Method for detecting content of chemical components in anchusa medicinal material

Abstract

The invention relates to the technical field of traditional Chinese medicine detection, in particular to a method for detecting the content of chemical components in an anchusa medicinal material, the method performs detection based on a specific HPLC chromatographic condition, and an extract of the anchusa medicinal material does not need to be purified in the detection process. The caffeic acid, the rutin, the kaempferol-3-O-rutinoside, the narcissoside and the rosmarinic acid in the medicinal material of the anchusa can be effectively separated, so that one or more of the caffeic acid, the rutin, the kaempferol-3-O-rutinoside, the narcissoside and the rosmarinic acid can be detected. Moreover, the HPLC chromatographic condition is adopted for detection, the peak appearance time is early, and rapid detection is facilitated.

Description

Technical Field

[0001] The present application relates to the technical field of traditional Chinese medicine detection, and in particular to a method for detecting the content of chemical components in ox tongue herb. Background Art

[0002] The Uyghur herb Anchusa italica Retz is the dried whole herb of the Boraginaceae plant Anchusa italica Retz. It is listed in the "Xinjiang Uyghur Autonomous Region Traditional Chinese Medicine Preparation Specifications for Uyghur Medicinal Pieces" but not in the Chinese Pharmacopoeia. It has the effects of removing dampness and heat, increasing black bile, refreshing the body and mind, enhancing the function of internal and sensory organs, strengthening natural forces, and relieving constipation. It is used to treat palpitations, insomnia, restlessness, headaches, slow reflexes, and constipation. The "Chinese Materia Medica: Uyghur Medicinal Volume" states that Anchusa is primarily used to treat dry-cold or black bile-based diseases, such as dry encephalitis, cold-related heart deficiency, palpitations, depression, dry pleurisy, pneumonia, and tuberculosis, as well as cold-related coughs, colds, and asthma.

[0003] The 2020 edition of the "Xinjiang Uyghur Autonomous Region Traditional Chinese Medicine and Uyghur Medicinal Pieces Preparation Specifications" stipulates the properties, microscopic identification, impurities, and moisture determination of Cnidium monnieri. Without thin layer identification and content identification, it is impossible to accurately and comprehensively control the quality of Cnidium monnieri materials.

[0004] HPLC technology can effectively determine the content of one or more chemical components in traditional Chinese medicine. Traditional HPLC technology is used to detect ox tongue herb, such as the following content recorded in CN108685968A:

[0005] Take the dried aerial part of Italian bugloss, remove impurities such as sand and stones, crush it through a 20-mesh sieve, weigh 100 g of the obtained Italian bugloss powder, add 20 times the volume fraction of 75% ethanol, reflux extract twice in a 100°C water bath, each time for 2 hours, combine the extracts, filter out the medicinal residue through double gauze, concentrate under reduced pressure, and place it in a 70°C vacuum drying oven to constant weight to obtain a crude extract powder of total flavonoids from Italian bugloss; take 20 g of the crude extract powder of total flavonoids from Italian bugloss, place it in a 1000 mL volumetric flask, dilute to the scale with distilled water, ultrasonically dissolve it, centrifuge it at 5000 r / min for 10 minutes, take the supernatant, and obtain a crude extract solution of Italian bugloss (20 mg / mL);

[0006] The polyamide was soaked in 95% ethanol by volume for 24 hours, wet packed into a column, eluted with 95% ethanol by volume until the eluate was no longer white and turbid when mixed with distilled water in a ratio of 1:5, washed with distilled water until there was no alcohol smell, first soaked in 5% NaOH solution by mass concentration for 4 hours, washed with water until neutral, then soaked in 3% HCl solution by mass concentration for 4 hours, washed with water until neutral; finally, washed with 5BV distilled water, filtered to dryness, and obtained a polyamide column;

[0007] The crude extract solution of Italian bugloss is loaded onto a polyamide column at a rate of 2 BV / h and subjected to static adsorption for 30 minutes to obtain a polyamide column adsorbed with the crude extract of total flavonoids from Italian bugloss. The polyamide column adsorbed with the crude extract of total flavonoids from Italian bugloss is then firstly decontaminated with 1 BV of distilled water, and then eluted with 20 BV of 70% ethanol by volume at a rate of 5 BV / h. The column liquid is collected, concentrated under reduced pressure, and vacuum dried to obtain a purified Italian bugloss total flavonoid product, i.e., an effective fraction of Italian bugloss total flavonoids.

[0008] The present invention further conducts component separation research on the obtained purified total flavonoids from Herba Bolognese using high performance liquid chromatography.

[0009] 1) Chromatographic conditions: chromatographic column: πNAP COSMOSIL C18 column (250 mm × 4.6 mm, 5 μm); mobile phase: acetonitrile (A)-2 mL / L phosphoric acid solution (B); gradient elution program: 0-10 min, 85% B; 10-15 min, 85% B→80% B; 15-25 min, 80% B→70% B; 25-33 min, 70% B→45% B; 33-40 min, 45% B→85% B; flow rate: 1 mL / min; column temperature: 30°C; detection wavelength: 354 nm; injection volume: 20 μL.

[0010] 2) Preparation of the test solution: Weigh 20 mg of the powder of the effective fraction of total flavonoids from Herba Lycopersicum var. Italicum into a 100-mL stoppered conical flask, add 50 mL of methanol, weigh the mixture, and extract under ultrasonic conditions at 70°C and 200W for 10 min. Allow to cool, weigh the mixture again, make up the loss with methanol, shake well, and filter through a 0.45 μm microporous filter membrane. The filtrate is collected to obtain the Herba Lycopersicum var. Italicum total flavonoid test solution.

