Figure 14-15 As shown in this scheme, chloroform, diethyl ether, and petroleum ether were used as Soxhlet extraction solvents in the preparation of the test solution, as detailed in Table 1:

[0163]

[0164] As shown in Table 1, the content of each component is better when chloroform is used as the Soxhlet extraction solvent, and there is no significant difference between the characteristic peaks at 203 nm and 320 nm.

[0165] See appendix Figure 16-17 As shown in this scheme, methanol and n-butanol were used as Soxhlet extraction solvents in the preparation of the test solution. See Table 2 for details.

[0166]

[0167] Table 2 shows that methanol and n-butanol are the optimal extraction solvents for each component, and there is no significant difference in the number of characteristic peaks at 203 nm and 320 nm. However, because the methanol extraction solution is viscous and prone to operational errors, n-butanol was chosen as the extraction solvent.

[0168] See appendix Figure 18-19 As shown in Table 3, the ultrasonic extraction time used in this scheme is 6 hours, and the value was obtained through comparison.

[0169]

[0170] As shown in Table 3, the ultrasonic extraction times were 0.5 hours, 2 hours, 4 hours, 6 hours, and 8 hours. There was no significant difference in the extraction times of saponin components from 0.5 to 8 hours, while phenolic acid components were better extracted from 6 to 8 hours. Therefore, the optimal extraction time was determined to be 6 hours.

[0171] See appendix Figure 20-21 As shown in the figure, in this scheme, the extraction method of the test sample solution is ultrasonic extraction, and the method has been verified by experiments. See Table 4 for details:

[0172]

[0173] As shown in Table 4, reflux extraction causes the interconversion of saponin components, so it is not applicable.

[0174] See appendix Figure 22-25 As shown, based on the characteristic spectra of 15 batches of ginseng medicinal materials at a wavelength of 203 nm, the HPLC standard characteristic spectra of ginseng medicinal materials at a wavelength of 203 nm were constructed by importing them into the Chinese medicine chromatographic fingerprint spectrum similarity evaluation system. The constructed standard characteristic spectra of ginseng medicinal materials at a wavelength of 203 nm should have 12 characteristic peaks, of which peak 4 is ginsenoside Rb1 (S peak); peak 1 is ginsenoside Rg1; peak 2 is ginsenoside Re; peak 3 is ginsenoside Rf; peak 6 is ginsenoside Rc; peak 9 is ginsenoside Rb2; and peak 11 is ginsenoside Rd.

[0175] Based on the characteristic chromatograms of 15 batches of ginseng medicinal materials at a wavelength of 320 nm, the chromatographic fingerprint similarity evaluation system for traditional Chinese medicine was introduced to construct the HPLC standard characteristic chromatogram of ginseng medicinal materials at a wavelength of 320 nm. It should have 5 characteristic peaks, of which peak 3 is 4-coumaric acid, which is the S peak; peak 2 is 3-O-feruloylquinic acid; and peak 5 is 8-O-4 / 8-O-4 dehydrotriferulic acid.

[0176] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it.

Claims

1. A method for quality analysis of ginseng medicinal materials, characterized in that, Includes the following steps: S100. Preparation of reference solution: Take appropriate amounts of ginsenoside Rg1 reference, ginsenoside Re reference, ginsenoside Rf reference, ginsenoside Rb1 reference, ginsenoside Rc reference, ginsenoside Rb2 reference, ginsenoside Rd reference, 3-O-feruloylquinic acid reference, 4-coumaric acid reference, and 8-O-4 / 8-O-4 dehydrotriferulic acid reference, add methanol to prepare a mixed solution, shake well, and the solution is obtained. S200. Preparation of the test solution: Weigh approximately 1-2 g of ginseng powder accurately, place it in a Soxhlet extractor, add chloroform and heat under reflux for 3 hours, discard the petroleum ether solution, evaporate the solvent from the residue, and transfer it along with the filter paper tube into a 100 ml conical flask. Accurately add 50 ml of water-saturated n-butanol, seal tightly, and let stand overnight. Sonicate for 6 hours, filter, discard the initial filtrate, accurately measure 25 ml of the subsequent filtrate, evaporate it to dryness in an evaporating dish, dissolve the residue in methanol and transfer it to a 5 ml volumetric flask, dilute with methanol to the mark, shake well, filter, and collect the subsequent filtrate to obtain the test solution. S300, Chromatographic conditions: C18 reversed-phase column; mobile phase A is acetonitrile and mobile phase B is a gradient eluent consisting of 0.2-1.0% phosphoric acid solution; detection wavelengths are 203 nm and 320 nm; column temperature is 33-37℃; flow rate is 0.8 ml / min-1.0 ml / min; determination time is 110 minutes. S400. Determination: Accurately pipette the reference solution and the test solution into the high performance liquid chromatograph, and determine the content of ten components by high performance liquid chromatography. Determine the content of ten components by external standard method and obtain characteristic chromatograms. Analyze the obtained chromatograms and evaluate the quality of ginseng based on the analysis results. Gradient elution requirements are as follows: From 0 to 35 minutes, mobile phase A was 19% and mobile phase B was 81%. 35-55 min, mobile phase A is 19%-29%, mobile phase B is 81%-71%; 55-70 min, mobile phase A is 29%, mobile phase B is 71%; For 70-110 min, mobile phase A is 29%-38% and mobile phase B is 71%-62%.

