Determination of content of Dangguiyinzi

Abstract

The present invention provides a method for determining the content of Danggui Yinzi, which belongs to the field of drug detection technology. The method comprises preparing a test solution from Danggui Yinzi, performing high performance liquid chromatography detection by gradient elution using a 0.08-0.12wt% phosphoric acid aqueous solution (A)-acetonitrile (B) as the mobile phase, and then calculating the contents of the nine components in Danggui Yinzi using an external standard point method or a standard curve substitution method. The present invention quantitatively analyzes the nine components in Danggui Yinzi at one time, effectively controlling the quality of Danggui Yinzi, and providing a basis for component and activity research in the later stage of Danggui Yinzi material basis research, thereby enabling it to exert greater medicinal value and providing strong support for the secondary development of Danggui Yinzi preparations.

Description

Technical Field

[0001] The present invention relates to the technical field of drug detection, and in particular to a method for determining the content of Dangguiyinzi. Background Art

[0002] Danggui Yinzi, from the Revised Yan's Prescriptions for Preserving Life, is suitable for treating stagnant blood in the heart, internal wind-heat, skin sores and scabies, swelling, itching, pus-filled skin, or rashes and tumors. Its ingredients, including Siwu herbal medicine and Shouwu, nourish Yin and blood, making it suitable for those with blood deficiency and wind-dryness. Therefore, this prescription can be considered for any skin condition with prolonged damage to Yin and blood, resulting in swelling or itching.

[0003] Danggui Yinzi is a compound preparation made from eleven traditional Chinese medicines (TCMs): angelica sinensis, white peony root, Chuanxiong rhizome, raw rehmannia root, tribulus terrestris, siler, schizonepeta spicate, polygonum multiflorum, astragalus root, roasted liquorice root, and ginger. Each of these herbs contains multiple components, resulting in a complex overall composition. Testing a single component or indicator is difficult to fully characterize the quality of Danggui Yinzi, and the interactions between the various components further hinder quantitative analysis of Danggui Yinzi. Due to the high content, difficulty in separating, and wide variations in the content of the various herbs in Danggui Yinzi, it is difficult to investigate the components in practice. Currently, there is no definitive method for quantitatively analyzing even a single ingredient in Danggui Yinzi to assess its quality. Therefore, it is even more impossible to quantitatively analyze multiple ingredients at once to evaluate the overall quality of Danggui Yinzi. Summary of the Invention

[0004] In view of the above problems, the present invention provides a method for determining the content of Dangguiyinzi.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] A method for determining the content of Dangguiyinzi, comprising the following steps:

[0007] Prepare a mixed reference solution by taking paeoniflorin, ammonium glycyrrhizate, liquiritin, hesperidin, 6-gingerol, cimicifugoside, 5-O-methylvisamidoside, ferulic acid, and 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside;

[0008] Prepare the test solution with Dangguiyinzi;

[0009] The mixed reference solution and the test solution were respectively subjected to HPLC detection, and the contents of paeoniflorin, glycyrrhizic acid, liquiritin, hesperidin, 6-gingerol, cimicifugoside, 5-O-methylvisamidoside, ferulic acid, and 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside in Dangguiyinzi were calculated using the external standard one-point method.

[0010] During the high performance liquid chromatography detection process, 0.08-0.12 wt% phosphoric acid aqueous solution was used as mobile phase A, acetonitrile was used as mobile phase B, and elution was performed in a gradient elution manner;

[0011] Among them, the elution procedure is:

[0012] The mobile phase is, the elution mode is gradient elution, and the elution program is:

[0013] 0-5 min: 95% mobile phase A, 5% mobile phase B;

[0014] 5-7 min: 95% → 88% mobile phase A, 5% → 12% mobile phase B;

[0015] 7-15 min: 88% mobile phase A, 12% mobile phase B;

[0016] 15-23 min: 88% → 85% mobile phase A, 12% → 15% mobile phase B;

[0017] 23-28 min: 85% → 83% mobile phase A, 15% → 17% mobile phase B;

[0018] 28-38 min: 83% → 80% mobile phase A, 17% → 20% mobile phase B;

[0019] 38-43 min: 80% mobile phase A, 20% mobile phase B;

[0020] 43-53 min: 80% → 70% mobile phase A, 20% → 30% mobile phase B;

[0021] 53-60 min: 70% → 60% mobile phase A, 30% → 40% mobile phase B;

[0022] 60-65 min: 60% mobile phase A, 40% mobile phase B;

[0023] 65-70 min: 60% → 40% mobile phase A, 40% → 60% mobile phase B;

[0024] 70-80 min: 40% → 0% mobile phase A, 60% → 100% mobile phase B.

[0025] Furthermore, during the high performance liquid chromatography detection process, the chromatographic column is an Agilent ZORBAX EclipsePlus C18 column.

[0026] Furthermore, during the high performance liquid chromatography detection process, the detection wavelengths are 230-235 nm, 248-252 nm, 273-278 nm, 280-285 nm, 296-300 nm and 320-325 nm;

[0027] Specifically, paeoniflorin is detected at a wavelength of 230-235 nm, glycyrrhizic acid is detected at a wavelength of 248-252 nm, liquiritin is detected at a wavelength of 273-278 nm, hesperidin and 6-gingerol are detected at a wavelength of 280-285 nm, cimicifugoside and 5-O-methylvisaminol are detected at a wavelength of 296-300 nm, and ferulic acid and 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside are detected at a wavelength of 320-325 nm;

[0028] Column temperature is 30-40°C;

[0029] The flow rate is 0.9~1.1mL / min.

[0030] Furthermore, in the process of preparing the test solution, the test solution is prepared by taking 0.8-1.2 mL of Dangguiyinzi extract and adding 10 mL of 65-75 vol% methanol aqueous solution, ultrasonically treating for more than 20 minutes, and filtering.

[0031] Furthermore, the content of paeoniflorin in the mixed reference solution is 12.52-237 μg / mL, the content of glycyrrhizic acid is 9.48-193 μg / mL, the content of liquiritin is 10.53-176 μg / mL, the content of hesperidin is 15.425-241 μg / mL, the content of 6-gingerol is 18.33-290 μg / mL, the content of cimicifugoside is 11.02-188 μg / mL, the content of 5-O-methylvisaminol glycoside is 11.42-205 μg / mL, the content of ferulic acid is 10.69-189 μg / mL, and the content of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside is 12.25-233 μg / mL.

[0032] A method for determining the content of Dangguiyinzi, comprising the following steps:

[0033] Prepare the test solution with Dangguiyinzi;

[0034] The test solution was subjected to high performance liquid chromatography, and the peak areas corresponding to the components were used to calculate the contents of paeoniflorin, glycyrrhizic acid, liquiritin, hesperidin, 6-gingerol, cimicifugoside, 5-O-methylvisamidoside, ferulic acid, and 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside in Danggui Yinzi by the standard curve method.

