Method for simultaneously determining the contents of ten components in Wenjing Decoction composition

Abstract

The present invention provides a method for simultaneously determining the contents of ten components in Wenjing Decoction composition, belonging to the technical field of drug detection. The method for determining the contents is as follows: prepare a reference substance standard solution by taking ginsenoside Rg1, ginsenoside Rb1, ginsenoside Re, ferulic acid, cinnamaldehyde, paeoniflorin, paeonol, liquiritin, β-ecdysterone and curcumol; prepare a test solution by taking Wenjing Decoction composition; then perform high performance liquid chromatography detection on the reference substance standard solution and the test solution respectively, and then use the external standard method to calculate the contents of the ten components in Wenjing Decoction composition. Through high performance liquid chromatography detection, the present invention can simultaneously determine the contents of the main ten components in 9 kinds of medicinal materials involved in Wenjing Decoction composition, especially can perform quantitative analysis on curcumol in Rhizoma Curcumae, and realize the comprehensive control of multiple index components in the classical famous prescription Wenjing Decoction composition.

Description

Technical Field

[0001] The invention relates to drug detection technology, in particular to a method for simultaneously determining the contents of ten components in a Wenjing Decoction composition. Background Art

[0002] Wenjing Decoction is a classic Chinese medicine prescription for "regulating menstruation and relieving pain". It comes from the "Furen Daquan Liangfang" by Chen Ziming, a famous doctor in the Southern Song Dynasty. It is now included in the "Ancient Classic Prescription Catalog (First Batch)". This prescription is composed of 9 Chinese herbs, including angelica, Chuanxiong, peony root, cinnamon heart, peony bark, curcuma, ginseng, liquorice, and Achyranthes bidentata. It has the effects of warming the meridians and dispelling cold, promoting blood circulation and regulating menstruation. It is mainly used for amenorrhea, dysmenorrhea, endometriosis and other symptoms. In the formula, curcumol in Curcuma has anti-cancer, antiviral, antibacterial, blood circulation, blood stasis and analgesic effects; ferulic acid in Angelica sinensis and Chuanxiong has obvious antioxidant, anti-inflammatory and antiplatelet aggregation effects, and is effective in the treatment of cardiovascular diseases; paeoniflorin in Paeonia lactiflora has obvious immunomodulatory effects on the human body; cinnamaldehyde in cinnamon has multiple pharmacological effects such as antipyretic, analgesic, antibacterial and anti-tumor; paeonol, the active ingredient in Paeonia suffruticosa moutan, has spasmolytic, anti-allergic, blood circulation and blood stasis effects; liquiritin in licorice exhibits estrogen-like effects when the body lacks estrogen, and is the main active ingredient of licorice in the treatment of dysmenorrhea and reproductive organ tumors; β-ecdysterone in Achyranthes bidentata belongs to insect growth regulating hormone, which has significant immunomodulatory, uterine excitation, anti-inflammatory and analgesic effects; ginsenoside Rg1, ginsenoside Rb1 and ginsenoside Re in ginseng have the effects of inducing cancer cell apoptosis, enhancing the body's immunity, improving immunity, relieving toxins, etc. However, the current research on Wenjing Decoction mainly focuses on the quantitative analysis of the active ingredients of some Chinese medicinal materials, and there is no comprehensive quantitative analysis of the different active ingredients in the nine Chinese medicinal materials, which makes it difficult to comprehensively evaluate the quality of the Wenjing Decoction composition, and is not conducive to the quality control of Wenjing Decoction and its subsequent development. In particular, there is no research and analysis on the curcumin in Wenjing Decoction, let alone a quantitative analysis of it. Summary of the invention

[0003] In view of the above problems, the present invention provides a method for simultaneously determining the contents of ten components in a Wenjing Decoction composition.

[0004] To achieve the above purpose, the technical solution adopted by the present invention is:

[0005] A method for simultaneously determining the contents of ten components in a Wenjing Decoction composition, the method comprising the following steps:

[0006] A total of 10 ingredients, including ginsenoside Rg1, ginsenoside Rb1, ginsenoside Re, ferulic acid, cinnamaldehyde, paeoniflorin, paeonol, licorice glycoside, β-ecdysterone and curcumol, were used to prepare a reference standard solution;

[0007] Prepare a test solution by taking the Wenjing Decoction composition;

[0008] The reference standard solution and the test solution were respectively subjected to high performance liquid chromatography detection, and then the external standard method was used to calculate the contents of ginsenoside Rg1, ginsenoside Rb1, ginsenoside Re, ferulic acid, cinnamaldehyde, paeoniflorin, paeonol, licorice glycoside, β-ecdysterone and curcumol in the Wenjing Decoction composition;

[0009] In the high performance liquid chromatography detection process, acetonitrile is used as mobile phase A, and 0.1 wt % acetic acid-ammonium acetate buffer is used as mobile phase B.

[0010] Furthermore, the elution mode of the high performance liquid chromatography detection is gradient elution, and the elution conditions are:

[0011] 0-5 min, 0% mobile phase A, 100% mobile phase B;

[0012] 5-28 min, 0%→51.5% mobile phase A, 100%→48.5% mobile phase B.

