A method for constructing a fingerprint of trichosanthes and burdock soup

Abstract

The present invention discloses a method for constructing a fingerprint of Gualou Niu Bang decoction, relating to the technical field of analytical chemistry. The method is simple and has high precision, stability, and repeatability. Determination revealed that seven components, including geniposide, liquiritin, hesperidin, arctiin, baicalin, melaleucalyptol A-7-O-β-D-glucuronide, and wogonin, can be used as common characteristic peaks in the fingerprint of Gualou Niu Bang decoction for identification research.

Description

Technical Field

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

[0002] Gualou Niubang Decoction is known for its effects on relieving liver depression, detoxifying and eliminating carbuncles. As a classic prescription for treating breast diseases, it is widely used in clinical Chinese medicine. The composition of this prescription was first recorded in Volume 3 of "Niubangzi Decoction", a surgical monograph written by Chen Shigong in the Ming Dynasty, "The Authentic Surgery": "Dried tangerine peel, burdock seed, gardenia, honeysuckle, licorice, Gualou kernel, scutellaria, radix trichosanthis, forsythia, soapberry thorn (one qian each), bupleurum, and green peel (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 has not yet formed pus." It was later renamed "Gualou Niubang Decoction" in "The Golden Mirror of the Medical Ancestral". The prescription is the same and is used to treat the initial stage of breast abscesses, redness, swelling, heat and pain, or chills and fever. In modern Chinese clinical practice, it is mainly used to treat acute mastitis and other diseases, and has a significant therapeutic effect in the early inflammatory stage.

[0003] At present, the research content of Trichosanthes and Burdock Decoction is still mostly focused on the modern clinical application and mechanism of action of the prescription. Providing a method for constructing the fingerprint of Trichosanthes and Burdock Decoction is extremely necessary to achieve the efficient determination of the reference substances of Trichosanthes and Burdock 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 kirilowii and burdock root;

[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] Fingerprint construction: HPLC method was used for analysis and determination to obtain the fingerprint of the Trichosanthes and Arctium Decoction.

[0010] Furthermore, the preparation method of the freeze-dried powder of the Trichosanthes and Burdock Decoction decoction comprises the following steps:

[0011] The raw materials of the trichosanthes and burdock soup are added with water for decocting, and the obtained decoction is freeze-dried to obtain the freeze-dried powder of the trichosanthes and burdock soup decoction;

[0012] The raw materials are dried tangerine peel, burdock fruit, gardenia, honeysuckle, liquorice, trichosanthes seed, scutellaria, radix trichosanthis, forsythia, thorn of honeysuckle, 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 radix trichosanthis, 3.73g of forsythia, 3.73g of soapberry spines, 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 as follows: gradient elution with methanol-acetonitrile as phase A and 0.2% formic acid aqueous solution as phase B; 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 to acetonitrile used in phase A is 10:1.

[0018] Furthermore, 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 present invention provides a method for constructing a fingerprint of Gualou Niu Bang decoction. The determination method is simple and has the characteristics of high precision, stability and repeatability. According to the determination, seven components, namely, geniposide, liquiritin, hesperidin, arctiin, baicalin, melaleuca alternifolia A-7-O-β-D-glucuronide and wogonin, can be used as common characteristic peaks of the fingerprint of Gualou Niu Bang decoction 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 following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[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 kirilowii and Arctium lappa in Example 1 of the present invention at 254 nm;

[0027] Figure 3 This is the HPLC comparison chart of the freeze-dried powder of the water decoction of Trichosanthes kirilowii and Arctium lappa in Example 1 of the present invention at 280 nm;

[0028] Figure 4 HPLC fingerprints at 254 nm for 15 batches of reference materials for Trichosanthes kirilowii and burdock soup in Example 1 of the present invention;

[0029] Figure 5 This is the HPLC fingerprint at 280 nm of 15 batches of reference substances for the Gualou Niubang decoction in Example 1 of the present invention. 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 rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

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

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice 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 any conflict with any incorporated document, the contents 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 described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative 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: Micsphere C 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 analysis were chromatographic grade reagents from Fisher; formic acid used in liquid phase analysis was chromatographic grade (Batch No. C14547531, Shanghai MacLean Biochemical Technology Co., Ltd.); water was high-purity water; and methanol used in sample preparation was analytical grade (Batch No. C16694794, Shandong Keyuan Biochemical Co., Ltd.).

[0039] Reference substances: Gardeniaside (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 purchased from the China Food and Drug Administration; melastomerein A-7-O-β-D-glucuronide (batch number: 23111396) and wogonin (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 ingredients of Trichosanthes and Burdock Soup are 12 herbs in total, including dried tangerine peel, burdock fruit, gardenia, honeysuckle, licorice, Trichosanthes seed, scutellaria, radix trichosanthis, forsythia, soapberry thorn, bupleurum, and green peel; among them, the burdock fruit is fried, the forsythia is cored, and the other 10 herbs are all used raw.

