A HPLC method for simultaneous determination of six active ingredients in Sophora flavescens

Abstract

The present invention proposes a method for simultaneously determining the contents of six active ingredients of the Zhuang medicine Sophora flavescens by HPLC, which belongs to the technical field of traditional Chinese medicine analysis. The method comprises the following steps: S1. Preparation of standard stock solution; S2. Preparation of standard mixed solution; S3. Preparation of test solution; S4. Chromatographic conditions; S5. Determination. The present invention compares and analyzes the content determination results of 10 batches of samples and finds that there are differences in the average contents of the six components of neochlorogenic acid, chlorogenic acid, caffeic acid, cryptochlorogenic acid, isochlorogenic acid A, and isochlorogenic acid C in different batches of Sophora flavescens medicinal materials. The present invention establishes a method for simultaneously determining the contents of the six components of neochlorogenic acid, chlorogenic acid, caffeic acid, cryptochlorogenic acid, isochlorogenic acid A, and isochlorogenic acid C in the Zhuang medicine Sophora flavescens, which will provide a theoretical basis for the quality control and standardized planting of Sophora flavescens.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine analysis, and in particular to a method for simultaneously determining the contents of six active ingredients in the Zhuang medicine Sophora flavescens by HPLC. Background Art

[0002] The Zhuang medicinal herb, Streptocaulon grif fithii Hook.f., is the dried root of the Asclepiadaceae plant, Streptocaulon grif fithii Hook.f., distributed in Guangxi and Yunnan, China, India, Vietnam, Myanmar, Laos, Cambodia, and Malaysia. Also known as vine sophora flavescens, old crow's beak, hairy green twig, southern sophora flavescens, milk vine, motor, red horse lian'an, tiger-yin vine, and old crow's beak, its woody roots are used to treat colds, fevers, enteritis, dysentery, stomachache, bruises, swelling, and snake bites. A Guangxi specialty and staple Zhuang and Yao medicinal herb, vine sophora flavescens is included in the national standard ethnic medicine and the main ingredient in the well-known Zhuang medicine Wai Gan Feng Sha Granules (Tablets). It is also one of the main ingredients in the Dai medicine classic formula called Hatun San. Zhang Lin isolated 19 compounds from the ethanol extract of the root of Sophora flavescens, of which 9 were triterpenoid sapogenins and 8 were cardiac glycosides. The compounds were α-amyrin acetate, lupeol acetate, 11-ethoxy-3-acetyl-12-ursene-3-ol, 11-keto-α-amyrin acetate, α-amyrin, lupeol, 24-methylenecycloxylbolanol, cycloxylbol-23-ene-3β, 25-diol, β-guosterol, periwinklein digitotoxin glycoside, Sophora flavescens aglycone, cycloxylbolane-3, 24,25-triol, nicotinic acid, digitotoxin aglycone, 16-O-acetylhydroxydigitotoxin aglycone, periwinklein, 16-O-acetylhydroxyperipterigin, periwinklein glucoside, and Sophora flavescens aglycone A. The research team conducted a preliminary analysis of the liquid-to-mass ratio of the Sophora flavescens extract and found that the Sophora flavescens extract also contains active ingredients such as neochlorogenic acid, chlorogenic acid, caffeic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid C, 4-methoxysalicylaldehyde, and psoralen. According to literature reports, chlorogenic acid components have good antiviral, liver-protecting and jaundice-reducing, antioxidant, anti-inflammatory, and antibacterial activities. Summary of the Invention

[0003] The purpose of the present invention is to propose a HPLC method for simultaneously determining the contents of six active ingredients of the Zhuang medicine Sophora flavescens. The present invention establishes a method for simultaneously determining the contents of six components of the Zhuang medicine Sophora flavescens, including neochlorogenic acid, chlorogenic acid, caffeic acid, cryptochlorogenic acid, isochlorogenic acid A, and isochlorogenic acid C, which will provide a theoretical basis for the quality control and standardized cultivation of Sophora flavescens.