[0011] 3) Preparation of Reference Solution: Accurately weigh appropriate amounts of the reference substances caffeic acid, rutin, kaempferol-3-O-rutinoside, narcissin, and rosmarinic acid, and completely dissolve them in methanol to prepare reference solutions. Accurately measure an appropriate amount of each reference solution and dilute with methanol to obtain a mixed reference solution with mass concentrations of 3.90 μg / mL for caffeic acid, 59.78 μg / mL for rutin, 110.92 μg / mL for kaempferol-3-O-rutinoside, 47.00 μg / mL for narcissin, and 518.69 μg / mL for rosmarinic acid.

[0012] 4) Measure the mixed reference solution and the test solution separately according to the above chromatographic conditions.

[0013] However, the traditional technology mentioned above requires the components in the herb to be extracted and purified before HPLC detection, which is complicated and time-consuming.

[0014] Therefore, a simple and quick HPLC-based method for detecting the chemical components in Herba Lycopodii is necessary for the quality control of Herba Lycopodii. Summary of the Invention

[0015] Based on this, one or more embodiments of the present application provide a method for detecting the content of chemical components in Herba Ox-tonguee. The method includes the following technical solutions:

[0016] One or more embodiments of the present application provide a method for detecting the content of chemical components in a herbal ox tongue herb, the method comprising the following steps:

[0017] Providing a reference solution, wherein the reference substance in the reference solution includes one or more of caffeic acid, rutin, kaempferol-3-O-rutinoside, narcissin, and rosmarinic acid;

[0018] preparing the Herba Lycopodii Herba samples to be tested into a test solution; and

[0019] The reference solution and the test solution are tested by high performance liquid chromatography to determine the content of one or more of caffeic acid, rutin, kaempferol-3-O-rutinoside, narcissin and rosmarinic acid in the tested ox tongue herb sample;

[0020] The high performance liquid chromatography method meets the following conditions:

[0021] The stationary phase is C18 column.

[0022] The mobile phase includes a mobile phase A and a mobile phase B, wherein the mobile phase A includes acetonitrile and the mobile phase B includes a phosphoric acid aqueous solution;

[0023] Gradient elution was used, and the elution procedure included:

[0024] 0min-17min, the volume proportion of the mobile phase A is 16%,

[0025] From 17min to 18min, the volume proportion of mobile phase A increased from 16% to 22%.

[0026] From 18min to 23min, the volume proportion of the mobile phase A was maintained at 22%.

[0027] From 23min to 27min, the volume proportion of mobile phase A increased from 22% to 40%.

[0028] From 27min to 29min, the volume proportion of mobile phase A decreased from 40% to 16%.

[0029] From 29 min to 35 min, the volume proportion of the mobile phase A was maintained at 16%.

[0030] In some embodiments of the present application, the C18 chromatographic column includes a Hypersil Gold C18 chromatographic column, 250 mm×4.6 mm, 5 μm.

[0031] In some embodiments of the present application, the mobile phase B comprises a phosphoric acid aqueous solution having a phosphoric acid concentration of 0.08 wt % to 0.12 wt %.

[0032] In some embodiments of the present application, the high performance liquid chromatography method further satisfies the following conditions: the column temperature is 25°C-35°C.

[0033] In some embodiments of the present application, the high performance liquid chromatography method further satisfies the following conditions: the injection volume is 5 μL-15 μL.

[0034] In some embodiments of the present application, the high performance liquid chromatography method further satisfies the following condition: the detection wavelength is 350 nm-360 nm.

[0035] In some embodiments of the present application, the high performance liquid chromatography method further satisfies the following conditions: the flow rate is 0.9 mL / min-1.1 mL / min.

[0036] In some embodiments of the present application, the reference solution satisfies one or more of the following conditions:

[0037] (1) The solvent in the reference solution includes methanol; and

[0038] (2) The concentrations of caffeic acid, rutin, kaempferol-3-O-rutinoside, narcissin and rosmarinic acid in the reference solution are independently 0.8 μg / mL to 140 μg / mL.

[0039] In some embodiments of the present application, the step of preparing the test solution includes: mixing Herba Cynanchifoliae powder and an extraction solvent, and performing reflux extraction to prepare the test solution.

[0040] In some embodiments of the present application, the preparation step of the test solution meets one or more of the following conditions:

[0041] 1) The Herba Lysimachiae powder is passed through a No. 3 to No. 9 sieve;

[0042] 2) The ratio of the Herba Lycopersici powder to the extraction solvent is 1 g: 20 mL-100 mL;

[0043] 3) the extraction solvent comprises an ethanol-water solution with an ethanol volume ratio of 60% to 75%; and

[0044] 4) The conditions for reflux extraction include: temperature of 80°C-90°C and time of 15 min-30 min.

