Method for determining contents of five components in white jade ointment based on quantitative analysis of multi-components by single marker

Abstract

The invention discloses a method for determining the content of five components in a white jade ointment based on quantitative analysis of multi-components by single marker, and relates to the technical field of medicine detection. According to the method, tanshinone IIA is used as an internal standard substance for quantitative analysis of multi-components by single marker, relative correction factors of 1, 4-di [4-(glucose oxygen) benzyl]-2-isobutyl malate, tanshinone I, 6-gingerol and beta, beta '-dimethylacryloyl alkannin are established, and the content of each component in the white jade ointment is calculated through the relative correction factors. The method comprises the following specific steps: preparing a test solution; preparing a reference substance solution; performing chromatographic condition and system adaptability test; and measuring and calculating. According to the method, the oneness and one-sidedness of quality control of the white jade ointment are overcome, and the method has important significance in further improving the quality control level of the white jade ointment, ensuring the clinical curative effect and improving a related evaluation system.

Description

Technical Field

[0001] The present invention relates to the technical field of drug detection, and particularly relates to a method for determining the contents of 5 components in Baiyu Ointment based on the multiple components by single marker method. Background Art

[0002] Baiyu Ointment is a product of Guizhou Green Sun Pharmaceutical Co., Ltd., which has the effects of promoting blood circulation to dispel wind and nourishing blood and moistening the skin. It is used for skin chaps caused by wind-dryness of blood deficiency, and has significant clinical treatment effects and is deeply favored by patients.

[0003] The prescription of Baiyu Ointment is composed of nine herbs, namely Bletilla striata, Polygonatum odoratum, Ligusticum sinense, Polygonum hydropiper, Geranium wilfordii, Salvia miltiorrhiza, Lithospermum erythrorhizon, Dry Ginger, and Angelica sinensis. The preparation method is as follows: For the above nine herbs, take Salvia miltiorrhiza, Angelica sinensis, and Dry Ginger, use 80% ethanol as the solvent, soak for 12 h, and then slowly percolate at a speed of 1-3 mL / min to collect 1200 mL of percolate. Recover ethanol under reduced pressure and concentrate to a clear paste with a relative density of 1.15 (50 °C) for standby; the remaining six herbs, such as Bletilla striata, are decocted with water twice, 2 h for the first time and 1.5 h for the second time. Combine the decoction liquids, filter, and concentrate the filtrate to a clear paste with a relative density of 1.05 (60 °C), and combine it with the above clear paste; add 60 g of sorbitol, 60 g of betacyclodextrin, 60 g of glycerol, 20 g of titanium dioxide (titanium white), and 3 g of sodium hyaluronate, heat (80 °C) to dissolve for standby; separately take 10 g of mink oil, 30 g of lanolin, 60 g of white petrolatum (petrolatum), 12 g of mono- and diglycerides of fatty acids (monoglyceride), 10 g of sucrose stearate (sucrose ester), 100 g of liquid paraffin (paraffin oil), 50 g of polysorbate 80, 50 g of sorbitan oleate (span 80), and 20 g of emulsifying silicone oil, heat (80 °C) to melt, mix with the above clear paste, emulsify for 15 min, keep warm at 80 °C for 30 min, cool to 40 °C, and then add 2 g of elastase (elastase), 6 g of benzoic acid, and 1.5 g of ethylparaben, mix well to make 1000 g, and that's it. In the current quality standard of Baiyu Ointment, only the content of tanshinone IIA is used as the detection standard, which cannot comprehensively reflect the quality of Baiyu Ointment.

[0004] Based on this, on the basis of deeply investigating the quality detection method of Baiyu Ointment, the research team established a method for determining the contents of 5 components in Baiyu Ointment based on the multiple components by single marker method. This method uses tanshinone IIA as the internal standard for multiple components by single marker, establishes the relative correction factors of 1,4-bis[4-(glucosyloxy)benzyl]-2-isobutylmalate, tanshinone I, 6-gingerol, and β,β ′ -dimethylacryloylalkanin, and calculates the contents of each component in Baiyu Ointment through the relative correction factors. The purpose of the present invention is to achieve more comprehensive quality control of Baiyu Ointment. Summary of the Invention

[0005] The object of the present invention is to provide a method for determining the contents of 5 components in Baiyu Ointment based on the method of multi-component determination by single marker.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] The method for determining the contents of 5 components in Baiyu Ointment based on the method of multi-component determination by single marker of the present invention uses tanshinone ⅡA as the internal standard substance to establish the relative correction factors of 1,4-bis[4-(gluconoyloxy)benzyl]-2-isobutyl malate, tanshinone Ⅰ, 6-gingerol, β,β ′ -dimethylacryloyl alkannin, and calculates the contents of the above components through the relative correction factors. The specific steps include: preparation of the test solution; preparation of the reference solution; chromatographic conditions and system suitability test; determination and calculation.

[0008] Preferably, in the method for determining the contents of 5 components in Baiyu Ointment based on the method of multi-component determination by single marker of the present invention, the preparation of the test solution is specifically as follows: Take 1.8 - 4.2 g of Baiyu Ointment, accurately weigh it, accurately add 15 - 35 mL of 70% - 100% methanol solution or 60% - 75% ethanol solution, weigh it; ultrasonically treat for 30 - 60 min, cool it, make up the lost weight with the above methanol solution or ethanol solution, shake well, filter; extract the filtrate with chloroform by shaking for 1 - 4 times, 20 - 40 mL each time; combine the extraction solutions, evaporate to near dryness, add an appropriate amount of methanol to dissolve and transfer to a 5 mL volumetric flask, make up the volume, shake well, filter, and obtain the solution.

[0009] More preferably, in the method for determining the contents of 5 components in Baiyu Ointment based on the method of multi-component determination by single marker of the present invention, the preparation of the test solution is specifically as follows: Take 2.4 - 4.2 g of Baiyu Ointment, accurately weigh it, accurately add 20 - 35 mL of 60% - 75% ethanol solution, weigh it; ultrasonically treat for 30 - 45 min, cool it, make up the lost weight with the above ethanol solution, shake well, filter; extract the filtrate with chloroform by shaking for 3 - 4 times, 30 - 40 mL each time; combine the extraction solutions, evaporate to near dryness, add an appropriate amount of methanol to dissolve and transfer to a 5 mL volumetric flask, make up the volume, shake well, filter through a 0.45 μm microporous membrane, and obtain the solution.