[0035] During the high performance liquid chromatography detection process, 0.08-0.12 wt% phosphoric acid aqueous solution was used as mobile phase A, acetonitrile was used as mobile phase B, and elution was performed in a gradient elution manner;

[0036] Among them, the elution procedure is:

[0037] The mobile phase is, the elution mode is gradient elution, and the elution program is:

[0038] 0-5 min: 95% mobile phase A, 5% mobile phase B;

[0039] 5-7 min: 95% → 88% mobile phase A, 5% → 12% mobile phase B;

[0040] 7-15 min: 88% mobile phase A, 12% mobile phase B;

[0041] 15-23 min: 88% → 85% mobile phase A, 12% → 15% mobile phase B;

[0042] 23-28 min: 85% → 83% mobile phase A, 15% → 17% mobile phase B;

[0043] 28-38 min: 83% → 80% mobile phase A, 17% → 20% mobile phase B;

[0044] 38-43 min: 80% mobile phase A, 20% mobile phase B;

[0045] 43-53 min: 80% → 70% mobile phase A, 20% → 30% mobile phase B;

[0046] 53-60 min: 70% → 60% mobile phase A, 30% → 40% mobile phase B;

[0047] 60-65 min: 60% mobile phase A, 40% mobile phase B;

[0048] 65-70 min: 60% → 40% mobile phase A, 40% → 60% mobile phase B;

[0049] 70-80 min: 40% → 0% mobile phase A, 60% → 100% mobile phase B.

[0050] Furthermore, in the standard curve method, the standard curves corresponding to each component are:

[0051] The standard curve corresponding to paeoniflorin is Y=11.454X-8.1114, R 2 =0.9998;

[0052] The standard curve corresponding to glycyrrhizic acid is Y=7.498X-20.537, R 2 =0.9994;

[0053] The standard curve corresponding to liquiritin is Y=15.685X-38.552, R 2 =0.999;

[0054] The standard curve corresponding to hesperidin is Y=16.271X-38.608, R 2 =0.9992;

[0055] The standard curve corresponding to 6-gingerol is Y=5.6889X-9.0822, R 2 =0.9994;

[0056] The standard curve corresponding to cimicifuga glycosides is Y=12.811X-31.983, R 2 =0.998;

[0057] The standard curve corresponding to 5-O-methylvisamidoside is Y=14.165X+24.178, R 2 =0.9993;

[0058] The standard curve corresponding to ferulic acid is Y=29.453X+81.971, R 2 =0.9983;

[0059] The standard curve corresponding to 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside is Y=23.549X-111.66, R 2 =0.9998.

[0060] Furthermore, in the standard curve method, the method for obtaining the standard curve corresponding to each component is as follows:

[0061] Paeoniflorin, ammonium glycyrrhizate, liquiritin, hesperidin, 6-gingerol, cimicifugoside, 5-O-methylvisamidoside, ferulic acid, and 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside were respectively taken to prepare mixed reference solutions of different concentrations;

[0062] In the mixed reference solution with different concentrations, the content of paeoniflorin was between 12.52 and 237 μg / mL, the content of glycyrrhizic acid was between 9.48 and 193 μg / mL, the content of liquiritin was between 10.53 and 176 μg / mL, the content of hesperidin was between 15.425 and 241 μg / mL, the content of 6-gingerol was between 18.33 and 290 μg / mL, the content of cimicifugoside was between 11.02 and 188 μg / mL, the content of 5-O-methylvisamidoside was between 11.42 and 205 μg / mL, the content of ferulic acid was between 10.69 and 189 μg / mL, and the content of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside was between 12.25 and 233 μg / mL.

[0063] Mixed reference solution of different concentrations was taken for high performance liquid chromatography detection, and the standard curve corresponding to each component was drawn according to the concentration of each component and the peak area of ​​the corresponding component at the corresponding concentration.

[0064] Furthermore, during the high performance liquid chromatography detection process, the chromatographic column was an Agilent ZORBAX EclipsePlus C18 column;

[0065] Detection wavelengths are 230-235nm, 248-252nm, 273-278nm, 280-285nm, 296-300nm and 320-325nm;

[0066] Specifically, paeoniflorin is detected at a wavelength of 230-235 nm, glycyrrhizic acid is detected at a wavelength of 248-252 nm, liquiritin is detected at a wavelength of 273-278 nm, hesperidin and 6-gingerol are detected at a wavelength of 280-285 nm, cimicifugoside and 5-O-methylvisaminol are detected at a wavelength of 296-300 nm, and ferulic acid and 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside are detected at a wavelength of 320-325 nm;

[0067] Column temperature is 30-40°C;

[0068] The flow rate is 0.9~1.1mL / min.

[0069] Furthermore, in the process of preparing the test solution, the test solution is prepared by taking 0.8-1.2 mL of Dangguiyinzi extract and adding 10 mL of 65-75 vol% methanol aqueous solution, ultrasonically treating for more than 20 minutes, and filtering.

[0070] The beneficial effects of the Dangguiyinzi content determination method of the present invention are:

[0071] The present invention quantitatively analyzes nine components in Danggui Yinzi at one time, which can effectively control the quality of Danggui Yinzi. In the later research process of the material basis of Danggui Yinzi, it provides a basis for the research of components and activities, so as to maximize its medicinal value and provide strong support for the secondary development of Danggui Yinzi preparations.

[0072] Since the quality of Danggui Yinzi is affected by the source of medicinal materials, preparation of medicinal pieces, compatibility ratio, preparation process, etc., and the ingredients in Danggui Yinzi are complex, quantifying the ingredients in Danggui Yinzi is conducive to ensuring the quality and efficacy of Danggui Yinzi. The Danggui Yinzi content determination method of the present invention can achieve the control of multiple index components (9 components) in Danggui Yinzi, effectively ensuring the quality and efficacy of Danggui Yinzi.

[0073] The method for determining the content of Danggui Yinzi of the present invention can simultaneously quantitatively analyze nine components in Danggui Yinzi by adjusting the extraction process and chromatographic conditions. The determination results are accurate, the operation is simple, rapid, stable and reliable, the precision is high, and it is easy to master. This effectively saves process steps and labor costs, and can more comprehensively control the quality of Danggui Yinzi, providing a reference for the development and quality standard formulation of Danggui Yinzi.

[0074] The present invention achieves the separation of a total of nine components, including paeoniflorin, glycyrrhizic acid, liquiritin, hesperidin, 6-gingerol, cimicifugoside, 5-O-methylvisamidoside, ferulic acid, and 2,3,5,4′-tetrahydroxystilbene-2-OBD-glucoside, by adjusting high-performance liquid chromatography conditions and screening suitable process parameters such as mobile phase and elution procedure. The components are effectively separated from other impurity peaks without mutual interference.

[0075] Different extraction processes have different extraction efficiencies for the active ingredients in traditional Chinese medicine. The present invention effectively improves the extraction efficiency of Danggui Yinzi by selecting a suitable extraction process, thereby accurately determining the contents of the nine ingredients in Danggui Yinzi.