[0004] The technical solution of the present invention is achieved as follows:

[0005] The present invention provides a method for simultaneously determining the contents of six active ingredients of the Zhuang medicinal herb Sophora flavescens by HPLC, comprising the following steps:

[0006] S1. Preparation of standard stock solutions: Accurately weigh appropriate amounts of neochlorogenic acid, chlorogenic acid, caffeic acid, cryptochlorogenic acid, isochlorogenic acid A, and isochlorogenic acid C standard reference substances, and add methanol to prepare standard stock solutions;

[0007] S2. Preparation of standard mixed solution: Accurately pipette an appropriate amount of each standard stock solution into a volumetric flask to prepare a standard mixed solution;

[0008] S3. Preparation of test solution: Accurately weigh fine powder of Sophora flavescens and place it in a flat-bottom flask. Heat under reflux for extraction. Filter and combine the filtrates. Evaporate to dryness. Add methanol to the residue and dilute to volume in a volumetric flask. Shake well and filter to obtain the test solution.

[0009] S4. Chromatographic conditions: X-PeonyxR C18 column; mobile phase A: 0.2-0.5% phosphoric acid, mobile phase B: acetonitrile, gradient elution; flow rate: 0.5-1.5 mL / min; column temperature: 30-40°C; detection wavelength: 300-350 nm; injection volume: 5-15 μL.

[0010] S5. Determination: Accurately aspirate the standard mixed solution described in step S2 and the test solution described in step S3, inject them into a high performance liquid chromatograph, and determine them according to the chromatographic conditions described in step S4.

[0011] As a further improvement of the present invention, the specification of the X-PeonyxR C18 chromatographic column is 250×4.6 mm.

[0012] As a further improvement of the present invention, the gradient elution conditions are: 0-20 min, 5%-6.5% phase B, 20-45 min, 6.5% phase B, 45-50 min, 6.5%-13% phase B, 50-52 min, 13-15% phase B, 52-90 min, 15% phase B, 90-92 min, 15%-16.5% phase B, 92-125 min, 16.5% phase B, 125-127 min, 16.5%-5% phase B, 127-130 min, 5% phase B.

[0013] As a further improvement of the present invention, the chromatographic conditions are as follows: X-PeonyxR C18 chromatographic column; mobile phase A is 0.3% phosphoric acid, phase B is acetonitrile, gradient elution; volume flow rate is 1 mL / min; column temperature is 35°C; detection wavelength is 325 nm; injection volume is 10 μL.

[0014] As a further improvement of the present invention, the concentration of neochlorogenic acid in the standard stock solution is 2-2.2 mg / mL, chlorogenic acid is 2.1-2.3 mg / mlL, caffeic acid is 1.2-1.5 mg / mL, cryptochlorogenic acid is 1.5-1.7 mg / mL, isochlorogenic acid A is 1.0-1.2 mg / mL, and isochlorogenic acid C is 1.7-2.0 mg / mL.

[0015] As a further improvement of the present invention, the concentration of neochlorogenic acid in the standard mixed solution is 0.02-0.03 mg / mL, chlorogenic acid is 0.03-0.04 mg / mL, caffeic acid is 0.006-0.009 mg / mL, cryptochlorogenic acid is 0.02-0.03 mg / mL, isochlorogenic acid A is 0.004-0.005 mg / mL, and isochlorogenic acid C is 0.02-0.03 mg / mL.

[0016] As a further improvement of the present invention, the retention times of the neochlorogenic acid, chlorogenic acid, caffeic acid, cryptochlorogenic acid, isochlorogenic acid A, and isochlorogenic acid C are 18, 33, 36, 44, 91, and 110 min, respectively.

[0017] As a further improvement of the present invention, the heating reflux extraction is performed 1-3 times, and the heating time is 0.5-1.5 hours.