[0010] Even more preferably, in the method for determining the contents of 5 components in Baiyu Ointment based on the method of multi-component determination by single marker of the present invention, the preparation of the test solution is specifically as follows: Take 3 g of Baiyu Ointment, accurately weigh it, accurately add 25 mL of 75% ethanol solution, weigh it; ultrasonically treat for 30 min, cool it, make up the lost weight with the above ethanol solution, shake well, filter; extract the filtrate with chloroform by shaking for 3 times, 30 mL each time; combine the extraction solutions, evaporate to near dryness, add an appropriate amount of methanol to dissolve and transfer to a 5 mL volumetric flask, make up the volume, shake well, filter through a 0.45 μm microporous membrane, and obtain the solution.

[0011] Preferably, in the method for determining the contents of 5 components in Baiyu Ointment based on the one-measure-multiple-evaluation method of the present invention, the preparation of the reference substance solution is specifically as follows: Take an appropriate amount of tanshinone IIA reference substance, weigh it accurately and place it in a brown volumetric flask, add methanol to make a reference substance solution containing 0.02 mg per 1 mL, and that's it.

[0012] Preferably, in the method for determining the contents of 5 components in Baiyu Ointment based on the one-measure-multiple-evaluation method of the present invention, the chromatographic conditions and system suitability test are specifically as follows:

[0013] The chromatographic column is packed with octadecylsilane chemically bonded silica gel; the column temperature is 25 - 35 °C; acetonitrile is used as mobile phase A, and a 0.1% phosphoric acid solution containing 0.1% sodium dodecyl sulfate is used as mobile phase B for gradient elution; the flow rate of the mobile phase is 0.9 - 1.1 mL / min; the injection volume is 10 μL; the detection wavelength is 270 nm; the number of theoretical plates calculated based on the tanshinone IIA peak should be not less than 4000;

[0014] Among them, the gradient elution program is specifically as follows:

[0015]

[0016] More preferably, in the method for determining the contents of 5 components in Baiyu Ointment based on the one-measure-multiple-evaluation method of the present invention, in the chromatographic conditions and system suitability test, the column temperature is 30 °C and the flow rate of the mobile phase is 1.0 mL / min.

[0017] Preferably, in the method for determining the contents of 5 components in Baiyu Ointment based on the one-measure-multiple-evaluation method of the present invention, the determination and calculation are specifically as follows: Inject the test solution and the reference substance solution into a high performance liquid chromatograph for determination, and calculate the contents of each component according to the relative correction factors of tanshinone IIA and 1,4-bis[4-(gluconoyloxy)benzyl]-2-isobutylmalic acid ester, tanshinone I, 6-gingerol, β,β ′ -dimethylacryloylalkanin.

[0018] More preferably, in the method for determining the contents of 5 components in Baiyu Ointment based on the one-measure-multiple-evaluation method of the present invention, in the determination and calculation, the relative retention times of tanshinone IIA and 1,4-bis[4-(gluconoyloxy)benzyl]-2-isobutylmalic acid ester, tanshinone I, 6-gingerol, β,β ′ -dimethylacryloylalkanin are 0.33, 0.44, 0.83, 1.53 respectively, and the relative retention times are within ±10% of the specified values.

[0019] Further preferably, in the method for determining the contents of 5 components in Baiyu Ointment based on the multi-component assay by single marker method of the present invention, in the said determination and calculation, the relative correction factors of tanshinone ⅡA, 1,4-bis[4-(gluconoyloxy)benzyl]-2-isobutyl malate, tanshinone Ⅰ, 6-gingerol, and β,β ′ -dimethylacryloylalkanin are 2.65, 1.37, 0.68, and 1.12 respectively.

[0020] Advantages of the present invention:

[0021] 1. The present invention firstly establishes a method for determining the contents of 5 components in Baiyu Ointment based on the multi-component assay by single marker method. As long as the content of tanshinone ⅡA is determined, the simultaneous analysis of the contents of 1,4-bis[4-(gluconoyloxy)benzyl]-2-isobutyl malate, tanshinone Ⅰ, 6-gingerol, and β,β ′ -dimethylacryloylalkanin can be realized, and the overall quality of the product is reflected by the simultaneous quality control of the above 5 components. The present invention overcomes the singleness and one-sidedness of the quality control of Baiyu Ointment, and is of great significance for further improving the quality control level of Baiyu Ointment, ensuring clinical efficacy and the improvement of relevant evaluation systems.

[0022] 2. The present invention investigates the factors affecting the extraction of 5 active components in Baiyu Ointment, and finally obtains the optimal extraction scheme for 5 active components in Baiyu Ointment as follows: the extraction solvent is 75% ethanol solution; the ultrasonic extraction time is 30 min; the number of chloroform extractions is 3 times; the amount of chloroform used each time is 30 mL.

[0023] 3. Through methodological verification experiments, it is confirmed that the method for determining the contents of 5 components in Baiyu Ointment established by the present invention based on the multi-component assay by single marker method has the advantages of high accuracy, good repeatability, strong stability, and good durability. On the basis of improving the overall quality control standard of Baiyu Ointment, it greatly improves its detection efficiency, and at the same time saves the detection cost of the quality control of Baiyu Ointment. Description of the Drawings

[0024] Figure 1 is the chromatogram for mobile phase investigation (eluted according to gradient elution program 1);

[0025] Figure 2 is the chromatogram for mobile phase investigation (eluted according to gradient elution program 2);

[0026] Figure 3 is the chromatogram for mobile phase investigation (eluted according to gradient elution program 3);

[0027] Figure 4 is the chromatogram for extraction method investigation (extracting active components according to method 1);

[0028] Figure 5 Chromatogram for the investigation of the extraction method (extracting the active ingredients according to Method 2);

[0029] Figure 6 Chromatogram for the investigation of the extraction method (extracting the active ingredients according to Method 3);

[0030] Figure 7 Chromatogram for the investigation of the extraction method (extracting the active ingredients according to Method 4);

[0031] Figure 8 Chromatogram of reference substances for each component (in the figure: 1 is 1,4 - bis[4-(glucosyloxy)benzyl]-2-isobutylmalate; 2 is Tanshinone I; 3 is 6-Gingerol, 4 is Tanshinone IIA; 5 is β,β ′ -Dimethylacryloylalkannin);

[0032] Figure 9 Chromatogram of the sample and the mixed reference substances (in the figure: A is the chromatogram of the sample; B is the chromatogram of the mixed reference substances). Specific Embodiments

[0033] The technical solutions of the present invention will be described in detail below in conjunction with specific embodiments. The following embodiments are only for explanation and illustration, and do not constitute a limitation to the technical solutions of the present invention.