[0076] The method for determining the content of Dangguiyinzi of the present invention has high repeatability, good precision and stability, excellent specificity and good linear relationship. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 is a standard curve diagram of paeoniflorin in Example 6 of the present invention;

[0078] Figure 2 is a standard curve diagram of glycyrrhizic acid in Example 6 of the present invention;

[0079] Figure 3 is a standard curve diagram of liquiritin in Example 6 of the present invention;

[0080] Figure 4 is a standard curve diagram of hesperidin in Example 6 of the present invention;

[0081] Figure 5 is a standard curve diagram of 6-gingerol in Example 6 of the present invention;

[0082] Figure 6 is a standard curve diagram of cimicifuga glycosides in Example 6 of the present invention;

[0083] Figure 7 This is a standard curve diagram of 5-O-methylvisamidoside in Example 6 of the present invention;

[0084] Figure 8 is a standard curve diagram of ferulic acid in Example 6 of the present invention;

[0085] Figure 9 This is a standard curve diagram of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside in Example 6 of the present invention;

[0086] Figure 10 1 is a graph showing the specificity of paeoniflorin in Example 11 of the present invention; wherein Figure a is a chromatogram comparison of the single decoction, yin decoction, and whole decoction of paeoniflorin in white peony root, wherein the blank represents the chromatogram of the blank solution, the control represents the chromatogram of the mixed reference solution, the single decoction represents the chromatogram of the single decoction of white peony root, the yin decoction represents the chromatogram of the yin decoction of white peony root, and the whole decoction represents the chromatogram of Danggui Yinzi; Figure b is a spectrum of paeoniflorin in the mixed reference substance, and Figure c represents the spectrum of paeoniflorin in the single decoction of white peony root;

[0087] Figure 11 11 is a graph showing the specificity of glycyrrhizic acid in Example 11 of the present invention; wherein Figure a is a chromatogram comparison of glycyrrhizic acid in single decoction, Yin decoction, and whole decoction of licorice, wherein the blank represents the chromatogram of the blank solution, the control represents the chromatogram of the mixed reference solution, the single decoction represents the chromatogram of single decoction of licorice, the Yin decoction represents the chromatogram of Yin decoction of licorice, and the whole decoction represents the chromatogram of Danggui Yinzi; Figure b is a spectrum of glycyrrhizic acid in the mixed reference, and Figure c represents the spectrum of glycyrrhizic acid in single decoction of licorice;

[0088] Figure 12 1 is a graph showing the specificity of liquiritin in Example 11 of the present invention; wherein Figure a is a chromatogram comparison of the single decoction, Yin decoction, and whole decoction of liquiritin in licorice, wherein the blank represents the chromatogram of the blank solution, the control represents the chromatogram of the mixed reference solution, the single decoction represents the chromatogram of the single decoction of licorice, the Yin decoction represents the chromatogram of the Yin decoction of licorice, and the whole decoction represents the chromatogram of Danggui Yinzi; Figure b is a spectrum of liquiritin in the mixed reference, and Figure c represents the spectrum of liquiritin in the single decoction of licorice;

[0089] Figure 131 is a graph showing the specificity of hesperidin in Example 11 of the present invention; wherein Figure a is a chromatogram comparison of the single decoction, the Yin decoction, and the whole decoction of hesperidin in Schizonepeta spicata. In the figure, the blank represents the chromatogram of the blank solution, the control represents the chromatogram of the mixed reference solution, the single decoction represents the chromatogram of the single decoction of Schizonepeta spicata, the Yin decoction represents the chromatogram of the Yin decoction of Schizonepeta spicata, and the whole decoction represents the chromatogram of Danggui Yinzi; Figure b is a spectrum of hesperidin in the mixed reference, and Figure c represents the spectrum of hesperidin in the single decoction of Schizonepeta spicata.

[0090] Figure 14 11 is a graph showing the specificity of 6-gingerol in Example 11 of the present invention; wherein FIG a is a chromatogram comparison of single decoction, yin decoction, and whole decoction of 6-gingerol in ginger, wherein the blank represents the chromatogram of the blank solution, the control represents the chromatogram of the mixed reference solution, the single decoction represents the chromatogram of single decoction of ginger, the yin decoction represents the chromatogram of yin decoction of ginger, and the whole decoction represents the chromatogram of Danggui Yinzi; FIG b is a spectrum of 6-gingerol in the mixed reference substance, and FIG c represents the spectrum of 6-gingerol in single decoction of ginger;

[0091] Figure 15 11 of the present invention; wherein Figure a is a chromatogram comparison of the single decoction, yin decoction, and whole decoction of cimicifuga and 5-O-methylvisaminol in Saposhnikovia divaricata. In the figure, the blank represents the chromatogram of the blank solution, the control represents the chromatogram of the mixed reference solution, the single decoction represents the chromatogram of the single decoction of Saposhnikovia divaricata, the yin decoction represents the chromatogram of the yin decoction of Saposhnikovia divaricata, and the whole decoction represents the chromatogram of Danggui Yinzi; Figure b is a spectrum of cimicifuga in the mixed reference, Figure c represents a spectrum of cimicifuga in the single decoction of Saposhnikovia divaricata; Figure d is a spectrum of 5-O-methylvisaminol in the mixed reference, and Figure e represents a spectrum of 5-O-methylvisaminol in the single decoction of Saposhnikovia divaricata;

[0092] Figure 16 11 is a graph showing the specificity of ferulic acid in Example 11 of the present invention; wherein Figure a is a chromatogram comparison of single decoction, Yin decoction, and whole decoction of ferulic acid in Chuanxiong and Angelica sinensis, wherein the blank represents the chromatogram of the blank solution, the control represents the chromatogram of the mixed reference solution, Chuanxiong single decoction represents the chromatogram of Chuanxiong single decoction, Angelica sinensis single decoction represents the chromatogram of Angelica sinensis single decoction, Angelica sinensis Chuanxiong combined decoction represents the chromatogram of Angelica sinensis Chuanxiong combined decoction, Chuanxiong Yin decoction represents the chromatogram of Chuanxiong Yin decoction, Angelica sinensis Yin decoction represents the chromatogram of Angelica sinensis Yin decoction, Angelica sinensis Chuanxiong Yin decoction represents the chromatogram of Angelica sinensis Chuanxiong Yin decoction, and the whole decoction represents the chromatogram of Angelica sinensis Yin decoction; Figure b is a spectrum of ferulic acid in the mixed reference, Figure c represents the spectrum of ferulic acid in Angelica sinensis single decoction; Figure d is a spectrum of ferulic acid in Chuanxiong single decoction, and Figure e represents the spectrum of ferulic acid in Angelica sinensis Chuanxiong combined decoction;

[0093] Figure 1711 of the present invention; wherein Figure a is a chromatogram comparison of the single decoction, yin decoction, and whole decoction of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside in Polygonum multiflorum, wherein the blank in the figure represents the chromatogram of the blank solution, the control represents the chromatogram of the mixed reference solution, the single decoction represents the chromatogram of the single decoction of Polygonum multiflorum, the yin decoction represents the chromatogram of the yin decoction of Polygonum multiflorum, and the whole decoction represents the chromatogram of Danggui Yinzi; Figure b is a spectrum of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside in the mixed reference substance, and Figure c represents the spectrum of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside in the single decoction of Polygonum multiflorum;

[0094] Figure 18 This is a sample recovery chromatogram of paeoniflorin at a wavelength of 232 nm in Example 11 of the present invention;

