Method for detecting flavonoid components in pogonatherum crinitum medicinal material

Abstract

The invention relates to the field of traditional Chinese medicine, in particular to a method for detecting the content of flavonoid components in a pogonatherum crinitum medicinal material, which comprises the following steps: providing a reference substance solution, reference substances in the reference substance solution comprising flavonoid components; preparing a test solution by using the pogonatherum crinitum medicinal material sample to be detected; and detecting the reference substance solution and the test solution by adopting a high performance liquid chromatography, and determining the content of the flavonoid component corresponding to the reference substance in the pogonatherum crinitum medicinal material sample to be detected, the high performance liquid chromatography meets the following conditions: the stationary phase takes octadecylsilane chemically bonded silica as a filler; the mobile phase comprises a mobile phase A and a mobile phase B, the mobile phase A comprises acetonitrile, and the mobile phase B comprises a phosphoric acid aqueous solution. The method is simple, reliable and high in objectivity, has good precision, repeatability, specificity and stability, can be used for comprehensively evaluating the quality of the pogonatherum crinitum medicinal material, and can provide a method and basis for formulating the quality standard of the high-quality pogonatherum crinitum medicinal material and stabilizing the production place of the raw material.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of traditional Chinese medicine, in particular to a detection method of flavonoid components in Cuscuta chinensis. BACKGROUND

[0002] Cuscuta chinensis, also known as Ai Fei Timon, is the dry aboveground part of Cuscuta chinensis Lam. of Convolvulaceae. Cuscuta chinensis was first recorded in Shennong Bencao Jing and listed as the top grade. It is distributed in various parts of China and likes to grow in fields, wastelands or shrubs. It generally relies on nutrients from host plants of Leguminosae, Asteraceae, Urticaceae and Linaceae to survive and belongs to parasitic plants.

[0003] Cuscuta chinensis plays an important role in traditional Chinese medicine. Modern medical research shows that Cuscuta chinensis contains flavonoids, sterols, polysaccharides, lignans, alkaloids and terpenoids, which have antioxidant, anti-inflammatory, immunoregulatory, hepatoprotective, anticancer, nerve-protective and lipid-lowering activities.

[0004] The quality control of Cuscuta chinensis medicinal material has not been included in Chinese Pharmacopoeia. Therefore, it is necessary to establish a detection method of flavonoid components in Cuscuta chinensis to better control the quantity and quality transmission changes of effective components in the production process. SUMMARY

[0005] Therefore, one or more embodiments of the present application provide a detection method for determining flavonoid components in Cuscuta chinensis medicinal material. The technical solutions are as follows:

[0006] One or more embodiments of the present application provide a detection method for determining flavonoid components in Cuscuta chinensis medicinal material. The detection method comprises the following steps:

[0007] A control solution is provided, and the control in the control solution includes flavonoid components;

[0008] A test sample solution is prepared from the Cuscuta chinensis medicinal material to be tested; and

[0009] The control solution and the test sample solution are detected by high performance liquid chromatography to determine the content of the flavonoid components corresponding to the control in the Cuscuta chinensis medicinal material to be tested;

[0010] The high performance liquid chromatography meets the following conditions:

[0011] The stationary phase is octadecylsilane bonded silica gel as the filler;

[0012] The mobile phase includes mobile phase A and mobile phase B, the mobile phase A includes acetonitrile, and the mobile phase B includes phosphoric acid aqueous solution;

[0013] The elution procedure is as follows:

[0014] 0-8 min, the volume ratio of mobile phase A is increased from 7% to 16%,

[0015] 8-30 min, the volume ratio of mobile phase A is kept at 16%,

[0016] 30-32 min, the volume ratio of mobile phase A is increased from 16% to 20%,

[0017] 32-42 min, the volume ratio of mobile phase A is kept at 20%,

[0018] 42-48 min, the volume ratio of mobile phase A is increased from 20% to 25%,

[0019] 48-68 min, the volume ratio of mobile phase A is increased from 25% to 39%,

[0020] 68-76 min, the volume ratio of mobile phase A is increased from 39% to 90%,

[0021] 76-78 min, the volume ratio of mobile phase A is decreased from 90% to 7%,

[0022] 78-85 min, the volume ratio of mobile phase A is kept at 7%.

[0023] In some embodiments of the present application, the stationary phase comprises a Pgrand L STC C18 chromatographic column, 250 mm x 4.6 mm, 5 μm.

[0024] In some embodiments of the present application, the mobile phase B comprises an aqueous solution of phosphoric acid with a concentration of 0.008%-0.012% (w / v) of phosphoric acid.

[0025] In some embodiments of the present application, the flavonoid component comprises one or more of rutin, hyperoside, isoquercitrin, astragalin, quercetin and kaempferol.

[0026] In some embodiments of the present application, the high performance liquid chromatography further satisfies one or more of the following conditions:

[0027] (1) the column temperature is 25-35 °C;

[0028] (2) the injection volume is 5-20 μL;

[0029] (3) the detection wavelength is 350-370 nm; and,

[0030] (4) the flow rate is 0.7-1.3 mL / min.

[0031] In some embodiments of the present application, the control solution satisfies one or more of the following conditions:

[0032] (A) the solvent in the control solution comprises methanol; and,

[0033] (B) the concentration of each of the controls in the control solution is independently 30-160 μg / mL.