[0013] Furthermore, the content of the Wenjing Decoction composition in the test solution is 8-12 mg / mL; and the solvent for preparing the test solution is 70 wt % methanol aqueous solution.

[0014] Furthermore, in the standard solution of the reference substance, the concentration of ginsenoside Rg1 is between 7.084 and 226.7 μg / mL, the concentration of ginsenoside Rb1 is between 6.528 and 208.9 μg / mL, the concentration of ginsenoside Re is between 5.819 and 186.2 μg / mL, the concentration of ferulic acid is between 1.024 and 32.76 μg / mL, and the concentration of cinnamaldehyde is between 0.8938 and 2 The concentration of paeoniflorin was between 6.356 and 203.4 μg / mL, the concentration of paeonol was between 1.150 and 36.80 μg / mL, the concentration of liquiritin was between 9.586 and 306.8 μg / mL, the concentration of β-ecdysterone was between 1.143 and 36.58 μg / mL, and the concentration of curcumol was between 0.3975 and 12.72 μg / mL.

[0015] Furthermore, the chromatographic conditions of the HPLC detection are: the chromatographic column is HSS T3, the detection wavelength is 254 nm, the flow rate is 0.2 mL / min, and the column temperature is 35°C.

[0016] A method for simultaneously determining the contents of ten components in a Wenjing Decoction composition, the method comprising the following steps:

[0017] A total of 10 ingredients, including ginsenoside Rg1, ginsenoside Rb1, ginsenoside Re, ferulic acid, cinnamaldehyde, paeoniflorin, paeonol, licorice glycoside, β-ecdysterone and curcumol, were used to prepare a series of reference standard solutions of different concentrations;

[0018] Take standard series solutions of reference substances of different concentrations for high performance liquid chromatography detection, and draw standard curves corresponding to different components according to the concentrations of different components and the peak areas of the corresponding components at the corresponding concentrations;

[0019] Prepare a test solution by taking the Wenjing Decoction composition;

[0020] The test solution is subjected to high performance liquid chromatography detection, and the peak area of ​​the corresponding component obtained is substituted into the standard curve corresponding to the corresponding component, and the content of the corresponding component in the Wenjing Decoction composition is obtained by calculation;

[0021] In the high performance liquid chromatography detection process, acetonitrile is used as mobile phase A, and 0.1 wt % acetic acid-ammonium acetate buffer is used as mobile phase B.

[0022] Furthermore, the elution mode of the high performance liquid chromatography detection is gradient elution, and the elution conditions are:

[0023] 0-5 min, 0% mobile phase A, 100% mobile phase B;

[0024] 5-28 min, 0%→51.5% mobile phase A, 100%→48.5% mobile phase B.

[0025] Further, the content of the Wenjing Decoction composition in the test solution is 8-12 mg / mL; the solvent for preparing the test solution is 70 wt % methanol aqueous solution;

[0026] The chromatographic conditions of the HPLC detection are: the chromatographic column is HSS T3, the detection wavelength is 254 nm, the flow rate is 0.2 mL / min, and the column temperature is 35°C.

[0027] Furthermore, in the standard series of reference substance solutions of different concentrations, the concentration of ginsenoside Rg1 in the reference substance standard solution is between 7.084 and 226.7 μg / mL, the concentration of ginsenoside Rb1 is between 6.528 and 208.9 μg / mL, the concentration of ginsenoside Re is between 5.819 and 186.2 μg / mL, the concentration of ferulic acid is between 1.024 and 32.76 μg / mL, and the concentration of cinnamaldehyde is between 1.024 and 32.76 μg / mL. The concentration of glyceryl-4-nitropropene was between 0.8938 and 28.60 μg / mL, the concentration of paeoniflorin was between 6.356 and 203.4 μg / mL, the concentration of paeonol was between 1.150 and 36.80 μg / mL, the concentration of liquiritin was between 9.586 and 306.8 μg / mL, the concentration of β-ecdysterone was between 1.143 and 36.58 μg / mL, and the concentration of curcumol was between 0.3975 and 12.72 μg / mL.

[0028] Furthermore, the standard curves corresponding to the different components are as follows:

[0029] The standard curve corresponding to ginsenoside Rg1 is y=62.821x-309.62, R 2 =0.9996;

[0030] The standard curve corresponding to ginsenoside Rb1 is y=19.915x+61.716, R 2 =0.9996;

[0031] The standard curve corresponding to ginsenoside Re is y=48.288x+3.5014, R 2 =0.9998;

[0032] The standard curve corresponding to ferulic acid is y=388.97x-315.75, R 2 =0.9994;

[0033] The standard curve corresponding to cinnamaldehyde is y=140.68x+247.37, R 2 =0.9998;

[0034] The standard curve corresponding to paeoniflorin is y=50.701x-165.0378, R 2 =0.9998;

[0035] The standard curve corresponding to paeonol is y=290.35x-96.299, R 2 =0.9995;

[0036] The standard curve corresponding to liquiritin is y=30.713+25.592, R 2 =0.9998;

[0037] The standard curve corresponding to β-ecdysterone is y=106.13x-12.566, R 2 =0.9997;

[0038] The standard curve corresponding to curcumol is y=728.23x-88.254, R 2 =0.9996.