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

[0043] Take 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 spinosa, 1.85g of bupleurum, and 1.85g of citrus aurantium, add 400mL of water, and boil until the volume is reduced to 160mL. The resulting decoction is lyophilized to obtain a lyophilized powder, which is the reference material for trichosanthes and burdock soup (lyophilized 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 amounts of gardeniain, glycyrrhizin, hesperidin, arctiin, baicalin, melaleuca alternifolia A-7-O-β-D-glucuronide, and wogonin reference substances, accurately weigh them, and add methanol to prepare reference substance stock 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 a 10 mL volumetric flask to prepare a mixed standard solution.

[0054] (3) Preparation of test solution

[0055] Take 0.25 g of freeze-dried powder of the decoction of Trichosanthes kirilowii and Arctium lappa, accurately weigh it, accurately add 5 mL of 80% methanol aqueous solution, weigh the mass, treat it with ultrasound (53 kHz, 350 W) 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 0.22 μm microporous filter membrane to obtain the product.

[0056] (4) Methodological investigation

[0057] a. Precision

[0058] An appropriate amount of the Gualou Niubang decoction test solution was sampled six times. The calculated peak area RSDs (%) for geniposide, liquiritin, hesperidin, arctiin, baicalin, melaleuca alternifolia 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 good instrument 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 calculated peak area RSDs (%) for geniposide, liquiritin, hesperidin, arctiin, baicalin, melaleuca alternifolia A-7-O-β-D-glucuronide, and wogonin were 0.26, 1.41, 0.85, 0.70, 2.66, 0.44, and 0.41, respectively, indicating that the sample solution had good stability within 24 hours.

[0061] c. Repeatability

[0062] Six portions of freeze-dried powder from the same batch of Gualou Niu Bang decoction were accurately weighed to prepare the test solution and injected. The calculated RSDs (%) for the peak areas of geniposide, liquiritin, hesperidin, arctiin, baicalin, melaleuca alternifolia A-7-O-β-D-glucuronide, and wogonin were 1.43, 1.08, 2.61, 1.37, 2.90, 1.73, and 0.57, respectively, demonstrating good reproducibility of the method.

[0063] (5) Sample measurement

[0064] Freeze-dried powder samples of the decoction of Trichosanthes kirilowii and Arctium lappa were taken respectively and prepared according to the method under the test solution. The samples were measured according to the sample determination 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 fingerprints of 15 batches of freeze-dried powder of Trichosanthes kirilowii and burdock decoction

[0066]

[0067] The test results showed that the similarities of the fingerprints of 15 batches of freeze-dried powder of Trichosanthes kirilowii and Arctium lappa 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 sample is shown in Figure 1 ; The 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 (280 nm) (S1-S15: samples, R: control fingerprints).

[0069] Figure 1-Figure 5 In the spectrum: 1. Gardenia jasminoides, 2. Glycyrrhizin, 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 geniposide, liquiritin, hesperidin, arctiin, baicalin, melaleuca alternifolia A-7-O-β-D-glucuronide and wogonin, could be used as the common characteristic peaks of the fingerprint of Gualou Niubang decoction for identification research.

[0071] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for constructing a fingerprint of Trichosanthes kirilowii and burdock soup, characterized in that: The following steps are involved: Preparation of test solution: freeze-dried powder of Trichosanthes kirilowii and Arctium lappa decoction was added to methanol-water solution and ultrasonicated to prepare test solution; 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; Fingerprint construction: HPLC method was used for analysis and determination to obtain the fingerprint of the Trichosanthes and Arctium Decoction; The HPLC analysis was performed using a Microsphere C column. 18 Chromatographic column, 5 μm, 4.6 × 150 mm; The chromatographic conditions for HPLC analysis and determination are as follows: methanol-acetonitrile as phase A, 0.2% formic acid aqueous solution as phase B for gradient elution; the volume ratio of methanol to acetonitrile used in phase A is 10:1; the detection wavelength is 254 nm or 280 nm; The gradient elution procedure is as follows:

2. The method for constructing the fingerprint of Trichosanthes and Burdock Decoction according to claim 1, characterized in that: The preparation method of the freeze-dried powder of the trichosanthes and burdock decoction comprises the following steps: The raw materials of the trichosanthes and burdock soup are added with water for decocting, and the obtained decoction is freeze-dried to obtain the freeze-dried powder of the trichosanthes and burdock soup decoction; The raw materials are dried tangerine peel, burdock fruit, gardenia, honeysuckle, liquorice, trichosanthes seed, scutellaria, radix trichosanthis, forsythia, thorn of honeysuckle, bupleurum and green peel.

3. The method for constructing the fingerprint of Gualou Niubang decoction 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 Gualou Niubang decoction 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 tricholoma, 3.73g of forsythia, 3.73g of soapberry spines, 1.85g of bupleurum and 1.85g of green peel.

5. The method for constructing the fingerprint of Gualou Niubang decoction according to claim 1, characterized in that: When the HPLC method was used for analysis and determination, the flow rate was 1.0 mL / min; the injection volume was 10 μL; and the column temperature was 30°C.

6. The method for constructing the fingerprint of Gualou Niubang Decoction according to claim 5, characterized in that: The solvent used to prepare the reference solution was methanol.

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