[0034] In some embodiments of the present application, the preparation step of the test solution comprises:

[0035] mixing the sample of the test Cuscuta medicinal material and an extraction solvent to extract, collecting the extract, and preparing the test solution.

[0036] In some embodiments of the present application, the extraction is performed by heating reflux extraction.

[0037] In some embodiments of the present application, the heating reflux extraction satisfies one or more of the following conditions:

[0038] (I) the temperature of the heating reflux extraction is 85-95℃;

[0039] (II) the time of the heating reflux extraction is 0.5-1.5 h; and,

[0040] (III) the extraction solvent of the heating reflux extraction comprises an ethanol aqueous solution;

[0041] Optionally, the volume percentage of ethanol in the ethanol aqueous solution is 65-75%.

[0042] In some embodiments of the present application, the ratio of the amount of the sample of the test Cuscuta medicinal material to the extraction solvent is 5 g: (150-200) mL; or / and, the sample of the test Cuscuta medicinal material is passed through a No. 3 sieve.

[0043] The details of one or more embodiments of the present application are set forth in the accompanying description, which makes apparent to those skilled in the art other features, purposes and advantages of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0044] In order to more clearly illustrate the technical solutions in the embodiments of the present application, more completely understand the present application and its beneficial effects, the drawings needed to be used in the description of the embodiments will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0045] Figures 1 to 4 The results of the mobile phase schemes one to four are shown in the following table.

[0046] Figures 5 to 17 Results of the elution program for program 1 to program 13.

[0047] Figures 18 to 24 Results of different flow rates.

[0048] Figures 25 to 28 Results of different injection volumes.

[0049] Figures 29 to 31 Chromatograms of test sample solution, mixed control solution, and negative sample solution in specificity test. DETAILED DESCRIPTION

[0050] The present application will be further described in conjunction with the drawings, embodiments, and examples. It should be understood that these embodiments and examples are only used to explain the present application and not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to make the disclosure of the present application more thorough and comprehensive. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or changes without departing from the spirit and scope of the present application, and the equivalent forms thus fall within the protection scope of the present application. In addition, in the following description, a large number of specific details are given in order to provide a more complete understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for the purpose of describing embodiments and examples only and is not intended to be limiting of the present application.

[0052] Terminology

[0053] Unless otherwise indicated or unless the context clearly indicates otherwise, the terms or phrases used in this document have the following meanings:

[0054] The selection range of the terms "and / or", "or / and", "and / or" used in the present application includes any one of two or more relevant listed items, and also includes any and all combinations of the relevant listed items, which includes any two relevant listed items, any more relevant listed items, or a combination of all relevant listed items. It should be noted that when at least two conjunctions selected from "and / or", "or / and", "and / or" are combined to connect at least three items, it should be understood that in the present application, the technical solution undoubtedly includes the technical solution connected by "logical and", and also undoubtedly includes the technical solution connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C and D (i.e. the technical solution connected by "logical or"), and also includes any and all combinations of A, B, C and D, i.e. includes the combination of any two or any three of A, B, C and D, and also includes the four-item combination of A, B, C and D (i.e. the technical solution connected by "logical and").

[0055] In the present application, "multiple", "various", "multiple times", "multiple" and the like are used without specific limitation, which means greater than or equal to 2 in quantity. For example, "one or more" means one or greater than or equal to two.

[0056] As used herein, "combinations thereof", "any combination thereof", "any combination manner thereof" and the like include all suitable combination manners of any two or more listed items.

[0057] In the present application, "suitable", "suitable", "any suitable manner" and the like are described in the context of being able to implement the technical solutions of the present application, solve the technical problems of the present application, and achieve the intended technical effects of the present application.

[0058] In the present application, "preferably", "better", "better", "as appropriate" are only used to describe the implementation manner or embodiment with better effect, and it should be understood that it does not constitute a limitation on the protection scope of the present application.

[0059] In the present application, "further", "more further", "in particular" and the like are used for description purposes to indicate differences in content, but should not be understood as a limitation on the protection scope of the present application.

[0060] In the present application, "optionally", "optional", "optional" means optional, i.e. selected from two parallel solutions of "yes" or "no". If there are multiple "optional" in a technical solution, and there is no specific description, and there is no contradiction or mutual restriction relationship, each "optional" is independent.

[0061] In the present application, the terms "first", "second", "third", "fourth" and the like in the "first aspect", "second aspect", "third aspect", "fourth aspect" and the like are used only for descriptive purposes, and cannot be construed as indicating or implying relative importance or quantity, nor can they be construed as implicitly indicating the importance or quantity of the technical features indicated. Moreover, "first", "second", "third", "fourth" and the like only serve the purpose of non-exhaustive enumeration and description, and should be understood as not constituting a closed limitation on the quantity.

[0062] In the present application, among the technical features described in an open manner, a closed technical solution composed of the listed features is also included, as well as an open technical solution containing the listed features.

