Quality analysis method of ginseng medicinal material

Abstract

The invention relates to the technical field of compound analysis, in particular to a quality analysis method of a ginseng medicinal material. Comprising the following steps: S100, preparation of a reference substance solution: taking a proper amount of a ginsenoside Rg1 reference substance, a ginsenoside Re reference substance, a ginsenoside Rf reference substance, a ginsenoside Rb1 reference substance, a ginsenoside Rc reference substance, a ginsenoside Rb2 reference substance, a ginsenoside Rd reference substance, a 3-O-feruloylquinic acid reference substance, a 4-coumaric acid reference substance and an 8-O-4 / 8-O-4 dehydrotriferulic acid reference substance; and adding methanol to prepare a mixed solution, and uniformly shaking. According to the method, the saponin and phenolic acid components are synchronously monitored under one detection method, the contents of ten components including seven saponin components and three polyphenol components are monitored, the specific chromatograms of the saponin components and the polyphenol components are obtained, and the monitoring quality is more comprehensive and efficient.

Description

Technical Field

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

[0002] Modern medical research has shown that ginseng has a variety of biological and pharmacological properties, such as immune stimulation, anti-cancer, anti-emetic, antioxidant, etc. 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 research on ginseng medicinal materials quality control: The 2020 edition of the Chinese Pharmacopoeia stipulates the content determination of three components, ginsenosides Rg1, Re, and Rb1, under the ginseng medicinal materials. Some scholars have studied and monitored the fingerprint of ginseng slices, monitored 9 common peaks, and identified five components, ginsenosides Rg1, Re, Rf, Rb1, and Rd. Some scholars monitor nine components of wild ginseng ginsenosides Rg1, Re, Rf, Rb1, Rc, F1, Rb2, Rb3, and Rd. Some scholars monitor eleven components of ginsenosides Rb1, Rg1, Re, Rf, Rh1, Rc, Ro, Rb2, Rb3, Rd, and Rg3. Some scholars have established ginseng characteristic spectrum monitoring, monitored 11 characteristic peaks, and monitored the contents of eight components, ginsenosides Rg1, Re, Rb1, Rf, Rc, Rb2, Rb3, and Rd. Some scholars have analyzed the polyphenolic compounds of ginseng medicinal materials, and found 13 components in the raw sun-dried ginseng roots, including protocatechuic acid, p-hydroxybenzoic acid, syringic acid, gentisic acid, p-coumaric acid, ferulic acid, m-coumaric acid, resveratrol, cinnamic acid, maltol, catechin, naringenin, and formononetin. Some scholars have also determined the content of seven components in the wild ginseng, including fumaric acid, citric acid, maleic acid, succinic acid, tartaric acid, vanillic acid, and cinnamic acid.

[0004] However, the technical research conducted by these scholars only monitored or studied one category of ingredients in ginseng medicinal materials, focusing on saponins or polyphenols, and the monitoring was relatively single and incomplete. Summary of the invention

[0005] The present invention aims at the deficiencies in the prior art and provides the following technical solutions:

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

[0007] S100, preparation of reference solution: take appropriate amount of ginsenoside Rg1 reference substance, ginsenoside Re reference substance, ginsenoside Rf reference substance, ginsenoside Rb1 reference substance, ginsenoside Rc reference substance, ginsenoside Rb2 reference substance, ginsenoside Rd reference substance, 3-O-feruloylquinic acid reference substance, 4-coumaric acid reference substance, 8-O-4 / 8-O-4 dehydrotriferulic acid reference substance, add methanol to make a mixed solution, shake well to obtain;

[0008] S200, preparation of test solution: take about 1-2g of ginseng powder, accurately weigh it, put it in a Soxhlet extractor, add chloroform and heat to reflux for 3 hours, discard the petroleum ether solution, evaporate the solvent from the residue, move it into a 100ml conical flask together with the filter paper tube, accurately add 50ml of saturated n-butanol with water, plug it tightly, leave it overnight, ultrasonically treat it for 6 hours, filter it, discard the initial filtrate, accurately measure 25ml of the subsequent filtrate, put it in an evaporating dish and evaporate it to dryness, dissolve the residue in methanol and transfer it to a 5ml volumetric flask, dilute it to the scale with methanol, shake it well, filter it, and take the subsequent filtrate to obtain;