[0039] The beneficial effects of the method for simultaneously determining the contents of ten components in the Wenjing Decoction composition of the present invention are:

[0040] Since the quality of the Wenjing Decoction composition is affected by the sources of various medicinal materials, the preparation of the decoction pieces, the compatibility ratio, the preparation process, etc., and the components of the prepared Wenjing Decoction composition are complex, quantifying the components in the Wenjing Decoction composition is conducive to ensuring the quality and efficacy of the Wenjing Decoction composition; the present invention can simultaneously determine the contents of the ten main components in the nine medicinal materials involved in the Wenjing Decoction composition through high performance liquid chromatography detection, especially can quantitatively analyze the curcumol in Curcuma zedoaria, and realize comprehensive control of multiple index components in the classic Wenjing Decoction composition;

[0041] The content determination method of the present invention can achieve one-time determination of 10 components including curcumin by adjusting process parameters and pretreatment conditions, and can quickly and easily know the quality of the Wenjing Decoction composition, thereby solving the problem that the detection method in the prior art cannot determine the content of curcumin in Wenjing Decoction, thereby achieving the purpose of controlling the quality of the Wenjing Decoction composition by determining the contents of 10 components;

[0042] The content determination method of the present invention has the advantages of being simple, rapid, stable, reliable, highly precise, and easy to master, and can examine the quality of all medicinal materials used in the Wenjing Decoction composition, providing a reference for the development of the Wenjing Decoction composition and the formulation of quality standards for the ancient classic prescription;

[0043] The present invention achieves complete separation of 10 components, including ginsenoside Rg1, ginsenoside Rb1, ginsenoside Re, ferulic acid, cinnamaldehyde, paeoniflorin, paeonol, licorice glycoside, β-ecdysterone and curcumol, by controlling high performance liquid chromatography conditions and screening suitable process parameters. The components are effectively separated from other impurity peaks without mutual interference, thereby simplifying the pretreatment steps of the Wenjing Decoction composition.

[0044] Different solvents or different concentrations of the same solvent have different extraction efficiencies for the active ingredients in traditional Chinese medicine substances. The present invention improves the extraction efficiency of the Wenjing Decoction composition by selecting a suitable extraction solvent, and then accurately determines the contents of the ten ingredients in the Wenjing Decoction composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1is a high performance liquid chromatogram of the Wenjing Decoction composition in Example 1 of the present invention;

[0046] Figure 2 is a standard curve of ginsenoside Rg1 at a wavelength of 254 nm in Example 6 of the present invention;

[0047] Figure 3 is a standard curve of ginsenoside Rb1 at a wavelength of 254 nm in Example 6 of the present invention;

[0048] Figure 4 is a standard curve of ginsenoside Re at a wavelength of 254 nm in Example 6 of the present invention;

[0049] Figure 5 is a standard curve of ferulic acid at a wavelength of 254 nm in Example 6 of the present invention;

[0050] Figure 6 is a standard curve of cinnamaldehyde at a wavelength of 254 nm in Example 6 of the present invention;

[0051] Figure 7 is a standard curve of paeoniflorin at a wavelength of 254 nm in Example 6 of the present invention;

[0052] Figure 8 is a standard curve of paeonol at a wavelength of 254 nm in Example 6 of the present invention;

[0053] Fig. 9 is a standard curve of liquiritin at a wavelength of 254 nm in Example 6 of the present invention;

[0054] Fig.10 is a standard curve of β-ecdysterone at a wavelength of 254 nm in Example 6 of the present invention;

[0055] Fig.11 is a standard curve diagram of curcumol at a wavelength of 254 nm in Example 6 of the present invention;

[0056] Fig.12 It is a high performance liquid chromatogram of the Wenjing Decoction composition in Comparative Example 1 of the present invention. DETAILED DESCRIPTION

[0057] The technical solutions in the embodiments of the present invention are described clearly and completely below. In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below.

[0058] Example 1 A method for simultaneously determining the contents of ten components in a Wenjing Decoction composition

[0059] This example uses the Wenjing Decoction composition material standard to determine the contents of ten components in the Wenjing Decoction composition. The specific preparation method of the Wenjing Decoction composition material standard is as follows:

[0060] Take 1.67g each of angelica, chuanxiong, peony root, cinnamon heart, peony bark, and curcuma, 3.33g each of ginseng, licorice, and Achyranthes bidentata, grind the medicinal materials and pass them through a No. 1 sieve without screening out fine powder, add them to a ceramic container, add 450mL of water, cover, soak for 30min, and then directly decoct with high heat for 15min, and decoct with slow heat for 60min. At this time, the remaining water volume is 240mL. After decoction, filter with a 100-mesh sieve (filtration takes 8s. The resulting medicinal solution is directly subjected to vacuum freeze drying (freeze drying), which is divided into pre-freezing and sublimation drying;

[0061] Prefreezing: Move the drug solution to a -40℃ refrigerator, cool it to -40℃, and maintain the -40℃ prefreezing for 16 hours;

[0062] Sublimation drying: evacuate the chamber to a pressure of 12 Pa, and perform sublimation drying at -55°C for 72 hours;

[0063] After drying is completed, the material basis of the Wenjing Decoction composition is obtained.

