Method for measuring contents of effective components in lotus leaves and preparations thereof

Abstract

The invention provides a method for determining the content of effective components in a lotus leaf medicinal material and a preparation thereof. The method comprises the following steps: determining a reference solution and a test solution by adopting a high performance liquid chromatography; a chromatographic column takes octadecylsilane chemically bonded silica as a filling agent, the detection wavelength is 265-275 nm, acetonitrile is taken as a mobile phase A, 0.05-0.2% phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out. The method can be used for simultaneously determining the contents of four active ingredients, namely hyperoside, isoquercitrin, quercetin-3-O-gluconate and nuciferine, in the lotus leaves, the lotus leaf aqueous extract and / or the lotus leaf aqueous extract preparation; the separation of four component peaks is effectively realized, and the method is high in accuracy, simple, convenient and rapid.

Description

Technical Field

[0001] The invention relates to the field of content determination of traditional Chinese medicines, and in particular to a method for determining the content of effective components in lotus leaves and preparations thereof. Background Art

[0002] Lotus leaf is the leaf of Nelumbo nuficera Gaertn, a plant of the Nymphaeaceae family. It is also known as lotus leaf and lotus leaf. It is planted in both the north and south of my country. It is bitter and cold in nature, and is neutral. It enters the liver, spleen, stomach, and heart meridians. It has the activity of clearing away heat and dampness, generating clear yang, clearing the heart and removing heat, and stopping bleeding and promoting diuresis. Lotus leaf mainly contains alkaloids, flavonoids, polysaccharides and other ingredients. Modern pharmacological research shows that lotus leaf has the effects of reducing fat and weight, anti-oxidation, anti-aging, inhibiting fatty liver, antibacterial, inhibiting HIV proliferation, anti-virus, anti-inflammatory, and anti-allergic.

[0003] The 2020 edition of the Chinese Pharmacopoeia only contains a method for determining the content of a single component, nuciferine, using high performance liquid chromatography under the lotus leaf item. Studies have shown that lotus leaf water extract contains a variety of active ingredients, with high levels of the main active ingredients quercetin-3-O-glucoside and isoquercetin. Quercetin-3-O-glucoside has anti-inflammatory and cytoprotective effects, preventing the cytotoxicity of cisplatin (a platinum-containing anticancer compound) in renal tubular cells and inhibiting the proliferation of cancer cells. Isoquercetin can regulate the body's immune system, showing anti-inflammatory capabilities.

[0004] At present, the research on the determination method of the active ingredient content in lotus leaf water extract is mostly focused on the determination of a single component, and there is no suitable method for the simultaneous determination of multiple components in lotus leaf water extract. Summary of the invention

[0005] In view of the shortcomings of the prior art, the object of the present invention is to provide a method for determining the content of effective ingredients in lotus leaves and their preparations, which can simultaneously determine the contents of four active ingredients, namely hyperoside, isoquercetin, quercetin-3-O-glucoside and nuciferine, in lotus leaves, lotus leaf water extracts and / or lotus leaf water extract preparations.

[0006] To achieve the purpose of the invention, the present invention provides the following technical solutions:

[0007] A method for determining the content of active ingredients in lotus leaves and preparations thereof, comprising using high performance liquid chromatography to determine a reference substance solution and a solution of lotus leaf medicinal material and preparations thereof;

[0008] The chromatographic conditions are as follows: the chromatographic column is filled with octadecylsilane bonded silica gel, the detection wavelength is 265nm-275nm, acetonitrile is used as mobile phase A, 0.05%-0.2% phosphoric acid solution is used as mobile phase B, and gradient elution is performed as specified in the following table;

[0009]

[0010] The column temperature of the chromatographic column is 10°C-20°C, and the flow rate is 0.9ml / min-1.1ml / min.

[0011] Preferably, the mobile phase B is 0.1% phosphoric acid solution; and the detection wavelength is 270 nm.