[0045] Compared with traditional technologies, the advantages of this application include:

[0046] The present application provides a new HPLC-based method for detecting active ingredients in ox tongue herb. This method is based on specific HPLC chromatographic conditions and does not require purification of the ox tongue herb extract during the detection process. The method can effectively separate caffeic acid, rutin, kaempferol-3-O-rutinoside, narcissin, and rosmarinic acid from the ox tongue herb, thereby enabling the detection of one or more of caffeic acid, rutin, kaempferol-3-O-rutinoside, narcissin, and rosmarinic acid. Furthermore, the use of the disclosed HPLC chromatographic conditions results in an early peak emission time, which facilitates rapid detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more fully understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present application. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0048] Figures 1 to 4 The chromatograms obtained under the conditions of elution program schemes 1 to 4 are respectively;

[0049] Figures 5 to 7 The chromatograms were obtained under the flow rate conditions of 0.8 mL / min, 1.0 mL / min, and 1.2 mL / min respectively;

[0050] Figures 8 to 10 The chromatograms obtained under the conditions of injection volume of 5μL, 10μL, and 15μL respectively;

[0051] Figures 11 to 12 The chromatograms obtained under the conditions of ultrasonic extraction and reflux extraction are respectively;

[0052] Figures 13 to 17 The chromatograms obtained under the conditions of using ethanol, methanol, 80% ethanol, 80% methanol, and 70% ethanol as extraction solvents respectively;

[0053] Figures 18 to 20 The chromatograms obtained under the conditions of material ratios of 1.0g:10mL, 1.0g:50mL, and 1.0g:100mL respectively;

[0054] Figures 21 to 23These are the chromatograms obtained under the conditions of 15min, 30min, and 60min extraction time;

[0055] Figures 24 to 26 The chromatograms were obtained under column temperatures of 25°C, 30°C, and 35°C, respectively. DETAILED DESCRIPTION

[0056] Below in conjunction with accompanying drawing, embodiment and example, the application is described in further detail.It should be understood that these embodiment and example are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiment and example is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms and is not limited to the embodiment and example described herein. Those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description below, a large amount of specific details are given in order to provide a more complete understanding of the application.It should be understood that the application can be implemented without one or more of these details.

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing embodiments and examples only and are not intended to limit this application.

[0058] the term

[0059] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0060] The terms "and / or", "or / and", and "and / or" used herein include any one of two or more related listed items, and also include any and all combinations of the related listed items, wherein the arbitrary and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical and" and also undoubtedly includes technical solutions connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution of all being connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, 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 (that is, the technical solution of all being connected by "logical AND").

[0061] In this application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0062] As used herein, "combination thereof", "any combination thereof", "any combination thereof" and the like include all suitable combinations of any two or more of the listed items.

[0063] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc. shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0064] Herein, "preferred", "better", "more preferred" and "suitable" are merely used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of this application.

[0065] In this application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of this application.

[0066] In this application, the terms "optionally," "optional," and "optional" mean optional or dispensable, i.e., they refer to either option being selected from two parallel options: "with" or "without." If a technical solution contains multiple "optional" clauses, each "optional" clause is independent unless otherwise specified and there are no contradictions or constraints.

[0067] In this application, the terms "first," "second," "third," "fourth," etc. in "the first aspect," "the second aspect," "the third aspect," "the fourth aspect," etc. are used for descriptive purposes only and should not be understood as indicating or implying relative importance or quantity, nor should they be understood as implicitly indicating the importance or quantity of the indicated technical features. Furthermore, "first," "second," "third," "fourth," etc. serve only as non-exhaustive enumeration and description, and should be understood not to constitute a closed-ended limitation on quantity.

[0068] In this application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0069] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval and includes the two numerical endpoints of the numerical range (i.e., the minimum and maximum values), as well as each numerical value between the two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0070] Unless otherwise specified, the temperature parameters in this application allow for both constant temperature treatment and temperature fluctuations within a certain temperature range. It should be understood that the constant temperature treatment allows for temperature fluctuations within the accuracy range of instrument control. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are permitted.

[0071] In this application, % (w / w) and wt% both refer to weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0072] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited in their entirety and for all purposes. When cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When cited documents are involved in this application, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be adaptively amended according to the description in this application.

[0073] One or more embodiments of the present application provide a method for detecting the content of chemical components in a herbal ox tongue herb, the method comprising the following steps:

[0074] Providing a reference solution, wherein the reference substance in the reference solution includes one or more of caffeic acid, rutin, kaempferol-3-O-rutinoside, narcissin, and rosmarinic acid;

[0075] preparing the Herba Lycopodii Herba samples to be tested into a test solution; and

[0076] The reference solution and the test solution are tested by high performance liquid chromatography to determine the content of one or more of caffeic acid, rutin, kaempferol-3-O-rutinoside, narcissin and rosmarinic acid in the tested ox tongue herb sample;

[0077] The high performance liquid chromatography method meets the following conditions:

[0078] The stationary phase is C18 column.

[0079] The mobile phase includes a mobile phase A and a mobile phase B, wherein the mobile phase A includes acetonitrile and the mobile phase B includes a phosphoric acid aqueous solution;

[0080] Gradient elution was used, and the elution procedure included:

[0081] 0min-17min, the volume proportion of the mobile phase A is 16%,

[0082] From 17min to 18min, the volume proportion of mobile phase A increased from 16% to 22%.

[0083] From 18min to 23min, the volume proportion of the mobile phase A was maintained at 22%.

[0084] From 23min to 27min, the volume proportion of mobile phase A increased from 22% to 40%.

[0085] From 27min to 29min, the volume proportion of mobile phase A decreased from 40% to 16%.

[0086] From 29 min to 35 min, the volume proportion of the mobile phase A was maintained at 16%.

[0087] In some examples of the present application, the C18 chromatographic column includes a Hypersil Gold C18 chromatographic column, 250 mm×4.6 mm, 5 μm.

[0088] In some examples of the present application, the mobile phase B includes a phosphoric acid aqueous solution having a phosphoric acid concentration of 0.08 wt%-0.12 wt% (0.08 wt%, 0.09 wt%, 0.1 wt%, 0.11 wt%, 0.12 wt%).

[0089] In some examples of the present application, the high performance liquid chromatography method further meets the following conditions: the column temperature is 25°C-35°C, for example, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35°C.

[0090] In some examples of the present application, the high performance liquid chromatography method further satisfies the following conditions: the injection volume is 5 μL-15 μL, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 μL.

