Method for constructing fingerprint spectrum of trichosanthes kirilowii maxim and burdock soup

Abstract

The invention discloses a construction method of a trichosanthes kirilowii maxim and burdock soup fingerprint spectrum, and relates to the technical field of analytical chemistry. The determination method disclosed by the invention is simple and has the characteristics of high precision, stability and repeatability, and through determination, seven components including geniposide, liquiritin, hesperidin, arctiin, baicalin, oroxylin A-7-O-beta-D-glucuronide and wogonoside can be used as common characteristic peaks of the fingerprint spectrum of the trichosanthes kirilowii Maxim and arctium lappa decoction for identification and research.

Description

Technical Field

[0001] The invention relates to the technical field of analytical chemistry, and in particular to a method for constructing a fingerprint spectrum of trichosanthes and burdock soup. Background Art

[0002] Gualou Niubang Decoction is known for its functions of relieving liver depression, detoxifying and eliminating carbuncle. As a classic prescription for treating breast diseases, it is widely used in TCM clinics. The composition of this prescription was first recorded in Volume 3 of the surgical monograph "The Authentic Surgery" written by Chen Shigong in the Ming Dynasty, "Niubangzi Decoction": "Cited tangerine peel, burdock seed, gardenia, honeysuckle, licorice, Gualou Ren, Scutellaria, Radix Trichosanthis, Forsythiae, Gleditsiae Spinosa (one qian each), Bupleurum, Citrus aurantium (five fen each). It is mainly used to treat breast abscesses, breast carbuncle, swelling and pain, regardless of whether it is new or old, but not yet purulent." It was renamed "Gualou Niubang Decoction" in "The Golden Mirror of Medical Ancestral", with the same prescription, and is used to treat the initial stage of breast abscesses, redness, swelling, heat and pain, or chills and fever. In modern TCM clinics, it is mainly used to treat acute mastitis and other diseases, and has significant therapeutic effects in the early inflammatory stage.

[0003] At present, the research content of Gua Lou Niu Bang Decoction is still mainly focused on the modern clinical application and mechanism of action of the prescription. It is extremely necessary to provide a method for constructing the fingerprint of Gua Lou Niu Bang Decoction in order to achieve the efficient determination of the reference substance of Gua Lou Niu Bang Decoction. Summary of the invention

[0004] The purpose of the present invention is to provide a method for constructing a fingerprint of trichosanthes and burdock soup, so as to solve the problems existing in the above-mentioned prior art and realize the efficient determination of the reference substance of trichosanthes and burdock soup.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a method for constructing a fingerprint of trichosanthes and burdock soup, comprising the following steps:

[0007] Preparation of test solution: Prepare the test solution from the freeze-dried powder of the decoction of Trichosanthes and Burdock;

[0008] Preparation of reference solution: Gardenia jasminoides, liquiritin, hesperidin, arctiin, baicalin, melaleuca alternifolia A-7-O-β-D-glucuronide and wogonin were used as reference substances to prepare reference solution;

[0009] Construction of fingerprint: HPLC method was used for analysis and determination to obtain the fingerprint of the Trichosanthes and Burdock Decoction.

[0010] Furthermore, the preparation method of the freeze-dried powder of the water decoction of trichosanthes and burdock comprises the following steps:

[0011] Add water to the raw materials of the Gualou Niubang decoction for decoction, and freeze-dry the obtained decoction to obtain the Gualou Niubang decoction decoction freeze-dried powder;

[0012] The raw materials are dried tangerine peel, burdock fruit, gardenia, honeysuckle, liquorice, trichosanthes seed, scutellaria baicalensis, radix trichosanthis, forsythia suspensa, thorn of gleditsia sinensis, bupleurum and green peel.

[0013] Furthermore, the burdock seeds are stir-fried burdock seeds, and the forsythia suspensa is cored.

[0014] Furthermore, the mass ratio of the raw materials is: 3.73g of dried tangerine peel, 3.73g of burdock fruit, 3.73g of gardenia, 3.73g of honeysuckle, 3.73g of licorice, 3.73g of trichosanthes seed, 3.73g of scutellaria, 3.73g of trichosanthes root, 3.73g of forsythia, 3.73g of gleditsia spine, 1.85g of bupleurum and 1.85g of green peel.