[0009] S300, chromatographic conditions: the chromatographic column is a C18 reverse phase column; the mobile phase A is a gradient eluent consisting of acetonitrile and the mobile phase B is a 0.2-1.0% phosphoric acid solution; the detection wavelength is 203nm and 320nm, the column temperature is 33-37°C; the flow rate is 0.8ml / min-1.0ml / min; the measurement time is 110 minutes;

[0010] S400, determination: Accurately aspirate the reference solution and the test solution and inject them into the high performance liquid chromatograph, determine them according to the high performance liquid chromatography method, determine the contents of ten components by the external standard method, and obtain characteristic spectra, analyze the obtained chromatograms, and evaluate the quality of ginseng medicinal materials based on the analysis results.

[0011] As an improvement of the above technical solution, the gradient elution requirements are as follows:

[0012] 0-35min, mobile phase A was 19%, mobile phase B was 81%;

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

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

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

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

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

[0018] As an improvement of 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 of the above technical solution, according to the characteristic spectra of 15 batches of ginseng medicinal materials obtained, the characteristic spectrum at a wavelength of 203 nm should have 12 characteristic peaks, among 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; Peak 11 is ginsenoside Rd.

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

[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 a wavelength of 320 nm has 5 characteristic peaks, wherein peak 2 is 3-O-feruloylquinic acid; peak 3 is 4-coumaric acid as a reference peak; peak 5 is 8-O-4 / 8-O-4 dehydrotriferulic acid; the relative retention time and relative standard deviation of each characteristic peak are:

[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 of the above technical solution, the constructed HPLC standard characteristic spectrum of ginseng medicinal materials at a wavelength of 203nm is composed of 12 characteristic peaks. Compared with the reference substance, 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; peak 11 is ginsenoside Rd, ginsenoside Rb1 is used as a reference, peak 4 is used as a reference peak, and the peak corresponding to the reference peak is used as the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and 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 constructed HPLC standard characteristic spectrum of ginseng medicinal materials at a wavelength of 300nm is composed of 5 characteristic peaks. Compared with the reference substance, peak 2 is 3-O-feruloylquinic acid; peak 3 is 4-coumaric acid; peak 5 is 8-O-4 / 8-O-4 dehydrotriferulic acid, 4-coumaric acid is used as the reference, peak 3 is the reference peak, and peak 7 corresponding to the reference peak is used as the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time is within ±5% of the specified value, and 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] Beneficial effects of the present invention:

[0040] Under one detection method, the two major components, saponins and phenolic acids, are monitored simultaneously. The contents of a total of ten components, including seven saponin components, ginsenoside Rg1, Re, Rf, Rb1, Rc, Rb2, and Rd, and three polyphenol components, 3-O-feruloylquinic acid, 4-coumaric acid, and 8-O-4 / 8-O-4 dehydrotriferulic acid, are monitored, and the characteristic spectra of saponin components and polyphenol components are obtained, making the monitoring quality more comprehensive and efficient. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a comparison chart of the detection effects of the evaporative light scattering detector and the ultraviolet detector;

[0042] Figure 2 This is a chromatographic comparison diagram of saponins with different mobile phase compositions at a wavelength of 203 nm;

[0043] Figure 3 This is a chromatographic comparison diagram of phenolic acids at a wavelength of 320 nm with different mobile phase compositions;

[0044] Figure 4This is a chromatographic comparison chart of saponins at a wavelength of 203 nm with different proportions of phosphoric acid in the mobile phase;

[0045] Figure 5 The chromatogram comparison diagram of phenolic acids at a wavelength of 320 nm with different phosphoric acid ratios in the mobile phase;

[0046] Figure 6 The chromatographic comparison chart at 203 nm wavelength shows the effect of different column temperatures on the preparation of saponins;

[0047] Figure 7 The chromatographic comparison chart at 320nm wavelength shows the effect of different column temperatures on the preparation of phenolic acids.