[0091] In some examples of the present application, the high performance liquid chromatography method further satisfies the following conditions: the detection wavelength is 350 nm-360 nm, for example, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360 nm.

[0092] In some examples of the present application, the high performance liquid chromatography method further satisfies the following conditions: the flow rate is 0.9 mL / min-1.1 mL / min, for example, 0.9, 0.95, 1, 1.05, 1.1 mL / min.

[0093] In some examples of the present application, the reference solution satisfies one or more of the following conditions:

[0094] (1) The solvent in the reference solution includes methanol; and

[0095] (2) The concentrations of caffeic acid, rutin, kaempferol-3-O-rutinoside, narcissin and rosmarinic acid in the reference solution are each independently 0.8 μg / mL to 140 μg / mL, for example, 0.8, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140.

[0096] In some examples of the present application, the steps of preparing the test solution include: mixing Herba Lycopodii powder and an extraction solvent, performing reflux extraction, and preparing the test solution.

[0097] In some examples of the present application, the steps for preparing the test solution meet one or more of the following conditions:

[0098] 1) The Herba Lysimachiae powder is passed through a No. 3 to No. 9 sieve;

[0099] 2) The amount ratio of the herba serrata powder to the extraction solvent is 1g:20mL-100mL, for example, 1g:20mL, 1g:25mL, 1g:30mL, 1g:35mL, 1g:40mL, 1g:45mL, 1g:50mL, 1g:55mL, 1g:60mL, 1g:65mL, 1g:70mL, 1g:75mL, 1g:80mL, 1g:85mL, 1g:90mL, 1g:95mL, and 1g:100mL;

[0100] 3) the extraction solvent comprises an ethanol-water solution with a volume percentage of 60%-75% (e.g., 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%) of ethanol; and

[0101] 4) The conditions for reflux extraction include: a temperature of 80°C-90°C (for example, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90°C) and a time of 15 min-30 min (for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 min).

[0102] 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 intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manuals or conventional conditions in this area, or according to the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0103] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0104] The mobile phase selected in the embodiment of the present application is acetonitrile and phosphoric acid gradient elution. By using specific HPLC chromatographic conditions to detect and analyze the herbal medicine of ox tongue, a method for simultaneously determining the content of 5 chemical components in the herbal medicine of ox tongue was constructed. The method is simple, reliable, objective, and has good precision, repeatability, specificity, and stability. Further, the obtained liquid phase spectrum of the herbal medicine of ox tongue was analyzed. There were five absorption peaks with complete peak types and peak areas in the liquid phase spectrum of the herbal medicine of ox tongue, and the five peaks were identified as 5 index components including caffeic acid, rutin, kaempferol-3-O-rutinoside, narcissin, and rosmarinic acid. These five components have high response peak types in the herbal medicine of ox tongue, and are all peak types that can be detected under the liquid phase method of the herbal medicine of ox tongue, providing an overall quality control basis for the quality standard of the authentic herbal medicine of ox tongue.

[0105] (1) Reduction of interference factors by the extraction method of medicinal materials: Due to the rich variety of substances in the herbal medicine of ox tongue grass, there will be a lot of interference during the extraction process of the medicinal materials. Among them, the most interfering factor is the herbal polysaccharide of ox tongue grass. Since ox tongue grass contains polysaccharides and most of them are mucus and mucopolysaccharide components, during the extraction process, when the water content of the solvent increases, the herbal extract will become viscous. In this state, the extract cannot pass through the filter membrane with a pore size of 0.22μm for liquid phase sample pretreatment. If a non-aqueous organic solvent is used for extraction, the extract will not become viscous, but the content of rosmarinic acid will be reduced by about 50%. Therefore, through the investigation of the extraction solvent of Herba Cynanchifolia, combined with the reduction of interference of polysaccharide components in the extraction solvent and the efficient extraction rate of each substance content as indicators, extraction experiments with methanol, anhydrous ethanol, 80% methanol, 80% ethanol, 70% ethanol, 60% ethanol and 65% ethanol were carried out. The experimental results showed that the content of each substance in the aqueous ethanol solvent was higher than that in the aqueous methanol solvent. With the increase of water content, the content of rosmarinic acid will be effectively increased. However, when the alcohol concentration reaches below 65%, the liquid phase test sample cannot be prepared due to the interference of the Herba Cynanchifolia polysaccharide components.

[0106] (2) Effective separation of similar substances: Among the five chemical components of the herb, there are compounds with high similarity, kaempferol-3-O-rutinoside and narcissin, whose specific molecular structures are shown in the following formula:

[0107]

[0108] Both chemical compounds share a common nucleus and carry two glycosidic bonds at the same position. The only difference is that narcissin has an additional methoxy group compared to kaempferol-3-O-rutinoside. However, through effective adjustments to the liquid phase method, the two chemical components can be effectively separated, with a resolution exceeding 2.03, and the resolution remains stable in repeated experiments with the medicinal materials.

[0109] (3) Analysis speed and environmental friendliness: This liquid phase method can effectively separate the five chemical components in the herb of Ox tongue within 35 minutes, and includes the time for washing the column with high organic phase and balancing to the initial mobile phase ratio, which can effectively reduce the amount of mobile phase used, saving the amount of mobile phase used to achieve the purpose of environmentally friendly and efficient detection.

[0110] The embodiments of this application include the following:

[0111] 1. Materials and Instruments

[0112] Experimental medicinal materials: Herba Ox-tonguee Herba (provided by Xinjiang New Green Treasure Pharmaceutical Co., Ltd., batch number NSC-YP-240502).

