CN116068096B - Specific marker related to stiff silkworm section silk gland ring, screening method and stiff silkworm quality detection method - Google Patents
Specific marker related to stiff silkworm section silk gland ring, screening method and stiff silkworm quality detection method Download PDFInfo
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Abstract
The invention provides a stiff silkworm section silk gland ring related specific marker, a screening method and a stiff silkworm quality detection method, and belongs to the field of traditional Chinese medicine quality detection. The invention takes beauvericin, hypoxanthine and astragalin as markers. The invention provides a quality evaluation method related to the section silk gland ring of the stiff silkworm, which comprehensively evaluates the quality of the stiff silkworm from multiple indexes, has strong operability and reflects the scientificity of the differentiation theory of the stiff silkworm.
Description
Technical Field
The invention relates to a stiff silkworm section silk gland ring related specific marker, a screening method and a stiff silkworm quality detection method, belonging to the field of traditional Chinese medicine quality detection.
Background
The Bombyx Batryticatus is Bombyx mori of Bombycis moriBombyx moriLarva of Linnaeus 4-5 years old infects (or artificially inoculates) beauveria bassianaBeauveria bassiana(Bals.) Vuillant, has effects of calming endogenous wind, relieving spasm, dispelling pathogenic wind, relieving pain, eliminating phlegm, and resolving masses. As one of typical common animal medicines, the medicine has definite curative effect and wide application.
The silk gland is an organ for specifically synthesizing and secreting silk protein with high specificity of silkworms, reflects the growth condition and health degree of the silkworms, and can be used as an important cross-section property evaluation index after stiff silkworm medicinal materials are formed. The silk gland ring requirement of stiff silkworm is recorded from ancient times, the ' black and bright in the broken abdomen ' is considered as true ' by the ' De-compound herbal medicine ', and the ' materia medica-compound ' emphasizes that the stiff silkworm, which is a genuine product, is heavy, straight and big, has the like asphalt in the inside, is like butterfly powder in the outside, and is black, white and lovely. The "Ben Cao Meng Gao's tea indicates that the stiff silkworm should be selected from the group consisting of Cao Jiang silkworm, and Fu Lian Ji Zhu. Therefore, the appearance character of the high-quality stiff silkworm has the characteristics of white color, straight stripes and black and bright cross sections.
At present, the literature reporting the quality standard of stiff silkworm is mostly used for detecting certain index components or identifying counterfeit products, such as application number 202011537889.9, invention name: a qualitative and quantitative marker of a batryticated silkworm pseudo product and detection thereof are disclosed, and the qualitative and quantitative marker of the batryticated silkworm pseudo product and detection method thereof are disclosed, wherein the marker is borax or boric acid. The borax in the stiff silkworm pseudo product is rapidly enriched and extracted by microwave through the ethylhexyl glycol-n-butyl alcohol solution, the borax is converted into boric acid under the acidic condition, the boric acid can undergo a color reaction with curcumin protonated by sulfuric acid, and the borax content in the stiff silkworm pseudo product can be quantitatively determined by a colorimetric method.
Except for the stiff silkworm which is not carried in the Chinese pharmacopoeia of edition 1985, the other calendar plates all require 4 bright brown or bright black silk gland rings on the section. Only limited regulations on inspection items, extracts and mycotoxins of medicinal materials are carried out under the stiff silkworm item of Chinese pharmacopoeia of 2020 edition, and quality control indexes related to the properties and quality of the medicinal materials are lacking. From the process of forming medicinal materials, the characteristics of the section silk gland ring have close relation with the quality of the medicinal materials, but the quality difference of the medicinal materials with different section silk gland ring characteristics is not clear.
Disclosure of Invention
The technical scheme of the invention is to provide a specific marker related to the silk gland ring of the section of the stiff silkworm, and the other technical scheme of the invention is to provide a screening method of the marker and a quality control method of the stiff silkworm.
The invention provides a specific marker related to the silk gland ring of a stiff silkworm section, wherein 4 silk gland ring samples contain 0.092-0.369 mg/g of beauverin, 21.19-328.02 mug/g of hypoxanthine and 5.44-37.35 mug/g of astragalin;
the content of beauvericin in 3 silk gland ring samples is 0.083-0.391 mg/g, the content of hypoxanthine is 13.74-265.10 mug/g, and the content of astragalin is 6.80-39.63 mug/g;
2 silk gland ring samples have the beauvericin content of 0.060-0.331 mg/g, the hypoxanthine content of 20.03-234.02 mug/g and the astragalin content of 4.16-39.12 mug/g;
0 silk gland ring sample beauvericin content is 0.047-0.268 mg/g, hypoxanthine content is 9.02-176.41 mug/g, astragalin content is 3.22-33.28 mug/g;
the white silk gland ring sample has the beauvericin content of 0.036-0.267 mg/g, the hypoxanthine content of 5.13-142.28 mug/g and the astragalin content of 3.44-26.04 mug/g.