[0063] In the present application, with respect to a numerical interval (i.e. a numerical range), if no special instructions are given, the optional numerical distribution within the above-mentioned numerical interval is considered to be continuous, and includes the two numerical end points (i.e. the minimum value and the maximum value) of the numerical range, as well as every numerical value between the two numerical end points. If no special instructions are given, when the numerical interval only points to the integers within the numerical interval, including the two end point integers of the numerical range and every integer between the two end points, in this document, it is equivalent to directly listing every integer, for example, t is an integer selected from 1-10, which means that t is any integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe a feature or a characteristic, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed in this document should be understood to include any and all sub-ranges included therein.

[0064] In the present application, the temperature parameter, if not specifically limited, allows both constant temperature treatment and variation within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. It is allowed to fluctuate within the range of, for example, ±5°C, ±4°C, ±3°C, ±2°C, ±1°C.

[0065] In the present application, %(w / w) and wt% both represent weight percentage, %(v / v) refers to volume percentage, and %(w / v) refers to mass volume percentage.

[0066] All the documents mentioned in the present application are incorporated by reference into the present application as if each document was individually incorporated by reference. Unless and to the extent that the incorporated documents conflict with the application purpose and / or technical solutions of the present application, the incorporated documents are incorporated by reference in their entirety and for all purposes. When the present application refers to the incorporated documents, the definitions of the relevant technical features, terms, names, phrases, etc. in the incorporated documents are also incorporated by reference into the present application. When the present application refers to the incorporated documents, the examples and preferred modes of the relevant technical features that are incorporated by reference into the present application are also incorporated by reference into the present application, but are limited by the implementation of the present application. It should be understood that when the incorporated content conflicts with the description in the present application, the present application is used for reference or is modified according to the description in the present application.

[0067] Mao Yan et al. used acetonitrile-0.01% phosphoric acid solution for isocratic elution separation. A method for simultaneously determining the contents of hyperoside and quercetin in the original medicine of Cuscuta chinensis was established.

[0068] Salameti Eli et al. used acetonitrile-0.01% phosphoric acid solution for isocratic elution separation. A method for determining the content of hyperoside in Cuscuta chinensis by high performance liquid chromatography (HPLC) was established.

[0069] Naren Gao et al. used methanol-water solution for isocratic elution separation. A method for determining the contents of quercetin, hyperoside and kaempferol by high performance liquid chromatography (HPLC) was established.

[0070] Dai Le et al. used acetonitrile-0.3% phosphoric acid as the mobile phase for gradient elution separation. A method for determining the contents of rutin, hyperoside, luteolin, astrin and quercetin by high performance liquid chromatography (HPLC) was established.

[0071] The above elution procedure cannot simultaneously determine the contents of six index components, i.e. rutin, hyperoside, isoquercitrin, astrin, quercetin and kaempferol in Cuscuta chinensis medicinal materials.

[0072] The present application provides a detection method for flavonoid components in Cuscuta chinensis medicinal materials, which comprises the following steps:

[0073] A control solution is provided, and the control in the control solution comprises flavonoid components;

[0074] A sample solution is prepared from a Cuscuta chinensis medicinal material sample; and,

[0075] The control solution and the sample solution are detected by high performance liquid chromatography to determine the content of the flavonoid components corresponding to the control in the Cuscuta chinensis medicinal material sample;

[0076] The high performance liquid chromatography satisfies the following conditions:

[0077] The stationary phase comprises octadecylsilane-bonded silica gel as a filler;

[0078] The mobile phase comprises mobile phase A and mobile phase B, the mobile phase A comprises acetonitrile, and the mobile phase B comprises an aqueous phosphoric acid solution;

[0079] The elution procedure is as follows:

[0080] 0-8min, the volume ratio of mobile phase A is increased from 7% to 16%,

[0081] 8-30min, the volume ratio of mobile phase A is kept at 16%,

[0082] 30-32min, the volume ratio of mobile phase A is increased from 16% to 20%,

[0083] 32-42min, the volume ratio of mobile phase A is kept at 20%,

[0084] 42-48min, the volume ratio of mobile phase A is increased from 20% to 25%,

[0085] 48-68min, the volume ratio of mobile phase A is increased from 25% to 39%,

[0086] 68-76min, the volume ratio of mobile phase A is increased from 39% to 90%,

[0087] 76-78min, the volume ratio of mobile phase A is decreased from 90% to 7%,

[0088] 78-85min, the volume ratio of mobile phase A is kept at 7%.

[0089] In some examples of the present application, the stationary phase comprises a Pgrand L STC C18 chromatographic column, 250mm x 4.6mm, 5μm.

[0090] In some examples of the present application, the mobile phase B comprises an aqueous phosphoric acid solution with a concentration of phosphoric acid of 0.008%-0.012% (w / v). In the present application, the concentration of phosphoric acid in the aqueous phosphoric acid solution is for example 0.008%, 0.009%, 0.01%, 0.011%, 0.012% (w / v).

[0091] In some examples of the present application, the flavonoid component comprises one or more of rutin, hyperoside, isoquercitrin, astragalin, quercetin and kaempferol.

[0092] In some examples of the present application, the high-performance liquid chromatography further satisfies one or more of the following conditions:

[0093] (1) the column temperature is 25-35°C, for example, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35°C;

[0094] (2) the injection volume is 5-20 μL, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 μL;

[0095] (3) the detection wavelength is 350-370 nm, for example, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370 nm; and,

[0096] (4) the flow rate is 0.7-1.3 mL / min, for example, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3 mL / min.