[0113] Experimental reagents: methanol, acetonitrile, ethanol, phosphoric acid, water (ultrapure water); caffeic acid reference substance, rutin reference substance, kaempferol-3-O-rutinoside reference substance, narcissin reference substance, rosmarinic acid reference substance.

[0114] Instruments and equipment: analytical balance MSU205DU; high performance liquid chromatograph Vanquish (Thermo Fisher Scientific), including diode array detector, quaternary gradient pump, online degasser, autosampler; As series ultrasonic cleaning machine.

[0115] 2. Sample Preparation

[0116] (1) Preparation of reference substance stock solutions: Accurately weigh 10 mg each of caffeic acid reference, rutin reference, kaempferol-3-O-rutinoside reference, narcissin reference, and rosmarinic acid reference, and place them in 10 mL volumetric flasks. Dissolve them in methanol and dilute to the mark to prepare solutions containing 1 mg per mL. These solutions are referred to as stock solutions ①, ②, ③, ④, and ⑤, respectively.

[0117] (2) Preparation of mixed reference solution: Accurately measure 1 mL each of stock solution ①, stock solution ②, stock solution ③, stock solution ④, and stock solution ⑤, add 5 mL of methanol, and prepare 100 μg mL each of caffeic acid reference, rutin reference, kaempferol-3-O-rutinoside reference, narcissin reference, and rosmarinic acid reference. -1 The solution was used as the mixed reference solution.

[0118] (3) Preparation of test solution: Weigh about 0.5 g of Herba Cynanchifoliae powder (passed through No. 3 sieve), accurately weigh, place in a stoppered conical flask, accurately add 50 mL of 70% ethanol, stopper, weigh, heat in a water bath at 88°C, reflux for 15 min, cool, weigh again, make up the lost weight with 70% ethanol, shake well, filter, and take the filtrate.

[0119] (4) Preparation of blank solution: Accurately add 50 mL of 70% ethanol to a stoppered conical flask, stopper tightly, weigh the solution, reflux and extract for 15 min, cool, weigh the solution again, add 70% ethanol to make up for the loss, shake well, filter, and collect the filtrate.

[0120] 3. Determination of chromatographic conditions

[0121] (1) Chromatographic conditions: Hypersil Gold C18 column (250 mm × 4.6 mm, 5 μm), gradient elution according to the table below, column temperature 30°C, injection volume 10 μL, detection wavelength 354 nm.

[0122] (2) Elution procedure: Four gradient elution schemes, namely methanol-water, acetonitrile-water, methanol-0.1% phosphoric acid water, and acetonitrile-0.1% phosphoric acid water, were used to investigate the mobile phase. The optimal elution procedure was determined based on the experimental results such as the theoretical plate number, peak separation, and asymmetry.

[0123] Scheme 1: The mobile phase is methanol-water, the flow rate is 1.0 mL / min, the column temperature is 30°C, the injection volume is 10 μL, and the detection wavelength is 354 nm.

[0124] Table 1

[0125] Time (min) Mobile phase A: methanol Mobile phase B: water 0 16 84 17 16 84 18 22 78 23 22 78 27 40 60 29 16 84 35 16 84

[0126] Specific experimental results such as Figure 1 As shown in the methanol-water system, flavonoids such as rutin cannot be effectively detected, and the content of rosmarinic acid is also low.

[0127] Scheme 2: The mobile phase is acetonitrile-water, the flow rate is 1.0 mL / min, the column temperature is 30°C, the injection volume is 10 μL, and the detection wavelength is 354 nm.

[0128] Table 2

[0129]

[0130]

[0131] Specific experimental results such as Figure 2 As shown: Caffeic acid and rosmarinic acid cannot be detected in the acetonitrile-water system, and contain flat-top peaks, and the elution ability is poor.

[0132] Scheme 3: The mobile phase is methanol-0.1% phosphoric acid, the flow rate is 1.0 mL / min, the column temperature is 30°C, the injection volume is 10 μL, and the detection wavelength is 354 nm.

[0133] Table 3

[0134] time Mobile phase A: methanol Mobile phase B: 0.1% phosphoric acid 0 16 84 17 16 84 18 22 78 23 22 78 27 40 60 29 16 84 35 16 84

[0135] Specific experimental results such as Figure 3 As shown: In the methanol-0.1% phosphoric acid system, the substance separation is poor, the peak area is low, and the overall detection ability is low.

[0136] Scheme 4: The mobile phase is acetonitrile-0.1% phosphoric acid, the flow rate is 1.0 mL / min, the column temperature is 30°C, the injection volume is 10 μL, and the detection wavelength is 354 nm.

[0137] Table 4

[0138] time Mobile phase A: acetonitrile Mobile phase B: 0.1% phosphoric acid 0 16 84 17 16 84 18 22 78 23 22 78 27 40 60 29 16 84 35 16 84

[0139] Specific experimental results such as Figure 4 As shown: In the acetonitrile-0.1% phosphoric acid system, all five components can be detected with good separation and peak shape, with high detection ability and high resolution.

[0140] (3) Flow rate investigation

[0141] On the basis of “(1) Chromatographic conditions” and the elution procedure of Scheme 4, a C18 chromatographic column was used with acetonitrile-0.1% phosphoric acid water as the mobile phase. Other conditions were kept unchanged and the flow rates were set to 0.8 mL / min, 1.0 mL / min, and 1.2 mL / min, respectively. The effects of different flow rates on the separation of the components in the Herba Glechomae were investigated.