Further preferably, the average value of the content of beauvericin in 4 silk gland ring samples is 0.230 mg/g, the average value of the content of hypoxanthine is 126.76 mug/g, and the average value of the content of astragalin is 24.15 mug/g;
the average value of the content of beauvericin in 3 silk gland ring samples is 0.212mg/g, the average value of the content of hypoxanthine is 121.82 mug/g, and the average value of the content of astragalin is 21.65 mug/g;
2 silk gland ring samples have the average beauvericin content of 0.175mg/g, hypoxanthine content of 118.93 mug/g and astragalin content of 20.68 mug/g;
0 silk gland ring sample beauvericin content average value is 0.133mg/g, hypoxanthine content average value is 96.66 mug/g, astragalin content average value is 16.05 mug/g;
the white silk gland ring sample has the beauvericin content average value of 0.120mg/g, the hypoxanthine content average value of 80.22 mug/g and the astragalin content average value of 14.50 mug/g.
The invention also provides a method for screening the specific marker related to the stiff silkworm section silk gland ring, which comprises the following steps:
1) The stiff silkworm medicinal materials are classified according to the section silk gland rings, samples are randomly selected, and the stiff silkworm medicinal materials are cut off between the 1 st to 2 nd pairs of feet of the abdomen; 4/3/2 bright brown or bright black silk gland rings are arranged on the observation section, and S4/S3/S2 are respectively arranged on the observation section; the section of the hollow body is free of a silk gland ring or silk gland ring cavity, and S0 is adopted; the section is provided with a silk gland ring, but the color of the silk gland ring is white or the silk gland ring is accompanied by partial deletion, and the silk gland ring is B;
2) Measuring the content of beauvericin in different types of stiff silkworm medicinal materials;
3) Establishing a method for measuring the content of nucleoside components in the stiff silkworm medicinal materials;
4) Establishing a method for measuring the content of flavonoid components of the stiff silkworm medicinal material;
5) Analyzing the relation between the silk gland ring of the cross section of the stiff silkworm medicinal material and the component content of the stiff silkworm medicinal material, and searching for quality control indexes related to the cross section.
The content determination method of the nucleoside components is to use uracil, hypoxanthine, uridine, guanosine and adenine as reference substances, and adopts high performance liquid chromatography for detection, wherein the chromatographic conditions are as follows: xueaq-C using chromatographic column 18 Columns (4.6 mm. Times.250 mm,5 μm); the detection wavelength is 260 nm; the column temperature is 25 ℃; the sample injection amount is 20 mu L; the flow rate is 0.8 mL/min; water is used as a mobile phase A, and methanol is used as a mobile phase B; gradient elution (0-4 min,99% A, 4-6 min,99% -98% A, 6-6.1 min,98% -93.5% A, 6.1-14 min,93.5% -86% A, 14-14.1 min,86% -85.5% A, 14.1-18 min,85.5% A, 18-18.1 min,85.5% -80% A, 18.1-24 min,80% A, 24-28 min,80% -99% A, 28-30 min,99% A) is performed.
The content determination method of the flavonoid component takes rutin, astragalin, quercetin and kaempferol as reference substances, and the chromatographic conditions are as follows: waters-C using chromatographic column 18 Column (4.6 mm X50 mm,1.7 μm); the detection wavelength is 365 and nm; the column temperature is 30 ℃; the sample injection amount is 3 mu L; the flow rate is 0.3 mL/min; taking 0.1% phosphoric acid water as a mobile phase A and acetonitrile as a mobile phase B; gradient elution (0-2 min, 85-80% A; 2-3.5 min, 80-50% A; 3.5-4 min, 50-45% A; 4-5 min,45% A; 5-6 min, 45-10% A; 6-7 min,10% A; 7-7.5 min; 10-85% A; 7.5-10 min,85% A) is performed.
The invention also provides a quality detection method of the stiff silkworm, which takes the beauvericin, the hypoxanthine and the astragalin as markers, and determines the condition of the section silk gland ring of the stiff silkworm and the quality of the stiff silkworm through the contents of the three markers.
The quality detection method comprises the following steps:
1) Weighing stiff silkworm medicinal materials to be detected;
2) Detecting the beauvericin content of the stiff silkworm medicinal material by adopting a liquid chromatography;
3) Detecting the content of nucleoside components in the stiff silkworm medicinal material by adopting a liquid chromatography method;
4) Detecting the content of flavonoid components in the stiff silkworm medicinal material by adopting a liquid chromatography method;
5) And analyzing the detection result.
The detection method in the step 3) comprises the following steps:
the method for measuring the content of the nucleoside components takes hypoxanthine as a reference substance, adopts high performance liquid chromatography for detection, and has the following chromatographic conditions: by chromatographic columns Yuehou AQ-C 18 Columns (4.6 mm. Times.250 mm,5 μm); the detection wavelength is 260 nm; the column temperature is 25 ℃; the sample injection amount is 20 mu L; the flow rate is 0.8 mL/min; water is used as a mobile phase A, and methanol is used as a mobile phase B; gradient elution (0-4 min,99% A, 4-6 min,99% -98% A, 6-6.1 min,98% -93.5% A, 6.1-14 min,93.5% -86% A, 14-14.1 min,86% -85.5% A, 14.1-18 min,85.5% A, 18-18.1 min,85.5% -80% A, 18.1-24 min,80% A, 24-28 min,80% -99% A, 28-30 min,99% A) is performed.