[0048] Figure 8 This is a chromatographic comparison chart of the effect of different flow rates on the preparation of saponins at a wavelength of 203 nm;

[0049] Fig. 9 The chromatographic comparison chart at 320nm wavelength shows the effect of different flow rates on the preparation of phenolic acids;

[0050] Fig.10 The chromatographic comparison chart of the effect of different injection amounts on saponins at a wavelength of 203 nm;

[0051] Fig.11 The chromatographic comparison chart of the effect of different injection amounts on phenolic acids at a wavelength of 320 nm;

[0052] Fig.12 This is a comparison chart of the UV absorption spectra of saponins and phenolic acids;

[0053] Fig.13 It is the 3D chromatogram of the test solution;

[0054] Fig.14 The chromatogram comparison of the test solution using different Soxhlet extraction solvents at a wavelength of 203 nm;

[0055] Fig.15 The chromatogram comparison of the test solution using different Soxhlet extraction solvents at a wavelength of 320nm;

[0056] Fig.16 This is a chromatographic comparison chart of n-butanol at a wavelength of 203 nm used in the preparation of the test solution;

[0057] Fig.17 This is a chromatographic comparison chart of n-butanol at a wavelength of 320 nm used in the preparation of the test solution;

[0058] Fig.18 The chromatograms of the test solution prepared at different extraction times at a wavelength of 203 nm are compared;

[0059] Fig.19 The chromatograms of the test solution prepared at different extraction times at a wavelength of 320 nm are compared;

[0060] Fig. 20 This is a chromatogram comparison of the test solution under different extraction methods at a wavelength of 203 nm;

[0061] Fig.21 This is a chromatogram comparison of the test solution under different extraction methods at a wavelength of 320nm;

[0062] Fig. 22 This is the standard characteristic spectrum of ginseng medicinal materials at a wavelength of 203nm;

[0063] Fig.23 This is a chromatogram comparison of multiple batches of ginseng medicinal materials at a wavelength of 203nm;

[0064] Fig.24 This is the standard characteristic spectrum of ginseng medicinal materials at a wavelength of 320nm;

[0065] Fig.25 This is a chromatogram comparison of multiple batches of ginseng medicinal materials at a wavelength of 320nm. DETAILED DESCRIPTION

[0066] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

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

[0068] Preparation of S100, reference solution:

[0069] Take appropriate amount of ginsenoside Rg1 reference substance, ginsenoside Re reference substance, ginsenoside Rf reference substance, ginsenoside Rb1 reference substance, ginsenoside Rc reference substance, ginsenoside Rb2 reference substance, ginsenoside Rd reference substance, 3-O-feruloylquinic acid reference substance, 4-coumaric acid reference substance, and 8-O-4 / 8-O-4 dehydrotriferulic acid reference substance, add methanol to make a mixed solution, shake well to obtain.

[0070] The reference solution contains saponins and phenolic acids, so they need to be prepared separately. In this scheme, specific preparation methods of saponin and phenolic acid reference solutions are provided. It should be noted that the specific preparation method is only used to illustrate the preparation of the reference solution and should not be understood as a limitation on the preparation method.

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

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

[0073] The preparation method of the saponin reference substance is:

[0074] S110, take appropriate amounts of ginsenoside Rg1 reference substance, ginsenoside Re reference substance, ginsenoside Rf reference substance, ginsenoside Rb1 reference substance, ginsenoside Rc reference substance, ginsenoside Rb2 reference substance, and ginsenoside Rd reference substance, accurately weigh and set aside;

[0075] S120. Add methanol to prepare a mixed solution containing 0.2 mg each of ginsenosides Rg1, Re, Rf, Rb1, Rc, Rb2, and Rd per 1 ml. Shake well to obtain the saponin reference solution.

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

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

[0078] The preparation method of the polyphenol reference substance is:

[0079] S130, take appropriate amounts of 3-O-feruloylquinic acid reference substance, 4-coumaric acid reference substance, and 8-O-4 / 8-O-4 dehydrotriferulic acid, accurately weigh and set aside;

[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 a phenolic acid reference solution.