[0018] The present invention has the following beneficial effects: The present invention establishes a method for simultaneously determining the content of neochlorogenic acid, chlorogenic acid, caffeic acid, cryptochlorogenic acid, isochlorogenic acid A, and isochlorogenic acid C in Sophora flavescens by HPLC. The method is simple, precise, and reproducible, and can provide a theoretical basis for the standardized planting and quality control of Sophora flavescens. The present invention compares and analyzes the content determination results of 10 batches of samples, and it is known that there are differences in the average content of the six components of neochlorogenic acid, chlorogenic acid, caffeic acid, cryptochlorogenic acid, isochlorogenic acid A, and isochlorogenic acid C in different batches of Sophora flavescens medicinal materials. The present invention establishes a method for simultaneously determining the six components of neochlorogenic acid, chlorogenic acid, caffeic acid, cryptochlorogenic acid, isochlorogenic acid A, and isochlorogenic acid C in the Zhuang medicine Sophora flavescens, which will provide a theoretical basis for the quality control and standardized planting of Sophora flavescens. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0020] Figure 1HPLC chromatogram of mixed reference solution of neochlorogenic acid, chlorogenic acid, caffeic acid, cryptochlorogenic acid, isochlorogenic acid A and isochlorogenic acid C

[0021] Figure 2 is the HPLC spectrum of the sample solution. DETAILED DESCRIPTION

[0022] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described 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 making any creative efforts are within the scope of protection of the present invention.

[0023] The instruments, reagents, and medicinal materials used in the examples of the present invention are as follows:

[0024] LC-2030C 3D high-performance liquid chromatograph (Shimadzu Enterprise Management Co., Ltd.); ME104E electronic balance (Mettler-Toledo Instrument Co., Ltd.); MS205U 1 / 100,000 electronic balance (Mettler-Toledo Instrument Co., Ltd.); ultrasonic cleaner (SG250HE, Shanghai Guante Ultrasonic Instrument Co., Ltd.); electric constant temperature water bath (HH-S4, Jiangsu Jinyi Instrument Technology Co., Ltd.);

[0025] Chlorogenic acid reference substance (110753-202018, content 96.1%) and caffeic acid reference substance (110885-201703) were purchased from the China Food and Drug Inspection Institute; neochlorogenic acid reference substance (906-33-2, content 98%), cryptochlorogenic acid reference substance (905-99-7, content 98%), isochlorogenic acid A reference substance (2450-53-5, content 98%), and isochlorogenic acid C reference substance (57378-72-0, content 98%) were purchased from Chengdu Purifa Technology Development Co., Ltd.; acetonitrile (chromatographic grade; Fisher); methanol (chromatographic grade; Fisher);

[0026] The samples of Sophora flavescens used in the content determination institute were purchased from Yulin City and identified by Professor Wei Songji of Guangxi University of Chinese Medicine. They were found to be in compliance with the Sophora flavescens standards in the Compilation of Quality Standards for Zhuang Medicinal Materials in Guangxi Zhuang Autonomous Region (Volume 1) and were the dried roots of Streptocaulon juventas (Lour.) Merr. of the Asclepiadaceae family.

[0027] Example 1

[0028] This embodiment provides a method for simultaneously determining the contents of six active ingredients of the Zhuang herb Sophora flavescens by HPLC, comprising the following steps:

[0029] S1. Preparation of standard stock solutions: Accurately weigh appropriate amounts of neochlorogenic acid, chlorogenic acid, caffeic acid, cryptochlorogenic acid, isochlorogenic acid A, and isochlorogenic acid C standard reference substances, and add methanol to prepare standard stock solutions. The concentrations of neochlorogenic acid are 2 mg / mL, chlorogenic acid is 2.1 mg / mL, caffeic acid is 1.2 mg / mL, cryptochlorogenic acid is 1.5 mg / mL, isochlorogenic acid A is 1.0 mg / mL, and isochlorogenic acid C is 1.7 mg / mL.

[0030] S2. Preparation of standard mixed solution: Accurately pipette an appropriate amount of each standard stock solution into a volumetric flask to prepare a standard mixed solution, wherein the concentration of the standard mixed solution is 0.02 mg / mL for neochlorogenic acid, 0.03 mg / mL for chlorogenic acid, 0.006 mg / mL for caffeic acid, 0.02 mg / mL for cryptochlorogenic acid, 0.004 mg / mL for isochlorogenic acid A, and 0.02 mg / mL for isochlorogenic acid C.