[0034] Example 1

[0035] A method for determining the contents of 5 components in Baiyu Ointment based on the multi-component quantification by single marker method is as follows:

[0036] (1) Preparation of the test solution

[0037] Take 3 g of Baiyu Ointment, accurately weigh it, accurately add 25 mL of 75% ethanol solution, weigh it again; perform ultrasonic treatment for 30 min, cool it, make up the lost weight with the above ethanol solution, shake well, and filter; extract the filtrate with chloroform by shaking 3 times, 30 mL each time; combine the extraction solutions, evaporate to near dryness, add an appropriate amount of methanol to dissolve and transfer it to a 5 mL volumetric flask, make up the volume, shake well, and filter through a 0.45 μm microporous membrane to obtain the test solution.

[0038] (2) Preparation of the reference substance solution

[0039] Take an appropriate amount of Tanshinone IIA reference substance, accurately weigh it and place it in a brown volumetric flask, add methanol to make a reference substance solution containing 0.02 mg per 1 mL, and obtain the reference substance solution.

[0040] (3) Chromatographic conditions and system suitability test

[0041] The chromatographic column is packed with octadecylsilyl silica gel; the column temperature is 30 °C; acetonitrile is used as mobile phase A, and a 0.1% phosphoric acid solution containing 0.1% sodium dodecyl sulfate is used as mobile phase B for gradient elution; the flow rate of the mobile phase is 1.0 mL / min; the injection volume is 10 μL; the detection wavelength is 270 nm; the number of theoretical plates calculated based on the tanshinone IIA peak should be not less than 4000;

[0042] Among them, the specific gradient elution program is as follows:

[0043]

[0044] (4) Determination and calculation

[0045] Take the test solution and the reference solution and inject them into a high performance liquid chromatograph for determination. According to the relative correction factors of tanshinone IIA to 1,4-bis[4-(gluconoyloxy)benzyl]-2-isobutylmalic acid ester of 2.65, to tanshinone I of 1.37, to 6-gingerol of 0.68, and to β,β ′ -dimethylacryloylalkanin of 1.12, calculate the contents of each component respectively.

[0046] Example 2

[0047] A method for determining the contents of 5 components in Baiyu Ointment based on the multi-component quantification by single marker method is as follows:

[0048] (1) Preparation of the test solution

[0049] Take 1.8 g of Baiyu Ointment, weigh it accurately, add 15 mL of 75% ethanol solution accurately, weigh again; ultrasonically treat for 30 min, let it cool, make up the lost weight with the above ethanol solution, shake well, filter; extract the filtrate with chloroform by shaking 3 times, 30 mL each time; combine the extraction liquids, evaporate to nearly dryness, add an appropriate amount of methanol to dissolve and transfer to a 5 mL volumetric flask, make up the volume, shake well, and filter through a 0.45 μm microporous filter membrane to obtain the solution.

[0050] (2) Preparation of the reference solution

[0051] Take an appropriate amount of tanshinone IIA reference substance, weigh it accurately and place it in a brown volumetric flask, add methanol to make a reference solution containing 0.02 mg per 1 mL, and obtain the solution.

[0052] (3) Chromatographic conditions and system suitability test

[0053] The chromatographic column is packed with octadecylsilyl silica gel; the column temperature is 25°C; acetonitrile is used as mobile phase A, and a 0.1% phosphoric acid solution containing 0.1% sodium dodecyl sulfate is used as mobile phase B for gradient elution; the flow rate of the mobile phase is 0.9 mL / min; the injection volume is 10 μL; the detection wavelength is 270 nm; the number of theoretical plates calculated based on the tanshinone IIA peak should be not less than 4000;

[0054] Among them, the specific gradient elution program is as follows:

[0055]

[0056] (4) Determination and calculation

[0057] Take the test solution and the reference solution and inject them into a high performance liquid chromatograph for determination. Calculate the contents of each component according to the relative correction factors of tanshinone IIA to 1,4-bis[4-(glucosyloxy)benzyl]-2-isobutylmalic acid ester being 2.65, to tanshinone I being 1.37, to 6-gingerol being 0.68, and to β,β ′ -dimethylacryloylalkanin being 1.12 respectively.

[0058] Example 3

[0059] A method for determining the contents of 5 components in Baiyu Ointment based on the multi-component assay by single marker, the steps are as follows:

[0060] (1) Preparation of the test solution

[0061] Take 2.4 g of Baiyu Ointment, weigh it accurately, accurately add 20 mL of 75% ethanol solution, weigh again; ultrasonically treat for 30 min, let it cool, make up the lost weight with the above ethanol solution, shake well, filter; extract the filtrate with chloroform by shaking 3 times, 30 mL each time; combine the extraction solutions, evaporate to nearly dry, add an appropriate amount of methanol to dissolve and transfer to a 5 mL volumetric flask, make up the volume, shake well, and filter through a 0.45 μm microporous membrane to obtain.

[0062] (2) Preparation of the reference solution

[0063] Take an appropriate amount of tanshinone IIA reference substance, weigh it accurately and place it in a brown volumetric flask, add methanol to make a reference solution containing 0.02 mg per 1 mL, and obtain.

[0064] (3) Chromatographic conditions and system suitability test

[0065] The chromatographic column is packed with octadecylsilyl silica gel; the column temperature is 35°C; acetonitrile is used as mobile phase A, and a 0.1% phosphoric acid solution containing 0.1% sodium dodecyl sulfate is used as mobile phase B for gradient elution; the flow rate of the mobile phase is 1.1 mL / min; the injection volume is 10 μL; the detection wavelength is 270 nm; the number of theoretical plates calculated based on the tanshinone IIA peak should be not less than 4000;

[0066] Among them, the specific gradient elution program is as follows:

[0067]

[0068] (4) Determination and calculation

[0069] Take the test solution and the reference solution and inject them into a high performance liquid chromatograph for determination. Calculate the contents of each component according to the relative correction factors of tanshinone IIA to 1,4-bis[4-(gluconoyloxy)benzyl]-2-isobutylmalic acid ester being 2.65, to tanshinone I being 1.37, to 6-gingerol being 0.68, and to β,β ′ -dimethylacryloylalkanin being 1.12 respectively.