[0097] In some examples of the present application, the control solution satisfies one or more of the following conditions:

[0098] (A) the solvent in the control solution comprises methanol; and,

[0099] (B) the concentration of each control in the control solution is independently 30-160 μg / mL, for example, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160 μg / mL.

[0100] In some examples of the present application, the preparation step of the test solution comprises:

[0101] mixing the test sample of the Chinese foxglove herb and an extraction solvent to extract, collecting the extract to prepare the test solution.

[0102] In some embodiments of the present application, the extraction method comprises heating reflux extraction.

[0103] In some examples of the present application, the heating reflux extraction satisfies one or more of the following conditions:

[0104] (I) the temperature of the heating reflux extraction is 85-95°C, for example, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95°C;

[0105] (II) the time of the heating reflux extraction is 0.5-1.5 h, for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5 h; and,

[0106] (III) The extraction solvent for heating reflux extraction includes ethanol aqueous solution;

[0107] Optionally, the volume percentage of ethanol in the ethanol aqueous solution is 65%-75%, for example, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, or 75%.

[0108] In some examples of the present application, the dosage ratio of the to-be-tested Chinese foxglove herb sample to the extraction solvent is 5 g:(150-200) mL; or / and, the to-be-tested Chinese foxglove herb sample is passed through a No. 3 sieve.

[0109] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. The experimental methods not specified in the following examples are preferably referred to the guidelines given in the present application, and can also be performed according to the experimental manuals or conventional conditions in the art, or according to the conditions suggested by the manufacturers, or according to the known experimental methods in the art.

[0110] In the following specific examples, the measurement parameters of the raw material components may have slight deviations within the weighing accuracy range if not specifically stated. Acceptable deviations caused by instrument testing accuracy or operation accuracy are allowed for temperature and time parameters.

[0111] 1. Scheme implementation

[0112] 1.1 Experimental materials

[0113] The Chinese foxglove herb was purchased from Xinjiang Xinglubo Pharmaceutical Co., Ltd. (TSC-YP-240903); the rutin reference substance (batch number 100080-202012), hyperoside reference substance (batch number 111521-202310), isoquercitrin reference substance (batch number 111809-202205), quercetin reference substance (batch number 100081-201610), and kaempferol reference substance (batch number 110861-202214) were all purchased from China Institute for Drug Control; and astragalin reference substance (WP25011008) was purchased from Sichuan Weikexi Biological Technology Co., Ltd.

[0114] 1.2 Experimental reagents and materials

[0115] Methanol, acetonitrile, ethanol, phosphoric acid, ultrapure water, rutin reference substance, hyperoside reference substance, isoquercitrin reference substance, astragalin reference substance, quercetin reference substance, and kaempferol reference substance.

[0116] 1.3 Instruments and equipment

[0117] Analytical balance MSU205DU; high performance liquid chromatograph L820 (Beijing Purkinje General Instrument Co., Ltd.); ultrasonic cleaner As series; constant temperature water bath DZKW-S-6.

[0118] 1.4 Experimental steps

[0119] 1.4.1 Preparation of control substance stock solution

[0120] Accurately weigh 10 mg of rutin control substance, hyperoside control substance, isoquercitrin control substance, astragalin control substance, quercetin control substance and kaempferol control substance respectively into 10 mL volumetric flasks, dissolve with methanol, dilute to the mark, and prepare a solution containing 1 mg per 1 mL as a stock solution.

[0121] 1.4.2 Preparation of mixed control substance solution

[0122] Accurately take the above single control substance stock solution in an appropriate amount into the same 10 mL volumetric flask, dilute to the mark with methanol, shake well, and prepare a mixed control substance solution containing rutin, hyperoside, isoquercitrin, astragalin, quercetin and kaempferol at concentrations of 32, 160, 64, 160, 160 and 160 μg / mL respectively.

[0123] 1.4.3 Preparation of test sample solution

[0124] Weigh about 5 g of cuscute powder (passed through a No. 3 sieve), accurately weigh, and place in a round-bottom flask. Accurately add 70% (v / v) ethanol 200 mL, reflux extract at 90 ℃ for 1 h, filter, and take the filtrate to obtain.

[0125] 1.4.4 Negative sample solution

[0126] Accurately add 70% (v / v) ethanol 200 mL into a round-bottom flask, reflux extract at 90 ℃ for 1 h, filter, and take the filtrate to obtain.

[0127] 1.4.5 Chromatographic conditions

[0128] Use Pgrand L STC C18 chromatographic column (250 mm x 4.6 mm, 5 μm) and perform gradient elution according to the following table; column temperature 30 ℃; flow rate 1.0 mL / min, injection volume 10 μL, and detection wavelength 360 nm.

[0129] Table 1 Elution gradient (% by volume)

[0130]

[0131] 1.4.6 Investigation of mobile phase

[0132] The best mobile phase was determined by the results of the theoretical plate number, the separation degree of each peak, and the asymmetry of the four gradient elution schemes, methanol-water, acetonitrile-water, methanol-0.01% phosphoric acid water, and acetonitrile-0.01% phosphoric acid water. In the mobile phase investigation, each scheme only replaced the mobile phase, and the rest was the same as the chromatographic conditions in item 1.4.5.