[0142] When the flow rate is 0.8 mL / min, the results are as follows: Figure 5 As shown in the figure: At a flow rate of 0.8 mL / min, the separation degree of kaempferol-3-O-rutinoside and narcissin is reduced because their detection times are similar.

[0143] When the flow rate is 1.0 mL / min, the results are as follows: Figure 6 As shown: At a flow rate of 1.0 mL / min, the separation and peak shape between the peaks are ideal.

[0144] When the flow rate is 1.2 mL / min, the results are as follows: Figure 7 As shown in the figure: At a flow rate of 1.2 mL / min, the separation degree of kaempferol-3-O-rutinoside and narcissin is reduced because their detection times are similar.

[0145] (4) Sample injection volume investigation

[0146] On the basis of "(1) Chromatographic conditions" and the elution procedure of Scheme 4, a C18 chromatographic column was used with acetonitrile-0.1% phosphoric acid water as the mobile phase. Other conditions were kept unchanged, and the injection volumes were set to 5μL, 10μL, and 15μL, respectively. The effects of different injection volumes on the separation of the components of the Herba Glehniae were investigated.

[0147] When the injection volume is 5 μL, the results are as follows Figure 8 As shown, when the injection volume is 10μL, the results are as follows Figure 9 As shown, when the injection volume is 15μL, the results are as follows Figure 10 As shown in the figure, when the injection volumes were 5μL, 10μL, and 15μL, the peak shapes of the different injection volumes were slightly different. The method was stable under different injection volumes, so the instrument standard injection volume of 10μL was selected as the method injection volume.

[0148] 4. Determination of the test solution preparation method

[0149] (1) Investigation of different extraction methods

[0150] On the basis of "(1) Chromatographic conditions" and the elution procedure of Scheme 4, a C18 chromatographic column was used with acetonitrile-0.1% phosphoric acid water as the mobile phase, and other conditions were kept unchanged. The extraction methods of the Herba Lycopersici test samples were prepared by ultrasonic extraction and reflux extraction, respectively, to investigate the effects of different extraction methods on the separation of the various components of the Herba Lycopersici sample.

[0151] Reflux extraction: Weigh about 0.5 g of Herba Cynanchifoliae powder (passed through No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 50 mL of 70% ethanol, stopper it, weigh it, heat it in an 88°C water bath, reflux extract it for 15 minutes, let it cool, weigh it again, make up the lost weight with 70% ethanol, shake it well, filter it, and take the filtrate to obtain it.

[0152] Ultrasonic extraction: Weigh about 0.5 g of Herba Cynanchifoliae powder (passed through No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 50 mL of 70% ethanol, stopper it, weigh it, place it in an ultrasonic machine and ultrasonicate it at 50 Hz for 15 minutes, let it cool, weigh it again, make up for the lost weight with 70% ethanol, shake it well, filter it, and take the filtrate to obtain it.

[0153] When the extraction method is ultrasonic extraction, the results are as follows Figure 11 As shown, when the extraction method is ultrasonic extraction, the results are as follows Figure 12 As shown in the figure, the rosmarinic acid content of the reflux extract is twice that of the ultrasonic extract, and the changes of the other peaks are low under the two extraction methods, so the reflux extraction method is selected.

[0154] (2) Screening of different extraction solvents

[0155] On the basis of "(1) Chromatographic conditions" and the elution procedure of Scheme 4, a C18 chromatographic column was used, acetonitrile-0.1% phosphoric acid water was used as the mobile phase, and other conditions were kept unchanged. The extraction solvents of the Herba Lycopersici were extracted with methanol, 80% methanol, ethanol, and 70% ethanol, respectively, to investigate the effects of different extraction solvents on the separation of the various components of the Herba Lycopersici samples.

[0156] When 100% ethanol was used for extraction, the results were as follows: Figure 13 When 100% methanol was used for extraction, the results were as shown in Figure 14 As shown, when 80% ethanol was used for extraction, the results were as follows Figure 15 As shown, when 80% methanol was used for extraction, the results were as follows Figure 16 As shown, when 70% ethanol was used for extraction, the results were as follows Figure 17 As shown, by comparing different solvents, based on their peak shapes and substance concentrations, the content of 80% methanol is lower than that of 80% ethanol, and the content of 80% ethanol is lower than that of 70% ethanol. The content extracted by 70% ethanol is the highest, so 70% ethanol is selected as the solvent.

[0157] (3) Material ratio investigation

[0158] On the basis of "(1) Chromatographic conditions" and the elution procedure of Scheme 4, a C18 chromatographic column was used with acetonitrile-0.1% phosphoric acid water as the mobile phase, and other conditions were kept unchanged. The mass volume ratios of the Herba Lycopersici test sample and the extraction solvent were 1.0 g:10 mL, 1.0 g:50 mL, and 1.0 g:100 mL, respectively. The effects of different material ratios on the separation of the various components of the Herba Lycopersici sample were investigated.

[0159] When the material ratio is 1.0g:10mL, the results are as follows Figure 18 As shown, when the material ratio is 1.0g:50mL, the results are as follows Figure 19 As shown, when the material ratio is 1.0g:100mL, the results are as follows Figure 20 As shown in the figure, through the investigation and content conversion of different solid-liquid ratios, the extraction rate of 10 times the solid-liquid ratio is lower, and the extraction rates of 50 and 100 times the solid-liquid ratio are similar. For energy saving considerations, a 50-fold solid-liquid ratio is selected as the extraction solvent amount.

[0160] (4) Extraction time investigation

[0161] On the basis of "(1) Chromatographic conditions" and the elution procedure of Scheme 4, a C18 chromatographic column was used with acetonitrile-0.1% phosphoric acid water as the mobile phase, and other conditions were kept unchanged. The extraction time of the Herba Lycopersici sample was set to 15 min, 30 min, and 60 min, respectively, to investigate the effect of different extraction times on the separation of the various components of the Herba Lycopersici sample.