[0012] The lotus leaf medicinal material and its preparation are at least one selected from lotus leaf medicinal material, lotus leaf decoction pieces, lotus leaf extract, and lotus leaf granule preparation.

[0013] When the lotus leaf medicinal material and the preparation thereof are lotus leaf medicinal material or lotus leaf decoction pieces, the preparation process of the solution of the lotus leaf medicinal material and the preparation thereof is: taking the test sample, accurately weighing, adding a solvent, pretreating, filtering, combining the filtrate, recording the volume, taking a small amount, and obtaining the solution.

[0014] When the lotus leaf medicinal material and its preparation are lotus leaf granules or lotus leaf extract, the preparation process of the solution of the lotus leaf medicinal material and its preparation is: take the test sample, accurately weigh it, put it in a volumetric flask, add solvent, seal it, pretreat it, then add the same solvent to make up the volume, shake it well, and obtain it.

[0015] Furthermore, in the preparation process of the solution of the lotus leaf medicinal material or lotus leaf decoction pieces and the lotus leaf medicinal material preparation thereof, the solvent added is water, and the pretreatment process is first soaking in water and then heating and refluxing.

[0016] Furthermore, the preparation process of the solution of the lotus leaf medicinal material or lotus leaf decoction pieces and the lotus leaf medicinal material and its preparation is as follows: take 2g of the coarse powder of the test sample, accurately weigh it, put it in a beaker, add 20ml of water, soak for 30min, transfer the decoction pieces and water to a round-bottom flask, rinse the beaker with 30ml of water, transfer the rinsing solution to the flask, heat and reflux for 40min, filter, add 46ml of water to the residue, heat and reflux for 30min, filter, combine the filtrate, record the volume, take a small amount, and obtain.

[0017] Furthermore, in the preparation process of the lotus leaf granules or lotus leaf extract and the solution of the lotus leaf medicinal material and the preparation thereof, the solvent added is water, the pretreatment is ultrasonic treatment, and the solution concentration is 10 mg / ml.

[0018] Furthermore, the reference substances are hyperoside, isoquercetin, quercetin-3-O-glucoside, and nuciferine reference substances, and the reference substance solution is prepared by taking the reference substances and dissolving them in solvents respectively.

[0019] Furthermore, the solvent added to the reference substance is methanol, and the concentrations of the prepared reference substance solutions are 37.96 μg / ml for hyperoside, 39.20 μg / ml for isoquercetin, 78.40 μg / ml for quercetin-3-O-glucoside, and 31.36 μg / ml for nuciferine.

[0020] The advantages of the technical solution of the present invention include:

[0021] 1. The present invention provides a method for determining the content of effective ingredients in lotus leaves and their preparations, which can simultaneously determine the contents of four active ingredients, namely, hyperoside, isoquercetin, quercetin-3-O-glucoside, and nuciferine, in lotus leaves, lotus leaf water extracts, and / or lotus leaf water extract preparations.

[0022] 2. This method effectively separated the four component peaks of hyperoside, isoquercetin, quercetin-3-O-glucoside and nuciferine. The method was highly accurate, simple and rapid. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is the chromatogram in Example 4 of the present invention. DETAILED DESCRIPTION

[0024] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0026] instrument:

[0027] High performance liquid chromatography (Thermo Fisher, Ultimate3000),

[0028] UV-Vis spectrophotometer (Thermo Fisher, Evqutionz01),

[0029] Ultrasonic cleaning instrument (Tianjin Aotesense Instrument Co., Ltd., AS20100BT).

[0030] Chromatographic column: ZORBAX SB-C18 250mm*4.6mm, 5μm.

[0031] Reagents and Drugs:

[0032] Isoquercetin (Chengdu Aifa, 98%, AZ21080601),

[0033] Quercetin-3-O-glucoside (Chengdu Aifa, 98%, AZ22010653),

[0034] Nuciferine (China Food and Drug Administration, 111566-201907, 99.8%),

[0035] Hyperoside (China Food and Drug Inspection Institute, 111521-201809, 94.9%);

[0036] Phosphoric acid (Tianjin Yongda),

[0037] Distilled water (Watsons, 20220607C),

[0038] Acetonitrile (Supelco, 11163729130);

[0039] Lotus leaf slices (Hunan 20231019),

[0040] Lotus leaf decoction pieces (HYZS20231201, HYZS20231202, HYZS20231203).