[0095] Figure 19 This is a sample recovery chromatogram of glycyrrhizic acid at a wavelength of 250 nm in Example 11 of the present invention;

[0096] Figure 20 This is a sample recovery chromatogram of liquiritin at a wavelength of 275 nm in Example 11 of the present invention;

[0097] Figure 21 This is a sample recovery chromatogram of hesperidin and 6-gingerol at a wavelength of 283 nm in Example 11 of the present invention;

[0098] Figure 22 This is a sample recovery chromatogram of cimicifugoside and 5-O-methylvisaminol glycoside at a wavelength of 298 nm in Example 11 of the present invention;

[0099] Figure 23 This is a sample recovery chromatogram of ferulic acid and 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside at a wavelength of 322 nm in Example 11 of the present invention;

[0100] Figure 24 These are chromatograms of Danggui Yinzi detected using different chromatographic columns in Example 12 of the present invention; Figure A is a chromatogram of Danggui Yinzi detected using a Waters Sunfire C18 column, Figure B is a chromatogram of Danggui Yinzi detected using an Agilent ZORBAX Eclipse Plus C18 column, and Figure C is a chromatogram of Danggui Yinzi detected using a Phenomenex Luna Su C18 column;

[0101] Figure 25These are chromatograms of Danggui Yinzi detected using different mobile phases in Example 12 of the present invention; wherein, Figure A is a chromatogram of Danggui Yinzi detected using a 0.1 wt % formic acid aqueous solution (A)-acetonitrile (B), Figure B is a chromatogram of Danggui Yinzi detected using a 0.1 wt % acetic acid aqueous solution (A)-acetonitrile (B), and Figure C is a chromatogram of Danggui Yinzi detected using a 0.1 wt % phosphoric acid aqueous solution (A)-acetonitrile (B);

[0102] Figure 26 This is a chromatogram of Danggui Yinzi detected at a wavelength of 210 nm using different elution procedures in Example 12 of the present invention; wherein, Figure A is a chromatogram of Danggui Yinzi detected using elution procedure one, and Figure B is a chromatogram of Danggui Yinzi detected using elution procedure two. DETAILED DESCRIPTION

[0103] The technical solutions in the embodiments of the present invention are described clearly and completely below. Many specific details are set forth in the following description to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0104] Example 1 A method for determining the content of Dangguiyinzi

[0105] In this embodiment, Dangguiyinzi extract is used as the sample to be tested, wherein the preparation method of Dangguiyinzi extract is as follows:

[0106] The prescription is: Angelica sinensis 5.84g, White Peony 5.84g, Chuanxiong 5.84g, Rehmannia glutinosa 5.84, Tribulus terrestris 5.84g, Saposhnikovia divaricata 5.84g, Schizonepeta tenuifolia 5.84g, Polygonum multiflorum 2.92g, Astragalus membranaceus 2.92g, Roasted Licorice 2.92g and Ginger 15g.

[0107] Crush / powder all the herbs into the coarsest powder (5mm).

[0108] Take 16.52g of the coarsest powder of medicinal materials (1.95g each of angelica, white peony root, Chuanxiong, raw rehmannia, white tribulus, siler, and schizonepeta spicate, 0.97g each of polygonum multiflorum, astragalus, and roasted liquorice), add 450mL of water and 5g of ginger, boil over high heat, and simmer until the volume is 80% (240mL). Filter out the residue while hot, and concentrate the resulting liquid under reduced pressure at 60°C to an extract with a density of 1.15-1.22, thereby obtaining the angelica drink extract.

[0109] In this example, Dangguiyinzi extract was used as the sample to be tested, and the contents of 9 components in Dangguiyinzi were determined. The specific method is as follows:

[0110] 1) Preparation of mixed reference solution and test solution

[0111] 11) Preparation of mixed reference solution

[0112] Take paeoniflorin, ammonium glycyrrhizate, liquiritin, hesperidin, 6-gingerol, cimicifugoside, 5-O-methylvisaminol glycoside, ferulic acid, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, add methanol to dissolve, filter, make per milliliter containing 20.03μg paeoniflorin, 15.17μg glycyrrhizic acid, 16.85μg liquiritin, 24.68μg hesperidin, 29.33μg 6-gingerol, 17.63μg cimicifugoside, 18.27μg 5-O-methylvisaminol glycoside, 17.10μg ferulic acid, 19.60μg 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside mixed reference solution, standby. It should be noted that the reference substance used in the present invention is ammonium glycyrrhizate, but the component measured is glycyrrhizic acid.

[0113] 12) Preparation of test solution

[0114] Accurately measure 1 mL of Dangguiyinzi extract (i.e., v = 1 mL) and place it in a 10 mL volumetric flask (i.e., V = 10 mL). Add 70 vol% methanol aqueous solution to dilute to the scale, shake well, ultrasonically treat for 20 min, filter, and take the filtrate to obtain the test solution.

[0115] 2) HPLC quantitative analysis

[0116] 21) Take the mixed reference solution and perform high performance liquid chromatography to obtain the high performance liquid chromatogram of the mixed reference solution, and obtain the corresponding peak area A in the high performance liquid chromatogram at the corresponding concentration of each component. 对照品 , that is, A 对照品-芍药苷 、A 对照品-甘草酸 、A 对照品-甘草苷 、A 对照品-橙皮苷 、A 对照品-6-姜辣素 、A 对照品-升麻素苷 、A 对照品-5-O-甲基维斯阿米醇苷 、A 对照品-阿魏酸 and A 对照品-2，3，5，4’-四羟基二苯乙烯-2-O-β-D-葡萄糖苷 .

[0117] Among them, the chromatographic conditions for detecting the mixed reference solution are:

[0118] Chromatographic column: C18 (Agilent ZORBAX Eclipse Plus C18, 4.6×250 mm Column, 5 μm, PN959990-902, SNUSUXA32938);

[0119] Column temperature: 35°C;

[0120] Flow rate: 1.0 mL / min;

[0121] Detection wavelengths: 232nm, 250nm, 275nm, 283nm, 298nm, 322nm;

[0122] In the case of a single injection, different detection wavelengths were used for detection, specifically: 232 nm for detection of paeoniflorin, 250 nm for detection of glycyrrhizic acid, 275 nm for detection of liquiritin, 283 nm for detection of hesperidin and 6-gingerol, 298 nm for detection of cimicifugoside and 5-O-methylvisaminol, and 322 nm for detection of ferulic acid and 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside.

