Construction method and application of characteristic chromatogram of jingcan medicinal materials, pieces, standard decoction and dispensing granules

Abstract

The application discloses a kind of jieseng medicinal materials, decoction piece, standard decoction and the construction method of characteristic chromatogram of formula granule and its application, it is related to traditional Chinese medicine identification technical field.The specific construction method includes: the first jieseng sample is extracted using water or 10vol%~50vol% methanol, to obtain test solution;Jieseng control medicinal materials and / or control product is extracted using water, to obtain reference solution;Wherein, control product includes uric acid control product, hypoxanthine control product, xanthine control product, uridine control product, adenine control product, guanosine control product, adenosine control product;Test solution, reference solution is determined using liquid chromatograph.The liquid chromatograph is with octadecylsilane bonded silica gel as stationary phase, with acetonitrile as mobile phase A, with 1mmol / L~10mmol / L ammonium acetate solution as mobile phase B gradient elution.The characteristic peak information of the characteristic chromatogram of the application is rich, which can provide reference for the identification of jieseng.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine identification, and particularly relates to a characteristic spectrum construction method of medicinal material, decoction piece, standard decoction and formula granules of Bombyx Batryticatus and application thereof. BACKGROUND

[0002] Bombyx Batryticatus is the dried body of Bombyx mori Linnaeus 4-5 larvae infected (or artificially inoculated) with Beauveria bassiana (Bals.) Vuillant. It is produced in spring and autumn, and the dead silkworms infected with Beauveria bassiana are dried. Bombyx Batryticatus tastes salty and pungent, and is flat. It belongs to the liver, lung and stomach channels, and has the effects of calming wind and stopping convulsion, dispelling wind and relieving pain, and resolving phlegm and removing mass. It is used for treating wind-phlegm syndrome, convulsion, infantile convulsion, tetanus, stroke, headache due to wind-heat, sore throat, red eyes, wind rashes, and itching, and so on. Modern studies have shown that Bombyx Batryticatus has various chemical components, including proteins, amino acids, oxalic acid, enzymes, nucleoside bases, steroids, volatile oils, and trace elements, among which the content of proteins is the highest. The pharmacological effects of Bombyx Batryticatus mainly include anticoagulation, antithrombosis and anticonvulsion, and it also has other effects such as lowering blood pressure, lowering blood sugar, antibiosis, and enhancing immunity.

[0003] At present, the characteristic spectrum of Bombyx Batryticatus medicinal material is not included in the 2020 edition of Chinese Pharmacopoeia. There are literatures reporting that the fingerprint spectrum of Bombyx Batryticatus mainly has nucleosides, amino acids and flavonoids, and for the nucleosides, there are studies reporting that the HPLC method is used to establish the fingerprint spectrum of Bombyx Batryticatus, the characteristic spectrum has 14 common peaks, and 6 compounds are identified. However, the above-mentioned methods have the problems of poor separation degree and small peak area of some common peaks. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a characteristic spectrum construction method of Bombyx Batryticatus medicinal material, decoction piece, standard decoction and formula granule, which has high separation degree of each characteristic peak and rich information.

[0005] The technical problem to be solved by the present application is to provide a characteristic spectrum construction method of Bombyx Batryticatus medicinal material, decoction piece, standard decoction and formula granule, which has high separation degree of each characteristic peak and rich information.

[0006] The technical problem to be solved by the present application is to provide a characteristic spectrum construction method of Bombyx Batryticatus medicinal material, decoction piece, standard decoction and formula granule, which has high separation degree of each characteristic peak and rich information.

[0007] In order to solve the above technical problems, the present application provides a characteristic spectrum construction method of Bombyx Batryticatus medicinal material, decoction piece, standard decoction and formula granule, which comprises:

[0008] A first sample of the Bombyx Batryticatus is extracted with water or 10vol%-50vol% methanol to obtain a first test solution; the first sample of the Bombyx Batryticatus includes Bombyx Batryticatus medicinal materials, Bombyx Batryticatus decoction pieces, Bombyx Batryticatus standard decoction or Bombyx Batryticatus formula granules;

[0009] A control Bombyx Batryticatus medicinal material and / or a control sample is extracted with water to obtain a reference solution; wherein the control sample includes a uric acid control sample, a hypoxanthine control sample, a xanthine control sample, a uridine control sample, an adenine control sample, a guanosine control sample and an adenosine control sample;

[0010] The first test solution and the reference solution are determined by using a liquid chromatograph, and thus the characteristic chromatogram of the Bombyx Batryticatus is obtained;

[0011] The liquid chromatograph uses octadecylsilane-bonded silica gel as a stationary phase, acetonitrile as a mobile phase A, and a 3mmol / L-10mmol / L ammonium acetate solution as a mobile phase B for gradient elution, and the gradient elution curve is as follows:

[0012] 0min-16min, the mobile phase A is 0% and the mobile phase B is 100%;

[0013] 16min-40min, the mobile phase A is from 0% to 3% and the mobile phase B is from 100% to 97%;

[0014] 40min-47min, the mobile phase A is from 3% to 6% and the mobile phase B is from 97% to 94%;

[0015] 47min-55min, the mobile phase A is from 6% to 15% and the mobile phase B is from 94% to 85%.

