Method for determining content of 2, 6-dipicolinic acid in bombyx batryticatus and preparation thereof and application of method

Abstract

The invention discloses a method for determining the content of 2, 6-dipicolinic acid in bombyx batryticatus and a preparation thereof and application of the method, and belongs to the technical field of traditional Chinese medicine chemistry. According to the determination method, the content of the 2, 6-dipicolinic acid in the bombyx batryticatus and the preparation thereof is determined through a high performance liquid chromatography method, and the content of the 2, 6-dipicolinic acid serves as a characteristic peak in a liquid chromatography characteristic spectrum; therefore, the method is applied to quality detection of stiff silkworms and preparations thereof. The 2, 6-dipicolinic acid is used as an index component under quality detection items such as chemical identification and content determination of the stiff silkworm and the traditional Chinese medicine preparation containing the stiff silkworm, and the defect that the existing pharmacopoeia standard is lack of index components is overcome; the characteristic of the 2, 6-dipicolinic acid is obviously better than that of uracil, adenosine, guanosine and other nucleoside components, detection is facilitated, and a reference substance is cheap and easy to obtain and can be used as a characteristic peak for identification to improve the quality standard of the stiff silkworm formula granules.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine chemistry, and in particular to a method for determining the content of 2,6-pyridinedicarboxylic acid in silkworm pupa and a preparation thereof, and application thereof. Background Art

[0002] Bombyx Batryticatus, also known as white silkworm, dead insect, and heavenly insect, is a commonly used traditional Chinese medicine in my country. It was first recorded in the "Shennong Bencao Jing" of the Eastern Han Dynasty and has a long history of use. The 2020 edition of the "Chinese Pharmacopoeia" records that Bombyx Batryticatus is an insect of the Bombycidae family, the silkworm. Bombyx mori Linnaeus 4th to 5th instar larvae infected (or artificially inoculated) with Beauveria bassiana Beauveria bassiana The dried body of Bals. Vuillant has the effects of calming wind and stopping spasms, dispelling wind and relieving pain, and resolving phlegm and dispersing knots. It is used to treat liver wind with phlegm, convulsions due to epilepsy, acute convulsions in children, tetanus, stroke, wind-heat headache, red eyes and sore throat, itching due to urticaria, and mumps. Bombyx mori contains chemical components such as proteins, polypeptides, organic acids and ammonium oxalate, steroids, flavonoids, amino acids, and nucleosides. Pharmacological studies have shown that Bombyx mori has anticonvulsant, anticoagulant, anti-inflammatory, anticancer, and antibacterial effects. Bombyx mori is mainly used in the treatment of convulsions, epilepsy, stroke, nephritis, asthma and other diseases, especially in the treatment of pediatric diseases such as high fever, bronchial asthma, and tics. The preparations containing Bombyx mori included in the 2020 edition of the Chinese Pharmacopoeia include more than 30 kinds of preparations such as Ginseng Zaizao Pills, Zhongfeng Huichun Tablets, and Yaotongning Capsules.

[0003] Decoction pieces refer to Chinese medicinal materials that have been processed and prepared under the guidance of traditional Chinese medicine theory and can be directly used for preparation or formulation. The 2020 edition of the Chinese Pharmacopoeia stipulates that silkworm slices are silkworm slices that have been washed, dried, and impurities removed. Stir-fried silkworm slices are made by taking clean silkworms and frying them according to the bran frying method until the surface turns yellow. Chinese medicine formula granules are a new type of decoction piece. They are granules made from single Chinese medicine slices that are processed according to traditional standards and then extracted and concentrated for use in Chinese medicine clinical prescriptions.

[0004] The inventors of this application have noticed that Bombyx batryticatus medicinal materials and decoction pieces and Chinese medicine preparations containing Bombyx batryticatus still have obvious deficiencies in quality inspection and quality standards. Specifically, for example, the quality standards for Bombyx batryticatus medicinal materials and decoction pieces included in the 2020 edition of the Chinese Pharmacopoeia do not include chemical composition-based detection methods and quality standards such as content determination, thin-layer chromatography identification, and characteristic spectrum identification, and only stipulate that its alcohol-soluble extract shall not be less than 20.0%.