[0031] S3. Preparation of test solution: Accurately weigh 2.0 g of Sophora flavescens fine powder (passed through a No. 3 sieve) and place in a flat-bottom flask. Heat and reflux twice, adding 100 mL of methanol each time for 1 h. Filter, combine the filtrates, evaporate to dryness, and dilute the residue to volume with methanol in a 10 mL volumetric flask. Shake well and filter through a 0.45 μm microporous filter to obtain the test solution.

[0032] S4. Chromatographic conditions: X-PeonyxR C18 column (250 × 4.6 mm); mobile phase A: 0.2% phosphoric acid, mobile phase B: acetonitrile, gradient elution; flow rate: 0.5 mL / min; column temperature: 30°C; detection wavelength: 300 nm; injection volume: 5 μL.

[0033] The gradient elution conditions are: 0-20 min, 5%-6.5% phase B, 20-45 min, 6.5% phase B, 45-50 min, 6.5%-13% phase B, 50-52 min, 13-15% phase B, 52-90 min, 15% phase B, 90-92 min, 15%-16.5% phase B, 92-125 min, 16.5% phase B, 125-127 min, 16.5%-5% phase B, 127-130 min, 5% phase B;

[0034] S5. Determination: Accurately aspirate the standard mixed solution described in step S2 and the test solution described in step S3, inject them into a high performance liquid chromatograph, and determine them according to the chromatographic conditions described in step S4.

[0035] Example 2

[0036] This embodiment provides a method for simultaneously determining the contents of six active ingredients of the Zhuang herb Sophora flavescens by HPLC, comprising the following steps:

[0037] S1. Preparation of standard stock solutions: Accurately weigh appropriate amounts of neochlorogenic acid, chlorogenic acid, caffeic acid, cryptochlorogenic acid, isochlorogenic acid A, and isochlorogenic acid C standard reference substances, and add methanol to prepare standard stock solutions. The concentrations of neochlorogenic acid are 2.2 mg / mL, chlorogenic acid is 2.3 mg / mL, caffeic acid is 1.5 mg / mL, cryptochlorogenic acid is 1.7 mg / mL, isochlorogenic acid A is 1.2 mg / mL, and isochlorogenic acid C is 2.0 mg / mL.

[0038] S2. Preparation of standard mixed solution: Accurately pipette an appropriate amount of each standard stock solution into a volumetric flask to prepare a standard mixed solution, wherein the concentration of the standard mixed solution is 0.03 mg / mL for neochlorogenic acid, 0.04 mg / mL for chlorogenic acid, 0.009 mg / mL for caffeic acid, 0.03 mg / mL for cryptochlorogenic acid, 0.005 mg / mL for isochlorogenic acid A, and 0.03 mg / mL for isochlorogenic acid C.

[0039] S3. Preparation of test solution: Accurately weigh 2.0 g of Sophora flavescens fine powder (passed through a No. 3 sieve) and place in a flat-bottom flask. Heat and reflux for 3 times, adding 100 mL of methanol each time for 1.5 h. Filter, combine the filtrates, evaporate to dryness, and dilute the residue to volume with methanol in a 10 mL volumetric flask. Shake well and filter through a 0.45 μm microporous filter to obtain the test solution.

[0040] S4. Chromatographic conditions: X-PeonyxR C18 column (250 × 4.6 mm); mobile phase A: 0.5% phosphoric acid, mobile phase B: acetonitrile, gradient elution; flow rate: 1.5 mL / min; column temperature: 40°C; detection wavelength: 350 nm; injection volume: 15 μL.

[0041] The gradient elution conditions are: 0-20 min, 5%-6.5% phase B, 20-45 min, 6.5% phase B, 45-50 min, 6.5%-13% phase B, 50-52 min, 13-15% phase B, 52-90 min, 15% phase B, 90-92 min, 15%-16.5% phase B, 92-125 min, 16.5% phase B, 125-127 min, 16.5%-5% phase B, 127-130 min, 5% phase B;

[0042] S5. Determination: Accurately aspirate the standard mixed solution described in step S2 and the test solution described in step S3, inject them into a high performance liquid chromatograph, and determine them according to the chromatographic conditions described in step S4.