The detection method in the step 4) comprises the following steps:
the content determination method of the flavonoid component takes astragalin as a reference substance, and the chromatographic conditions are as follows: a column was used as Waters-C18 column (4.6 mm X50 mm,1.7 μm); the detection wavelength is 365 and nm; the column temperature is 30 ℃; the sample injection amount is 3 mu L; the flow rate is 0.3 mL/min; taking 0.1% phosphoric acid water as a mobile phase A and acetonitrile as a mobile phase B; gradient elution (0-2 min, 85-80% A; 2-3.5 min, 80-50% A; 3.5-4 min, 50-45% A; 4-5 min,45% A; 5-6 min, 45-10% A; 6-7 min,10% A; 7-7.5 min; 10-85% A; 7.5-10 min,85% A) is performed.
The analysis result is obtained by detecting the contents of three markers of beauvericin, hypoxanthine and astragalin, and determining the quality of the stiff silkworm.
The invention provides a method for measuring the content of nucleoside and flavonoid components of a stiff silkworm medicinal material, which is used for determining the correlation between the silk gland ring of the section of the stiff silkworm and the quality, and provides a method for evaluating the quality of the stiff silkworm medicinal material by correlating the silk gland ring of the section of the stiff silkworm medicinal material.
Drawings
FIG. 1 HPLC chromatogram of nucleoside components of Bombyx Batryticatus (A: control, B: sample, C: blank solution) (1 uracil, 2 hypoxanthine, 3 uridine, 4 guanosine, 5 adenine).
FIG. 2 HPLC chromatogram of flavonoid components of Bombyx Batryticatus (A: control, B: sample, C: blank solution) (1 rutin, 2 astragalin, 3 quercetin, 4 kaempferol).
Detailed Description
EXAMPLE 1 method of screening for specific markers related to the silk gland ring of Bombyx Batryticatus section
1. Materials and instruments
1.1 Experimental materials
35 batches of stiff silkworm medicinal materials are purchased from different producing places and markets in 5 months 2020 to 1 month 2022, and sample information is shown in table 1, and are identified as stiff silkworm Bombyx Batryticatus through teaching of a Chinese medicine identification teaching and research room Li Min of Chengdu university.
Table 1 sample information table
1.2 Instrument and reagent
An Ulti Mate 3000 high performance liquid chromatograph (Thermo Fisher Co., USA); BP-121s precision electronic balance (one ten thousandth, zhejiang precision instruments Co., ltd.); BS-200S-WEI precision electronic balance (thousandth, beijing cerdolis instrument systems limited); KQ-500DE type digital controlled ultrasonic cleaner (Kunshan ultrasonic instruments Co., ltd.); DZKW-4 type electronic thermostat water bath (Beijing Zhongxing Wei industry instruments Co., ltd.); intelligent electrothermal blast drying oven (Shanghai Lang laboratory equipment Co., ltd.); muffle furnace (Lindberg/Blue M);
control: beauverine (lot number: 419469, beijing Baoling technologies Co., ltd., purity: 99%); uracil, hypoxanthine, guanosine, uridine, adenine (lot numbers: wkq21101205, wkq21042902, wkq21042810, wkq21081905, wkq21060210, sichuan Wick Biotechnology Co., ltd., purity not less than 98%); rutin (batch number: MUST-19010202, purity. Gtoreq.98%, chengdoman Biotechnology Co., ltd.); quercetin, kaempferol, astragalin (lot numbers wkq20061112, wkq21041507, MUST-20070414, purity not less than 98%, sichuan Uygur craving Biotechnology Co., ltd.);
sodium chloride (GR), acetonitrile, phosphoric acid, methanol are all chromatographically pure; the water is ultrapure water.
2 Experimental methods
2.1 Classification of traits
200 g-500 g of samples are randomly selected, and the samples are divided into stiff silkworm medicinal materials of different S4, S3, S2, S0 and B silk gland ring types according to the silk gland ring conditions of the section.
Note that: cutting between the 1 st to 2 nd feet of the abdomen, wherein the observation section is provided with 4/3/2 bright brown or bright black silk gland rings which are respectively S4/S3/S2; the section of the hollow body is free of a silk gland ring or silk gland ring cavity, and S0 is adopted; the section has silk gland ring, but the color of silk gland ring is white or with partial deletion, and B.
2.2 Beauverine content determination
The content of beauvericin is determined by reference to literature methods (Chen Hui, he Jinxiao, liu Jialing, etc.) A high performance liquid method for safely and rapidly determining the content of beauvericin in stiff silkworm is studied [ J ]. Shizhen national medicine, 2018,29 (4): 823-825.).
2.3 Determination of the content of nucleoside Components
2.3.1 Chromatographic conditions
Chromatographic column: xueaq-C18 column (4.6 mm×250 mm,5 μm); gradient elution was performed with water (a) -methanol (B), the gradient elution procedure is shown in table 2; the detection wavelength is 260 nm; the column temperature is 25 ℃; the flow rate was 0.8mL/min.