[0015] Furthermore, the chromatographic column used for the HPLC analysis was Micsphere C 18 Chromatographic column, 5 μm, 4.6 × 150 mm.

[0016] Furthermore, the chromatographic conditions for HPLC analysis were: methanol-acetonitrile as phase A and 0.2% formic acid aqueous solution as phase B for gradient elution; flow rate 1.0 mL / min; injection volume: 10 μL; column temperature 30° C.; detection wavelength: 254 nm or 280 nm.

[0017] Furthermore, the volume ratio of methanol and acetonitrile used in phase A is 10:1.

[0018] Further, the gradient elution procedure is as follows:

[0019]

[0020] Furthermore, the solvent used to prepare the test solution is methanol aqueous solution.

[0021] Furthermore, the solvent used to prepare the reference solution is methanol.

[0022] The present invention discloses the following technical effects:

[0023] The invention provides a method for constructing a fingerprint of trichosanthes and burdock soup. The determination method is simple and has the characteristics of high precision, stability and repeatability. According to determination, seven components, namely, gardenia glycoside, liquiritin, hesperidin, arctiin, baicalin, melaleucatin A-7-O-β-D-glucuronide and wogonin, can be used as common characteristic peaks of the fingerprint of trichosanthes and burdock soup for identification research. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0025] Figure 1 HPLC spectrum of the mixed standard reference substance in Example 1 of the present invention;

[0026] Figure 2 This is the HPLC comparison chart of the freeze-dried powder of the water decoction of Trichosanthes and Burdock Decoction of Example 1 of the present invention at 254 nm;

[0027] Figure 3 The HPLC comparison chart of the freeze-dried powder of the water decoction of Trichosanthes and Burdock Decoction of Example 1 of the present invention at 280 nm;

[0028] Figure 4 The HPLC fingerprints of 15 batches of reference materials of Gualou Niubang Decoction in Example 1 of the present invention at 254 nm;

[0029] Figure 5 This is the HPLC fingerprint of 15 batches of reference substances in Trichosanthes and Burdock Decoction in Example 1 of the present invention at 280 nm. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] Example 1

[0036] 1. Instruments and reagents:

[0037] High performance liquid chromatograph: Shimadzu LC-20AT (AIL-20A, SPD-M20A, CT0-20A); Chromatographic column: MicsphereC 18 Chromatographic column (5 μm, 4.6 × 150 mm); Analytical electronic balance: Shanghai Liangping Instrument Co., Ltd., FA1004; Ultrasonic cleaner: Shanghai Kedao Ultrasonic Instrument Co., Ltd., SK7210HP;

[0038] Acetonitrile and methanol used in liquid phase were chromatographic grade reagents from Fisher Company; formic acid used in liquid phase was chromatographic grade (batch number: C14547531, Shanghai MacLean Biochemical Technology Co., Ltd.); water was high-purity water; methanol used in sample preparation was analytical grade (batch number: C16694794, Shandong Keyuan Biochemical Co., Ltd.);

[0039] Reference substances: Gardenia glycoside (batch number: 110749-202320), liquiritin (batch number: 110749-202320), hesperidin (batch number: 110721-202220), arctiin (batch number: 110819-202213), and baicalin (batch number: 110715-201720) were all purchased from China Food and Drug Inspection Institutes; melaleuca alternifolia A-7-O-β-D-glucuronide (batch number: 23111396) and baicalin (batch number: 23080601) were purchased from Beijing BetterKang Biotechnology Co., Ltd.

[0040] Test sample: Trichosanthes and Burdock Decoction reference material, 15 batches in total (numbered S1-S15).

[0041] The raw materials of Trichosanthes and Burdock Soup are 12 kinds of herbs, including dried tangerine peel, burdock fruit, gardenia, honeysuckle, licorice, trichosanthes seed, scutellaria, radix trichosanthis, forsythia, sapodilla spine, bupleurum, and green peel; among them, the burdock fruit is fried, the forsythia is cored, and the other 10 herbs are all raw.