[0081] S200, Preparation of test solution:

[0082] Take about 1-2g of ginseng powder, accurately weigh it, put it in a Soxhlet extractor, add chloroform and heat to reflux for 3 hours, discard the petroleum ether liquid, evaporate the solvent from the residue, transfer it together with the filter paper tube into a 100ml conical flask, accurately add 50ml of water-saturated n-butanol, plug it tightly, leave it overnight, ultrasonically treat it for 6 hours, filter, discard the initial filtrate, accurately measure 25ml of the subsequent filtrate, put it in an evaporating dish and evaporate it to dryness, dissolve the residue in methanol and transfer it to a 5ml volumetric flask, add methanol to dilute to the scale, shake well, filter, and take the subsequent filtrate.

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

[0084] Place 1 g 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, transfer it together with the filter paper tube into a 100 ml conical flask, accurately add 50 ml of water-saturated n-butanol, plug it tightly, leave it overnight, treat it with ultrasound 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 it to the scale with methanol, shake it well, filter it, and take the subsequent filtrate to obtain the test solution.

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

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

[0087] S300, chromatographic conditions:

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

[0089] S400, determination:

[0090] Accurately pipette the reference solution and the test solution into a high performance liquid chromatograph, determine them by high performance liquid chromatography, determine the contents of ten components by the external standard method, and obtain characteristic spectra. Analyze the obtained chromatograms, and evaluate the quality of ginseng medicinal materials based on the analysis results.

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

[0092] Inject the reference solution and the test solution into the liquid chromatograph, determine the characteristic spectrum under chromatographic conditions, and analyze and compare the characteristic spectrum.

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

[0094] Wherein, the chromatographic conditions are:

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

[0096] Acetonitrile was used as mobile phase A;

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

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

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

[0100] 0-35min, mobile phase A was 19%, mobile phase B was 81%;

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

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

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

[0104] It should be noted that, in stage 1, the concentration of mobile phase A was 19% and that of mobile phase B was 81% during the period from 0 to 35 min.

[0105] At this stage, the proportion of mobile phase A is 19% and that of mobile phase B is 81%. This fixed ratio allows the components of the sample to be initially distributed and moved in the chromatographic column in accordance with the interaction between the stationary phase and the mobile phase under this specific flow environment, eluting those components that are relatively easy to separate under this ratio.

[0106] Stage 2: 35-55 min, mobile phase A is 19%-29%, mobile phase B is 81%-71%.

[0107] The proportion of mobile phase A gradually increased from 19% to 29%, and correspondingly, the proportion of mobile phase B gradually decreased from 81% to 71%. The gradual change of the proportion can change the distribution coefficient of each component of the sample between the stationary phase and the mobile phase, so that some components that are difficult to elute or have not been completely separated at the previous proportion can be further separated in the chromatographic column as the mobile phase proportion changes, and thus eluted in sequence.

[0108] Stage three: 55-70 min, mobile phase A is 29%, mobile phase B is 71%.

[0109] Mobile phase A was kept at a fixed ratio of 29% and mobile phase B at 71%. After the ratio changes in the previous stage, this stage used a stable ratio to ensure that some specific components could be stably eluted under this environment, further improving the entire separation process and ensuring that each component had a suitable peak elution time and separation effect.

[0110] Stage 4: 70-110 min, mobile phase A is 29%-38%, mobile phase B is 71%-62%.

[0111] The proportion of mobile phase A increased from 29% to 38%, and the proportion of mobile phase B decreased from 71% to 62%. The mobile phase proportion gradually changes, and for those substances that are different from the previously eluted components in polarity and other properties and more difficult to be eluted, such proportion changes can promote them to be eluted from the chromatographic column, and finally achieve a relatively complete separation of multiple components in the sample.

[0112] The mobile phase ratio change setting is suitable for the analysis of samples with more complex components. It improves the separation degree by changing the mobile phase ratio reasonably, so that the chromatographic peak shape of each component is good and the separation is complete, so as to carry out subsequent accurate qualitative and quantitative analysis.

[0113] This scheme simultaneously monitors the two major components, saponins and phenolic acids, under one detection method, and monitors the contents of a total of ten components, including seven saponin components, including ginsenoside Rg1, Re, Rf, Rb1, Rc, Rb2, and Rd, and three polyphenol components, including 3-O-feruloylquinic acid, 4-coumaric acid, and 8-O-4 / 8-O-4 dehydrotriferulic acid, to obtain characteristic maps of saponin components and polyphenol components, making the monitoring quality more comprehensive and efficient.