TABLE 2 gradient elution procedure
2.3.2 Preparation of the mixed control solution
Precisely weighing the control substances of uracil, hypoxanthine, uridine, guanosine and adenine, placing in a 20mL volumetric flask, dissolving with 0.9% sodium chloride solution, and fixing volume to obtain mother liquor of the control substances of the nucleoside components. And then taking a proper amount of each nucleoside mother solution, placing the nucleoside mother solution into a 10mL volumetric flask, diluting with 0.9% sodium chloride solution, and fixing the volume to obtain a mixed reference solution.
2.3.3 Preparation of test solution
Taking 1.0g of the product powder, placing into a conical flask with a plug, precisely adding 20mL of 0.9% sodium chloride solution, sealing, weighing, performing ultrasonic extraction for 30min, cooling, adding the weight, shaking uniformly, centrifuging for 5min at 8000r/min, filtering with a microporous filter membrane of 0.45 μm, and collecting the subsequent filtrate.
2.3.4 Methodology investigation
2.3.4.1 System applicability test
Taking mixed reference substance solution and sample solution, respectively performing sample injection measurement according to the chromatographic conditions under the item of 2.3.1, and recording the chromatograms. The result shows that the separation degree between each component to be detected and each adjacent peak is larger than 1.5, the theoretical plate number is not lower than 4000 according to uracil peak, and the chromatographic peak symmetry factor is between 0.95 and 1.05. The chromatogram is shown in FIG. 1.
2.3.4.2 Linear relationship investigation
Accurately sucking the mixed reference substance solutions 1000, 600, 300, 200 and 50 mu L to 1mL measuring flask under the item "2.3.2", respectively, adding 0.9% sodium chloride solution to the scale mark, and shaking uniformly. Peak areas were recorded as determined by chromatographic conditions under item "2.3.1". A standard curve was plotted with the peak area on the ordinate (y) and the mass concentration of each control on the abscissa (x, μg/mL) and linear regression analysis was performed, and the results are shown in Table 3.
TABLE 3 Linear relationship of five components
2.3.4.3 Precision investigation
Mixing the reference substance solutions under the item "2.3.2", continuously sampling and measuring for 6 times according to the chromatographic conditions under the item "2.3.1", and recording the peak area of each chromatographic peak. As a result, the RSD values of the peak areas of uracil, hypoxanthine, uridine, guanosine and adenine were 0.13%, 1.77%, 0.51%, 0.72% and 0.29% (n=6), respectively, and the RSD values of the peak areas of the components all meet the standard, indicating that the instrument precision is good.
2.3.4.4 Repeatability investigation
Taking the same stiff silkworm sample, preparing 6 parts of test solution according to the item of 2.3.3, detecting according to the chromatographic condition of the item of 2.3.1, and recording the peak area of each chromatographic peak. As a result, the peak areas RSD of uracil, hypoxanthine, uridine, guanosine and adenine were 2.50%, 2.67%, 2.34% and 2.49% (n=6), respectively, and the peak areas RSD of the respective components were the same as the standard, indicating that the reproducibility of the method was good.
2.3.4.5 Stability investigation
Taking stiff silkworm sample powder, preparing a test solution according to the item 2.3.3, standing at room temperature, respectively injecting samples at 0, 2, 4, 6, 8 and 12 h, detecting according to the chromatographic conditions under the item 2.3.1, and recording the peak area of each chromatographic peak. As a result, the peak areas RSD of uracil, hypoxanthine, uridine, guanosine and adenine were 1.17%, 1.93%, 2.36%, 1.89% and 2.94%, respectively, and the peak areas RSD of the components were all in accordance with the standard, indicating that the test sample solution was stable in 12 h.
2.3.4.6 Sample addition recovery test
Taking 0.5 parts of stiff silkworm sample powder g, adding a certain amount of each reference substance solution precisely, preparing a test substance solution according to the item "2.3.3", measuring according to the chromatographic condition under the item "2.3.1", and calculating the recovery rate, wherein the result is shown in Table 4. The result shows that the sample recovery rate of each component is within 95-110%, and the RSD value accords with the standard, which proves that the method has good accuracy.
TABLE 4 sample recovery test results
2.4 Flavonoid component content determination
2.4.1 Chromatographic conditions
Chromatographic column: waters-C 18 Column (4.6 m)m.times.50 mm,1.7 μm); gradient elution was performed with acetonitrile (a) -0.1% phosphoric acid water (B), and the gradient elution procedure is shown in table 5; the detection wavelength is 365 and nm; the column temperature is 30 ℃; the flow rate was 0.3 mL/min. The sample injection amount was 3. Mu.L.
TABLE 5 gradient elution procedure
2.4.2 Preparation of mixed control solution
Accurately weighing rutin, astragalin, quercetin and kaempferol, respectively, placing in 20mL volumetric flask, dissolving in 80% methanol solution, and fixing volume to obtain mother liquor of flavonoid. And then taking a proper amount of each flavonoid mother liquor, placing the flavonoid mother liquor into a 10mL volumetric flask, diluting with water, and fixing the volume to scale marks, namely mixing the reference substance solutions.