[0042] The preparation steps of each batch of Trichosanthes and Burdock Soup reference material are as follows:

[0043] Take 3.73g of dried orange peel, 3.73g of burdock seeds, 3.73g of gardenia, 3.73g of honeysuckle, 3.73g of liquorice, 3.73g of trichosanthes seeds, 3.73g of scutellaria, 3.73g of trichosanthes root, 3.73g of forsythia, 3.73g of licorice, 1.85g of bupleurum, 1.85g of green peel, add 400mL of water, and boil until 160mL. The obtained decoction is freeze-dried to obtain freeze-dried powder, which is the reference substance of trichosanthes and burdock soup (freeze-dried powder of trichosanthes and burdock soup decoction).

[0044] 2. Construction of fingerprint

[0045] (1) Chromatographic conditions

[0046] Column: Micsphere C 18 Chromatographic column (5 μm, 4.6×150 mm).

[0047] Mobile phase: organic phase (phase A) is methanol-acetonitrile with a volume ratio of 10:1, and aqueous phase (phase B) is 0.2% formic acid aqueous solution; two-phase gradient elution; flow rate 1.0 ml / min; injection volume: 10 μL; column temperature 30°C; detection wavelength: 254 nm or 280 nm.

[0048] The gradient elution program is shown in Table 1.

[0049] Table 1

[0050]

[0051] (2) Preparation of reference solution

[0052] Take appropriate amount of gardenia glycoside, glycyrrhizin, hesperidin, arctiin, baicalin, melaleuca alternifolia A-7-O-β-D-glucuronide and wogonin reference substances respectively, weigh accurately, and add methanol to prepare reference substance mother solutions with mass concentrations of 1120, 534, 438, 166, 1702, 342 and 624 μg / mL, respectively.

[0053] Take 1 mL of each of the above mother solutions and dilute to 10 mL volumetric flask to prepare mixed standard solutions.

[0054] (3) Preparation of test solution

[0055] Take 0.25 g of freeze-dried powder of the decoction of Trichosanthes and Arctium, weigh it accurately, add 5 mL of 80% methanol aqueous solution, weigh it, treat it with ultrasound (53KHz, 350W) for 30 min, let it cool to room temperature, weigh it again, make up the lost weight with 80% methanol aqueous solution, shake it well, filter it, take the filtrate, and filter it through a 0.22μm microporous filter membrane to obtain it.

[0056] (4) Methodological investigation

[0057] a. Precision

[0058] Take an appropriate amount of the test solution of Gualou Niubang Decoction and inject it continuously for 6 times. The calculated peak area RSD (%) values ​​of gardenia glycoside, liquiritin, hesperidin, arctiin, baicalin, melaleuca A-7-O-β-D-glucuronide, and wogonin were 0.16, 0.70, 0.45, 0.25, 3.23, 0.29, and 0.20, respectively, indicating that the instrument has good precision.

[0059] b. Stability

[0060] The sample solution of Gualou Niubang Decoction was injected within 0, 4, 8, 12, 16, 20, and 24 hours. The peak area RSD (%) values ​​of gardenia glycoside, liquiritin, hesperidin, arctiin, baicalin, melaleuca A-7-O-β-D-glucuronide, and wogonin were calculated to be 0.26, 1.41, 0.85, 0.70, 2.66, 0.44, and 0.41, respectively, indicating that the sample solution has good stability within 24 hours.

[0061] c. Repeatability

[0062] Weigh 6 portions of freeze-dried powder of the same batch of Gualou Niubang decoction decoction to prepare the test solution and inject it. The peak area RSD (%) values ​​of gardenia glycoside, liquiritin, hesperidin, arctiin, baicalin, melaleuca A-7-O-β-D-glucuronide and wogonin were calculated to be 1.43, 1.08, 2.61, 1.37, 2.90, 1.73 and 0.57, respectively, indicating that the method has good repeatability.

[0063] (5) Sample measurement

[0064] The freeze-dried powder samples of the decoction of Trichosanthes and Arctium were taken respectively, prepared according to the method under the test solution, measured according to the sample measurement method, the chromatograms were recorded, and the similarity was calculated using similarity software. The similarity was calculated based on 7 characteristic peaks. The results are shown in Table 2.

[0065] Table 2 Similarity calculation results of fingerprint spectra of 15 batches of freeze-dried powder of Trichosanthes and Burdock Decoction decoction

[0066]

[0067] The test results showed that the similarities of the fingerprints of 15 batches of freeze-dried powder of Trichosanthes and Arctium decoction were calculated based on 7 common peaks, all of which were greater than 0.985, indicating that the similarity between the 15 batches of reference substances was good.