[0123] Injection volume: 10 μL;

[0124] The mobile phase was 0.1 wt% phosphoric acid aqueous solution (A)-acetonitrile (B), the elution mode was gradient elution, and the elution program was:

[0125] 0-5 min: 95% mobile phase A, 5% mobile phase B;

[0126] 5-7 min: 95% → 88% mobile phase A, 5% → 12% mobile phase B;

[0127] 7-15 min: 88% mobile phase A, 12% mobile phase B;

[0128] 15-23 min: 88% → 85% mobile phase A, 12% → 15% mobile phase B;

[0129] 23-28 min: 85% → 83% mobile phase A, 15% → 17% mobile phase B;

[0130] 28-38 min: 83% → 80% mobile phase A, 17% → 20% mobile phase B;

[0131] 38-43 min: 80% mobile phase A, 20% mobile phase B;

[0132] 43-53 min: 80% → 70% mobile phase A, 20% → 30% mobile phase B;

[0133] 53-60 min: 70% → 60% mobile phase A, 30% → 40% mobile phase B;

[0134] 60-65 min: 60% mobile phase A, 40% mobile phase B;

[0135] 65-70 min: 60% → 40% mobile phase A, 40% → 60% mobile phase B;

[0136] 70-80 min: 40% → 0% mobile phase A, 60% → 100% mobile phase B.

[0137] 22) Take the test solution and perform high performance liquid chromatography under the above-mentioned chromatographic conditions to obtain a high performance liquid chromatogram of the test solution;

[0138] At the same time, the peak area A corresponding to each component in the test solution was obtained from the high performance liquid chromatogram of the test solution. 供试品 , that is, A 供试品-芍药苷 、A 供试品-甘草酸 、A 供试品-甘草苷 、A 供试品-橙皮苷 、A 供试品-6-姜辣素 、A 供试品-升麻素苷 、A 供试品-5-O-甲基维斯阿米醇苷 、A 供试品-阿魏酸 and A 供试品-2，3，5，4’-四羟基二苯乙烯-2-O-β-D-葡萄糖苷 .

[0139] 23) The content of each component in Dangguiyinzi extract was determined using the external standard method as follows:

[0140] According to the formula: the content of each component in Dangguiyinzi extract = A 供试品-相应成分 ×C 对照品-相应成分 ×V / (v×A 对照品-相应成分 ), calculate the content of each component in Dangguiyinzi extract;

[0141] Or according to C 供试品-相应成分 =(A 供试品-相应成分 ×C 对照品-相应成分 ) / A 对照品-相应成分 , calculate the concentration C of each component in the corresponding test solution 供试品-相应成分 ;

[0142] Then use the content of each component in Dangguiyinzi extract = (C 供试品-相应成分 × V) / v, and calculate the content of each component in Dangguiyinzi extract;

[0143] Among them, A 供试品-相应成分 ——Peak area of ​​the corresponding component in the test solution;

[0144] A 对照品-相应成分 ——Peak area of ​​the corresponding component in the mixed reference solution;

[0145] C 对照品-相应成分 ——Concentration of the corresponding component in the mixed reference solution, μg / mL;

[0146] C 供试品-相应成分 ——Concentration of the corresponding component in the test solution, μg / mL;

[0147] V——the volume of the prepared test solution, mL;

[0148] v——The volume of Dangguiyinzi extract used to prepare the test solution, mL.

[0149] Taking paeoniflorin as an example:

[0150] The content of paeoniflorin in Dangguiyinzi extract = = A 供试品-芍药苷 ×C 对照品-芍药苷 ×10mL / (1mL×A 对照品-芍药苷 ).

[0151] The calculation methods for other components are similar.

[0152] Example 2-5 Dangguiyinzi content determination method

[0153] Examples 2 to 5 are respectively a method for determining the content of Dangguiyinzi. The steps thereof are substantially the same as those of Example 1, and the only difference is that some parameters are different. See Table 1 for details:

[0154] Table 1 Summary of analysis parameters in Examples 2 to 5

[0155]

[0156] The rest of the contents of Examples 2 to 5 are the same as those of Example 1 and will not be repeated here.

[0157] Example 6: A method for determining the content of Dangguiyinzi

[0158] S1. Preparation of mixed reference solution and test solution of different concentrations

[0159] S11. Preparation of mixed reference solutions of different concentrations

[0160] Accurately measure appropriate amounts of paeoniflorin, ammonium glycyrrhizate, liquiritin, hesperidin, 6-gingerol, cimicifugoside, 5-O-methylvisamidoside, ferulic acid and 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, dissolve them in methanol to prepare mixed reference solutions of different concentrations, i.e., linear 1 solution to linear 5 solution; the concentrations of the components in linear 1 solution to linear 5 solution are shown in the following table:

[0161] Table 2 Specific concentrations of each component in linear 1 solution to linear 5 solution (μg / mL)

[0162]

[0163] S12. Preparation of test solution

[0164] Prepare the test solution according to the method in step 12) of Example 1.

[0165] S2. Quantitative analysis by high performance liquid chromatography

[0166] S21. Taking mixed reference solution of different concentrations (i.e., linear 1 solution to linear 5 solution) and performing high performance liquid chromatography under the chromatographic conditions of Example 1, respectively, to obtain high performance liquid chromatograms corresponding to the mixed reference solution of different concentrations, and obtain the corresponding peak area of ​​each component corresponding to the concentration measurement;

[0167] Based on the concentration of each component and the corresponding peak area of ​​the corresponding component, draw the standard curve corresponding to the different components, and use the concentration of each component in the mixed reference solution as the abscissa (i.e., X, in μg / mL) and the peak area (A) of the corresponding component as the ordinate (i.e., Y) to find its linear regression equation. See the table below for details:

[0168] Table 3 Linear relationships of different components

[0169]

[0170]

[0171] It can be seen that the standard curve corresponding to paeoniflorin is Y=11.454X-8.1114, R 2 =0.9998, see Figure 1 , which has a good linear relationship in the concentration range of 12.52 to 237 μg / mL;

[0172] The standard curve corresponding to glycyrrhizic acid is Y=7.498X-20.537, R 2 =0.9994, see Figure 2 , which has a good linear relationship in the concentration range of 9.48 to 193 μg / mL;

[0173] The standard curve corresponding to liquiritin is Y=15.685X-38.552, R 2 =0.999, see Figure 3 , which has a good linear relationship in the concentration range of 10.53 to 176 μg / mL;

[0174] The standard curve corresponding to hesperidin is Y=16.271X-38.608, R 2 =0.9992, see Figure 4 , which has a good linear relationship in the concentration range of 15.425-241 μg / mL;

[0175] The standard curve corresponding to 6-gingerol is Y=5.6889X-9.0822, R 2 =0.9994, see Figure 5 , which has a good linear relationship in the concentration range of 18.33 to 290 μg / mL;

[0176] The standard curve corresponding to cimicifuga glycosides is Y=12.811X-31.983, R 2 =0.998, see Figure 6 , which has a good linear relationship in the concentration range of 11.02 to 188 μg / mL;

[0177] The standard curve corresponding to 5-O-methylvisamidoside is Y=14.165X+24.178, R 2 =0.9993, see Figure 7 , which has a good linear relationship in the concentration range of 11.42 to 205 μg / mL;

[0178] The standard curve corresponding to ferulic acid is Y=29.453X+81.971, R 2 =0.9983, see Figure 8 , which has a good linear relationship in the concentration range of 10.69 to 189 μg / mL;

[0179] The standard curve corresponding to 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside is Y=23.549X-111.66, R 2 =0.9998, see Figure 9 , which has a good linear relationship in the concentration range of 12.25~233μg / mL.