2. The method for quality analysis of ginseng medicinal material according to claim 1, characterized in that, In step S400, the injection volume of the reference solution and the test solution into the high performance liquid chromatograph is precisely 10-20 μl.

3. The method for quality analysis of ginseng medicinal material according to claim 1, characterized in that, The content of seven saponin components—ginsenoside Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rb1, ginsenoside Rc, ginsenoside Rb2, and ginsenoside Rd—can be determined at a wavelength of 203 nm.

4. The method for quality analysis of ginseng medicinal material according to claim 1, characterized in that, The content of three phenolic acid components, namely 3-O-feruloylquinic acid, 4-coumaric acid, and 8-O-4 / 8-O-4 dehydrotriferulic acid, can be determined at a wavelength of 320 nm.

5. The method for quality analysis of ginseng medicinal material according to claim 1, characterized in that, Based on the characteristic spectra of 15 batches of ginseng medicinal materials, the characteristic spectra at a wavelength of 203nm should have 12 characteristic peaks, of which peak 1 is ginsenoside Rg1; peak 2 is ginsenoside Re; peak 3 is ginsenoside Rf; peak 4 is ginsenoside Rb1 as a reference peak; peak 6 is ginsenoside Rc; peak 9 is ginsenoside Rb2; and peak 11 is ginsenoside Rd.

6. The method for quality analysis of ginseng medicinal material according to claim 1, characterized in that, The relative retention times and relative standard deviations of each characteristic peak are as follows: Peak 1: Relative retention time 0.455±0.002, relative standard deviation 0.36%; Peak 2: Relative retention time 0.490±0.003, relative standard deviation 0.40%; Peak 3: Relative retention time 0.814 ± 0.002, relative standard deviation 0.12%; Peak 4: Relative retention time 1.000±0.000, relative standard deviation 0.00%; Peak 5: Relative retention time 1.051 ± 0.002, relative standard deviation 0.15%; Peak 6: Relative retention time 1.063 ± 0.002, relative standard deviation 0.09%; Peak 7: Relative retention time 1.089 ± 0.003, relative standard deviation 0.13%; Peak 8: Relative retention time 1.109 ± 0.002, relative standard deviation 0.15%; Peak 9: Relative retention time 1.129 ± 0.001, relative standard deviation 0.09%; Peak 10: Relative retention time 1.169 ± 0.003, relative standard deviation 0.15%; Peak 11: Relative retention time 1.240 ± 0.002, relative standard deviation 0.11%; Peak 12: Relative retention time 1.279 ± 0.003, relative standard deviation 0.14%; The characteristic spectrum at 320 nm wavelength has five characteristic peaks, where peak 2 is 3-O-feruloylquinic acid; peak 3 is 4-coumaric acid as a reference peak; and peak 5 is 8-O-4 / 8-O-4 dehydrotriferulic acid. The relative retention time and relative standard deviation of each characteristic peak are as follows: Peak 1: Relative retention time 0.655±0.000, relative standard deviation 0.03%; Peak 2: Relative retention time 0.735±0.001, relative standard deviation 0.04%; Peak 3: Relative retention time 7.211 ± 0.013, relative standard deviation 0.11%; Peak 4: Relative retention time 7.524 ± 0.013, relative standard deviation 0.10%.

7. The method for quality analysis of ginseng medicinal material according to claim 1, characterized in that, The constructed HPLC standard characteristic spectrum of ginseng at a wavelength of 203 nm consists of 12 characteristic peaks, with reference standards as the standard. Peak 1 is ginsenoside Rg1; peak 2 is ginsenoside Re; peak 3 is ginsenoside Rf; peak 4 is ginsenoside Rb1; peak 6 is ginsenoside Rc; peak 9 is ginsenoside Rb2; and peak 11 is ginsenoside Rd. Ginsenoside Rb1 is used as the reference, peak 4 is used as the reference peak, and the corresponding peak of the reference peak is used as the S peak. The relative retention time of each characteristic peak and the S peak is calculated. The relative retention time is within ±5% of the specified value, and the specified value is peak 1. 0.46, Peak 2: 0.49, Peak 3: 0.81, Peak 4: 1.00, Peak 5: 1.05, Peak 6: 1.06, Peak 7: 1.09, peak 8: 1.11, peak 9: 1.13, peak 10: 1.17, peak 11: 1.24, peak 12: 1.28; The HPLC standard characteristic chromatogram of ginseng at 300 nm wavelength obtained by construction consists of 5 characteristic peaks. Using the reference standard, peak 2 is 3-O-feruloylquinic acid; peak 3 is 4-coumaric acid; peak 5 is 8-O-4 / 8-O-4 dehydrotriferulic acid. Using 4-coumaric acid as the reference, peak 3 is the reference peak, and peak 7 corresponding to the reference peak is the S peak. The relative retention time of each characteristic peak and the S peak is calculated. The relative retention time is within ±5% of the specified value. The specified value is peak 1: Peak 1: 0.66, Peak 2: 0.74, Peak 3: 1.00, Peak 4: 7.21, Peak 5: 7.52.

Images