[0133] Scheme one: the mobile phase was methanol (mobile phase A)-water (mobile phase B), the elution gradient referred to Table 1, the flow rate was 1.0 mL / min, the column temperature was 30°C; the injection amount was 10 μL, and the detection wavelength was 360 nm.

[0134] Scheme two: the mobile phase was acetonitrile (mobile phase A)-water (mobile phase B), the elution gradient referred to Table 1, the flow rate was 1.0 mL / min, the column temperature was 30°C; the injection amount was 10 μL, and the detection wavelength was 360 nm.

[0135] Scheme three: the mobile phase was methanol (mobile phase A)-0.01% phosphoric acid (mobile phase B), the elution gradient referred to Table 1, the flow rate was 1.0 mL / min, the column temperature was 30°C; the injection amount was 10 μL, and the detection wavelength was 360 nm.

[0136] Scheme four: the mobile phase was acetonitrile (mobile phase A)-0.01% phosphoric acid (mobile phase B), the elution gradient referred to Table 1, the flow rate was 1.0 mL / min, the column temperature was 30°C; the injection amount was 10 μL, and the detection wavelength was 360 nm.

[0137] The mobile phase investigation (scheme one: methanol-water) was shown in Figure 1. Figure 1 The figure showed: 1-rutin; 2-jinshetao glycoside; 3-isorhamnetin; 4-tengwarin glycoside; 5-quercetin; 6-kamferol.

[0138] The mobile phase investigation (scheme two: acetonitrile-water) was shown in Figure 2. Figure 2 The figure showed: 1-rutin; 2-jinshetao glycoside; 3-isorhamnetin; 4-tengwarin glycoside; 5-quercetin; 6-kamferol.

[0139] The mobile phase investigation (scheme three: methanol-0.01% phosphoric acid) was shown in Figure 3. Figure 3 The figure showed: 1-rutin; 2-jinshetao glycoside; 3-isorhamnetin; 4-tengwarin glycoside; 5-quercetin; 6-kamferol.

[0140] The mobile phase investigation (scheme four: acetonitrile-0.01% phosphoric acid) was shown in Figure 4. Figure 4 The figure showed: 1-rutin; 2-jinshetao glycoside; 3-isorhamnetin; 4-tengwarin glycoside; 5-quercetin; 6-kamferol.

[0141] According to the above results, using scheme four down flow phase can detect all chromatographic peaks, while using other flow phase has some chromatographic peaks cannot be detected.

[0142] 1.4.7 Elution program investigation

[0143] With acetonitrile-0.01% phosphoric acid as the mobile phase, the flow rate was 1.0 mL / min, the column temperature was 30℃, the injection volume was 10 μL, and the detection wavelength was 360 nm. Thirteen elution schemes were used to set the mobile phase, and the best elution program was determined from the test results of theoretical plate number, peak separation, and asymmetry.

[0144] Table 2

[0145]

[0146] Table 3

[0147]

[0148] Table 4

[0149]

[0150] Table 5

[0151]

[0152] Table 6

[0153]

[0154] Table 7

[0155]

[0156] Table 8

[0157]

[0158] Table 9

[0159]

[0160] Table 10

[0161]

[0162] Table 11

[0163]

[0164] Table 12

[0165]

[0166] Table 13

[0167]

[0168] Table 14

[0169]

[0170] Elution procedure investigation: Scheme I, as shown in the figure: 1- Rutin; 2-Hyperoside; 3-Isoquercitrin; 4-Astragalin; 5-Quercetin; 6-Kaempferol. Figure 5 Elution procedure investigation: Scheme II, as shown in the figure: 1- Rutin; 2-Hyperoside; 3-Isoquercitrin; 4-Astragalin; 5-Quercetin; 6-Kaempferol.

[0171] Figure 6 Elution procedure investigation: Scheme III, as shown in the figure: 1- Rutin; 2-Hyperoside; 3-Isoquercitrin; 4-Astragalin; 5-Quercetin; 6-Kaempferol.

[0172] Elution procedure investigation: Scheme IV, as shown in the figure: 1- Rutin; 2-Hyperoside; 3-Isoquercitrin; 4-Astragalin; 5-Quercetin; 6-Kaempferol. Figure 7 Elution procedure investigation: Scheme V, as shown in the figure: 1- Rutin; 2-Hyperoside; 3-Isoquercitrin; 4-Astragalin; 5-Quercetin; 6-Kaempferol.

[0173] Figure 8 Elution procedure investigation: Scheme VI, as shown in the figure: 1- Rutin; 2-Hyperoside; 3-Isoquercitrin; 4-Astragalin; 5-Quercetin; 6-Kaempferol.

[0174] Elution procedure investigation: Scheme VII, as shown in the figure: 1- Rutin; 2-Hyperoside; 3-Isoquercitrin; 4-Astragalin; 5-Quercetin; 6-Kaempferol. Figure 9 Elution procedure investigation: Scheme VIII, as shown in the figure: 1- Rutin; 2-Hyperoside; 3-Isoquercitrin; 4-Astragalin; 5-Quercetin; 6-Kaempferol.