[0070] Example 4

[0071] A method for determining the contents of 5 components in Baiyu Ointment based on the multi-component assay by single marker approach is as follows:

[0072] (1) Preparation of the test solution

[0073] Take 4.2 g of Baiyu Ointment, weigh it precisely, add 35 mL of 75% ethanol solution precisely, weigh again; ultrasonically treat for 30 min, cool to room temperature, make up the lost weight with the above ethanol solution, shake well, filter; extract the filtrate with chloroform by shaking for 3 times, 30 mL each time; combine the extraction solutions, evaporate to nearly dryness, add an appropriate amount of methanol to dissolve and transfer to a 5 mL volumetric flask, make up the volume, shake well, and filter through a 0.45 μm microporous membrane to obtain the solution.

[0074] (2) Preparation of the reference solution

[0075] Take an appropriate amount of tanshinone IIA reference substance, weigh it precisely and place it in a brown volumetric flask, add methanol to make a reference solution containing 0.02 mg per 1 mL, and obtain the solution.

[0076] (3) Chromatographic conditions and system suitability test

[0077] The chromatographic column is packed with octadecylsilyl silica gel; the column temperature is 30 °C; acetonitrile is used as mobile phase A, and a 0.1% phosphoric acid solution containing 0.1% sodium dodecyl sulfate is used as mobile phase B for gradient elution; the flow rate of the mobile phase is 1.0 mL / min; the injection volume is 10 μL; the detection wavelength is 270 nm; the number of theoretical plates calculated based on the tanshinone IIA peak should be not less than 4000;

[0078] Among them, the specific gradient elution program is as follows:

[0079]

[0080] (4) Determination and calculation

[0081] Inject the test solution and the reference solution into a high performance liquid chromatograph for determination. Calculate the contents of each component according to the relative correction factors of tanshinone IIA to 1,4-bis[4-(glucosyloxy)benzyl]-2-isobutylmalic acid ester being 2.65, to tanshinone I being 1.37, to 6-gingerol being 0.68, and to β,β ′ -dimethacryloylalkanin being 1.12 respectively.

[0082] Example 5

[0083] A method for determining the contents of 5 components in Baiyu Ointment based on the multi-component quantification by single marker method is as follows:

[0084] (1) Preparation of the test solution

[0085] Take 3 g of Baiyu Ointment, weigh accurately, accurately add 25 mL of 60% ethanol solution, weigh again; ultrasonically treat for 60 min, cool to room temperature, make up the lost weight with the above ethanol solution, shake well, filter; extract the filtrate with 40 mL of chloroform by shaking; evaporate the extract to near dryness, add an appropriate amount of methanol to dissolve and transfer to a 5 mL volumetric flask, make up the volume, shake well, and filter through a 0.45 μm microporous membrane to obtain the solution.

[0086] (2) Preparation of the reference solution

[0087] Take an appropriate amount of tanshinone IIA reference substance, weigh accurately and place it in a brown volumetric flask, add methanol to make a reference solution containing 0.02 mg per 1 mL, and obtain the solution.

[0088] (3) Chromatographic conditions and system suitability test

[0089] The chromatographic column is packed with octadecylsilyl silica gel; the column temperature is 30 °C; acetonitrile is used as mobile phase A, and a 0.1% phosphoric acid solution containing 0.1% sodium dodecyl sulfate is used as mobile phase B for gradient elution; the flow rate of the mobile phase is 1.0 mL / min; the injection volume is 10 μL; the detection wavelength is 270 nm; the number of theoretical plates calculated based on the tanshinone IIA peak should be not less than 4000;

[0090] Among them, the specific gradient elution program is as follows:

[0091]

[0092]

[0093] (4) Determination and calculation

[0094] Take the test solution and the reference solution and inject them into a high performance liquid chromatograph for determination. Calculate the contents of each component according to the relative correction factors of tanshinone IIA to 1,4-bis[4-(glucosyloxy)benzyl]-2-isobutylmalic acid ester of 2.65, to tanshinone I of 1.37, to 6-gingerol of 0.68, and to β,β ′ -dimethylacryloylalkanin of 1.12 respectively.

[0095] Example 6

[0096] A method for determining the contents of 5 components in Baiyu Ointment based on the multi-component quantification by single marker method is as follows:

[0097] (1) Preparation of the test solution

[0098] Take 3 g of Baiyu Ointment, weigh it precisely, add 25 mL of 70% methanol solution precisely, weigh again; ultrasonically treat for 45 min, let it cool, make up the lost weight with the above methanol solution, shake well, filter; extract the filtrate with chloroform by shaking 3 times, 30 mL each time; combine the extraction solutions, evaporate to nearly dryness, add an appropriate amount of methanol to dissolve and transfer to a 5 mL volumetric flask, make up the volume, shake well, and filter through a 0.45 μm microporous filter membrane to obtain.

[0099] (2) Preparation of the reference solution

[0100] Take an appropriate amount of tanshinone IIA reference substance, weigh it precisely and place it in a brown volumetric flask, add methanol to make a reference solution containing 0.02 mg per 1 mL, and obtain.

[0101] (3) Chromatographic conditions and system suitability test

[0102] The chromatographic column is packed with octadecylsilyl silica gel; the column temperature is 30 °C; acetonitrile is used as mobile phase A, and a 0.1% phosphoric acid solution containing 0.1% sodium dodecyl sulfate is used as mobile phase B for gradient elution; the flow rate of the mobile phase is 1.0 mL / min; the injection volume is 10 μL; the detection wavelength is 270 nm; the number of theoretical plates calculated based on the tanshinone IIA peak should be not less than 4000;

[0103] Among them, the gradient elution program is specifically as follows:

[0104]

[0105] (4) Determination and calculation

[0106] Take the test solution and the reference solution and inject them into a high performance liquid chromatograph for determination. According to the relative correction factors of tanshinone IIA to 1,4-bis[4-(glucosyloxy)benzyl]-2-isobutylmalic acid ester of 2.65, to tanshinone I of 1.37, to 6-gingerol of 0.68, and to β,β ′ -dimethylacryloylalkanin of 1.12, calculate the contents of each component respectively.