[0114] The specific explanation will be based on the characteristic spectra of the 15 batches of ginseng medicinal materials obtained.

[0115] The characteristic spectrum at a wavelength of 203 nm should have 12 characteristic peaks, wherein 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 time and relative standard deviation of each characteristic peak are:

[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 a wavelength of 320 nm has 5 characteristic peaks, wherein peak 2 is 3-O-feruloylquinic acid; peak 3 is 4-coumaric acid as a reference peak; peak 5 is 8-O-4 / 8-O-4 dehydrotriferulic acid; the relative retention time and relative standard deviation of each characteristic peak are:

[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 medicinal materials at a wavelength of 203 nm is composed of 12 characteristic peaks, which are compared with the reference substance; 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; peak 11 is ginsenoside Rd, ginsenoside Rb1 is used as a reference, peak 4 is used as a reference peak, and the peak corresponding to the reference peak is used as the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and 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 constructed HPLC standard characteristic spectrum of ginseng medicinal materials at a wavelength of 300nm is composed of 5 characteristic peaks, compared with the reference substance, peak 2 is 3-O-feruloylquinic acid; peak 3 is 4-coumaric acid; peak 5 is 8-O-4 / 8-O-4 dehydrotriferulic acid, 4-coumaric acid is used as a reference, peak 3 is a reference peak, and peak 7 corresponding to the reference peak is taken as S peak, and the relative retention time of each characteristic peak and S peak is calculated, and the relative retention time is within ±5% of the specified value, and the specified value 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 the present application, step S300 is further described. The characteristic spectra measured under chromatographic conditions are compared and described in detail below, and the analysis and comparison results of the characteristic spectra are given. The specific analysis is as follows:

[0136] See attached Figure 1 As shown, the selection of detection instruments is analyzed in this plan.

[0137] In this scheme, the detector used does not use an evaporative light scattering detector or an ultraviolet detector because the evaporative light scattering detector can only monitor saponin components and is not applicable. See the attached figure for details.

[0138] See attached 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 used:

[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] Use group: acetonitrile (A)-0.5% phosphoric acid (B).

[0145] Through experimental comparison, it can be seen that when control group 1 and control group 2 are used as mobile phase, the baseline fluctuation of 203nm saponin components is large, so control group 1 and control group 2 are not applicable.

[0146] When the control group 3 was used as the mobile phase, the peak shape of some saponin components at 203nm was abnormal, and the polyphenol components at 320nm could not be detected, so the control group 3 was not applicable. In summary, the effect was the best when the group was used as the mobile phase.

[0147] See attached 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 is obtained through experimental comparison.

[0149] Through the experimental comparison, it can be seen that compared with 0.1%-1.0% phosphoric acid solution, 0.2%-1.0% phosphoric acid has better chromatographic peak separation at 203nm, and there is no significant difference at 320nm. Therefore, 0.5% phosphoric acid is selected as mobile phase B.

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

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

[0152] Through experimental comparison, it can be seen that compared with the column temperature of 25℃-40℃, the chromatographic peak separation at 203nm is better when the column temperature is 35℃, and there is no significant difference at 320nm. Therefore, the column temperature of 35℃ is selected.

[0153] See attached Figure 8-9 As shown, the selection of flow rate is analyzed in this scheme.

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

[0155] Through the experimental comparison, it can be seen that the flow rates of 0.8ml / min, 1.0ml / min and 1.2ml / min are compared, and the separation of each chromatographic peak at 203nm wavelength is better at 0.8-1.0m / min, and there is no significant difference at 320nm. Therefore, the flow rate of 1.0ml per minute is selected.

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

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

[0158] Through the experimental comparison, it can be seen that compared with the injection volume of 10μl-50μl, the peak shape of each chromatographic peak is better with 10-20μl, and the peak shape of 50μl is abnormal and not applicable. Compared with the injection volume of 20μl, the effect of using the injection volume of 10μl is basically the same, and the amount of reference solution and test solution required is less, and the efficiency is higher.

[0159] See attached Fig.12 As shown in a, 12b, 12c, 12d, 12e, 12f, 12h, 12i, and 12j, in this scheme, the selection of the optimal absorption wavelengths of saponins and phenolic acids was analyzed.