[0043] Example 3

[0044] This embodiment provides a method for simultaneously determining the contents of six active ingredients of the Zhuang herb Sophora flavescens by HPLC, comprising the following steps:

[0045] S1. Preparation of standard stock solutions: Accurately weigh appropriate amounts of neochlorogenic acid, chlorogenic acid, caffeic acid, cryptochlorogenic acid, isochlorogenic acid A, and isochlorogenic acid C standard reference substances, and add methanol to prepare standard stock solutions. The concentrations of neochlorogenic acid are 2.016 mg / mL, chlorogenic acid is 2.217 mg / mL, caffeic acid is 1.289 mg / mL, cryptochlorogenic acid is 1.503 mg / mL, isochlorogenic acid A is 1.053 mg / mL, and isochlorogenic acid C is 1.801 mg / mL.

[0046] S2. Preparation of standard mixed solution: Accurately pipette an appropriate amount of each standard stock solution into a volumetric flask to prepare a standard mixed solution. The concentration of the standard mixed solution is 0.0272 mg / mL for neochlorogenic acid, 0.0399 mg / mL for chlorogenic acid, 0.0070 mg / mL for caffeic acid, 0.0293 mg / mL for cryptochlorogenic acid, 0.0047 mg / mL for isochlorogenic acid A, and 0.0288 mg / mL for isochlorogenic acid C.

[0047] S3. Preparation of test solution: Accurately weigh 2.0 g of Sophora flavescens fine powder (passed through a No. 3 sieve) and place in a flat-bottom flask. Heat and reflux twice, adding 100 mL of methanol each time for 1 h. Filter, combine the filtrates, evaporate to dryness, and dilute the residue to volume with methanol in a 10 mL volumetric flask. Shake well and filter through a 0.45 μm microporous filter to obtain the test solution.

[0048] S4. Chromatographic conditions: X-PeonyxR C18 column (250 × 4.6 mm); mobile phase A: 0.3% phosphoric acid, mobile phase B: acetonitrile, gradient elution; flow rate: 1 mL / min; column temperature: 35°C; detection wavelength: 325 nm; injection volume: 10 μL.

[0049] The gradient elution conditions are: 0-20 min, 5%-6.5% phase B, 20-45 min, 6.5% phase B, 45-50 min, 6.5%-13% phase B, 50-52 min, 13-15% phase B, 52-90 min, 15% phase B, 90-92 min, 15%-16.5% phase B, 92-125 min, 16.5% phase B, 125-127 min, 16.5%-5% phase B, 127-130 min, 5% phase B;

[0050] S5. Determination: Accurately draw the standard mixed solution described in step S2 and the test solution described in step S3, respectively, inject them into the high performance liquid chromatograph, and determine them according to the chromatographic conditions described in step S4. The retention times of the neochlorogenic acid, chlorogenic acid, caffeic acid, cryptochlorogenic acid, isochlorogenic acid A, and isochlorogenic acid C are 18, 33, 36, 44, 91, and 110 min, respectively. The number of theoretical plates is not less than 5000, and the separation degree of each component is greater than 1.5. The chromatographic results are shown in FIG. Figure 1 and Figure 2 Among them, 1 is neochlorogenic acid; 2 is chlorogenic acid; 3 is caffeic acid; 4 is cryptochlorogenic acid; 5 isochlorogenic acid A; 6 isochlorogenic acid C.

[0051] Example 4 Linear range investigation

[0052] Accurately pipette appropriate amount of standard stock solution, dilute step by step, and prepare 6 standard series working solutions with different concentrations.

[0053] A series of standard working solutions were sequentially injected into an ultra-high performance liquid chromatograph at an injection volume of 10 μL. The chromatographic conditions in step S4 of Example 3 were used for measurement, and the peak area of ​​each component was recorded. A standard curve was plotted with the injection concentration (mg / mL) as the abscissa (X) and the corresponding peak area as the ordinate (Y). The regression equation was obtained. The results are shown in Table 1.