[0064] A specific method for simultaneously determining the contents of ten components in a Wenjing Decoction composition comprises the following specific steps:

[0065] 1) Preparation of test solution and reference solution

[0066] 11) Preparation of test solution

[0067] Accurately weigh 100 mg (i.e., m = 100 mg) of the Wenjing Decoction composition, place it in a 10 mL volumetric flask, add 70 wt% methanol aqueous solution to dissolve, sonicate for 30 min at 40 kHz, 300 W, add 70 wt% methanol aqueous solution to the scale after sonication to obtain a test solution with a concentration of C = 10 mg / mL, shake well, filter, and take the filtrate to obtain the test solution, with a volume of V. 试 =10mL.

[0068] 12) Preparation of reference substance stock solution

[0069] Take appropriate amount of ginsenoside Rg1, ginsenoside Rb1, ginsenoside Re, ferulic acid, cinnamaldehyde, paeoniflorin, paeonol, licorice glycoside, β-ecdysterone and curcumol reference substances, accurately weigh them, dissolve them in methanol and dilute them to make concentrations of 226.7 μg / mL (concentration of ginsenoside Rg1), 208.9 μg / mL (concentration of ginsenoside Rb1), 186.2 μg / mL (concentration of ginsenoside Re), and 14.8 μg / mL (concentration of ginsenoside Rg1). The control substance stock solutions were 32.76 μg / mL (concentration of ferulic acid), 28.60 μg / mL (concentration of cinnamaldehyde), 203.4 μg / mL (concentration of paeoniflorin), 36.80 μg / mL (concentration of paeonol), 306.75 μg / mL (concentration of liquiritin), 36.58 μg / mL (concentration of β-ecdysterone) and 12.72 μg / mL (concentration of curcumol).

[0070] 13) Preparation of reference standard solution

[0071] Accurately measure 6 mL of reference substance stock solution and place it in a 10 mL volumetric flask. Use methanol to make up the volume as the reference substance standard solution with a concentration of C. 对-相应成分 Among them, C 对-人参皂苷Rg1 =136.02μg / mL, C 对-人参皂苷Rb1 =125.34μg / mL, C 对-人参皂苷Re =111.72μg / mL, C 对-阿魏酸 =19.656μg / mL, C 对-桂皮醛 =17.16μg / mL, C 对-芍药苷 =122.04μg / mL, C 对-丹皮酚 =22.08μg / mL, C 对-甘草苷 =184.05μg / mL, C 对-β-蜕皮甾酮 =21.948 μg / mL and C 对-莪术醇 =7.632μg / mL.

[0072] 2) High performance liquid chromatography detection

[0073] 21) Take the standard solution of the reference substance and perform high performance liquid chromatography to obtain the peak area A at the corresponding concentration of different components. 对-相应成分 Among them, A 对-人参皂苷Rg1 =8267, A 对-人参皂苷Rb1 =2563, A 对-人参皂苷Re =5471, A 对-阿魏酸 =7254, A 对-桂皮醛 =2680, A 对-芍药苷 =5959, A 对-丹皮酚 =6321, A 对-甘草苷 =5604, A 对-β-蜕皮甾酮 =2346 and A 对-莪术醇 =5385.

[0074] Among them, the HPLC conditions for detecting the standard solution of the reference substance are:

[0075] Chromatographic column: HSS T3 (2.1x100mm, 1.8μm);

[0076] Mobile phase: acetonitrile (A)-0.1wt% acetic acid-ammonium acetate buffer solution (B);

[0077] Detection wavelength: 254nm;

[0078] Injection volume: 5 μL;

[0079] Flow rate: 0.2 mL / min; column temperature: 35 °C;

[0080] Sample chamber temperature: 5°C;

[0081] The elution mode is gradient elution;

[0082] Elution program: 0-5 min, 0% mobile phase A, 100% mobile phase B;

[0083] 5-28 min, 0%→51.5% mobile phase A, 100%→48.5% mobile phase B.

[0084] 22) Take the test solution and perform HPLC under the above chromatographic conditions. The obtained chromatogram is shown in Figure 1 Among them, peak 6 is ferulic acid, peak 7 is paeoniflorin, peak 9 is liquiritigenin, peak 10 is β-ecdysterol, peak 11 is ginsenoside Rg1, peak 13 is ginsenoside Re, peak 14 is ginsenoside Rb1, peak 16 is curcumol, peak 20 is cinnamaldehyde, and peak 21 is paeonol;

[0085] From the obtained chromatographic peak, it can be known that the peak area of ​​the corresponding component contained in the test solution is A 试-相应成分 Among them, A 试-人参皂苷Rg1 =816, A 试-人参皂苷Rb1 =332, A 试-人参皂苷Re =598, A 试-阿魏酸 =7628, A 试-桂皮醛 =2183, A 试-芍药苷 =3052, A 试-丹皮酚 =3991, A 试-甘草苷 =1455, A 试-β-蜕皮甾酮 =1035 and A 试-莪术醇 =352.