[0180] S22, take the test solution and perform high performance liquid chromatography under the chromatographic conditions of Example 1 to obtain a high performance liquid chromatogram of the test solution, and obtain the peak area corresponding to each component, substitute the peak area of ​​the corresponding component into the corresponding standard curve, and calculate the concentration C of each component in the test solution. 供试品-相应成分 .

[0181] The content of the corresponding components in Dangguiyinzi extract = C 供试品-相应成分 × V / v, calculate the content of each component in Dangguiyinzi extract;

[0182] Among them, C 供试品-相应成分 ——Concentration of the corresponding component in the test solution, μg / mL;

[0183] V——the volume of the prepared test solution, mL;

[0184] v——The volume of Dangguiyinzi extract used to prepare the test solution, mL.

[0185] Taking paeoniflorin as an example:

[0186] The peak area (A) of paeoniflorin measured under the chromatographic conditions of Example 1 was 供试品-芍药苷) was substituted into the standard curve corresponding to paeoniflorin to calculate the concentration of paeoniflorin in the test solution (C 供试品-芍药苷 );

[0187] The content of paeoniflorin in Dangguiyinzi extract = C 供试品-芍药苷 ×10mL / 1mL.

[0188] The calculation methods for other components are similar.

[0189] In addition, in actual application, if the standard curve is known, the test solution can be directly prepared for HPLC detection, and the peak area of ​​each component corresponding to the concentration measurement can be directly substituted into the standard curve for calculation. There is no need to repeatedly prepare mixed reference solution of different concentrations for measurement and draw the standard curve.

[0190] Example 7-10 Dangguiyinzi Content Determination Method

[0191] Examples 7 to 10 are respectively a method for determining the content of Dangguiyinzi. The steps thereof are substantially the same as those of Example 6, the only difference being the different analysis parameters. Detailed details are shown in Table 4:

[0192] Table 4 Summary of analytical parameters in Examples 7 to 10

[0193]

[0194]

[0195] The process parameters and steps of other parts of Examples 7 to 10 are the same as those of Example 6 and will not be repeated here.

[0196] Example 11 Methodological Investigation

[0197] In this example, the content determination method of Dangguiyinzi was investigated, mainly focusing on the precision, stability, repeatability and specificity of the content determination method.

[0198] 1. Precision inspection

[0199] A mixed standard solution containing 221.84 μg of paeoniflorin, 130.32 μg of ammonium glycyrrhizate, 84.24 μg of liquiritin, 25.064 μg of hesperidin, 20.5296 μg of 6-gingerol, 39.672 μg of cimicifugoside, 45.68 μg of 5-O-methylvisaminol glycoside, 29.932 μg of ferulic acid, and 58.8 μg of 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside per milliliter was taken and subjected to high performance liquid chromatography under the chromatographic conditions in Example 1. The sample was injected six times continuously and the RSD was calculated. The specific results are shown in Table 5.

[0200] Table 5 Summary of precision inspection results

[0201]

[0202]

[0203] As can be seen from Table 5, the method of the present invention has good precision.

[0204] 2. Stability Investigation

[0205] The Dangguiyinzi extract was used to prepare a test solution according to the method in Example 1. The sample was injected and analyzed according to the chromatographic conditions in Example 1 at 0, 2, 4, 8, 12, and 24 hours, and the peak area RSD was calculated. The results are shown in the table below.

[0206] Table 6 Summary of stability investigation results (peak area)

[0207]

[0208] As can be seen from Table 6, the RSD values ​​of the peak areas of the various index components were all within 2.414%, indicating that the components in the test solution were stable within 24 h.

[0209] 3. Repeatability Investigation

[0210] Accurately weigh 1 mL of Dangguiyinzi extract into a 10 mL volumetric flask, add 70 vol% methanol aqueous solution, and ultrasonically extract for 20 min. After cooling, adjust the volume, filter, and filter through a membrane for later use. Prepare six replicates in parallel, and then inject and analyze according to the chromatographic conditions in Example 1. Calculate the content RSD. The results are shown in the table below.

[0211] Table 7 Summary of repeatability test results (content, mg / mL)

[0212]

[0213]

[0214] As can be seen from Table 7, the present invention has good repeatability.

[0215] 4. Specificity Inspection

[0216] (1) Comparison of chromatograms and spectra of single decoction, Yin decoction, and whole decoction of each medicinal ingredient

[0217] Take a mixed reference solution containing 257 μg paeoniflorin, 188 μg glycyrrhizic acid, 100 μg liquiritin, 20 μg hesperidin, 37 μg 6-gingerol, 33 μg cimicifugo glycoside, 65 μg 5-O-methylvisamidoside, 24 μg ferulic acid, and 75 μg 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside per ml, and pass it through a membrane for later use.

[0218] The dosage for each herb is calculated as follows: Angelica sinensis 1.95g, White Peony Root 1.95g, Chuanxiong Rhizome 1.95g, Rehmannia Glutinosa 1.95g, Tribulus Terrestris 1.95g, Saposhnikovia Divaricata 1.95g, Schizonepeta Tenuifolia 1.95g, Polygonum Multiflorum 0.97g, Astragalus 0.97g, and Licorice 0.97g. Add 5g of ginger.

[0219] Weigh white peony root, licorice root, schizonepeta spicate, ginger, siler, angelica root, ligusticum chuanxiong, and polygonum multiflorum according to the converted amount for one dose, place them separately in a casserole, add 450mL of water and boil until the volume reaches 240mL, and concentrate under reduced pressure to the corresponding volume of angelica extract, thus obtaining single decoctions of white peony root, licorice root, schizonepeta spicate, ginger, siler, angelica root, ligusticum chuanxiong, and polygonum multiflorum.

[0220] Weigh 1.95 g each of coarse granules of Angelica sinensis slices and coarse granules of Chuanxiong slices, place them in a casserole, add 450 mL of water and boil until the volume reaches 240 mL, and concentrate under reduced pressure to the corresponding volume of Angelica sinensis extract to obtain the Angelica sinensis and Chuanxiong decoction.

[0221] Take 1.95g each of coarse-grained Angelica sinensis, Chuanxiong, Rehmannia root, Tribulus terrestris, Saposhnikovia divaricata, and Schizonepeta tenuifolia, 0.97g each of Polygonum multiflorum, Astragalus membranaceus, and Licorice, and 5g of ginger, except for white peony root. Put them in a casserole, add 450mL of water and boil until the volume reaches 240mL. Concentrate under reduced pressure to the corresponding volume of Angelica sinensis extract to obtain White Peony Yin Decoction (i.e., the missing medicinal material is white peony root).

[0222] The preparation methods of the other Yin decoctions (i.e., Licorice Yin Decoction, Schizonepeta Spica Yin Decoction, Ginger Yin Decoction, Saposhnikovia Yin Decoction, Angelica Yin Decoction, Chuanxiong Yin Decoction, Angelica and Chuanxiong Yin Decoction, and Polygonum multiflorum Yin Decoction) are the same as those of White Peony Yin Decoction, the only difference being the different medicinal materials used.