[0054] Table 1 Regression equation and linear range

[0055] Compound Regression equation <![CDATA[R 2 ]]> <![CDATA[Linear range mg.mL -1 > Neochlorogenic acid Y=27353200.89X+63725.81 0.9991 0.0105-0.1008 Chlorogenic acid Y=27480812.83X+212946.68 0.9998 0.0089-1.2859 Caffeic acid Y=51234553.40X+7193073 0.9993 0.0026-0.0902 Cryptochlorogenic acid Y=26013330.77X+13834.11 0.9990 0.0120-0.1585 Isochlorogenic acid A Y=31816639.32X+150252.12 0.9999 0.0018-0.0632 Isochlorogenic acid C Y=31816639.32X+150252.12 0.9994 0.0180-0.8645

[0056] Example 5 Precision Test

[0057] The standard mixed solution in step S2 in Example 3 was taken and sampled and measured 6 times according to the chromatographic conditions in step S4 in Example 3. The RSDs of the peak areas of neochlorogenic acid, chlorogenic acid, caffeic acid, cryptochlorogenic acid, isochlorogenic acid A, and isochlorogenic acid C were calculated to be 0.11%, 0.13%, 0.14%, 0.14%, 1.9%, and 0.50%, respectively, indicating that the instrument had good precision.

[0058] Example 6 Repeatability Test

[0059] 6 portions of 4.0 g fine powder of Sophora flavescens (batch number 20220317) were taken and accurately weighed. 6 portions of the test solution were prepared according to the test solution preparation method in step S3 of Example 3. The contents were determined according to the chromatographic conditions in step S4. The RSDs of the contents of neochlorogenic acid, chlorogenic acid, caffeic acid, cryptochlorogenic acid, isochlorogenic acid A, and isochlorogenic acid C were 0.5%, 0.56%, 1.17%, 1.12%, and 0.51%, respectively. The results showed that the method had good reproducibility.

[0060] Example 7 Stability Test

[0061] The same test solution (batch number 20220317) was taken and 10 μL was injected at 0, 4, 8, 12, 24, and 48 h under the chromatographic conditions in step S4. The results showed that the peak areas RSDs of neochlorogenic acid, chlorogenic acid, caffeic acid, cryptochlorogenic acid, isochlorogenic acid A, and isochlorogenic acid C were 0.36%, 0.35%, 0.75%, 1.46%, 1.17%, and 0.52%, respectively, indicating that the test solution had good stability within 48 h.

[0062] 2.7 Sample recovery test

[0063] Six portions of Sophora flavescens fine powder (batch number 20220317), 2.0 g each, were accurately weighed and added to a control solution at a 100% level. The test solution was prepared according to the test solution preparation method in step S3 of Example 3. 10 μL of each sample was injected for analysis under the chromatographic conditions in step S4, and the recovery was calculated. The average recoveries of neochlorogenic acid, chlorogenic acid, caffeic acid, cryptochlorogenic acid, isochlorogenic acid A, and isochlorogenic acid C were 98.07%, 99.11%, 99.16%, 99.34%, 100.84%, and 100.50%, respectively, with RSDs of 2.37%, 2.40%, 1.85%, 2.34%, 2.70%, and 2.36%, respectively. The results are shown in Table 2.

[0064] Table 2 Results of sample recovery test (n=6)

[0065]

[0066]

[0067] Example 6 Sample Determination

[0068] About 4.0 g of each of 10 batches of Sophora flavescens fine powder was taken and accurately weighed. The test solution was prepared according to the test solution preparation method in step S3 of Example 3. The chromatographic conditions were determined in step S4, and the contents of neochlorogenic acid, chlorogenic acid, caffeic acid, cryptochlorogenic acid, isochlorogenic acid A, and isochlorogenic acid C in each batch of medicinal materials were calculated. The results are shown in Table 3.

[0069] Table 3 Content determination results

[0070]

[0071] By comparing and analyzing the content determination results of 10 batches of samples, it can be seen that there are differences in the average contents of the six components of neochlorogenic acid, chlorogenic acid, caffeic acid, cryptochlorogenic acid, isochlorogenic acid A, and isochlorogenic acid C in different batches of Sophora flavescens medicinal materials. This paper establishes a method for simultaneously determining the content of the six components of neochlorogenic acid, chlorogenic acid, caffeic acid, cryptochlorogenic acid, isochlorogenic acid A, and isochlorogenic acid C in the Zhuang medicine Sophora flavescens, which will provide a theoretical basis for the quality control and standardized cultivation of Sophora flavescens.