[0086] According to C 试-相应成分 =(A 试-相应成分 ×C 对-相应成分 ) / A 对-相应成分, calculate the concentration C of each component in the test solution 试-相应成分 ;

[0087] Then, according to the weight-volume ratio of the material standard of the Wenjing Decoction composition to the test solution, the content of each component in the Wenjing Decoction composition = (C 对-相应成分 ×V 试 ) / m, and calculate the content of each component in the Wenjing Decoction composition;

[0088] Alternatively, the content of each component in the Wenjing Decoction composition = A 试-相应成分 ×C 对-相应成分 ×V 试 / (m×A 对-相应成分 ), calculate the content of each component in the Wenjing Decoction composition;

[0089] Take curcuminol as an example:

[0090] Calculation method 1: First, calculate the concentration of glycyrrhizic acid in the test solution C 试-莪术醇 =(A 试-莪术醇 ×C 对-莪术醇 ) / A 对-莪术醇 =(352×7.632μg / mL) / 5385=0.4989μg / mL;

[0091] Then, the content of curcumol in the Wenjing Decoction composition is calculated = (C 对-莪术醇 ×V 试 ) / m=(0.4989μg / mL×10mL) / 100mg=0.04989μg / mg.

[0092] The same applies to other ingredients.

[0093] Calculation method 2: the content of curcumol in the Wenjing Decoction composition = A 试-莪术醇 ×C 对-莪术醇 ×V 试 / (m×A 对-莪术醇 )=352×7.632μg / mL×10mL / (100mg×5385)=0.04989mg / g.

[0094] The same applies to other ingredients.

[0095] Calculation method 3: the content of curcumol in the Wenjing Decoction composition = A 试-莪术醇 ×C 对-莪术醇 / (C×A 对-莪术醇 )=352×7.632μg / mL×10mL / (100mg×5385)=0.04989mg / g.

[0096] The same applies to other ingredients.

[0097] In this embodiment, the Wenjing Decoction composition contains ginsenoside Rg1 1.3426 mg / g, ginsenoside Rb1 1.6236 mg / g, ginsenoside Re 1.2211 mg / g, ferulic acid 2.0669.mg / g, cinnamaldehyde 1.3978 mg / g, paeoniflorin 6.2505 mg / g, paeonol 1.3941 mg / g, glycyrrhizin 4.7786 mg / g, β-ecdysterone 0.9683 mg / g, and curcumol 0.04989 mg / g.

[0098] Example 2-5 Method for simultaneously determining the contents of ten components in the Wenjing Decoction composition

[0099] Examples 2 to 5 are respectively a method for simultaneously determining the contents of ten components in the Wenjing Decoction composition. The steps thereof are substantially the same as those of Example 1, and the only difference is the difference in the process parameters, as shown in Table 1 for details:

[0100] Table 1 List of process parameters in Examples 2 to 5

[0101]

[0102]

[0103] The process parameters and steps of other parts of Examples 2 to 5 are the same as those of Example 1; the test results are also similar to those of Example 1, and will not be repeated here.

[0104] Example 6 A method for simultaneously determining the contents of ten components in the Wenjing Decoction composition

[0105] b1) Preparation of test solution and reference solution

[0106] b11) Preparation of test solution

[0107] The test solution was prepared according to the method of step 11) in Example 1, wherein the steps and amounts were the same as in Example 1.

[0108] b12) Preparation of reference substance stock solution

[0109] The reference substance stock solution was prepared according to the method of step 12) in Example 1, wherein the steps and amounts were the same as in Example 1.

[0110] b13) Preparation of standard series solutions of reference substances with different concentrations

[0111] Accurately measure 1mL, 1mL, 2mL, 4mL, 6mL, and 10mL of the reference substance stock solution, respectively, and dilute to 32mL, 10mL, 10mL, 10mL, 10mL, 10mL, and 10mL with 70wt% methanol aqueous solution as a series of reference substance standard solutions of different concentrations, where the concentrations of different components are shown in Table 1.

[0112] b2) High performance liquid chromatography detection

[0113] b21) Take the reference substance standard series solution of different concentrations and carry out HPLC detection under the chromatographic conditions of Example 1, and obtain the peak area of ​​the corresponding concentration measurement of the corresponding component. The specific peak area is shown in Table 1. Then, according to the concentration of different components and the peak area of ​​the corresponding concentration measurement of the corresponding component, the standard curve corresponding to the different components is drawn, and the concentration of the different components is used as the abscissa (i.e., x, in μg / mL), and the peak area of ​​the corresponding concentration measurement of the corresponding component is used as the ordinate (i.e., y). The concentration and peak area of ​​each component in the reference substance standard series solution of specific different concentrations are shown in the following table:

[0114] Table 2 List of experimental results of standard series solutions of reference substances with different concentrations

[0115]

[0116] The standard curve drawn using a series of standard solutions of reference substances with different concentrations is as follows:

[0117] The standard curve corresponding to ginsenoside Rg1 is y=62.821x-309.62, R 2 =0.9996, standard curve see Figure 2 ;