[0041] Example 1

[0042] Determination of the content of active ingredients in lotus leaf medicinal materials

[0043] Determined by high performance liquid chromatography (General Rule 0512).

[0044] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel as filler; acetonitrile as mobile phase A, 0.05% phosphoric acid solution as mobile phase B, gradient elution as specified in the table below; column temperature 15°C, flow rate 0.9ml / min; detection wavelength 265nm. The theoretical plate number calculated based on the quercetin-3-O-glucoside peak should be no less than 3000.

[0045] Time program:

[0046]

[0047] Preparation of reference solution: Take appropriate amount of hyperoside, isoquercetin, quercetin-3-O-glucoside and nuciferol reference substances and add methanol to make solutions containing 37.96 μg of hyperoside, 39.20 μg of isoquercetin, 78.40 μg of quercetin-3-O-glucoside and 31.36 μg of nuciferol per 1 ml.

[0048] Preparation of test solution: Take about 2g of crude powder of this product, weigh it accurately, put it in a beaker, add 20ml of water, soak for 30min, transfer the pieces and water to a round-bottom flask, rinse the beaker with 30ml of water, transfer the rinse solution to the flask, heat and reflux for 40min, filter, add 46ml of water to the residue, heat and reflux for 30min, filter, combine the filtrate, record the volume, take a small amount, and obtain.

[0049] Determination method: Accurately pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0050] Example 2

[0051] Determination of the content of active ingredients in lotus leaf extract

[0052] [Preparation of lotus leaf extract] Take lotus leaves, spray water to moisten them slightly, cut them into shreds, dry them, add water and boil them, filter the decoction, and concentrate the filtrate into a thick paste.

[0053] Determined by high performance liquid chromatography (General Rule 0512).

[0054] Chromatographic conditions and system suitability test

[0055] Octadecylsilane bonded silica gel was used as filler; acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in the following table; column temperature was 10°C, flow rate was 1.1 ml / min, and detection wavelength was 275 nm. The theoretical plate number calculated based on the quercetin-3-O-glucoside peak should be no less than 3000.

[0056] Time program:

[0057]

[0058] Preparation of reference solution

[0059] Take appropriate amounts of hyperoside, isoquercetin, quercetin-3-O-glucoside and nuciferol reference substances and add methanol to prepare solutions containing 37.96 μg of hyperoside, 39.20 μg of isoquercetin, 78.40 μg of quercetin-3-O-glucoside and 31.36 μg of nuciferol per 1 ml.

[0060] Preparation of lotus leaf preparation solution

[0061] Take 0.5g of this product, weigh it accurately, put it in a 50ml volumetric flask, add appropriate amount of water to dissolve it by ultrasonic, make up to volume with pure water, shake well, and you have it.

[0062] Determination method

[0063] Accurately pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, and measure to obtain the result.

[0064] Example 3

[0065] Determination of the content of active ingredients in lotus leaf preparations

[0066]

Preparation method of lotus leaf water extraction granules

[0067] Take lotus leaves, add water and boil, filter the decoction, concentrate the filtrate under reduced pressure, add appropriate amount of maltodextrin, mix well, and make granules.

[0068] Determined by high performance liquid chromatography (General Rule 0512).

[0069] Chromatographic conditions and system suitability test

[0070] Octadecylsilane bonded silica gel was used as filler; acetonitrile was used as mobile phase A, 0.2% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in the following table; column temperature was 20°C, flow rate was 0.9ml / min, and detection wavelength was 270nm. The theoretical plate number calculated based on the quercetin-3-O-glucoside peak should be no less than 3000.