[0162] When the extraction time is 15 min, the results are as follows Figure 21 As shown in Figure 2, when the extraction time is 30 min, the results are as follows: Figure 22 As shown in Figure 2, when the extraction time is 60 min, the results are as follows: Figure 23 As shown in the figure, through the investigation of extraction time, it was found that the content of each substance was not much different when the extraction time was 15 minutes and 30 minutes, and the content of rosmarinic acid decreased when the extraction time was 60 minutes, so 15 minutes was selected as the extraction time.

[0163] 5. Column temperature inspection

[0164] On the basis of "(1) Chromatographic conditions" and the elution procedure of Scheme 4, a C18 chromatographic column was used with acetonitrile-0.1% phosphoric acid water as the mobile phase. Other conditions were kept unchanged and the column temperature was set to 25℃, 30℃, and 35℃, respectively. The effects of different column temperatures on the separation of the components of the Herba Glechomae were investigated.

[0165] When the column temperature is 25°C, the results are as follows: Figure 24 As shown in the figure, when the column temperature is 30℃, the results are as follows: Figure 25 As shown in Figure 2, when the column temperature is 35°C, the results are as follows: Figure 26 As shown in the figure, by investigating different column temperatures, the method was stable at different temperatures, so 30°C was selected as the final column temperature.

[0166] 6. Verify the reliability of the method

[0167] (1) Precision

[0168] Take the reference solution and the test solution respectively, and inject them continuously for 6 times for detection and analysis according to the chromatographic conditions under item 3. Record the peak area and calculate the relative standard deviation (RSD) of each peak area.

[0169] The results of the precision test of the samples are shown in the following table:

[0170] Table 5

[0171] name Caffeic acid Rutin Kaempferol-3-O-rutinoside Narcissusin Rosmarinic acid Bugloss Precision 1 1.0899 15.5819 17.9397 15.8706 58.2320 Bugloss Precision 2 1.1016 15.2967 17.744 15.8481 58.2187 Bugloss Precision 3 1.0982 15.5066 17.957 15.9552 58.3798 Bugloss Precision 4 1.1078 15.3068 17.9725 15.885 58.3712 Bugloss Precision 5 1.1013 15.3576 17.8264 15.8267 58.2427 Bugloss Precision 6 1.0808 15.4582 17.9306 16.0216 58.2889 Average sample size 1.097 15.418 17.895 15.901 58.289 Relative standard deviation 0.885％ 0.751％ 0.504％ 0.462％ 0.122％

[0172] The test results show that the precision of the five index components are all qualified.

[0173] (2) Repeatability

[0174] Take samples from the same batch number and prepare 6 test solutions in parallel according to the method under item 2. Measure and record the peak areas according to the chromatographic conditions under item 3.

[0175] The results of the repeatability test of the samples are shown in the following table:

[0176] Table 6

[0177]

[0178]

[0179] The test results show that the repeatability of the five index components are all qualified.

[0180] (3) Durability

[0181] Take the same test solution, place it at room temperature, and inject 10 μL of it at 0, 2, 4, 8, 12, 18, and 24 hours according to the chromatographic conditions under item 3 for detection and analysis, and record the peak areas of the five component peaks. Calculate the relative standard deviation (RSD) of the peak area corresponding to each component within 24 hours.

[0182] The durability test of the samples shows the results in the following table:

[0183] Table 7

[0184] name Caffeic acid Rutin Kaempferol-3-O-rutinoside Narcissusin Rosmarinic acid 24h 2.4172 38.7465 41.7603 36.9069 129.9846 12h 2.3611 38.9613 42.2805 36.7991 132.6855 8h 2.3386 38.5087 41.9516 36.1422 132.8766 4h 2.4074 38.5791 41.9925 35.9911 133.3616 2h 2.4045 38.284 42.1595 36.1085 133.459 0h 2.3535 38.0072 41.9361 36.5112 133.6185 Average sample size 2.38038 38.51446 42.01341 36.40983 132.6643 Relative standard deviation 1.394％ 0.875％ 0.435％ 1.061％ 1.026％

[0185] The test results show that the durability of the five index components are all qualified.

[0186] (4) Linear relationship

[0187] Accurately pipette 1 μL, 2 μL, 4 μL, 6 μL, 8 μL, and 10 μL of the mixed reference solution, inject them into the liquid chromatograph for detection and analysis according to the chromatographic conditions, and draw a standard curve with the peak area Y as the ordinate and the injection volume X (μg) as the abscissa.

[0188] Through the linear relationship test of the samples, the results are shown in the following table:

[0189] Table 8

[0190]

[0191]

[0192] Results The linearity of the five index components was qualified.

[0193] (5) Sample recovery rate

[0194] Take 5 portions of the sample with known determination, take about 0.5 g of each portion, accurately weigh, and place them in conical flasks respectively. Accurately add appropriate amounts of reference stock solutions of caffeic acid, rutin, kaempferol-3-O-rutinoside, narcissin and rosmarinic acid. Prepare the test solution according to the method under item 2 above. Inject 10 μL of each sample for determination under the chromatographic conditions under item 3 and calculate the recovery.

[0195] The recovery rate of the sample spiked was tested and the results are shown in the following table:

[0196] Table 9

[0197]

[0198] According to the above table, the recovery rates of each component are within the effective range, and the RSD values ​​are within the qualified range. The recovery data are true, valid and stable.