[0118] The standard curve corresponding to ginsenoside Rb1 is y=19.915x+61.716, R 2 =0.9996, standard curve see Figure 3 ;

[0119] The standard curve corresponding to ginsenoside Re is y=48.288x+3.5014, R 2 =0.9998, standard curve see Figure 4 ;

[0120] The standard curve corresponding to ferulic acid is y=388.97x-315.75, R 2 =0.9994, standard curve see Figure 5 ;

[0121] The standard curve corresponding to cinnamaldehyde is y=140.68x+247.37, R 2 =0.9998, standard curve see Figure 6 ;

[0122] The standard curve corresponding to paeoniflorin is y=50.701x-165.0378, R 2 =0.9998, standard curve see Figure 7 ;

[0123] The standard curve corresponding to paeonol is y=290.35x-96.299, R 2 =0.9995, standard curve see Figure 8 ;

[0124] The standard curve corresponding to liquiritin is y=30.713+25.592, R 2 =0.9998, standard curve see Fig. 9 ;

[0125] The standard curve corresponding to β-ecdysterone is y=106.13x-12.566, R 2 =0.9997, standard curve see Fig.10 ;

[0126] The standard curve corresponding to curcumol is y=728.23x-88.254, R 2 =0.9996, standard curve see Fig.11 .

[0127] b23) Take the test solution and perform HPLC under the above chromatographic conditions to obtain the peak areas corresponding to the different components contained in the test solution, and substitute the peak areas of the corresponding components into the corresponding standard curve to obtain the concentration (C 试-相应成分 ).

[0128] Then the concentration of the corresponding component in the test solution (C 试-相应成分 ) multiplied by the total volume of the prepared test solution (V 试 =10mL) divided by the amount of the Wenjing Decoction composition substance used to prepare the test solution (m = 100mg), the content of the corresponding component in the Wenjing Decoction composition is obtained, that is, the content of the corresponding component in the Wenjing Decoction composition = (C 对-相应成分 ×V 试 ) / m.

[0129] Taking curcumol as an example, the peak area (A 对-莪术醇 =352) was substituted into the standard curve corresponding to curcuminol to calculate the concentration of curcuminol in the test solution I (C 试-莪术醇 =59.26 μg / mL);

[0130] The content of curcumol in the Wenjing Decoction composition = (C 对-莪术醇 ×V I-莪术醇) / m=59.26μg / mL×50mL / 0.1g=29630μg / g=29.63mg / g.

[0131] In this embodiment, the Wenjing Decoction composition contains ginsenoside Rg1 1.7917 mg / g, ginsenoside Rb1 1.3572 mg / g, ginsenoside Re 1.2312 mg / g, ferulic acid 2.0423 mg / g, cinnamaldehyde 1.3759 mg / g, paeoniflorin 6.3451 mg / g, paeonol 1.4077 mg / g, licorice 4.6541 mg / g, β-ecdysterone 0.9871 mg / g, and curcumol 0.06046 mg / g. It should be noted that due to the standard deviation of the standard curve, the content calculated by the external standard method will be different from the content calculated by the standard curve, which is common knowledge in the art.

[0132] In addition, in actual application, when the standard curve is known, the test solution can be directly prepared for HPLC detection and then directly substituted into the standard curve for calculation. There is no need to repeatedly prepare a series of standard solutions of reference substances with different concentrations for determination and draw a standard curve.

[0133] Examples 7 to 10 Method for simultaneously determining the contents of ten components in the Wenjing Decoction composition

[0134] Examples 7 to 10 are respectively a method for simultaneously determining the contents of ten components in the Wenjing Decoction composition. The steps thereof are substantially the same as those of Example 6, and the only difference is the difference in the process parameters, as shown in Table 3 for details:

[0135] Table 3 List of process parameters in Examples 7 to 10

[0136]

[0137] The process parameters and steps of other parts of Examples 7 to 10 are the same as those of Example 1; the test results are also similar to those of Example 1, and will not be repeated here.

[0138] Experimental Example 1 Methodological Investigation

[0139] This experimental example simultaneously determines the content of ten components in the Wenjing Decoction composition and examines the method for determining the content, mainly focusing on the system applicability, accuracy, precision and stability of the content determination method.

[0140] 1. System Applicability

[0141] The reference standard solution prepared in step 13) of Example 1 was tested six times continuously to calculate the RSD value of the peak area of ​​each component.

[0142] Table 4 Summary of system suitability results

[0143] Injection times 1 2 3 4 5 6 RSD value (%) <![CDATA[Ginsenoside Rg1]]> 816 815 816 817 815 819 0.18 <![CDATA[Ginsenoside Rb1]]> 334 332 330 335 336 329 0.84 Ginsenoside Re 596 599 598 597 595 596 0.25 Ferulic acid 7627 7629 7624 7628 7627 7631 0.03 Cinnamaldehyde 2186 2182 2184 2185 2187 2186 0.08 Paeoniflorin 3056 3049 3052 3053 3057 3051 0.1 Paeonol 3995 3988 3992 3991 3994 3996 0.07 Licorice glycoside 1456 1452 1457 1453 1454 1458 0.16 β-Ecdysteroid 1037 1034 1032 1038 1035 1036 0.21 Curcumol 351 352 350 349 352 353 0.42

[0144] It can be seen from Table 4 that, in the content determination method of the present invention, the peak area RSD value of each component is less than 0.84%, and the system applicability is good.