[0071] Time program:

[0072]

[0073] Preparation of reference solution

[0074] Take appropriate amounts of hyperoside, isoquercetin, quercetin-3-O-glucoside and nuciferol reference substances and add methanol to prepare solutions containing 37.96 μg of hyperoside, 39.20 μg of isoquercetin, 78.40 μg of quercetin-3-O-glucoside and 31.36 μg of nuciferol per 1 ml.

[0075] Preparation of test solution

[0076] Take 1.0g of this product, weigh it accurately, put it in a 100ml volumetric flask, add appropriate amount of water to dissolve it by ultrasonic, make up to volume with pure water, shake well, and you have it.

[0077] Determination method

[0078] Accurately pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, and measure to obtain the result.

[0079] Example 4

[0080] Determination method of active ingredient content in lotus leaf preparation granules and intermediates

[0081] Determined by high performance liquid chromatography (General Rule 0512).

[0082] Chromatographic conditions and system suitability test

[0083] Octadecylsilane bonded silica gel was used as filler; acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in the following table; column temperature was 15°C, flow rate was 1.0 ml / min, and detection wavelength was 270 nm. The theoretical plate number calculated based on the quercetin-3-O-glucoside peak should be no less than 3000.

[0084] Time program:

[0085]

[0086] Preparation of reference solution

[0087] Take appropriate amounts of hyperoside, isoquercetin, quercetin-3-O-glucoside and nuciferol reference substances and add methanol to prepare solutions containing 37.96 μg of hyperoside, 39.20 μg of isoquercetin, 78.40 μg of quercetin-3-O-glucoside and 31.36 μg of nuciferol per 1 ml.

[0088] Preparation of test solution

[0089] Three batches of lotus leaf slices (HYZS20231201, HYZS20231202, HYZS20231203) were taken respectively, decocted twice with water, the decoction was filtered, and the filtrate was combined to obtain lotus leaf extract. The obtained extract was concentrated under reduced pressure to a relative density of 1.15-1.25 (30°C) to obtain lotus leaf extract. The lotus leaf extract was made into granules with maltodextrin as an auxiliary material to obtain lotus leaf granules.

[0090] Take lotus leaf slices (Hunan 20231019), three batches of lotus leaf extracts (HYZS20231201, HYZS20231202, HYZS20231203), three batches of lotus leaf extracts (HYZS20231201, HYZS20231202, HYZS20231203), and three batches of lotus leaf granules (HYZS20231201, HYZS20231202, HYZS20231203), and prepare test sample solutions according to the test sample type, respectively, referring to the test sample preparation methods in Examples 1 to 3. The lotus leaf extract was directly used as the test sample solution.

[0091] Determination method

[0092] Accurately pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, and measure to obtain the result.

[0093] Results: The content determination results of three batches of lotus leaf extract and three batches of lotus leaf granules are shown in Table 1; the chromatograms of all samples are shown in Figure 1It was found that in each batch of test samples, the peaks of the four components were well separated, and the retention time, wavelength, peak area and other parameters of each batch of samples were basically consistent, verifying the reliability of the test method. The specific markings in the figure are as follows:

[0094] A: mixed standard;

[0095] B&C: Medicinal pieces Hunan 20231019;

[0096] D: HYZS20231201 extract;

[0097] E: HYZS20231202 extract;

[0098] F: HYZS20231203 extract;

[0099] G:HYZS20231201 extract;

[0100] H:HYZS20231202 extract;

[0101] I: HYZS20231203 extract;

[0102] J: HYZS20231201 granules;

[0103] K: HYZS20231202 particles;

[0104] L: HYZS20231203 particles;

[0105] Table 1 Content determination results of three batches of lotus leaf extract and three batches of lotus leaf granules

[0106]

[0107] The examples given in the invention are not intended to limit the implementation methods of the invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here, and the obvious changes or modifications derived from them are still within the scope of protection of the invention.