[0160] Through experimental comparison, it can be seen that the maximum absorption wavelength of saponin components is 190nm. Considering the stability of the baseline, 203nm is selected; the maximum absorption wavelength of polyphenol components is around 320nm, so 320nm is selected.

[0161] See attached Fig.13 As shown in the attached figure, it can be seen that the main chromatographic peaks are concentrated in 200-210nm, and the peaks show a decreasing trend; there are more typical chromatographic peaks in 300-360nm. Considering the consistency with the content determination, the wavelengths are determined to be 200nm (characteristic spectrum of saponin components) and 320nm (characteristic spectrum of polyphenol components).

[0162] See attached Figure 14-15 As shown in this scheme, the test solution was prepared using chloroform, ether and petroleum ether as Soxhlet extraction solvents, as shown in Table 1:

[0163]

[0164] It can be seen from Table 1 that the content of each component is better when chloroform is used as the Soxhlet extraction solvent, and there is no significant difference in the characteristic peaks at 203nm and 320nm.

[0165] See attached 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] It can be seen from Table 2 that the content of each component is better when methanol and n-butanol are used as extraction solvents, and there is no significant difference in the number of characteristic peaks at 203nm and 320nm. Because the methanol extraction solution is more viscous and easy to cause operational errors, n-butanol was selected as the extraction solvent.

[0168] See attached Figure 18-19 As shown, in this scheme, the ultrasonic extraction time used is 6h, and its value is obtained by comparison, as shown in Table 3:

[0169]

[0170] It can be seen from Table 3 that the ultrasonic extraction times are 0.5 hours, 2 hours, 4 hours, 6 hours and 8 hours respectively. There is no significant difference in the extraction time of each saponin component from 0.5 to 8 hours, while the extraction time of phenolic acid components is better at 6 to 8 hours. Therefore, 6 hours is determined to be the optimal extraction time.

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

[0172]

[0173] As can be seen from Table 4, reflux extraction will cause the mutual conversion of saponin components, so it is not applicable.

[0174] See attached Figure 22-25 As shown, based on the characteristic spectra of 15 batches of ginseng medicinal materials at a wavelength of 203 nm, the Chinese medicine chromatographic fingerprint similarity evaluation system was introduced to construct the HPLC standard characteristic spectrum of ginseng medicinal materials at a wavelength of 203 nm, which should have 12 characteristic peaks, of which Peak 4 is ginsenoside Rb1, which is the 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; Peak 11 is ginsenoside Rd.

[0175] According to the characteristic spectra of the 15 batches of ginseng medicinal materials obtained at a wavelength of 320nm, the traditional Chinese medicine chromatographic fingerprint similarity evaluation system was introduced to construct the HPLC standard characteristic spectrum of ginseng medicinal materials at a wavelength of 320nm, which should have 5 characteristic peaks, among which peak 3 is 4-coumaric acid, which is the S peak; peak 2 is 3-O-feruloylquinic acid; 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, rather than to limit them.

Claims

1. A method for quality analysis of ginseng medicinal materials, characterized in that: The following steps are involved: S100, preparation of reference solution: take appropriate amount of ginsenoside Rg1 reference substance, ginsenoside Re reference substance, ginsenoside Rf reference substance, ginsenoside Rb1 reference substance, ginsenoside Rc reference substance, ginsenoside Rb2 reference substance, ginsenoside Rd reference substance, 3-O-feruloylquinic acid reference substance, 4-coumaric acid reference substance, 8-O-4 / 8-O-4 dehydrotriferulic acid reference substance, add methanol to make a mixed solution, shake well to obtain; S200, preparation of test solution: take about 1-2g of ginseng powder, accurately weigh it, put it in a Soxhlet extractor, add chloroform and heat to reflux for 3 hours, discard the petroleum ether solution, evaporate the solvent from the residue, move it into a 100ml conical flask together with the filter paper tube, accurately add 50ml of saturated n-butanol with water, plug it tightly, leave it overnight, ultrasonically treat it for 6 hours, filter it, discard the initial filtrate, accurately measure 25ml of the subsequent filtrate, put it in an evaporating dish and evaporate it to dryness, dissolve the residue in methanol and transfer it to a 5ml volumetric flask, dilute it to the scale with methanol, shake it well, filter it, and take the subsequent filtrate to obtain; S300, chromatographic conditions: the chromatographic column is a C18 reverse phase column; the mobile phase A is a gradient eluent consisting of acetonitrile and the mobile phase B is a 0.2-1.0% phosphoric acid solution; the detection wavelength is 203nm and 320nm, the column temperature is 33-37°C; the flow rate is 0.8ml / min-1.0ml / min; the measurement time is 110 minutes; S400, determination: Accurately aspirate the reference solution and the test solution and inject them into the high performance liquid chromatograph, determine them according to the high performance liquid chromatography method, determine the contents of ten components by the external standard method, and obtain characteristic spectra, analyze the obtained chromatograms, and evaluate the quality of ginseng medicinal materials based on the analysis results.