[0199] (6) Limit of quantification

[0200] According to the chromatographic conditions under item 3, collect a blank baseline and measure the baseline noise. Take the reference solution under item 2 and measure the peak heights of each peak under the chromatographic conditions under item 3. Record the peak heights when the peak heights of the five components in the reference solution are approximately 10 times the baseline noise. The specific experimental results are shown in the following table:

[0201] Table 10

[0202]

[0203] According to the table above, the quantitative limit and detection limit of each component can be known, and the content of each component detected in the ox tongue herb is above the quantitative limit. Therefore, the content data deviation is small, which can reflect the true component content of the medicinal material itself, and the data is authentic.

[0204] 7. Detection Application

[0205] Accurately weigh 0.5g of ox tongue herbal powder in three portions and perform liquid chromatography according to the extraction and detection methods under this method. The specific data are shown in the following table:

[0206] Table 11. Contents of five chemical components in the herbal medicine of ox tongue (μg / mL)

[0207]

[0208] According to the above table, this detection method is stable and effective and can be used normally.

[0209] The various technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. In order to make the description concise, not all possible combinations of the various technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of the description in this specification.

[0210] The above-described embodiments only express several implementation methods of the present application, which facilitate a specific and detailed understanding of the technical solutions of the present application, but cannot be understood as limiting the scope of protection of the patent application. It should be pointed out that, for ordinary technicians in this field, without departing from the concept of the present application, several variations and improvements can be made, which all fall within the scope of protection of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the scope of protection of the present application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the scope of protection of the claims attached to the present application. Therefore, the scope of protection of the patent application of the present application shall be based on the content of the attached claims, and the description and drawings can be used to interpret the content of the claims.

Claims

1. A method for detecting the content of chemical components in Herba Ox-tonguee, characterized in that: The detection method comprises the following steps: Providing a reference solution, wherein the reference substance in the reference solution includes one or more of caffeic acid, rutin, kaempferol-3-O-rutinoside, narcissin, and rosmarinic acid; preparing the Herba Lycopodii Herba samples to be tested into a test solution; and The reference solution and the test solution are tested by high performance liquid chromatography to determine the content of one or more of caffeic acid, rutin, kaempferol-3-O-rutinoside, narcissin and rosmarinic acid in the tested ox tongue herb sample; The high performance liquid chromatography method meets the following conditions: The stationary phase is C18 column. The mobile phase includes a mobile phase A and a mobile phase B, wherein the mobile phase A includes acetonitrile and the mobile phase B includes a phosphoric acid aqueous solution; Gradient elution was used, and the elution procedure included: 0min-17min, the volume proportion of the mobile phase A is 16%, From 17min to 18min, the volume proportion of mobile phase A increased from 16% to 22%. From 18min to 23min, the volume proportion of the mobile phase A was maintained at 22%. From 23min to 27min, the volume proportion of mobile phase A increased from 22% to 40%. From 27min to 29min, the volume proportion of mobile phase A decreased from 40% to 16%. From 29 min to 35 min, the volume proportion of the mobile phase A was maintained at 16%.

2. The method for detecting the content of chemical components in the herb Herba Ox-tongue according to claim 1, characterized in that: The C18 chromatographic column includes a Hypersil Gold C18 chromatographic column, 250 mm×4.6 mm, 5 μm.

3. The method for detecting the content of chemical components in the herb Herba Ox-tongue according to claim 1, characterized in that: The mobile phase B includes a phosphoric acid aqueous solution with a phosphoric acid concentration of 0.08 wt% to 0.12 wt%.

4. The method for detecting the content of chemical components in the herb Herba Ox-tongue according to any one of claims 1 to 3, characterized in that: The high performance liquid chromatography method also meets the following conditions: the column temperature is 25°C-35°C.

5. The method for detecting the content of chemical components in the herb of Ox tongue according to any one of claims 1 to 3, characterized in that: The high performance liquid chromatography method also meets the following conditions: the injection volume is 5 μL-15 μL.

6. The method for detecting the content of chemical components in the herb Herba Ox-tongue according to any one of claims 1 to 3, characterized in that: The high performance liquid chromatography method also meets the following conditions: the detection wavelength is 350nm-360nm.

7. The method for detecting the content of chemical components in the herb Herba Ox-tongue according to any one of claims 1 to 3, characterized in that: The high performance liquid chromatography method also meets the following conditions: the flow rate is 0.9 mL / min-1.1 mL / min.

8. The method for detecting the content of chemical components in the herb Herba Ox-tongue according to any one of claims 1 to 3, characterized in that: The reference solution meets one or more of the following conditions: (1) The solvent in the reference solution includes methanol; and (2) The concentrations of caffeic acid, rutin, kaempferol-3-O-rutinoside, narcissin and rosmarinic acid in the reference solution are independently 0.8 μg / mL to 140 μg / mL.

9. The method for detecting the content of chemical components in the herb of Ox tongue according to any one of claims 1 to 3, characterized in that: The steps of preparing the test solution include: mixing the herba serrata powder and the extraction solvent, and performing reflux extraction to prepare the test solution.

10. The method for detecting the content of chemical components in the herb Herba Ox-tongue according to claim 9, characterized in that: The preparation steps of the test solution meet one or more of the following conditions: 1) The Herba Lysimachiae powder is passed through a No. 3 to No. 9 sieve; 2) The ratio of the Herba Lycopersici powder to the extraction solvent is 1 g: 20 mL-100 mL; 3) The extraction solvent comprises an ethanol aqueous solution with an ethanol volume ratio of 60% to 75%; as well as, 4) The conditions for reflux extraction include: temperature of 80°C-90°C and time of 15 min-30 min.

Images