[0175] Figure 10 Elution procedure investigation: Scheme IX, as shown in the figure: 1- Rutin; 2-Hyperoside; 3-Isoquercitrin; 4-Astragalin; 5-Quercetin; 6-Kaempferol.

[0176] Elution procedure investigation: Scheme X, as shown in the figure: 1- Rutin; 2-Hyperoside; 3-Isoquercitrin; 4-Astragalin; 5-Quercetin; 6-Kaempferol. Figure 11 Elution procedure investigation: Scheme XI, as shown in the figure: 1- Rutin; 2-Hyperoside; 3-Isoquercitrin; 4-Astragalin; 5-Quercetin; 6-Kaempferol.

[0177] Figure 12 Elution procedure investigation: Scheme XII, as shown in the figure: 1- Rutin; 2-Hyperoside; 3-Isoquercitrin; 4-Astragalin; 5-Quercetin; 6-Kaempferol.

[0178] Elution procedure investigation: Scheme XIII, as shown in the figure: 1- Rutin; 2-Hyperoside; 3-Isoquercitrin; 4-Astragalin; 5-Quercetin; 6-Kaempferol. Figure 13 Elution procedure investigation: Scheme XIV, as shown in the figure: 1- Rutin; 2-Hyperoside; 3-Isoquercitrin; 4-Astragalin; 5-Quercetin; 6-Kaempferol.

[0179] Figure 14 ​​​​​As shown in the figure: 1-rutin; 2- hyperoside; 3- isoquercitrin; 4- astragalin; 5- quercetin; 6-kaempferol.

[0180] Elution program investigation: scheme eleven, as shown in Figure 15 As shown in the figure: 1-rutin; 2- hyperoside; 3- isoquercitrin; 4- astragalin; 5- quercetin; 6-kaempferol.

[0181] Elution program investigation: scheme twelve, as shown in Figure 16 As shown in the figure: 1-rutin; 2- hyperoside; 3- isoquercitrin; 4- astragalin; 5- quercetin; 6-kaempferol.

[0182] Elution program investigation: scheme thirteen, as shown in Figure 17 As shown in the figure: 1-rutin; 2- hyperoside; 3- isoquercitrin; 4- astragalin; 5- quercetin; 6-kaempferol.

[0183] Combined with the above results: use different elution programs (table 2- table 14) for content determination, according to Figures 5-17 It can be seen that the elution program under item 1.4.5 can detect all chromatographic peaks, and the separation degree is good, while using other elution programs, part of the chromatographic peaks cannot be detected, or there is a very poor separation degree, resulting in very low accuracy.

[0184] 1.4.8 Flow rate investigation

[0185] Using C18 chromatographic column, with acetonitrile-0.01% phosphoric acid water as mobile phase, relative to the chromatographic conditions recorded in item 1.4.5, keeping other conditions unchanged, only setting the flow rate to multiple levels, that is, setting the flow rate to 0.7mL / min, 0.8mL / min, 0.9mL / min, 1.0mL / min, 1.1mL / min, 1.2mL / min, 1.3mL / min to investigate the influence of different flow rates on the separation of cuscute samples.

[0186] Flow rate investigation: 0.7mL / min. The results are shown in 18, and the figure: 1-rutin; 2- hyperoside; 3- isoquercitrin; 4- astragalin; 5- quercetin; 6-kaempferol.

[0187] Flow rate investigation: 0.8mL / min. The results are shown in Figure 19 As shown in the figure: 1-rutin; 2- hyperoside; 3- isoquercitrin; 4- astragalin; 5- quercetin; 6-kaempferol.

[0188] Flow rate investigation: 0.9mL / min. The results are shown in Figure 20 As shown in the figure: 1-rutin; 2- hyperoside; 3- isoquercitrin; 4- astragalin; 5- quercetin; 6-kaempferol.

[0189] Flow rate investigation: 1.0 mL / min. The results are shown in the figure below: 1 - Rutin; 2 - Hyperoside; 3 - Isoquercitrin; 4 - Astragalin; 5 - Quercetin; 6 - Kaempferol Figure 21

[0190] Flow rate investigation: 1.1 mL / min. The results are shown in the figure below: 1 - Rutin; 2 - Hyperoside; 3 - Isoquercitrin; 4 - Astragalin; 5 - Quercetin; 6 - Kaempferol Figure 22

[0191] Flow rate investigation: 1.2 mL / min. The results are shown in the figure below: 1 - Rutin; 2 - Hyperoside; 3 - Isoquercitrin; 4 - Astragalin; 5 - Quercetin; 6 - Kaempferol Figure 23

[0192] Flow rate investigation: 1.3 mL / min. The results are shown in the figure below: 1 - Rutin; 2 - Hyperoside; 3 - Isoquercitrin; 4 - Astragalin; 5 - Quercetin; 6 - Kaempferol Figure 24

[0193] 1.4.9 Injection amount investigation

[0194] Using a C18 column, acetonitrile-0.01% phosphoric acid water as the mobile phase, and keeping other conditions unchanged relative to the chromatographic conditions described in item 1.4.5, only the injection amount was set to multiple levels, i.e., the injection amount was set to 5 μL, 10 μL, 15 μL, and 20 μL to investigate the influence of different injection amounts on the separation of the cuscute sample.