[0005] In addition, there is currently no national standard for Bombyx Batryticatus Formula Granules. The provincial local standards of each province usually use nucleoside components as characteristic peaks for characteristic spectrum identification and indicator components for content determination. For example, in the Guangdong Province's Bombyx Batryticatus Formula Granule Quality Standard (Guangdong PFKL20220049) and the Fried Bombyx Batryticatus Formula Granule Quality Standard (Guangdong PFKL2022008), the characteristic spectrum uses adenine, uridine, and adenosine as characteristic peaks, and the content determination uses the total amount of adenine and adenosine as a quantitative control indicator. In the Beijing Bombyx Batryticatus Formula Granule Quality Standard (BJ-PFKL-2022104) and the Fried Bombyx Batryticatus Formula Granule Quality Standard (BJ-PFKL-2022004), the characteristic spectrum uses uracil, uridine, and guanosine as characteristic peaks, and the content determination uses uracil as a quantitative control indicator. It is known to those skilled in the art that nucleoside components are small molecule compounds commonly found in animals and plants. There are obvious deficiencies in the rationality of using such components as chemical identification characteristics or content determination index components of Bombyx mori. Summary of the invention

[0006] The purpose of the present invention is to provide a method for determining the content of 2,6-pyridinedicarboxylic acid in silkworm pupa and its preparations to solve the above problems.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows: a method for determining the content of 2,6-pyridinedicarboxylic acid in silkworm pupa and its preparation, comprising the following steps: (1) Preparation of test solution: Take Bombyx batryticatus medicinal materials, decoction pieces or preparations, crush them, add solvent for extraction, filter, and use the filtrate as the test solution; (2) Preparation of reference solution: Take 2,6-pyridinedicarboxylic acid and add solvent to prepare reference solutions of different concentrations; (3) Preparing a standard curve: using high performance liquid chromatography, preparing a standard curve using the reference solution of different concentrations prepared in step (2); (4) Sample content determination: Take the test sample solution in step (1) and perform determination using the same method and chromatographic conditions as in step (3).

[0008] As a preferred technical solution, in step (1), the specific preparation method is: the sample is crushed and then sieved; then 0.5 g of the sample powder is taken, accurately weighed, 25 mL of 20 vol.% methanol is added, the weight is weighed, ultrasonic treatment is performed for 30 minutes, cooled, weighed, the lost weight is supplemented with 20 vol.% methanol, shaken, filtered, and the filtrate is obtained.

[0009] As a preferred technical solution, in step (2), 2,6-pyridinedicarboxylic acid is accurately weighed and added with 20 vol.% methanol to prepare a 1.09 mg / mL solution as a reference stock solution; the stock solution is diluted with 20 vol.% methanol to a series of concentrations, namely 218, 109, 54.5, 27.3, and 13.6 mg / mL.

[0010] As a preferred technical solution, in step (3), the HPLC conditions for determining the Bombyx Batryticatus medicinal materials or decoction pieces are as follows: using a UPLC chromatography system; acetonitrile is chromatographically pure, and water is ultrapure water; the chromatographic column is a C18 column; the mobile phase is acetonitrile-0.2 vol.% phosphoric acid aqueous solution (5:95); the flow rate is 0.4 mL / min; the column temperature is 30°C; and the detection wavelength is 270 nm.

[0011] As a preferred technical solution, the preparation is Jisheng Wumei Tablets, and the chromatographic conditions during its determination are: using a UPLC chromatography system, the chromatographic column is a C18 column; the mobile phase is acetonitrile (A)-0.2 vol.% phosphoric acid water (B); gradient elution 0-1.5 min (2 vol.% A), 1.5-3.0 min (2 vol.% A-3 vol.% A), 3.0-15.0 min (3 vol.% A-30vol.% A), 15.0-16.0 min (30 vol.% A-100 vol.% A); flow rate 0.4 mL / min; column temperature 30°C; detection wavelength 270 nm.

[0012] The second object of the present invention is to provide an application of 2,6-pyridinedicarboxylic acid in the quality detection of Bombyx batryticatus and its preparations.

[0013] As a preferred technical solution, 2,6-pyridinedicarboxylic acid is used as a characteristic peak in the characteristic spectrum of Bombyx batryticatus and its preparation.

[0014] As a preferred technical solution, the silkworm is a silkworm medicinal material or a silkworm decoction piece, and the preparation is Jisheng Wumei tablets.

[0015] In response to the above problems, the inventors of the present application conducted in-depth chemical research on Bombyx mori, and ultimately discovered a compound 2,6-pyridinedicarboxylic acid from Bombyx mori, which has strong characteristics, easy-to-detect chromatographic signals, and cheap and readily available reference substances. This provides a more suitable indicator component for improving the quality inspection methods and quality standards of Bombyx mori and traditional Chinese medicine preparations containing Bombyx mori.