[0068] The mixed standard reference material spectrum is shown in Figure 1 ; The reference fingerprint of the freeze-dried powder of Gualou Niubang decoction is shown in Figure 2 (254nm) and Figure 3 (280nm); The fingerprints of 15 batches of reference substances of Gualou Niubang Decoction are shown in Figure 4 (254nm) and Figure 5 (280nm) (S1-S15: samples, R: control fingerprints).

[0069] Figure 1-Figure 5 In the spectrum: 1. Gardenia glycoside, 2. Licorice glycoside, 3. Hesperidin, 4. Arctiin, 5. Baicalin, 6. Melaleuca alternifolia A-7-O-β-D-glucuronide, 7. Wogonin.

[0070] After determination of 15 batches of samples, 7 components, including gardenia glycoside, liquiritin, hesperidin, arctiin, baicalin, melaleucatin A-7-O-β-D-glucuronide and wogonin, could be used as the common characteristic peaks of the fingerprint of Trichosanthes and Burdock Decoction for identification research.

[0071] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for constructing a fingerprint of trichosanthes and burdock soup, characterized in that: The following steps are involved: Preparation of test solution: Prepare the test solution from the freeze-dried powder of the decoction of Trichosanthes and Burdock; Preparation of reference solution: Gardenia jasminoides, liquiritin, hesperidin, arctiin, baicalin, melaleuca alternifolia A-7-O-β-D-glucuronide and wogonin were used as reference substances to prepare reference solution; Construction of fingerprint: HPLC method was used for analysis and determination to obtain the fingerprint of the Trichosanthes and Burdock Decoction.

2. The method for constructing the fingerprint of Gualou Niubang decoction according to claim 1, characterized in that: The preparation method of the freeze-dried powder of the water decoction of trichosanthes and burdock comprises the following steps: Adding water to the raw materials of the Gualou Niubang decoction for decoction, and freeze-drying the obtained decoction to obtain the Gualou Niubang decoction decoction freeze-dried powder; The raw materials are dried tangerine peel, burdock fruit, gardenia, honeysuckle, liquorice, trichosanthes seed, scutellaria baicalensis, radix trichosanthis, forsythia suspensa, thorn of gleditsia sinensis, bupleurum and green peel.

3. The method for constructing the fingerprint of Trichosanthes and Burdock Soup according to claim 2, characterized in that: The burdock seeds are stir-fried burdock seeds, and the forsythia suspensa is cored.

4. The method for constructing the fingerprint of Trichosanthes and Burdock Soup according to claim 3, characterized in that: The mass ratio of the raw materials is: 3.73g of dried tangerine peel, 3.73g of burdock fruit, 3.73g of gardenia, 3.73g of honeysuckle, 3.73g of liquorice, 3.73g of trichosanthes seed, 3.73g of scutellaria, 3.73g of trichosanthes root, 3.73g of forsythia, 3.73g of gleditsia spine, 1.85g of bupleurum and 1.85g of green peel.

5. The method for constructing the fingerprint of Trichosanthes and Burdock Soup according to claim 1, characterized in that: The HPLC analysis was performed using a Micsphere C column. 18 Chromatographic column, 5 μm, 4.6 × 150 mm.

6. The method for constructing the fingerprint of Trichosanthes and Burdock Soup according to claim 1, characterized in that: The chromatographic conditions for HPLC analysis are: methanol-acetonitrile as phase A and 0.2% formic acid aqueous solution as phase B for gradient elution; flow rate 1.0 mL / min; injection volume: 10 μL; column temperature 30° C.; detection wavelength: 254 nm or 280 nm.

7. The method for constructing the fingerprint of Gualou Niubang decoction according to claim 6, characterized in that: The volume ratio of methanol to acetonitrile used in phase A was 10:

1.

8. The method for constructing the fingerprint of Gualou Niubang decoction according to claim 6, characterized in that: The gradient elution procedure is as follows:

9. The method for constructing the fingerprint of Gualou Niubang decoction according to claim 6, characterized in that: The solvent used to prepare the test solution is methanol-water solution.

10. The method for constructing the fingerprint of Gualou Niubang decoction according to claim 6, characterized in that: The solvent used to prepare the reference solution is methanol.

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