[0107] Example 7

[0108] A method for determining the contents of 5 components in Baiyu Ointment based on the multi-component quantification by single marker method is as follows:

[0109] (1) Preparation of the test solution

[0110] Take 3 g of Baiyu Ointment, weigh it accurately, add 25 mL of methanol accurately, weigh again; ultrasonically treat for 30 min, let it cool, make up the lost weight with methanol, shake well, filter; extract the filtrate with chloroform by shaking 4 times, 20 mL each time; combine the extraction solutions, evaporate to nearly dryness, add an appropriate amount of methanol to dissolve and transfer to a 5 mL volumetric flask, make up the volume, shake well, and filter through a 0.45 μm microporous filter membrane to obtain.

[0111] (2) Preparation of the reference solution

[0112] Take an appropriate amount of tanshinone IIA reference substance, weigh it accurately and place it in a brown volumetric flask, add methanol to make a reference solution containing 0.02 mg per 1 mL, and obtain.

[0113] (3) Chromatographic conditions and system suitability test

[0114] The chromatographic column is packed with octadecylsilyl silica gel; the column temperature is 30 °C; acetonitrile is used as mobile phase A, and a 0.1% phosphoric acid solution containing 0.1% sodium dodecyl sulfate is used as mobile phase B for gradient elution; the flow rate of the mobile phase is 1.0 mL / min; the injection volume is 10 μL; the detection wavelength is 270 nm; the number of theoretical plates calculated based on the tanshinone IIA peak should be not less than 4000;

[0115] Among them, the specific gradient elution program is as follows:

[0116]

[0117] (4) Determination and calculation

[0118] Inject the test solution and the reference solution into the high performance liquid chromatograph for determination. Calculate the contents of each component according to the relative correction factors of tanshinone IIA to 1,4-bis[4-(glucosyloxy)benzyl]-2-isobutylmalic acid ester being 2.65, to tanshinone I being 1.37, to 6-gingerol being 0.68, and to β,β ′ -dimethylacryloylalkanin being 1.12 respectively.

[0119] In order to further verify the reliability of the present invention and screen out the best solution, the inventor conducted a series of experiments as follows:

[0120] 1. Experimental instruments

[0121] Electronic balance (BSM-220.4, Shanghai Zhuojing Electronic Technology Co., Ltd.); ultrasonic cleaner (MH-0403, Dong'an Meihong Metal Processing Factory); electronic balance (AB-135-S, Mettler-Toledo); high performance liquid chromatograph (LC-2010AHT, Shimadzu, Japan). Chromatographic column: Welchrom-C 18 (4.6×250 mm, 5 μm); Agilent 5TC-C 18 (4.6×250 mm, 5 μm); Shim-pack GIST C 18 (4.6×250 mm, 5 μm).

[0122] 2. Materials and reagents

[0123] Reference substance of Tanshinone ⅡA (Lot No.: 110766 - 202323), reference substance of 1,4 - bis[4 - (gluconoyloxy)benzyl]-2 - isobutylmalate (Lot No.: 112061 - 202102), reference substance of Tanshinone Ⅰ (Lot No.: 110867 - 201607), reference substance of 6 - Gingerol (Lot No.: 111833 - 202408), reference substance of β,β’ - Dimethylacryloylalkannin (Lot No.: 111689 - 201805), all from the National Institutes for Food and Drug Control; water is Wahaha purified water; methanol (chromatographic grade, Thermo Fisher Scientific); acetonitrile (chromatographic grade, Thermo Fisher Scientific); phosphoric acid (analytical grade, Chongqing Wansheng Chuandong Chemical Co., Ltd.); sodium dodecyl sulfate (chromatographic grade, Shanghai Aladdin Biochemical Technology Co., Ltd.).

[0124] 3. Investigation of Detection Methods

[0125] (1) Investigation of Mobile Phase

[0126] Take 3 g of Baiyu Ointment, weigh accurately, add 25 mL of 60% ethanol solution accurately, weigh again; ultrasonically treat for 30 min, cool, make up the lost weight with 60% ethanol solution, shake well, filter; extract the filtrate with chloroform by shaking for 3 times, 30 mL each time; combine the extraction solutions, evaporate to nearly dry, add an appropriate amount of methanol to dissolve and transfer to a 5 - mL volumetric flask, make up the volume, shake well, filter through a 0.45 - μm microporous membrane to obtain the test solution, and perform gradient elution according to the elution procedures in Table 1 - Table 3 respectively.

[0127] Among them, the specific HPLC chromatographic conditions and system suitability test are as follows:

[0128] Chromatographic column: Shim - pack GIST C 18 (4.6×250 mm, 5 μm); column temperature: 30 °C; mobile phase flow rate: 1.0 mL / min; injection volume: 10 μL; detection wavelength: 270 nm; the number of theoretical plates calculated by the Tanshinone ⅡA peak should be not less than 4000.

[0129] Table 1 Gradient Elution Procedure 1

[0130]

[0131] Table 2 Gradient Elution Procedure 2

[0132]

[0133] Table 3 Gradient Elution Procedure 3

[0134]

[0135]

[0136] The chromatogram of the mobile phase investigation is as follows Figures 1 - 3 shown. The results show that: when gradient elution is carried out according to the elution program in Table 1, the resolution of each component fails to meet the requirements, and the chromatographic peak shape is not good; when gradient elution is carried out according to the elution program in Table 2, the resolution of each component fails to meet the requirements; when gradient elution is carried out according to the elution program in Table 3, the chromatographic peak shape is good and the resolution meets the requirements. Therefore, acetonitrile is selected as mobile phase A, and 0.1% phosphoric acid solution (containing 0.1% sodium dodecyl sulfate) is selected as mobile phase B, and subsequent research is carried out according to the elution program in Table 3.

[0137] (2) Investigation of extraction methods

[0138] Method 1: Take 3 g of Baiyu Ointment, weigh it accurately, accurately add 50 mL of 70% ethanol, weigh again; ultrasonically treat for 30 min, let it cool, make up the lost weight with the above ethanol solution, shake well, filter, and obtain the solution.

[0139] Method 2: Take 3 g of Baiyu Ointment, weigh it accurately, accurately add 50 mL of 70% ethanol, weigh again; heat under reflux for 30 min, let it cool, make up the lost weight with the above ethanol solution, shake well, filter, and obtain the solution.