[0016] Specifically, the inventor of the present application has isolated a pyridine dicarboxylic acid compound, namely 2,6-pyridine dicarboxylic acid (C7H5NO4), from silkworm slices by solvent extraction, chromatographic separation, crystallization and other methods after a large number of creative experiments. It is reported that the compound has inhibitory activity against phosphomannose isomerase, lipoamide dehydrogenase, glutamate dehydrogenase, etc., and also has the effect of killing nematodes. The inventors showed through liquid chromatography that the 2,6-pyridine dicarboxylic acid can be detected in silkworms and preparations containing silkworms. The only known natural sources of 2,6-pyridine dicarboxylic acid are Cordyceps militaris and the culture fluid of Penicillium and Streptomyces. Therefore, the compound has strong source specificity. Using it as a chemical identification feature or content determination indicator component of silkworms and their preparations has significant significance for the quality control of silkworms and their preparations.

[0017] Compared with the prior art, the advantages of the present invention are: 2,6-pyridinedicarboxylic acid is used as an indicator component in quality inspection items such as chemical identification and content determination of Bombyx mori and traditional Chinese medicine preparations containing Bombyx mori, making up for the deficiency of lack of indicator components in the current pharmacopoeia standards; the characteristic of 2,6-pyridinedicarboxylic acid is obviously better than that of nucleoside components such as uracil, adenosine, guanosine, etc., and it is conducive to detection, and the reference substance is cheap and easy to obtain, so it can be used as a characteristic peak for identification to improve the quality standard of Bombyx mori formula granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the HR-MS spectrum of 2,6-pyridinedicarboxylic acid isolated from Bombyx batryticatus; Figure 2 2,6-pyridinedicarboxylic acid isolated from Bombyx batryticatus 1 H-NMR spectrum; Figure 3 2,6-pyridinedicarboxylic acid isolated from Bombyx batryticatus 13 C-NMR spectrum; Figure 4 This is the standard curve of 2,6-pyridinedicarboxylic acid reference substance; Figure 5 This is the detection chromatogram of 2,6-pyridinedicarboxylic acid in Bombyx batryticatus; Figure 6 This is the detection chromatogram of 2,6-pyridinedicarboxylic acid in the traditional Chinese medicine preparation containing Bombyx batryticatus. DETAILED DESCRIPTION

[0019] The present invention will be further described below in conjunction with the accompanying drawings.

[0020] It should be noted that, in the following embodiments, all percentages (%) involved refer to volume percentages unless otherwise specified.

[0021] Example 1

[0022] The extraction and separation of 2,6-pyridinedicarboxylic acid from Bombyx batryticatus comprises the following steps: The dried Bombyx batryticatus medicinal material was crushed into coarse powder; 500 g of the coarse powder was weighed, 2 L of methanol was added, ultrasonic extraction was performed for 1 hour, filtration was performed, the filtrate was collected, and the drug residue was extracted twice; the filtrate was combined and concentrated to about 60 mL under reduced pressure. 120 mL of water was added to the concentrate, mixed, and extracted with ethyl acetate (40 mL × 3 times). The aqueous layer after ethyl acetate extraction was concentrated under reduced pressure to about 60 mL, applied to an MCI-Gel fine separation resin column (20 cm × 3 cm), eluted with 200 mL of pure water, and the water eluate was collected. After reduced pressure concentration, the YMC ODS-AQ reverse phase silica gel column (46 cm × 2.4 cm) was applied. The mobile phase consisted of 0.4% formic acid and 10% ethanol, and the gradient elution was performed at a flow rate of 20 mL / min. The eluate was collected (30 mL / tube); after UPLC-DAD detection, the eluates with high content of the target component were combined, concentrated under reduced pressure to a small volume, refrigerated and allowed to stand, and the precipitated solid was collected. After drying, 61 mg of white powder crystals (purity calculated by peak area normalization method was 96.7%) were obtained, and its structure was identified as 2,6-pyridinedicarboxylic acid by spectroscopy; Detection method: Waters Acquity UPLC chromatography system; acetonitrile was chromatographic grade, water was ultrapure water, and other reagents were analytical grade; the chromatographic column was Welch XB C18 (100mm×2.1mm id, 3μm); the mobile phase was acetonitrile-0.2% phosphoric acid aqueous solution (5:95); the flow rate was 0.4mL / min; the column temperature was 30˚C; the detection wavelength was 270nm; Spectral data: UV λ max 272nm; HR-MS (ESI) Figure 1 : m / z 168.0294 [M+H] + (calcd.for C7H6NO4, m / z 168.0297); 1 H NMR (600 MHz, methanol- d 4) See Figure 2 : δ 8.37 (2H, d), 8.22 (1H, t); 13 C NMR (150 MHz, methanol- d 4) See Figure 3 : δ 165.8, 147.4, 139.4, 127.7.