[0145] 2. Accuracy

[0146] Take 100 mg of the material standard of the Wenjing Decoction composition with known content (this experiment uses the same batch of Wenjing Decoction composition as in Example 2, and the content of each component in the Wenjing Decoction composition determined in Example 2 is used as the known content of each component), accurately weigh, add each component reference substance at 50%, 100%, and 150% of the known content of each component, respectively, and prepare three accuracy test solutions of low, medium, and high concentrations according to the preparation method of the test solution in Example 2, and prepare 3 copies of each concentration in parallel. Perform the corresponding high performance liquid chromatography detection according to the above chromatographic conditions, and calculate the average recovery rate and RSD value of each component. The specific results are as follows:

[0147] Table 5 Accuracy test results list

[0148]

[0149]

[0150] As can be seen from Table 5, the recoveries of each component were all within the range of 99.92% to 100.03%, and the method had good accuracy.

[0151] 3. Precision

[0152] Take the same batch of Wenjing Decoction composition material standard, accurately weigh 50 mg, 100 mg, and 150 mg respectively, and prepare three precision test solutions of low, medium, and high concentrations according to the preparation method of the test solution in Example 2 (except for the different amounts of the Wenjing Decoction composition material standard, the amounts of other solvents and the methods are the same as in Example 1), and prepare 3 copies of each concentration in parallel; on the same day, perform corresponding HPLC detection according to the above-mentioned chromatographic conditions, determine the content of each component, and calculate the RSD value of the content of each component as a repeatability experiment (i.e., intra-day precision experiment).

[0153] Take 0.1 g of the standard substance of the Wenjing Decoction composition from the same batch, prepare 3 portions of the test solution of the same concentration according to the method in the repeatability experiment, and perform corresponding HPLC detection according to the above-mentioned chromatographic conditions. Prepare 3 portions in parallel every day, and perform sampling and detection for three consecutive days. Calculate the RSD value of the content of each component to be tested, which is the inter-day precision experiment.

[0154] The specific results are as follows:

[0155] Table 6 List of precision test results

[0156]

[0157]

[0158] As can be seen from Table 6, the RSD values ​​of the 10 components on the same day were 0.07% to 0.86%, respectively, indicating that the method had good repeatability. The RSD values ​​of the inter-day precision were 0.15% to 0.72%, respectively, indicating that the method had good inter-day precision.

[0159] 4. Stability

[0160] The test solution was prepared according to the method of Example 2, and after being placed at room temperature for 0, 1, 2, 4, 8, 12, and 24 hours, HPLC detection was performed, and the RSD of each component was calculated, as follows:

[0161] Table 7 Stability test results list

[0162] project Stability (RSD,%) <![CDATA[Ginsenoside Rg1]]> 0.56 <![CDATA[Ginsenoside Rb1]]> 1.24 Ginsenoside Re 0.62 Ferulic acid 0.37 Cinnamaldehyde 0.82 Paeoniflorin 0.65 Paeonol 0.18 Licorice glycoside 0.43 β-Ecdysteroid 0.86 Curcumol 0.92

[0163] It can be seen from Table 7 that the 10 components were 0.18% to 1.24% within 24 hours, respectively. The results show that the test solution prepared by the present invention has good stability.

[0164] Comparative Example 1

[0165] Comparative Example 1 is a comparative test for simultaneously determining the contents of ten components in the Wenjing Decoction composition in Example 1, the only difference being that:

[0166] In Comparative Example 1, only mobile phase B was replaced with 0.1 wt % acetic acid aqueous solution, and the obtained chromatogram was shown in Fig.12 Among them, peak No. 16 corresponding to curcumol did not emerge well and could not be well separated from the impurity peaks, so the content could not be determined at one time.

[0167] Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Claims

1. A method for simultaneously determining the contents of ten components in a Wenjing Decoction composition, characterized in that: The content determination method comprises the following steps: A total of 10 ingredients, including ginsenoside Rg1, ginsenoside Rb1, ginsenoside Re, ferulic acid, cinnamaldehyde, paeoniflorin, paeonol, licorice glycoside, β-ecdysterone and curcumol, are used to prepare a reference standard solution; a test solution is prepared by taking the Wenjing Decoction composition; the content of the Wenjing Decoction composition in the test solution is 8-12 mg / mL; the solvent for preparing the test solution is a 70wt% methanol aqueous solution; The reference standard solution and the test solution are respectively taken for high performance liquid chromatography detection, and then the external standard method is used to calculate the contents of ginsenoside Rg1, ginsenoside Rb1, ginsenoside Re, ferulic acid, cinnamaldehyde, paeoniflorin, paeonol, licorice glycoside, β-ecdysterone and curcumol in the Wenjing Decoction composition; wherein, in the high performance liquid chromatography detection process, acetonitrile is used as mobile phase A, and 0.09-0.12wt% acetic acid-ammonium acetate buffer is used as mobile phase B; The elution mode of the high performance liquid chromatography detection is gradient elution, and the elution conditions are: 0-5 min, 0% mobile phase A, 100% mobile phase B; 5-28 min, 0% → 51.5% mobile phase A, 100% → 48.5% mobile phase B; The chromatographic conditions of the HPLC detection are: the chromatographic column is HSS T3, the detection wavelength is 254 nm, the flow rate is 0.2 mL / min, and the column temperature is 35°C.