2. The method for quality analysis of ginseng medicinal materials according to claim 1, characterized in that: The gradient elution requirements are as follows: 0-35min, mobile phase A was 19%, mobile phase B was 81%; 35-55min, mobile phase A is 19%-29%, mobile phase B is 81%-71%; 55-70min, mobile phase A is 29%, mobile phase B is 71%; 70-110min, mobile phase A is 29%-38%, mobile phase B is 71%-62%.

3. A method for analyzing the quality of ginseng medicinal materials according to claim 1, characterized in that: In step S400, the injection volume of the reference solution and the test solution accurately injected into the high performance liquid chromatograph is 10-20 μl.

4. A method for analyzing the quality of ginseng medicinal materials according to claim 1, characterized in that: At a wavelength of 203nm, the contents of seven saponin components, including ginsenoside Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rb1, ginsenoside Rc, ginsenoside Rb2, and ginsenoside Rd, can be determined.

5. A method for analyzing the quality of ginseng medicinal materials according to claim 1, characterized in that: The contents of three phenolic acid components, 3-O-feruloylquinic acid, 4-coumaric acid, and 8-O-4 / 8-O-4 dehydrotriferulic acid, can be determined at a wavelength of 320nm.

6. A method for analyzing the quality of ginseng medicinal materials according to claim 1, characterized in that: According to the characteristic spectra of the 15 batches of ginseng medicinal materials obtained, the characteristic spectrum at a wavelength of 203 nm should have 12 characteristic peaks, wherein 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; Peak 11 is ginsenoside Rd.

7. A method for analyzing the quality of ginseng medicinal materials according to claim 1, characterized in that: The relative retention time and relative standard deviation of each characteristic peak are: 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 a wavelength of 320 nm has 5 characteristic peaks, wherein peak 2 is 3-O-feruloylquinic acid; peak 3 is 4-coumaric acid as a reference peak; peak 5 is 8-O-4 / 8-O-4 dehydrotriferulic acid; the relative retention time and relative standard deviation of each characteristic peak are: 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%.

8. The method for quality analysis of ginseng medicinal materials according to claim 1, characterized in that: The constructed HPLC standard characteristic spectrum of ginseng medicinal materials at a wavelength of 203 nm is composed of 12 characteristic peaks, with reference substance as reference; 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; Peak 11 is ginsenoside Rd, with ginsenoside Rb1 as reference, Peak 4 as reference peak, and the peak corresponding to the reference peak as S peak, and the relative retention time of each characteristic peak and S peak is calculated, and 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 constructed HPLC standard characteristic spectrum of ginseng medicinal materials at a wavelength of 300nm is composed of 5 characteristic peaks, compared with the reference substance, peak 2 is 3-O-feruloylquinic acid; peak 3 is 4-coumaric acid; peak 5 is 8-O-4 / 8-O-4 dehydrotriferulic acid, 4-coumaric acid is used as a reference, peak 3 is a reference peak, and peak 7 corresponding to the reference peak is taken as S peak, and the relative retention time of each characteristic peak and S peak is calculated, and the relative retention time is within ±5% of the specified value, and the specified value is peak 1: 0.66, Peak 2: 0.74, Peak 3: 1.00, Peak 4: 7.21, Peak 5:7.52.

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