Claims

1. A method for determining the content of active ingredients in lotus leaf medicinal materials and their preparations, Features include: The solution of lotus leaf medicinal material and its preparation was determined by high performance liquid chromatography; The reference solution was measured by high performance liquid chromatography; in: The chromatographic conditions are as follows: the chromatographic column is filled with octadecylsilane bonded silica gel, the detection wavelength is 265nm-275nm, acetonitrile is used as mobile phase A, 0.05%-0.2% phosphoric acid solution is used as mobile phase B, and gradient elution is performed as specified in the following table:

2. A method for determining the content of active ingredients in a lotus leaf medicinal material and a preparation thereof according to claim 1, It is characterized in that The column temperature of the chromatographic column is 10°C-20°C, and the flow rate is 0.9ml / min-1.1ml / min.

3. A method for determining the content of active ingredients in a lotus leaf medicinal material and a preparation thereof according to claim 1, It is characterized in that The mobile phase B is 0.1% phosphoric acid solution; the detection wavelength is 270 nm.

4. A method for determining the content of active ingredients in a lotus leaf medicinal material and a preparation thereof according to claim 3, It is characterized in that The lotus leaf medicinal material and the preparation thereof are at least one selected from lotus leaf granules, lotus leaf extract, lotus leaf extract, lotus leaf medicinal material and lotus leaf decoction pieces.

5. A method for determining the content of active ingredients in a lotus leaf medicinal material and a preparation thereof according to any one of claims 1 to 4, It is characterized in that When the lotus leaf medicinal material and the preparation thereof are lotus leaf medicinal materials or lotus leaf decoction pieces, the preparation process of the solution of the lotus leaf medicinal material and the preparation thereof is: taking the test sample, accurately weighing, adding a solvent, pre-treating, filtering, combining the filtrate, recording the volume, taking a small amount, and obtaining, When the lotus leaf medicinal material and its preparation are lotus leaf granules or lotus leaf extract, the preparation process of the solution of the lotus leaf medicinal material and its preparation is: take the test sample, accurately weigh it, put it in a volumetric flask, add solvent, seal it, pretreat it, then add the same solvent to make up the volume, shake it well, and obtain it.

6. A method for determining the content of active ingredients in lotus leaf medicinal materials and preparations thereof according to claim 5, It is characterized in that In the preparation process of the solution of lotus leaf medicinal material or lotus leaf decoction pieces, the added solvent is water, and the pretreatment process is first soaking in water and then heating and refluxing.

7. A method for determining the content of active ingredients in lotus leaf medicinal materials and preparations thereof according to claim 6, It is characterized in that The preparation process of the solution of the lotus leaf medicinal material or lotus leaf slices is as follows: take 2g of the coarse powder of the test sample, accurately weigh it, put it in a beaker, add 20ml of water, soak for 30min, transfer the slices and water to a round-bottom flask, rinse the beaker with 30ml of water, transfer the rinse solution to the flask, heat and reflux for 40min, filter, add 46ml of water to the residue, heat and reflux for 30min, filter, combine the filtrate, record the volume, take a small amount, and obtain.

8. The method for determining the content of active ingredients in lotus leaf medicinal materials and preparations thereof according to claim 5, It is characterized in that During the preparation of the solution of the lotus leaf particles or lotus leaf extract, the added solvent is water, the pretreatment is ultrasonic treatment, and the solution concentration is 10 mg / ml.

9. The method for determining the content of active ingredients in lotus leaf medicinal materials and preparations thereof according to claim 1, Features: The reference substance preparation process is to mix hyperoside, isoquercetin, quercetin-3-O-glucoside, nuciferine, etc. The method for preparing the reference substance solution is to take the reference substance and dissolve it in a solvent respectively.

10. The method for determining the content of active ingredients in lotus leaf medicinal materials and preparations thereof according to claim 9, Features: The solvent added to the reference substance is methanol, and the concentrations of the prepared reference substance solutions are 37.96 μg / ml for hyperoside, 39.20 μg / ml for isoquercetin, 78.40 μg / ml for quercetin-3-O-glucoside, and 31.36 μg / ml for nuciferine.

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