2.4.3 Preparation of test solutions
2.0g of stiff silkworm powder is precisely weighed, 20mL of 80% methanol is added for ultrasonic extraction for 30min, filtration and precise suction of 10mL are carried out, vacuum concentration is carried out until the stiff silkworm powder is dried, 2mL of 80% methanol is added for redissolution, a 0.22 mu m microporous filter membrane is used for filtration, and subsequent filtrate is taken to obtain the stiff silkworm powder.
2.4.4 Methodology investigation
2.4.4.1 System applicability test
Taking mixed reference substance solution and sample solution to be tested, respectively carrying out sample injection measurement according to the chromatographic conditions under the item of 2.4.1, and recording a chromatogram. The result shows that the separation degree between each component to be detected and each adjacent peak is larger than 1.5, the theoretical plate number is not lower than 6000 according to the quercetin peak, the chromatographic peak symmetry factor is between 0.95 and 1.05, and the chromatogram is shown in figure 2.
2.4.4.2 Linear relationship investigation
Precisely sucking the reference substance solutions 1000, 500, 200, 100, 50 μl to 1mL measuring flask under item "2.4.2", respectively, adding 80% methanol solution to scale mark, and shaking. Peak areas were recorded as determined by chromatographic conditions under item "2.4.1". A standard curve was plotted with the peak area on the ordinate (y) and the mass concentration of each control on the abscissa (x, μg/mL) and linear regression analysis was performed, and the results are shown in Table 6.
TABLE 6 Linear relationship of four flavonoid components
2.4.4.3 Precision investigation
Taking the reference substance solution under the item "2.4.2", continuously sampling and measuring for 6 times according to the chromatographic condition under the item "2.4.1", and recording the peak area. As a result, the RSD values of rutin, astragalin, quercetin and kaempferol peak areas are respectively 0.40%, 0.62%, 0.71% and 2.09%, and the RSD values of the peak areas of all components meet the standard, so that the instrument precision is good.
2.4.4.4 Repeatability investigation
Taking the same part of stiff silkworm sample, preparing 6 parts of test solution according to the condition of 2.4.3, detecting according to the chromatographic condition of 2.4.1, and calculating the content of each component. As a result, the RSD values of rutin, astragalin, quercetin and kaempferol peak areas are 2.82%, 2.16%, 1.24% and 2.50% respectively, and the RSD values of the peak areas of all components meet the standard, which shows that the method is good in reproducibility.
2.4.4.5 Stability investigation
Taking stiff silkworm sample powder, preparing a test solution according to the item 2.4.3, standing at room temperature, sampling at 0, 2, 4, 6, 8, 12 and 24 h respectively, detecting according to the chromatographic conditions under the item 2.4.1, and recording the peak area of each chromatographic peak. As a result, the RSD values of rutin, astragalin, quercetin and kaempferol peak areas are respectively 0.92%, 1.64%, 0.84% and 2.58%, and the RSD values of the peak areas of the components meet the standard, so that the stability of the sample solution in 24 h is good.
2.4.4.6 Sample recovery rate investigation
Taking 1.0g parts of stiff silkworm sample powder, precisely adding a certain amount of each reference substance solution respectively, preparing a test substance solution according to the method under the item "2.4.3", measuring according to the chromatographic condition under the item "2.4.1", and calculating the recovery rate. The result shows that the sample recovery rate of each component is within 95-110%, and the RSD value accords with the standard, which proves that the method has good accuracy. The results are shown in Table 7.
TABLE 7 sample recovery test results
3 results and analysis
3.1 relation of silk gland ring character and content of medicinal material beauvericin
The results of the different silk gland ring samples are shown in Table 8. The content of beauvericin in 4 silk gland ring samples is between 0.092 and 0.369 and mg/g, and the average value is 0.230 mg/g; the content of beauvericin in 3 silk gland ring samples is 0.083-0.391 mg/g, and the average value is 0.212mg/g; the content of beauvericin in 2 silk gland ring samples is 0.060-0.331 mg/g, and the average value is 0.175 mg/g;0 silk gland ring sample beauvericin content is 0.047-0.268 mg/g, average value is 0.133mg/g; the white silk gland ring sample beauvericin content is 0.036-0.267 mg/g, and the average value is 0.120 mg/g. The content results of the beauvericin in samples of each batch are mainly S4, S3 and S2> S0> B. The significance analysis results show that the beauvericin content of the white silk gland ring samples of each batch of samples is obviously lower than that of S4.
TABLE 8 determination of the content of Viburnin in the samples of 35 batches [ (]
±s，n=3， mg·g-1）/>
Note that: "-" indicates that the corresponding silk gland ring ratio of the batch of samples is low, and no detection data exists;
different lowercase letters of the same row are significantly different, as follows.