[0195] Injection amount investigation: 5 μL. The results are shown in the figure below: 1 - Rutin; 2 - Hyperoside; 3 - Isoquercitrin; 4 - Astragalin; 5 - Quercetin; 6 - Kaempferol Figure 25 Injection amount investigation: 10 μL. The results are shown in the figure below: 1 - Rutin; 2 - Hyperoside; 3 - Isoquercitrin; 4 - Astragalin; 5 - Quercetin; 6 - Kaempferol

[0196] Figure 26 Injection amount investigation: 15 μL. The results are shown in the figure below: 1 - Rutin; 2 - Hyperoside; 3 - Isoquercitrin; 4 - Astragalin; 5 - Quercetin; 6 - Kaempferol

[0197] Injection amount investigation: 20 μL. The results are shown in the figure below: 1 - Rutin; 2 - Hyperoside; 3 - Isoquercitrin; 4 - Astragalin; 5 - Quercetin; 6 - Kaempferol Figure 27

[0198] Figure 28

[0199] ​​​​​​​​1.4.10 After the elution scheme is determined, the reliability of the method is verified in terms of specificity, precision, repeatability, robustness, limit of quantitation, etc.

[0200] (1) Specificity

[0201] Take 10 μL of the test solution, mixed control solution, and negative sample solution, respectively, and inject them into the HPLC chromatograph to confirm whether there is interference. The chromatograms of the test solution, mixed control solution, and negative sample solution are shown in Figures 29 to 31

[0202] (2) Precision

[0203] Take the test solution of the Cleavers prepared under 1.4.3, and continuously inject it 6 times according to the chromatographic conditions in 1.4.5, and record the peak areas of each component. Calculate the relative standard deviation (RSD) of the peak areas of each component. The results are shown in Table 19, and the RSD values are all less than 2.0%, indicating that the precision of the instrument is good.

[0204] Table 19 Precision

[0205]

[0206] (3) Repeatability

[0207] Take the same batch of sample, and prepare 6 test solutions in parallel under 1.4.3, and determine them according to the chromatographic conditions in 1.4.5, and record the peak areas of each component. Calculate the relative standard deviation (RSD) of the peak areas of each component. The results are shown in Table 20, and the RSD values are all less than 2.0%, indicating that the method has good repeatability.

[0208] Table 20 Repeatability

[0209]

[0210] (4) Robustness

[0211] Take the same test solution, and place it at room temperature, and inject 10 μL of it at 0, 3, 6, 9, 12, 18, 24, and 48 h according to the chromatographic conditions in 1.4.5, and detect and analyze it and record the peak areas of the 6 components, and calculate the sample content and relative standard deviation (RSD) within 36 h. The results are shown in the following table.

[0212] Table 21 Robustness

[0213]

[0214] (5) Linearity

[0215] ​The mixed reference solution under item "1.4.2" was precisely taken, and methanol was used for stepwise dilution to prepare a series of reference solutions with different concentrations. The samples were injected and analyzed according to the above chromatographic conditions, and the chromatograms were recorded. The standard curve was plotted with the concentration (X, mg / mL) of the reference solution as the abscissa and the peak area (Y) as the ordinate, and the regression equation and the correlation coefficient (r) were calculated to investigate the linear relationship of the six active ingredients within a certain concentration range. The results are shown in the following table. The correlation coefficients (r) of the six flavonoid ingredients within the corresponding linear range were all greater than 0.999, showing good linearity.

[0216] Table 22 Linear relationship of six flavonoid ingredients

[0217]

[0218] (6) Recovery rate of sample addition

[0219] Take 9 portions of the known content of the Chinese foxglove herb sample (TSC-YP-240903), each weighing 2.5 g, and precisely add the rutin, hyperoside, isoquercitrin, astragalin, quercetin, and kaempferol reference stock solutions, with the addition amount being about 50%, 100%, and 150% of the content of each component in the sample, with 3 portions for each concentration. Prepare the sample solution for addition according to the method under item 1.4.3, and then inject and measure according to the chromatographic conditions under item 1.4.5 above, and calculate the recovery rate and RSD value. The results are shown in Table 23.

[0220] Table 23 Recovery rate of sample addition

[0221]

[0222] (7) Quantitative limit and detection limit

[0223] According to the chromatographic conditions under item 1.4.5, the baseline noise was collected. Another reference solution under item 1.4.2 was measured according to the chromatographic conditions under item 1.4.5, and the peak height of each component in the reference solution was recorded when the peak height was about 10 times the baseline noise.

[0224] Table 24 Quantitative limit and detection limit

[0225]

[0226] Overall, the selected mobile phase of the application is acetonitrile and 0.01% phosphoric acid gradient elution, by using specific HPLC chromatographic conditions for detection and analysis of the herb of the plant, a method for simultaneously determining the content of 6 flavonoids in the herb of the plant is constructed, the method is simple, reliable, objective, and has good precision, repeatability, specificity and stability. Further, the obtained liquid chromatogram of the herb of the plant is analyzed, and 6 index components including rutin, hyperoside, isoquercitrin, astragalin, quercetin and kaempferol are identified, which provides overall quality control basis for the quality standard of the herb of the plant. In addition, the method is also suitable for the determination of the content of 6 flavonoids in the herb of the plant and the comprehensive evaluation of the quality of the herb, which can provide methods and basis for formulating the quality standard of high-quality herb of the plant and stabilizing the raw material producing area.