2. The method for simultaneously determining the contents of ten components in the Wenjing Decoction composition according to claim 1, characterized in that: The concentration of ginsenoside Rg1 in the standard solution of the reference substance is between 7.084 and 226.7 μg / mL, the concentration of ginsenoside Rb1 is between 6.528 and 208.9 μg / mL, the concentration of ginsenoside Re is between 5.819 and 186.2 μg / mL, the concentration of ferulic acid is between 1.024 and 32.76 μg / mL, the concentration of cinnamaldehyde is between 0.8938 and 28.6 0μg / mL, the concentration of paeoniflorin was between 6.356 and 203.4μg / mL, the concentration of paeonol was between 1.150 and 36.80μg / mL, the concentration of liquiritin was between 9.586 and 306.8μg / mL, the concentration of β-ecdysterone was between 1.143 and 36.58μg / mL, and the concentration of curcumol was between 0.3975 and 12.72μg / mL.

3. A method for simultaneously determining the contents of ten components in a Wenjing Decoction composition, characterized in that: The content determination method comprises the following steps: A total of 10 ingredients, including ginsenoside Rg1, ginsenoside Rb1, ginsenoside Re, ferulic acid, cinnamaldehyde, paeoniflorin, paeonol, licorice glycoside, β-ecdysterone and curcumol, were used to prepare a series of reference standard solutions of different concentrations; Take standard series solutions of reference substances of different concentrations for high performance liquid chromatography detection, and draw standard curves corresponding to different components according to the concentrations of different components and the peak areas of the corresponding components at the corresponding concentrations; The Wenjing Decoction composition is used to prepare a test solution; the content of the Wenjing Decoction composition in the test solution is 8-12 mg / mL; the solvent for preparing the test solution is a 70 wt % methanol aqueous solution; The test solution is subjected to high performance liquid chromatography detection, and the peak area of ​​the corresponding component obtained is substituted into the standard curve corresponding to the corresponding component, and the content of the corresponding component in the Wenjing Decoction composition is obtained by calculation; Wherein, in the high performance liquid chromatography detection process, acetonitrile is used as mobile phase A, and 0.09-0.12wt% acetic acid-ammonium acetate buffer is used as mobile phase B; The elution mode of the high performance liquid chromatography detection is gradient elution, and the elution conditions are: 0-5 min, 0% mobile phase A, 100% mobile phase B; 5-28 min, 0% → 51.5% mobile phase A, 100% → 48.5% mobile phase B; The chromatographic conditions of the HPLC detection are: the chromatographic column is HSS T3, the detection wavelength is 254 nm, the flow rate is 0.2 mL / min, and the column temperature is 35°C.

4. The method for simultaneously determining the contents of ten components in the Wenjing Decoction composition according to claim 3, characterized in that: In the standard series of reference solutions of different concentrations, the concentration of ginsenoside Rg1 is between 7.084 and 226.7 μg / mL, the concentration of ginsenoside Rb1 is between 6.528 and 208.9 μg / mL, the concentration of ginsenoside Re is between 5.819 and 186.2 μg / mL, the concentration of ferulic acid is between 1.024 and 32.76 μg / mL, the concentration of cinnamaldehyde is between 0.8938 and 0.916 μg / mL. The concentration of paeoniflorin was between 6.356 and 203.4 μg / mL, the concentration of paeonol was between 1.150 and 36.80 μg / mL, the concentration of liquiritin was between 9.586 and 306.8 μg / mL, the concentration of β-ecdysterone was between 1.143 and 36.58 μg / mL, and the concentration of curcumol was between 0.3975 and 12.72 μg / mL.

5. The method for simultaneously determining the contents of ten components in the Wenjing Decoction composition according to claim 3, characterized in that: The standard curves corresponding to the different components are as follows: The standard curve corresponding to ginsenoside Rg1 is y=62.821x-309.62, R 2 =0.9996; The standard curve corresponding to ginsenoside Rb1 is y=19.915x+61.716, R 2 =0.9996; The standard curve corresponding to ginsenoside Re is y=48.288x+3.5014, R 2 =0.9998; The standard curve corresponding to ferulic acid is y=388.97x-315.75, R 2 =0.9994; The standard curve corresponding to cinnamaldehyde is y=140.68x+247.37, R 2 =0.9998; The standard curve corresponding to paeoniflorin is y=50.701x-165.0378, R 2 =0.9998; The standard curve corresponding to paeonol is y=290.35x-96.299, R 2 =0.9995; The standard curve corresponding to liquiritin is y=30.713+25.592, R 2 =0.9998; The standard curve corresponding to β-ecdysterone is y=106.13x-12.566, R 2 =0.9997; The standard curve corresponding to curcumol is y=728.23x-88.254, R 2 =0.9996.

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