[0223] Accurately weigh 1 mL each of white peony root single decoction, licorice root single decoction, schizonepeta spicate single decoction, ginger single decoction, saposhnikovia root single decoction, angelica root single decoction, Chuanxiong single decoction, angelica and Chuanxiong combined decoction, Polygonum multiflorum single decoction, white peony root decoction in a yin-yang style, licorice root decoction in a yin-yang style, schizonepeta spicate decoction in a yin-yang style, ginger decoction in a yin-yang style, saposhnikovia root decoction in a yin-yang style, angelica root decoction in a yin-yang style, Chuanxiong decoction in a yin-yang style, and Polygonum multiflorum decoction in a yin-yang style. Add 70 vol% methanol aqueous solution, perform ultrasonic extraction for 15 min, cool, dilute to volume, filter, and pass through a membrane to obtain the corresponding single decoction test solution and each decoction test solution for later use.

[0224] The blank solution (70 vol% methanol aqueous solution), the mixed reference solution, each single decoction test solution, the angelica and Chuanxiong decoction, each Yin decoction test solution and the test solution prepared by using Angelica Decoction in Example 1 were respectively taken and subjected to HPLC detection according to the chromatographic conditions in Example 1. The results are as follows Figures 10 to 17 , it can be seen that the specificity of each component is good.

[0225] (2) Sample recovery rate

[0226] The recovery rate of the sample was calculated based on the average content of the repeatability test. 0.5 mL of the whole decoction was accurately weighed into a 10 mL volumetric flask, and a 70 vol% methanol aqueous solution and a reference substance of corresponding concentration were added. Ultrasonic extraction was performed for 20 min. After cooling, the volume was fixed, filtered, and passed through a membrane for standby use to prepare a 100% concentration sample. The sample was then injected and analyzed according to the chromatographic conditions in Example 1, and the RSD of the recovery rate of the sample was calculated. The specific results are shown in Table 8 and Figures 18 to 23 .

[0227] Table 8 Summary of sample recovery results

[0228]

[0229]

[0230] As can be seen from Table 8, the method of the present invention has a good recovery rate.

[0231] Example 12 Investigation of Chromatographic Conditions

[0232] (1) Chromatographic column inspection

[0233] Due to the influence of chromatographic column packing, particle size, pore size, density, and applicable range, the separation ability of chromatographic columns produced by different manufacturers for compounds of different properties in the sample varies to a certain extent. The present invention selected three different brands of chromatographic columns for investigation, namely Waters Sunfire C18 column, Agilent ZORBAX Eclipes Plus C18 column, and Phenomenex Luna Su C18 column, and investigated the same batch of test solution according to the chromatographic conditions in Example 1 (that is, only the chromatographic column in Example 1 was changed, and other process parameters were not changed). The results are shown as follows: Figure 24 shown.

[0234] from Figure 24 It can be seen that the response value and separation degree of the chromatographic peaks are better when using the Agilent ZORBAX Eclipse Plus C18 column, so this chromatographic column is selected in the present invention.

[0235] (2) Mobile phase investigation

[0236] The effects of three mobile phases, 0.1 wt% formic acid aqueous solution (A)-acetonitrile (B), 0.1 wt% acetic acid aqueous solution (A)-acetonitrile (B), and 0.1 wt% phosphoric acid aqueous solution (A)-acetonitrile (B), on the fingerprint separation were investigated. The same batch of test solution was investigated using the three mobile phases according to the chromatographic conditions in Example 1 (i.e., only the mobile phase in Example 1 was changed, and other process parameters were not changed). The results are shown in Figure 2. Figure 25 shown.

[0237] from Figure 25 It can be seen that when 0.1 wt % phosphoric acid aqueous solution (A)-acetonitrile (B) is used as the mobile phase, the baseline is more stable, the peak shape and separation effect are better, so 0.1 wt % phosphoric acid aqueous solution (A)-acetonitrile (B) is finally selected as the mobile phase.

[0238] (3) Elution procedure investigation

[0239] The chromatographic conditions in Example 1 were used to examine the separation effects of different elution programs under the condition of a detection wavelength of 210 nm (i.e., only the elution program and detection wavelength in Example 1 were changed, and other process parameters were not changed). The different elution programs are as follows:

[0240] Elution procedure 1:

[0241] 0-10 min: 95% → 85% mobile phase A, 5% → 15% mobile phase B;

[0242] 10-20 min: 85% mobile phase A, 15% mobile phase B;

[0243] 20-30 min: 85% → 80% mobile phase A, 15% → 20% mobile phase B;

[0244] 30-35 min: 80% mobile phase A, 80% mobile phase B;

[0245] 35-45 min: 80% → 70% mobile phase A, 20% → 30% mobile phase B;

[0246] 45-55 min: 70% mobile phase A, 30% mobile phase B;

[0247] 55-60 min: 70% → 60% mobile phase A, 30% → 40% mobile phase B;

[0248] 60-70 min: 60% mobile phase A, 40% mobile phase B;

[0249] 70-72 min: 60% → 40% mobile phase A, 40% → 60% mobile phase B;

[0250] 72-80 min: 40% mobile phase A, 60% mobile phase B;

[0251] 80-82 min: 40% → 20% mobile phase A, 60% → 80% mobile phase B;

[0252] 82-100 min: 20% → 0% mobile phase A, 80% → 100% mobile phase B.

[0253] Elution procedure 2 is the elution procedure in Example 1 of the present invention.

[0254] The chromatograms obtained by using two different elution procedures are shown in Figure 26 It can be seen that the chromatographic peaks are densely distributed under elution procedure one (i.e., the proportion of high-polarity mobile phase). Based on the elution procedure one, optimization was carried out to obtain the chromatogram of elution procedure two, in which the chromatographic peaks are evenly distributed and the separation is good. Elution procedure two was determined as the final elution procedure.

[0255] Example 13 Determination of the content of fifteen batches of Dangguiyinzi

[0256] Fifteen batches of Dangguiyinzi extract were used to prepare test solutions according to the method in Example 6, and the content of each component was determined according to the chromatographic conditions and calculation method in Example 6. The results are shown in the table below.

[0257] Table 9 Summary of the results of determination of the content of each component in different batches of Danggui Yinzi (content, mg / mL)

[0258]

[0259] As can be seen from Table 9, the contents of active ingredients in different batches of Dangguiyinzi extract are different. It can be seen that the current preparation method of Dangguiyinzi extract needs to be improved. The present invention provides a good technical means for the quality control of the subsequent production of Dangguiyinzi extract.