[0227] The technical features of the above-described embodiments and examples can be combined in any suitable manner. To make the description concise, not all possible combinations of the technical features in the above-described embodiments and examples are described, but it should be understood that any combination of the technical features that does not cause contradiction is within the scope of the present disclosure.

[0228] The above-described embodiments only express several embodiments of the present application, which are convenient for specifically and concretely understanding the technical solutions of the present application, but should not be understood as a limitation on the scope of patent protection. It should be noted that, for those skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of the present application. In addition, it should be understood that after reading the above description of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms also fall within the scope of the present application. It should also be understood that those skilled in the art can obtain technical solutions based on the technical solutions provided by the present application through logical analysis, reasoning or limited experiments, which are within the scope of the appended claims. Therefore, the scope of protection of the patent of the present application should be based on the contents of the appended claims, and the description and drawings can be used to explain the contents of the claims.

Claims

1. A method for detecting flavonoids in Cuscuta chinensis, characterized in that, The detection method includes the following steps: A reference solution is provided, wherein the reference standard in the reference solution includes flavonoids; Prepare a test solution from the sample of the dodder herb to be tested; and, The reference solution and the test solution were analyzed by high performance liquid chromatography to determine the content of flavonoids corresponding to the reference standard in the sample of Cuscuta chinensis. The high-performance liquid chromatography method meets the following conditions: The stationary phase uses octadecylsilane-bonded silica gel as a filler; The mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A includes acetonitrile and mobile phase B includes an aqueous solution of phosphoric acid; The elution procedure is as follows: From 0 to 8 minutes, the volume percentage of mobile phase A increased from 7% to 16%. For 8-30 minutes, the volume percentage of mobile phase A is maintained at 16%. Over 30-32 minutes, the volume percentage of mobile phase A increased from 16% to 20%. For 32-42 minutes, the volume percentage of mobile phase A is maintained at 20%. Over 42-48 minutes, the volume percentage of mobile phase A increased from 20% to 25%. Between 48 and 68 minutes, the volume percentage of mobile phase A increased from 25% to 39%. Between 68 and 76 minutes, the volume percentage of mobile phase A increased from 39% to 90%. Between 76 and 78 minutes, the volume percentage of mobile phase A decreased from 90% to 7%. For 78-85 minutes, the volume percentage of mobile phase A is maintained at 7%.

2. The method for detecting flavonoids in Cuscuta chinensis as described in claim 1, characterized in that, The stationary phase comprises a Pgrand L STC C18 column, 250 mm × 4.6 mm, 5 μm.

3. The method for detecting flavonoids in Cuscuta chinensis herbal medicine according to claim 1, characterized in that, The mobile phase B comprises an aqueous solution of phosphoric acid with a concentration of 0.008%-0.012% (w / v).

4. The method for detecting flavonoids in Cuscuta chinensis herbal medicine according to claim 1, characterized in that, The flavonoids include one or more of the following: rutin, hyperoside, isoquercitrin, astragaloside, quercetin, and kaempferol.

5. The method for detecting flavonoids in Cuscuta chinensis herbal medicine according to any one of claims 1 to 4, characterized in that, The high-performance liquid chromatography method also satisfies one or more of the following conditions: (1) The column temperature is 25-35℃; (2) The injection volume is 5-20 μL; (3) The detection wavelength is 350-370 nm; and, (4) The flow rate is 0.7-1.3 mL / min.

6. The method for detecting flavonoids in Cuscuta chinensis herbal medicine according to any one of claims 1 to 4, characterized in that, The reference solution satisfies one or more of the following conditions: (A) The solvent in the reference solution includes methanol; and, (B) The concentration of each reference standard in the reference solution is independently 30-160 μg / mL.

7. The method for detecting flavonoids in Cuscuta chinensis herbal medicine according to any one of claims 1 to 4, characterized in that, The preparation steps of the test solution include: The Cuscuta herbal sample to be tested is mixed with an extraction solvent for extraction, and the extract is collected to prepare the test solution.

8. The method for detecting flavonoids in Cuscuta chinensis herbal medicine according to claim 7, characterized in that, Extraction methods include heating and reflux extraction.

9. The method for detecting flavonoids in Cuscuta chinensis herbal medicine according to claim 8, characterized in that, Reflux extraction satisfies one or more of the following conditions: (I) The temperature for reflux extraction is 85-95℃; (II) The extraction time under reflux is 0.5-1.5 h; and, (III) The extraction solvent for heating and reflux extraction includes an aqueous solution of ethanol; Optionally, the volume percentage of ethanol in the ethanol-water solution is 65%-75%.

10. The method for detecting flavonoids in Cuscuta chinensis herbal medicine according to any one of claims 8 to 9, characterized in that, The ratio of the amount of the Cuscuta herbal sample to the amount of the extraction solvent is 5g:(150-200)mL; or / and, the Cuscuta herbal sample is passed through a No. 3 sieve.

Images