[0023] Example 2 The content of 2,6-pyridinedicarboxylic acid in Bombyx batryticatus is determined by the following steps: (1) Preparation of test solution: Crush the dried Bombyx batryticatus medicinal materials or slices and pass through a No. 3 sieve. Take 0.5 g of Bombyx batryticatus powder, weigh accurately, add 25 mL of 20% methanol, weigh the weight, ultrasonically treat for 30 minutes, cool, weigh, make up the lost weight with 20% methanol, shake well, filter, and take the filtrate to obtain; (2) Preparation of reference solution: Take an appropriate amount of 2,6-pyridinedicarboxylic acid (purchased from Aladdin Reagent Company, purity ≥99%), accurately weigh it, and add 20% methanol to make a 1.09 mg / mL solution as the reference stock solution; take an appropriate amount of the stock solution and dilute it with 20% methanol to a series of concentrations, namely 218, 109, 54.5, 27.3, and 13.6 mg / mL; (3) Preparation of standard curve: Under the above chromatographic conditions, take 1 μL of the reference solution of a series of concentrations and inject it. Draw the standard curve with concentration as the horizontal axis and peak area as the vertical axis, as shown in the following figure: Figure 4 As shown, the linear equation is: y = 4247.2x -9007 ( R 2 =1); The results showed that 2,6-pyridinedicarboxylic acid had a good linear relationship in the range of 13.6-218 μg / mL; (4) Precision test: Take the reference solution and inject it 5 times continuously, and record the peak area. The results show that the peak area RSD of 2,6-pyridinedicarboxylic acid is 0.38%, indicating that the instrument has good precision; (5) Repeatability test: Accurately weigh 5 portions of the same batch of test sample, prepare the test sample solution according to the method under (1), and determine the content; the results show that the RSD of the content of 2,6-pyridinedicarboxylic acid is 1.45%, indicating that this method has good repeatability; (6) Stability test: The same test solution was sampled and tested at 0, 1, 3, 6, and 12 h after preparation, and the stability was calculated by peak area. The results showed that the RSD of the peak area of ​​2,6-pyridinedicarboxylic acid was 1.12%, indicating that the test solution was basically stable within 12 h. (7) Sample recovery test: Accurately weigh 6 samples of known content, each weighing about 0.25 g, and add reference substances equivalent to 50%, 100% and 150% of the known content, respectively. Prepare the test solution according to the method under (1), determine the content and calculate the recovery. The results showed that the average recovery of 2,6-pyridinedicarboxylic acid was 99.2% (RSD = 1.21%), indicating that this method has good accuracy. (8) Sample testing: Take 1 μL of the test sample solution and inject it into UPLC for determination under the above chromatographic conditions; the results are shown in Table 1, where the test results of the sample of "Xinhehua Chinese Medicine Pieces Company, batch number 2403111" in Table 1 are shown in the figure Figure 5 .

[0024] Table 1 Determination results of 2,6-pyridinedicarboxylic acid content in Bombyx batryticatus Manufacturer Variety specifications batch number Content (mg / g) New Lotus Chinese Medicine Pieces Company Fried Silkworm Pieces 2309157 2.31 New Lotus Chinese Medicine Pieces Company Fried Silkworm Pieces 2306138 1.90 New Lotus Chinese Medicine Pieces Company Fried Silkworm Pieces 2403111 1.95 Sichuan Limin Chinese Medicine Pieces Company Fried Silkworm Pieces 240401 1.93 Chengdu Lotus Pond Chinese Herbal Medicine Market Silkworm medicinal materials —— 2.24 The above results show that the chromatographic peak separation of 2,6-pyridinedicarboxylic acid is good, the interference between adjacent chromatographic peaks is small, and the content of this component in Bombyx mori is relatively high and the content in samples from different sources is relatively stable, which is suitable for use as a quality detection indicator for Bombyx mori.