[0140] Method 3: Take 3 g of Baiyu Ointment, weigh it accurately, accurately add 25 mL of 70% ethanol, weigh again; ultrasonically treat for 30 min, let it cool, make up the lost weight with the above ethanol solution, shake well, filter; accurately measure 10 mL of the continuous filtrate, pass it through D101 macroporous adsorption resin (inner diameter 1.5 cm, column height 13 cm), elute successively with 50 mL of water and 35 mL of 20% ethanol, and discard the eluate; then elute with 15 mL of 40% ethanol, 15 mL of 60% ethanol and 50 mL of 75% ethanol; collect the above eluate and concentrate it, make up the volume to 10 mL with 75% ethanol, shake well, filter, and obtain the solution.

[0141] Method 4: Take 3 g of Baiyu Ointment, weigh it accurately, accurately add 25 mL of 60% ethanol, weigh again; ultrasonically treat for 30 min, let it cool, make up the lost weight with the above ethanol solution, shake well, filter; extract the filtrate with chloroform by shaking 3 times, 30 mL each time; combine the extraction solutions, evaporate to nearly dry, add an appropriate amount of methanol to dissolve and transfer to a 5 mL volumetric flask, make up the volume, shake well, filter, and obtain the solution.

[0142] The HPLC analysis was carried out on the test solution prepared by the above 4 methods under the chromatographic conditions and the optimal gradient elution program in item (1) "Mobile phase investigation", and the results are as follows Figures 4 - 7As shown. It can be seen from the results that when preparing the test solution by Method 1 or Method 2, there is a large interference from impurities in the obtained chromatogram, and the resolution of the chromatographic peaks does not meet the requirements; when preparing the test solution by Method 3, there is a large interference from impurities in the obtained chromatogram, the resolution of the chromatographic peaks does not meet the requirements, and component loss may be caused by adsorption and multiple elutions; when preparing the test solution by Method 4, there is no interference from impurities in the obtained chromatogram, and the chromatographic peaks have a good shape and the resolution meets the requirements. Therefore, Method 4 is selected as the extraction method for the active ingredients in Baiyu Ointment for subsequent research. The chromatograms of the reference substances for each component are as Figure 8 shown (in the figure, 1 is 1,4-bis[4-(glucosyloxy)benzyl]-2-isobutylmalate; 2 is tanshinone I; 3 is 6-gingerol, 4 is tanshinone IIA; 5 is β,β ′ -dimethylacryloylalkanin), and the chromatograms of the sample and the mixed reference substances under Method 4 are as Figure 9 shown (in the figure: A is the chromatogram of the sample; B is the chromatogram of the mixed reference substances).

[0143] (3) Investigation of extraction solvents

[0144] Take 3 g of Baiyu Ointment, accurately weigh 4 portions, and use methanol, 70% methanol, 60% ethanol, and 75% ethanol as extraction solvents respectively. Prepare the test solution according to Method 4 under item (2) "Investigation of extraction methods", and perform HPLC analysis according to the chromatographic conditions and the optimal gradient elution program under item (1) "Investigation of mobile phase", and determine and calculate the contents of each component. The results are shown in Table 4.

[0145] Table 4 Determination results of the contents of each component under different extraction solvents

[0146]

[0147] It can be seen from the results that when extracting with 75% ethanol, the contents of each target component are relatively high and the extraction is relatively complete. Therefore, 75% ethanol solution is selected as the extraction solvent for subsequent research.

[0148] (4) Investigation of extraction time

[0149] Take 3 g of Baiyu Ointment, accurately weigh 3 portions, accurately add 25 mL of 75% ethanol, weigh, and ultrasonically treat for 30 min, 45 min, and 60 min respectively. Prepare the test solution according to Method 4 under item (2) "Investigation of extraction methods", and perform HPLC analysis according to the chromatographic conditions and the optimal gradient elution program under item (1) "Investigation of mobile phase", and determine and calculate the contents of each component. The results are shown in Table 5.

[0150] Table 5 Determination results of the contents of each component under different extraction times

[0151]

[0152] As can be seen from the results, the ultrasonic time has little effect on the contents of the components to be measured. Considering energy conservation and emission reduction, the ultrasonic time of 30 min was selected for subsequent research.

[0153] (5) Investigation of extraction times

[0154] Take 3 g of Baiyu Ointment, accurately weigh 4 portions, use 75% ethanol solution as the extraction solvent, and prepare the test solution according to Method 4 under "(2) Investigation of extraction methods". Extract with 30 mL of chloroform by shaking for 1 time, 2 times, 3 times, and 4 times respectively, and perform HPLC analysis according to the chromatographic conditions and the optimal gradient elution program under "(1) Investigation of mobile phase", and determine and calculate the contents of each component. The results are shown in Table 6.

[0155] Table 6 Determination results of the contents of each component under different chloroform extraction times

[0156]

[0157] As can be seen from the results, when the extraction times are 3 times or 4 times, the determination results of the contents of each component are not significantly different, and both are greater than those when the extraction times are 1 time or 2 times. Therefore, the extraction times of 3 times were selected for subsequent research.

[0158] (6) Investigation of the dosage of extractant

[0159] Take 3 g of Baiyu Ointment, accurately weigh 3 portions, use 75% ethanol solution as the extraction solvent, and prepare the test solution according to the optimal effective component extraction method obtained under "(2) Investigation of extraction methods". Extract with 20 mL, 30 mL, and 40 mL of chloroform by shaking for 3 times respectively, and perform HPLC analysis according to the chromatographic conditions and the optimal gradient elution program under "(1) Investigation of mobile phase", and determine and calculate the contents of each component. The results are shown in Table 7.

[0160] Table 7 Determination results of the contents of each component under different dosages of chloroform

[0161]

[0162] As can be seen from the results, when the dosage of the extractant chloroform is 30 mL and 40 mL, the determination results of the contents of each component are not significantly different. Therefore, 30 mL of chloroform was selected for extraction.

[0163] (7) Selection of detection wavelength

[0164] The ultraviolet detector was used for full-wavelength scanning, and the chromatographic information at the collection wavelengths of 220 nm, 265 nm, 270 nm, 280 nm, and 330 nm was compared. The results showed that when the wavelength was 270 nm, the chromatographic peak shape and resolution were both better than those at other wavelengths, and the baseline was stable. Therefore, 270 nm was selected as the detection wavelength for this study.

[0165] 4. Methodological verification

[0166] (1) Investigation of linear relationship

[0167] Precisely pipette 2 μL, 6 μL, 10 μL, 14 μL, 18 μL, and 22 μL of the mixed reference solution respectively, and inject them for determination according to the optimal chromatographic separation conditions of this study. Using the concentration as the abscissa (x) and the peak area as the ordinate, perform linear regression. The results are shown in Table 8, indicating a good linear relationship.