3.2 relation of silk gland ring character and nucleoside component of medicinal material
3.2.1 Relationship between silk gland ring character and medicinal material hypoxanthine
The results of hypoxanthine assays for the various silk gland ring samples are shown in Table 9. The hypoxanthine content of 4 silk gland ring samples is between 21.19 and 328.02 mug/g, and the average value is 126.76 mug/g; 3 silk gland ring samples have hypoxanthine content of 13.74-265.10 mug/g, and average value of 121.82 mug/g; 2 silk gland ring samples have hypoxanthine content of 20.03-234.02 mug/g, and average value of 118.93 mug/g; 0 silk gland ring samples have hypoxanthine content of 9.02-176.41 mug/g, and average value of 96.66 mug/g; the hypoxanthine content of the white silk gland ring sample is 5.13-142.28 mug/g, and the average value is 80.22 mug/g. The hypoxanthine content results of samples of each batch are mainly S4, S3 and S2> S0> B. The significance analysis results show that the contents of 0 silk gland rings and white silk gland ring hypoxanthine in each batch of samples are obviously lower than those of S4, S3 and S2.
TABLE 9 determination of the amount of silk-gland cyclic hypoxanthine in 35 batches
±s，n=3，μg·g-1）
3.2.2 Relationship between silk gland ring character and guanosine content of medicinal material
The guanosine measurements for the different silk gland ring samples are shown in Table 10. The guanosine content of the 4 silk gland ring samples is 85.34-824.36 mug/g, and the average value is 488.20 mug/g; 3 silk gland ring samples have guanosine content of 84.8-820.27 mug/g and average value of 479.04 mug/g; the guanosine content of 2 silk gland ring samples is 80.57-1006.48 mug/g, and the average value is 490.22 mug/g; 0 silk gland ring samples have guanosine content of 95.47-1177.2 mug/g and average value of 498.62 mug/g; the guanosine content of the white silk gland ring sample is 86.95-891.22 mug/g, and the average value is 508.48 mug/g. The guanosine content of the samples of different silk gland ring types has no obvious rule.
TABLE 10 determination of guanosine content of medicinal materials with different silk gland ring characteristics [ ]
±s，n=3，μg·g-1）
3.2.3 Relationship between silk gland ring character and uracil content in medicinal material
Uracil measurements for different silk gland ring samples are shown in Table 11. The uracil content of 4 silk gland ring samples is 110.40-674.36 mug/g, and the average value is 301.61 mug/g; 3 silk gland ring samples have uracil content of 85.45-666.64 mug/g, and average value of 340.40 mug/g; the uracil content of 2 silk gland ring samples is 90.58-610.39 mug/g, the average value is 349.79 mug/g, the uracil content of 0 silk gland ring samples is 121.78-639.52 mug/g, and the average value is 408.08 mug/g; the uracil content of the white silk gland ring sample is 161.65-607.19 mug/g, and the average value is 419.62 mug/g. The uracil content of the white silk gland ring sample is generally higher, and the uracil content of different silk gland ring samples has no obvious rule.
TABLE 11 determination of uracil content of medicinal materials with different silk gland ring characteristics [ (]
±s，n=3， μg·g-1）
3.2.4 relationship between silk adenylate cyclase character and adenine content of medicinal material
Adenine measurements for the different silk gland ring samples are shown in Table 12. The adenine content of 4 silk gland ring samples is 130.06-570.13 mug/g, and the average value is 325.98 mug/g; 3 silk gland ring samples have adenine content of 93.07-618 mug/g, and average value of 345.86 mug/g; the adenine content of 2 silk gland ring samples is 112.2-666.22 mug/g, and the average value is 361.51 mug/g; 0 silk gland ring samples have adenine content of 148.27-625.87 mug/g and average value of 315.44 mug/g; the adenine content of the white silk gland ring sample is 116.79-674.25 mug/g, and the average value is 318.97 mug/g. The adenine content of the samples of different silk gland ring types is irregular.
TABLE 12 determination of adenine content of medicinal materials with different silk gland ring characteristics [ (]
±s，n=3， μg·g-1）
Relationship between 3.2.5 silk gland ring character and uridine content in medicinal material
The uridine measurements for the different silk gland ring samples are shown in Table 13. The uridine content of 4 silk gland ring samples is 77.64-769.64 mug/g, and the average value is 275.84 mug/g; 3 silk gland ring samples have uridine content of 45.15-924.39 mug/g and average value of 295.96 mug/g; 2 silk gland ring samples have uridine content of 92.77-746.77 mug/g, and average value of 285.09 mug/g; the uridine content of 0 silk gland ring samples is 26.20-577.56 mug/g, and the average value is 196.00 mug/g; the uridine content of the white silk gland ring sample is between 39.30 and 944.31 mug/g, and the average value is 220.71 mug/g. The uridine content of the samples of different silk gland ring types is irregular.