[0260] Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

Claims

1. A method for determining the content of Dangguiyinzi, characterized in that: The method for determining the content of Dangguiyinzi comprises the following steps: Prepare a mixed reference solution by taking paeoniflorin, ammonium glycyrrhizate, liquiritin, hesperidin, 6-gingerol, cimicifugoside, 5-O-methylvisamidoside, ferulic acid, and 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside; Take Danggui Yinzi and add methanol water solution, treat it by ultrasound, and filter it to prepare the test solution; The mixed reference solution and the test solution were respectively subjected to high performance liquid chromatography detection, and the contents of paeoniflorin, glycyrrhizic acid, liquiritin, hesperidin, 6-gingerol, cimicifugoside, 5-O-methylvisamidoside, ferulic acid, and 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside in Dangguiyinzi were calculated using the external standard one-point method. During the high performance liquid chromatography detection process, 0.08-0.12 wt% phosphoric acid aqueous solution was used as mobile phase A, acetonitrile was used as mobile phase B, and elution was performed in a gradient elution manner; Among them, the elution procedure is: The mobile phase is, the elution mode is gradient elution, and the elution program is: 0-5 min: 95% mobile phase A, 5% mobile phase B; 5-7 min: 95% → 88% mobile phase A, 5% → 12% mobile phase B; 7-15 min: 88% mobile phase A, 12% mobile phase B; 15-23 min: 88% → 85% mobile phase A, 12% → 15% mobile phase B; 23-28 min: 85% → 83% mobile phase A, 15% → 17% mobile phase B; 28-38 min: 83% → 80% mobile phase A, 17% → 20% mobile phase B; 38-43 min: 80% mobile phase A, 20% mobile phase B; 43-53 min: 80% → 70% mobile phase A, 20% → 30% mobile phase B; 53-60 min: 70% → 60% mobile phase A, 30% → 40% mobile phase B; 60-65 min: 60% mobile phase A, 40% mobile phase B; 65-70 min: 60% → 40% mobile phase A, 40% → 60% mobile phase B; 70-80 min: 40% → 0% mobile phase A, 60% → 100% mobile phase B; During the HPLC detection process, the chromatographic column was Agilent ZORBAX Eclipse Plus C18, 4.6×250 mm Column, 5 μm; Detection wavelength: 230~235nm for detection of paeoniflorin, 248~252nm for detection of glycyrrhizic acid, 273~278nm for detection of liquiritin, 280~285nm for detection of hesperidin and 6-gingerol, 296~300nm for detection of cimicifugoside and 5-O-methylvisamidoside, 320~325nm for detection of ferulic acid and 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside.

2. The method for determining the content of Dangguiyinzi according to claim 1, wherein: The content of paeoniflorin in the mixed reference solution is 12.52-237 μg / mL, the content of glycyrrhizic acid is 9.48-193 μg / mL, the content of liquiritin is 10.53-176 μg / mL, the content of hesperidin is 15.425-241 μg / mL, the content of 6-gingerol is 18.33-290 μg / mL, the content of cimicifugoside is 11.02-188 μg / mL, the content of 5-O-methylvisaminol glycoside is 11.42-205 μg / mL, the content of ferulic acid is 10.69-189 μg / mL, and the content of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside is 12.25-233 μg / mL.

3. A method for determining the content of Dangguiyinzi, characterized in that: The method for determining the content of Dangguiyinzi comprises the following steps: Take Danggui Yinzi and add methanol water solution, treat it by ultrasound, and filter it to prepare the test solution; The test solution was subjected to high performance liquid chromatography detection, and the peak areas corresponding to the components were calculated by the standard curve method to obtain the contents of paeoniflorin, glycyrrhizic acid, liquiritin, hesperidin, 6-gingerol, cimicifugoside, 5-O-methylvisamidoside, ferulic acid, and 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside in Danggui Yinzi. During the high performance liquid chromatography detection process, 0.08-0.12 wt% phosphoric acid aqueous solution was used as mobile phase A, acetonitrile was used as mobile phase B, and elution was performed in a gradient elution manner; Among them, the elution procedure is: The mobile phase is, the elution mode is gradient elution, and the elution program is: 0-5 min: 95% mobile phase A, 5% mobile phase B; 5-7 min: 95% → 88% mobile phase A, 5% → 12% mobile phase B; 7-15 min: 88% mobile phase A, 12% mobile phase B; 15-23 min: 88% → 85% mobile phase A, 12% → 15% mobile phase B; 23-28 min: 85% → 83% mobile phase A, 15% → 17% mobile phase B; 28-38 min: 83% → 80% mobile phase A, 17% → 20% mobile phase B; 38-43 min: 80% mobile phase A, 20% mobile phase B; 43-53 min: 80% → 70% mobile phase A, 20% → 30% mobile phase B; 53-60 min: 70% → 60% mobile phase A, 30% → 40% mobile phase B; 60-65 min: 60% mobile phase A, 40% mobile phase B; 65-70 min: 60% → 40% mobile phase A, 40% → 60% mobile phase B; 70-80 min: 40% → 0% mobile phase A, 60% → 100% mobile phase B; During the HPLC detection process, the chromatographic column was Agilent ZORBAX Eclipse Plus C18, 4.6×250 mm Column, 5 μm; Detection wavelength: 230~235nm for detection of paeoniflorin, 248~252nm for detection of glycyrrhizic acid, 273~278nm for detection of liquiritin, 280~285nm for detection of hesperidin and 6-gingerol, 296~300nm for detection of cimicifugoside and 5-O-methylvisamidoside, 320~325nm for detection of ferulic acid and 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside.

4. The method for determining the content of Dangguiyinzi according to claim 3, wherein: In the standard curve method, the standard curves corresponding to each component are: The standard curve corresponding to paeoniflorin is Y=11.454X-8.1114, R 2 =0.9998; The standard curve corresponding to glycyrrhizic acid is Y=7.498X-20.537, R 2 =0.9994; The standard curve corresponding to liquiritin is Y=15.685X-38.552, R 2 =0.999; The standard curve corresponding to hesperidin is Y=16.271X-38.608, R 2 =0.9992; The standard curve corresponding to 6-gingerol is Y=5.6889X-9.0822, R 2 =0.9994; The standard curve corresponding to cimicifuga glycosides is Y=12.811X-31.983, R 2 =0.998; The standard curve corresponding to 5-O-methylvisamidoside is Y=14.165X+24.178, R 2 =0.9993; The standard curve corresponding to ferulic acid is Y=29.453X+81.971, R 2 =0.9983; The standard curve corresponding to 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside is Y=23.549X-111.66, R 2 =0.9998.

5. The method for determining the content of Dangguiyinzi according to claim 3 or 4, characterized in that: In the standard curve method, the standard curve corresponding to each component is obtained as follows: Paeoniflorin, ammonium glycyrrhizate, liquiritin, hesperidin, 6-gingerol, cimicifugoside, 5-O-methylvisamidoside, ferulic acid, and 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside were respectively taken to prepare mixed reference solutions of different concentrations; The contents of paeoniflorin, glycyrrhizic acid, liquiritin, hesperidin, 6-gingerol, cimicifugoside, 5-O-methylvisamidoside, ferulic acid, and 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside in the mixed reference solution at different concentrations were between 12.52 and 237 μg / mL, 9.48 and 193 μg / mL, 10.53 and 176 μg / mL, 15.425 and 241 μg / mL, 18.33 and 290 μg / mL, 11.02 and 188 μg / mL, 11.42 and 205 μg / mL, 10.69 and 189 μg / mL, and 12.25 and 233 μg / mL, respectively. Mixed reference solution of different concentrations was taken for high performance liquid chromatography detection, and the standard curve corresponding to each component was drawn according to the concentration of each component and the peak area of ​​the corresponding component at the corresponding concentration.

Images