[0168] Table 8 Results of the investigation of linear relationship

[0169]

[0170] (2) Investigation of precision

[0171] Take 3 g of Baiyu Ointment, accurately weigh it, prepare the test solution according to the optimal extraction method of this study, inject it into the high-performance liquid chromatograph for HPLC analysis, inject continuously for 6 times, measure and calculate the contents of each component and calculate the RSD to investigate the precision of the instrument. The results are shown in Table 9.

[0172] Table 9 Results of the investigation of precision

[0173]

[0174]

[0175] It can be seen from the results that the RSD of each component to be measured is in the range of 0.06% - 0.81%, indicating good precision of the instrument.

[0176] (3) Investigation of repeatability

[0177] Take 3 g of Baiyu Ointment, accurately weigh 6 portions, prepare the test solution according to the optimal extraction method of this study, inject it into the high-performance liquid chromatograph for HPLC analysis, measure and calculate the contents of each component and calculate the RSD. The results are shown in Table 10.

[0178] Table 10 Results of the investigation of repeatability

[0179]

[0180] As can be seen from the results, the RSD values of the content determination results of each component in the six repeated experimental samples are in the range of 0.09%-1.19%, indicating good repeatability.

[0181] (4) Stability investigation

[0182] Take 3 g of Baiyu Ointment, weigh it accurately, prepare the test solution according to the best extraction method in this study, and inject it into the high performance liquid chromatograph for HPLC analysis at 0, 2, 4, 6, 8, 12, and 24 h respectively. Determine and calculate the content of each component and calculate the RSD. The results are shown in Table 11.

[0183] Table 11 Results of stability investigation

[0184]

[0185]

[0186] As can be seen from the results, the RSD values of tanshinone IIA, 1,4-bis[4-(glucosyloxy)benzyl]-2-isobutylmalate, tanshinone I, 6-gingerol, and β,β'-dimethylacryloylalkanin are 0.28%, 0.08%, 0.70%, 0.31%, and 1.30% respectively, indicating that the test solution has good stability within 24 h.

[0187] (5) Standard addition recovery test

[0188] Accurately weigh 6 portions of the sample, 1.5 g for each portion, add each reference substance according to the content ratio of 1:1 of each component in the sample, prepare the test solution, inject it into the high performance liquid chromatograph for HPLC analysis, calculate the average recovery rate and RSD value of each component. The results are shown in Tables 12-16.

[0189] Table 12 Recovery test of tanshinone IIA

[0190]

[0191] Table 13 Recovery test of 1,4-bis[4-(glucosyloxy)benzyl]-2-isobutylmalate

[0192]

[0193]

[0194] Table 14 Recovery test of tanshinone I

[0195]

[0196] Table 15 Recovery test of 6-gingerol

[0197]

[0198] Table 16 Recovery test of β,β'-dimethylacryloyl alkannin

[0199]

[0200] As can be seen from the results, the average recoveries of tanshinone ⅡA, 1,4-bis[4-(glucosyloxy)benzyl]-2-isobutylmalate, tanshinone Ⅰ, 6-gingerol, and β,β'-dimethylacryloyl alkannin were 99.77%, 99.28%, 99.76%, 98.93%, and 99.07% respectively, and the RSD values were 0.82%, 1.23%, 0.78%, 0.98%, and 0.75% respectively, indicating that the recovery of this method was good.

[0201] 5. Relative correction factor

[0202] (1) Calculation of relative correction factor

[0203] Take the mixed reference solution, inject it into the liquid chromatograph for determination. Using tanshinone ⅡA as the internal reference substance, calculate the relative correction factors of 1,4-bis[4-(glucosyloxy)benzyl]-2-isobutylmalate, tanshinone Ⅰ, 6-gingerol, and β,β'-dimethylacryloyl alkannin according to the following formula. The results are shown in Table 17.

[0204]

[0205] Among them, A s is the peak area of the internal reference substance, W S is the concentration (or mass) of the internal reference substance, A k is the peak area of the component to be measured, W k is the concentration (or mass) of the component to be measured.

[0206] Table 17 Relative correction factors

[0207]

[0208] (2) Investigation of the reproducibility of relative correction factors

[0209] ① Investigation using different instruments and different chromatographic columns

[0210] The relative correction factors were determined using different instruments and different chromatographic columns respectively. The results are shown in Table 18. The RSD values of the measured relative correction factors were between 0.44% and 1.38%, indicating that the relative correction factors of each component to be measured had good reproducibility between different instruments and different chromatographic columns.

[0211] Table 18 Influence of different instruments and different chromatographic columns on the relative correction factors of components to be measured

[0212]

[0213]

[0214] ② Investigation of different flow rates

[0215] The relative correction factors were measured at different flow rates respectively, and the results are shown in Table 19. It can be seen from the results that the RSD values of the measured relative correction factors are between 0.38% and 2.26%, indicating that the relative correction factors of each component to be measured have good reproducibility among different flow rates.

[0216] Table 19 Influence of different flow rates on the relative correction factors of components to be measured

[0217]

[0218] ③ Investigation of different column temperatures

[0219] The relative correction factors were measured at different column temperatures respectively, and the results are shown in Table 20. The measured RSD values of the relative correction factors are between 0.38% and 1.52%, indicating that the relative correction factors of each component to be measured have good reproducibility among different column temperatures.

[0220] Table 20 Influence of different column temperatures on the relative correction factors of components to be measured

[0221]

[0222] 6. Chromatographic peak localization of components to be measured

[0223] The relative retention values (r) of each component to be measured and the internal reference tanshinone IIA were calculated respectively, and their reproducibility among different instruments and different chromatographic columns was investigated. The results are shown in Table 21. The relative retention times fluctuate slightly. Finally, the average value of the relative retention time values of each component to be measured in different instruments and different chromatographic columns was selected as the basis for peak localization.