[0025] Example 3 The identification of the characteristic spectrum of the traditional Chinese medicine preparation with 2,6-pyridinedicarboxylic acid as the characteristic peak is carried out in the following steps: Preparation of test solution: Jisheng Wumei Tablets were purchased from Chongqing Traditional Chinese Medicine Hospital (batch number 20230702). It is a traditional Chinese medicine preparation made of Wumei, Bombyx Batryticatus, Carthamus tinctorius and Curcuma zedoaria. Take Jisheng Wumei Tablets, grind them into powder, weigh 1.0 g of powder, add 10 mL of 25% methanol, and ultrasonically treat for 30 minutes. The extract was filtered through a 0.22 μm filter membrane, and the filtrate was taken as the test solution; The detection method is as follows: Waters Acquity UPLC chromatography system; acetonitrile was chromatographically pure, water was ultrapure water, and other reagents were analytically pure; the chromatographic column was Welch XB C18 (100 mm×2.1 mm id, 3μm); the mobile phase was acetonitrile (A)-0.2% phosphoric acid water (B); gradient elution 0-1.5 min (2% A), 1.5-3.0 min (2% A-3% A), 3.0-15.0 min (3% A-30% A), 15.0-16.0 min (30% A-100% A); flow rate 0.4 mL / min; column temperature 30˚C; detection wavelength 270 nm; the results are shown in Figure 6 .

[0026] The results showed that 2,6-pyridinedicarboxylic acid could be identified in the characteristic spectrum of the traditional Chinese medicine preparation composed of black plum, silkworm, safflower and zedoaria. The characteristic peaks of hydroxysafflor yellow A from safflower and chlorogenic acid and neochlorogenic acid from black plum could also be identified in the characteristic spectrum; therefore, 2,6-pyridinedicarboxylic acid was suitable as the characteristic peak of silkworm and was used for the characteristic spectrum identification of silkworm in traditional Chinese medicine preparations.

[0027] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for determining the content of 2,6-pyridinedicarboxylic acid in silkworm pupa and its preparation, characterized in that: The steps include: (1) Preparation of test solution: Take Bombyx batryticatus medicinal materials, decoction pieces or preparations, crush them, add solvent for extraction, filter, and use the filtrate as the test solution; (2) Preparation of reference solution: Take 2,6-pyridinedicarboxylic acid and add solvent to prepare reference solutions of different concentrations; (3) Preparing a standard curve: using high performance liquid chromatography, preparing a standard curve using the reference solution of different concentrations prepared in step (2); (4) Sample content determination: Take the test sample solution in step (1) and perform determination using the same method and chromatographic conditions as in step (3).

2. The method according to claim 1, characterized in that In step (1), the specific preparation method is: the sample is crushed and then sieved; then 0.5 g of the sample powder is taken, accurately weighed, 25 mL of 20 vol.% methanol is added, the weight is weighed, ultrasonic treatment is performed for 30 minutes, cooled, weighed, the lost weight is supplemented with 20 vol.% methanol, shaken, filtered, and the filtrate is obtained.

3. The method according to claim 1, characterized in that In step (2), 2,6-pyridinedicarboxylic acid was accurately weighed and added with 20 vol.% methanol to prepare a 1.09 mg / mL solution as a reference stock solution; the stock solution was diluted with 20 vol.% methanol to form a series of concentrations, namely 218, 109, 54.5, 27.3, and 13.6 mg / mL.

4. The method according to claim 1, characterized in that In step (3), the HPLC conditions for determining the Bombyx Batryticatus medicinal materials or decoction pieces are as follows: using a UPLC chromatography system; the chromatographic column is a C18 column; the mobile phase is acetonitrile-0.2 vol.% phosphoric acid aqueous solution (5:95); the flow rate is 0.4 mL / min; the column temperature is 30°C; and the detection wavelength is 270 nm.

5. The method according to claim 1, characterized in that The preparation is Jisheng Wumei Tablets, and the chromatographic conditions for its determination are: using a UPLC chromatography system, the chromatographic column is a C18 column; the mobile phase is acetonitrile (A)-0.2 vol.% phosphoric acid water (B); gradient elution 0-1.5 min (2 vol.% A), 1.5-3.0 min (2 vol.% A-3 vol.% A), 3.0-15.0 min (3vol.% A-30 vol.% A), 15.0-16.0 min (30 vol.% A-100 vol.% A); flow rate 0.4 mL / min; column temperature 30°C; detection wavelength 270 nm.

6. Application of 2,6-pyridinedicarboxylic acid in quality inspection of silkworm and its preparations.

7. The use according to claim 6, characterized in that: 2,6-pyridinedicarboxylic acid is used as a characteristic peak in the characteristic spectrum of silkworm pupa and its preparations.

8. The use according to claim 6, characterized in that: The silkworm is a silkworm medicinal material or silkworm decoction pieces, and the preparation is Jisheng Wumei tablets.

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