[0072] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for simultaneously determining the contents of six active ingredients in the strong medicinal herb Sophora flavescens by HPLC, characterized in that: The following steps are involved: S1. Preparation of standard stock solutions: Accurately weigh appropriate amounts of neochlorogenic acid, chlorogenic acid, caffeic acid, cryptochlorogenic acid, isochlorogenic acid A, and isochlorogenic acid C standard reference substances, and add methanol to prepare standard stock solutions; S2. Preparation of standard mixed solution: Accurately pipette an appropriate amount of each standard stock solution into a volumetric flask to prepare a standard mixed solution; S3. Preparation of test solution: Accurately weigh fine powder of Sophora flavescens and place it in a flat-bottom flask. Heat under reflux for extraction. Filter and combine the filtrates. Evaporate to dryness. Add methanol to the residue and dilute to volume in a volumetric flask. Shake well and filter to obtain the test solution. S4. Chromatographic conditions: X-PeonyxR C18 column; mobile phase A: 0.2-0.5% phosphoric acid, mobile phase B: acetonitrile, gradient elution; flow rate: 0.5-1.5 mL / min; column temperature: 30-40°C; detection wavelength: 300-350 nm; injection volume: 5-15 μL; gradient elution: 0-20 min, 5%-6.5% phase B, 20-45 min, 6.5% phase B, 45-50 min, 6.5%-13% phase B, 50-52 min, 13-15% phase B, 52-90 min, 15% phase B, 90-92 min, 15%-16.5% phase B, 92-125 min, 16.5% phase B, 125-127 min, 16.5%-5% phase B, 127-130 min, 5% phase B. S5. Determination: Accurately aspirate the standard mixed solution described in step S2 and the test solution described in step S3, respectively, inject them into a high performance liquid chromatograph, and determine according to the chromatographic conditions described in step S4 to obtain; the retention times of the neochlorogenic acid, chlorogenic acid, caffeic acid, cryptochlorogenic acid, isochlorogenic acid A, and isochlorogenic acid C are 18, 33, 36, 44, 91, and 110 min, respectively.

2. The method for simultaneously determining the contents of 6 active ingredients of the strong medicinal herb Sophora flavescens by HPLC according to claim 1, characterized in that: The specification of the X-PeonyxR C18 chromatographic column is 250×4.6 mm.

3. The method for simultaneously determining the contents of 6 active ingredients of the strong medicinal herb Sophora flavescens by HPLC according to claim 1, characterized in that: The chromatographic conditions are as follows: an X-PeonyxR C18 column; mobile phase A is 0.3% phosphoric acid, phase B is acetonitrile, gradient elution; volume flow rate is 1 mL / min; column temperature is 35° C.; detection wavelength is 325 nm; and injection volume is 10 μL.

4. The method for simultaneously determining the contents of 6 active ingredients of the strong medicinal herb Sophora flavescens by HPLC according to claim 1, characterized in that: The concentration of neochlorogenic acid in the standard stock solution is 2-2.2 mg / mL, chlorogenic acid is 2.1-2.3 mg / mL, caffeic acid is 1.2-1.5 mg / mL, cryptochlorogenic acid is 1.5-1.7 mg / mL, isochlorogenic acid A is 1.0-1.2 mg / mL, and isochlorogenic acid C is 1.7-2.0 mg / mL.

5. The method for simultaneously determining the contents of 6 active ingredients of the strong medicinal herb Sophora flavescens by HPLC according to claim 1, characterized in that: The concentration of neochlorogenic acid in the standard mixed solution is 0.02-0.03 mg / mL, chlorogenic acid is 0.03-0.04 mg / mL, caffeic acid is 0.006-0.009 mg / mL, cryptochlorogenic acid is 0.02-0.03 mg / mL, isochlorogenic acid A is 0.004-0.005 mg / mL, and isochlorogenic acid C is 0.02-0.03 mg / mL.

6. The method for simultaneously determining the contents of six active ingredients of the strong medicinal herb Sophora flavescens by HPLC according to claim 1, characterized in that: The heating reflux extraction is performed 1-3 times, and the heating time is 0.5-1.5 hours.

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