TABLE 13 determination of uridine content of medicinal materials with different silk gland ring characteristics [ (]
±s，n=3， μg·g-1）/>
3.3 relation of silk gland ring character and flavonoid component content of medicinal materials
3.3.1 Relation between silk gland ring character and content of medicinal material astragalin
The results of the astragalin assay for the different silk gland ring samples are shown in Table 14. The content of astragalin in 4 silk gland ring samples is 5.44-37.35 mug/g, and the average value is 24.15 mug/g; 3 silk gland ring samples have astragalin content of 6.80-39.63 mug/g and average value of 21.65 mug/g; 2 silk gland ring samples have astragalin content of 4.16-39.12 mug/g and average value of 20.68 mug/g; the content of the astragalin in 0 silk gland ring samples is 3.22-33.28 mug/g, and the average value is 16.05 mug/g; the white silk gland ring sample has astragalin content of 3.44-26.04 mug/g and average value of 14.50 mug/g. The results of the astragalin content of each batch of samples are mainly S4, S3> S2> S0> B. The significance analysis result shows that the astragalin content of 0 silk gland rings and white silk gland ring samples of each batch of samples is obviously lower than that of S4.
TABLE 14 determination of the content of silk gland cycloastragalin in 35 batches of samples
±s，n=3， μg·g-1）
3.3.2 Relation between silk gland ring character and content of medicinal material quercetin
The results of the quercetin measurements for the different silk gland ring samples are shown in table 15. The content of quercetin in 4 silk gland ring samples is between 5.25 and 49.06 mug/g, and the average value is 21.78 mug/g; the quercetin content of the 3 silk gland ring samples is between 5.81 and 63.61 mug/g, and the average value is 23.20 mug/g; 2 silk gland ring samples have quercetin content of 2.90-48.57 mug/g, and average value of 24.36 mug/g; the content of quercetin in the 0 silk gland ring samples is 7.15-71.22 mug/g, and the average value is 28.59 mug/g; the content of quercetin in the white silk gland ring sample is between 4.03 and 60.45 mug/g, and the average value is 23.74 mug/g. The content of the quercetin in each batch of samples is irregular.
TABLE 15 determination of the content of silk gland cyclic quercetin in 35 batches of samples @
±s，n=3， μg·g-1）
3.3.3 Relation between silk gland ring character and content of kaempferol in medicinal material
The results of kaempferol measurements for the different silk gland ring samples are shown in Table 16. The content of kaempferol in 4 silk gland ring samples is 1.76-35.00 mug/g, and the average value is 7.84 mug/g; the content of kaempferol in the 3 silk gland ring samples is between 2.22 and 37.62 mug/g, and the average value is 7.89 mug/g; the content of kaempferol in 2 silk gland ring samples is between 2.12 and 27.82 mug/g, and the average value is 8.49 mug/g; the content of kaempferol in 0 silk gland ring samples is 1.35-43.97 mug/g, and the average value is 11.97 mug/g; the content of kaempferol in the white silk gland ring sample is between 1.84 and 29.51 mug/g, and the average value is 9.31 mug/g. The content of kaempferol in each batch of samples is irregular.
TABLE 16 determination of the content of kaempferol in the sample of 35 batches
±s，n=3，μg·g-1）
3.3.4 Relation between silk gland ring character and rutin content of medicinal material
The results of rutin measurements on the various silk gland ring samples are shown in Table 17. The rutin content of 4 silk gland ring samples is between 4.52 and 84.09 mug/g, and the average value is 30.09 mug/g; the rutin content of 3 silk gland ring samples is 7.30-77.32 mug/g, and the average value is 32.40 mug/g; the rutin content of 2 silk gland ring samples is 3.42-79.80 mug/g, and the average value is 32.46 mug/g; the rutin content of 0 silk gland ring samples is between 4.66 and 76.01 mug/g, and the average value is 31.50 mug/g; the rutin content of the white silk gland ring sample is 3.87-61.79 mug/g, and the average value is 25.91 mug/g. The rutin content of each batch of samples is not obvious.
TABLE 17 determination of the amount of silk gland Cyclorutin in 35 batches of samples
±s，n=3，μg·g-1）
3.4 Correlation analysis of silk gland ring character and medicinal material quality index
The correlation analysis results are shown in Table 18, the number of silk gland rings is obviously and positively correlated with the content of beauvericin, astragalin and hypoxanthine, and obviously and negatively correlated with the content of uracil and kaempferol. Considering that the stiff silkworm is a complex of fungus, insect and leaf, 3 components with strong positive correlation are selected from the main components as quality control indexes of stiff silkworm medicinal materials, namely beauvericin, hypoxanthine and astragalin.
TABLE 18 analysis results of the correlation between the number of silk gland rings and each quality index of the medicinal materials
Note that: * Indicating that the correlation was significant at the 0.01 level (double tail). * The correlation was shown to be significant at the 0.05 level (double-tailed).
4. Verification experiment
4.1 Materials and instruments
4.1.1 Experimental materials
The information of the stiff silkworm samples of 5 batches of different silkworm species is shown in Table 19
TABLE 19 sample information Table
4.1.2 Instrument and reagent
Same as 1.2.
4.2 Content determination of related components of different types of silk gland rings
As can be seen from Table 20, the overall trend of the content of the related components of the different types of silk gland rings was consistent with the foregoing, wherein the content of beauvericin, hypoxanthine and astragalin of YZ1 and YZ4 was relatively high, and it can be presumed that silk gland rings of the two samples were good. As is clear from Table 21, YZ1 and YZ4 had a high ratio of 4 silk gland rings, and 0 silk gland ring and white silk gland ring had a low ratio, and they were consistent with the content of the relevant components.