[0224] Table 21 Relative retention times of each component to be measured under different instruments and different chromatographic columns

[0225]

[0226] 7. Comparison of the determination results between the multi-component quantification method and the external standard method

[0227] Three batches of Baiyu Ointment were taken respectively, and the test solution was prepared according to the optimal extraction method of this study. The contents of tanshinone ⅡA, 1,4-bis[4-(glucosyloxy)benzyl]-2-isobutylmalate, tanshinone Ⅰ, 6-gingerol, and β,β'-dimethylacryloylalkanin were determined and calculated by the quantitative analysis of multi-components by single marker (QASM method) and external standard method (ESM method) respectively. The results are shown in Table 22. It can be seen from the results that the relative error between the two groups of contents is <3%, indicating that there is no significant difference in the contents measured by the two methods, which shows that the QASM method established in this study has good accuracy and can be used for the content determination of Baiyu Ointment.

[0228] Table 22 Results of the determination of the contents of 5 components by QASM method and ESM method

[0229]

[0230] Although the present invention has been described in detail with general descriptions, specific embodiments and experiments in the above text, some modifications or improvements can be made on the basis of the present invention, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. A method for determining the contents of 5 components in Baiyu Ointment based on the method of multi-component assay by single marker, characterized in that, The method is to establish the relative correction factors of 1,4-bis[4-(gluconoyloxy)benzyl]-2-isobutyl malate, tanshinone I, 6-gingerol, and β,β ′ -dimethylacryloylalkanin using tanshinone IIA as the internal standard, and calculate the contents of the above components through the relative correction factors. The specific steps include: preparation of the test solution; preparation of the reference solution; chromatographic conditions and system suitability test; determination and calculation.

2. The method for determining the contents of 5 components in Baiyu Ointment based on the multi-component simultaneous determination method according to claim 1, wherein, The preparation of the test solution is specifically as follows: Take 1.8 - 4.2 g of Baiyu Ointment, accurately weigh it, accurately add 15 - 35 mL of 70% - 100% methanol solution or 60% - 75% ethanol solution, weigh again; ultrasonically treat for 30 - 60 min, let it cool, make up the lost weight with the above methanol solution or ethanol solution, shake well, filter; extract the filtrate with chloroform by shaking for 1 - 4 times, 20 - 40 mL each time; combine the extraction solutions, evaporate to nearly dryness, add an appropriate amount of methanol to dissolve and transfer to a 5 mL volumetric flask, make up the volume, shake well, filter, and obtain the solution.

3. The method for determining the contents of 5 components in Baiyu Ointment based on the one-test-multi-evaluation method according to claim 2, wherein, The preparation of the test solution is specifically as follows: Take 2.4 - 4.2 g of Baiyu Ointment, accurately weigh it, accurately add 20 - 35 mL of 60% - 75% ethanol solution, weigh again; ultrasonically treat for 30 - 45 min, let it cool, make up the lost weight with the above ethanol solution, shake well, filter; extract the filtrate with chloroform by shaking for 3 - 4 times, 30 - 40 mL each time; combine the extraction solutions, evaporate to nearly dryness, add an appropriate amount of methanol to dissolve and transfer to a 5 mL volumetric flask, make up the volume, shake well, filter through a 0.45 μm microporous membrane, and obtain the solution.

4. The method for determining the contents of 5 components in Baiyu Ointment based on the multi-component simultaneous determination method according to claim 3, wherein, The preparation of the test solution is specifically as follows: Take 3 g of Baiyu Ointment, accurately weigh it, accurately add 25 mL of 75% ethanol solution, weigh again; ultrasonically treat for 30 min, let it cool, make up the lost weight with the above ethanol solution, shake well, filter; extract the filtrate with chloroform by shaking for 3 times, 30 mL each time; combine the extraction solutions, evaporate to nearly dryness, add an appropriate amount of methanol to dissolve and transfer to a 5 mL volumetric flask, make up the volume, shake well, filter through a 0.45 μm microporous membrane, and obtain the solution.

5. The method for determining the contents of 5 components in Baiyu Ointment based on the one-test-multiple-evaluation method according to claim 1, wherein The preparation of the reference solution is specifically as follows: Take an appropriate amount of tanshinone ⅡA reference substance, accurately weigh it and place it in a brown volumetric flask, add methanol to make a reference solution containing 0.02 mg per 1 mL, and obtain the solution.

6. The method for determining the contents of 5 components in Baiyu Ointment based on the one-measure multi-evaluation method according to claim 1, characterized in that, The chromatographic conditions and system suitability test are specifically as follows: The chromatographic column is packed with octadecylsilane chemically bonded silica gel; the column temperature is 25 - 35 °C; acetonitrile is used as mobile phase A, and 0.1% phosphoric acid solution containing 0.1% sodium dodecyl sulfate is used as mobile phase B for gradient elution; the flow rate of the mobile phase is 0.9 - 1.1 mL / min; the injection volume is 10 μL; the detection wavelength is 270 nm; the number of theoretical plates calculated based on the tanshinone ⅡA peak should be not less than 4000; Among them, the gradient elution program is specifically as follows:

7. The method for determining the contents of 5 components in Baiyu Ointment based on the one-test-multiple-evaluation method according to claim 6, wherein, In the chromatographic conditions and system suitability test, the column temperature is 30 °C, and the flow rate of the mobile phase is 1.0 mL / min.

8. The method for determining the contents of 5 components in Baiyu Ointment based on the one-test-multiple-evaluation method according to claim 1, wherein The determination and calculation are specifically as follows: Inject the test solution and the reference solution into a high-performance liquid chromatograph for determination, and calculate the contents of each component according to the relative correction factors of tanshinone IIA, 1,4-bis[4-(glucosyloxy)benzyl]-2-isobutylmalate, tanshinone I, 6-gingerol, and β,β ′ -dimethylacrylalkannin.

9. The method for determining the contents of 5 components in Baiyu Ointment based on the one-test-multiple-evaluation method according to claim 8, wherein, In the determination and calculation, the relative retention times of tanshinone IIA, 1,4-bis[4-(glucosyloxy)benzyl]-2-isobutyl malate, tanshinone I, 6-gingerol, and β,β ′ -dimethylacrylalkannin are 0.33, 0.44, 0.83, and 1.53, respectively, and the relative retention times are within ±10% of the specified values.

10. The method for determining the contents of 5 components in Baiyu Ointment based on the one-test-multiple-evaluation method according to claim 8, characterized in that, In the said determination and calculation, the relative correction factors of tanshinone IIA, 1,4-bis[4-(gluconoxy)benzyl]-2-isobutylmalate, tanshinone I, 6-gingerol, and β,β ′ -dimethylacryloylalkanin are 2.65, 1.37, 0.68, and 1.12 respectively.

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