TABLE 20 determination of content of related ingredients of Bombyx Batryticatus of different silk gland ring types
±s，n=3）/>
Note that: lower case letter designations in the same columnP<The difference of 0.05 was significant.
Table 21 statistics of sample silk gland ring proportion
4 knots
The stiff silkworm has the effects of calming endogenous wind, relieving spasm, resolving phlegm, resolving masses, dispelling pathogenic wind and relieving pain. The beauvericin as an active ingredient has the pharmacological effects of sedation and anticonvulsant, and can be used as one of the drug effect evaluation and quality control indexes of the stiff silkworm; the stiff silkworm and the earthworm have the effects of resolving phlegm and relieving cough, and the two are often combined for application, and researches show that the hypoxanthine is used as one of the medicinal components of the earthworm for relieving cough and asthma, so that the hypoxanthine can be deduced to be also used as one of the medicinal effect evaluation and quality control indexes of the stiff silkworm; astragaloside is derived from one of the metabolic components of stiff silkworm edible mulberry leaves, has pharmacological effects of anti-inflammatory and analgesic, and can also be used as one of the drug effect evaluation and quality control indexes of stiff silkworm.
In conclusion, the research shows that the silkworm section silk gland ring has stronger correlation with the quality of medicinal materials, the compound of fungus, worm and leaf of the stiff silkworm is fully considered, beauvericin, hypoxanthine and astragalin are selected as the quality control index of the stiff silkworm medicinal materials, the thought is provided for comprehensively evaluating the quality of the stiff silkworm medicinal materials by multiple indexes, and the scientific basis is provided for perfecting the quality control system of the stiff silkworm medicinal materials.
Claims (1)
1. A quality detection method of stiff silkworm is characterized in that: it comprises the following steps:
1) Weighing stiff silkworm medicinal materials to be detected;
2) Detecting the beauvericin content of the stiff silkworm medicinal material by adopting a liquid chromatography;
3) Detecting the content of nucleoside components in the stiff silkworm medicinal material by adopting a liquid chromatography method;
4) Detecting the content of flavonoid components in the stiff silkworm medicinal material by adopting a liquid chromatography method;
5) Analyzing the detection result;
the detection method of the step 3) comprises the following steps:
the content determination method of the nucleoside components takes uracil, hypoxanthine, uridine, guanosine and adenine as reference substances, and adopts high performance liquid chromatography for detection, and the chromatographic conditions are as follows: xueaq-C using chromatographic column 18 Columns 4.6mm by 250 mm,5 μm; the detection wavelength is 260 nm; the column temperature is 25 ℃; the sample injection amount is 20 mu L; the flow rate is 0.8 mL/min; water is used as a mobile phase A, and methanol is used as a mobile phase B; gradient elution is carried out for 0 to 4min, and the concentration of A is 99 percent; 4-6 min, 99-98% of A;6 to 6.1min,98 to 93.5 percent of A;6.1 to 14 minutes, 93.5 to 86 percent of A;14 to 14.1min,86 to 85.5 percent of A;14.1 to 18 minutes, 85.5 percent of A,18 to 18.1 minutes, 85.5 to 80 percent of A,18.1 to 24 minutes, 80 percent of A; 24-28 min, 80-99% A; 28-30 min,99% A; the preparation method of the sample solution comprises the following steps: placing 1.0g of the product powder into a conical bottle with a plug, precisely adding 20mL of 0.9% sodium chloride solution, sealing, weighing, performing ultrasonic extraction for 30min, cooling, adding the weight, shaking uniformly, centrifuging for 5min at 8000r/min, filtering with a 0.45 μm microporous filter membrane, and collecting the subsequent filtrate;
the detection method in the step 4) comprises the following steps:
the content determination method of the flavonoid component takes rutin, astragalin, quercetin and kaempferol as reference substances, and the chromatographic conditions are as follows: waters-C using chromatographic column 18 Column 4.6mm ×50 mm,1.7 μm; the detection wavelength is 365 and nm; the column temperature is 30 ℃; the sample injection amount is 3 mu L; the flow rate is 0.3 mL/min; taking 0.1% phosphoric acid water as a mobile phase A and acetonitrile as a mobile phase B; gradient elution is carried out for 0 to 2min, and the concentration of A is 85 to 80 percent; 2 to 3.5min,80 to 50 percent of A;3.5 to 4 minutes, 50 to 45 percent of A; 4-5 min,45% A; 5-6 min, 45-10% of A; 6-7 min,10% A;7 to 7.5 minutes; 10% -85% of A; 7.5-10 min,85% A; the preparation method of the sample solution comprises the following steps:
2.0g of stiff silkworm powder is precisely weighed, 20mL of 80% methanol is added for ultrasonic extraction for 30min, filtration and precise suction of 10mL are carried out, vacuum concentration is carried out until the stiff silkworm powder is dried, 2mL of 80% methanol is added for redissolution, a 0.22 mu m microporous filter membrane is used for filtration, and subsequent filtrate is taken to obtain the stiff silkworm powder.
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