[0016] Correspondingly, the application further discloses a method for constructing a characteristic chromatogram of fried Bombyx Batryticatus decoction pieces, standard decoction and formula granules, which comprises the following steps:

[0017] A second sample of the Bombyx Batryticatus is extracted with water or 10vol%-50vol% methanol to obtain a second test solution; the second sample of the Bombyx Batryticatus includes fried Bombyx Batryticatus decoction pieces, fried Bombyx Batryticatus standard decoction or fried Bombyx Batryticatus formula granules;

[0018] A control Bombyx Batryticatus medicinal material and / or a control sample is extracted with water to obtain a reference solution; wherein the control sample includes a uric acid control sample, a hypoxanthine control sample, a xanthine control sample, a uridine control sample, an adenine control sample, a guanosine control sample and an adenosine control sample;

[0019] The second test solution and the reference solution are determined by using a liquid chromatograph, and thus the characteristic chromatogram of the Bombyx Batryticatus is obtained;

[0020] The liquid chromatograph uses octadecylsilane bonded silica gel as a stationary phase, acetonitrile as a mobile phase A, and a 3mmol / L-10mmol / L ammonium acetate solution as a mobile phase B for gradient elution, and the gradient elution curve is:

[0021] 0min-16min, mobile phase A is 0%, mobile phase B is 100%;

[0022] 16min-40min, mobile phase A is from 0% to 3%, mobile phase B is from 100% to 97%;

[0023] 40min-47min, mobile phase A is from 3% to 6%, mobile phase B is from 97% to 94%;

[0024] 47min-55min, mobile phase A is from 6% to 15%, mobile phase B is from 94% to 85%.

[0025] As an improvement of the above technical solution, the pH of the mobile phase B is adjusted to 4.4-4.6 by a pH adjusting liquid, and the pH adjusting liquid is a 30vol%-98vol% acetic acid solution;

[0026] The particle size of the stationary phase is 1.5-6μm;

[0027] The column length of the chromatographic column of the liquid chromatograph is 100-300mm, the column diameter is 2-6mm, the column temperature is 28-32℃, and the flow rate is 0.9-1.1mL / min;

[0028] The detection wavelength of the liquid chromatograph is 260nm.

[0029] As an improvement of the above technical solution, the concentration of the ammonium acetate solution is 5mmol / L, and the pH is 4.5;

[0030] The particle size of the stationary phase is 5μm;

[0031] The column length of the chromatographic column of the liquid chromatograph is 250mm, the column diameter is 4.6mm, the column temperature is 30℃, and the flow rate is 1mL / min.

[0032] As an improvement of the above technical solution, the reference solution includes a control medicinal material reference solution and a control reference solution;

[0033] The preparation method of the control medicinal material reference solution is as follows: 0.5-1.2g of a control medicinal material of canthyris is added into 20-30mL of water, ultrasonic treatment is performed for 20-40min at a frequency of 20-50kHz, heating is performed at 95-100℃ for 2-10min, cooling is performed to 20-30℃, and then filtration is performed, and the filtrate is obtained.

[0034] The preparation method of the reference substance solution is as follows: take appropriate amounts of uric acid reference substance, hypoxanthine reference substance, xanthine reference substance, uridine reference substance, adenine reference substance, guanosine reference substance, and adenosine reference substance, add water to make a mixed solution containing 110μg~130μg of uric acid, 10μg~30μg of hypoxanthine, 20μg~40μg of xanthine, 10μg~20μg of uridine, 10μg~30μg of adenine, 10μg~20μg of guanosine, and 20μg~30μg of adenosine per 1mL, to obtain the result.

[0035] As an improvement of the above technical solution, the preparation method of the control medicinal material reference solution is as follows: take 1.0 g of the silkworm pupa control medicinal material, add 25 mL of water, use ultrasonic treatment at a frequency of 40 kHz for 30 minutes, heat in a water bath at 100°C for 5 minutes, cool to 20°C to 30°C, filter, and take the filtrate to obtain;

[0036] The preparation method of the reference substance solution is as follows: take appropriate amounts of uric acid reference substance, hypoxanthine reference substance, xanthine reference substance, uridine reference substance, adenine reference substance, guanosine reference substance, and adenosine reference substance, add water to make a mixed solution containing 120 μg of uric acid, 20 μg of hypoxanthine, 30 μg of xanthine, 15 μg of uridine, 20 μg of adenine, 15 μg of guanosine, and 25 μg of adenosine per 1 mL.

[0037] As an improvement of the above technical solution, the preparation method of the first test solution is: take 0.8g~1.5g of Bombyx batryticatus medicinal materials or Bombyx batryticatus decoction pieces, add 20mL~40mL of water or 10vol%~50vol% methanol, use ultrasonic treatment with a frequency of 20kHz~50kHz for 15min~45min, heat at 95℃~100℃ for 2min~10min, cool to 20℃~30℃, filter, and take the filtrate to obtain;

[0038] or

[0039] The preparation method of the first test solution is as follows: take 0.1g to 0.4g of standard decoction of Bombyx batryticatus or formula granules of Bombyx batryticatus, add 20mL to 30mL of water or 10vol% to 50vol% methanol, use ultrasonic treatment with a frequency of 20kHz to 50kHz for 20min to 40min, cool, shake well, filter, and take the filtrate to obtain the first test solution.

[0040] As an improvement of the above technical solution, the preparation method of the first test solution is as follows: take 1.0 g of Bombyx batryticatus medicinal materials or Bombyx batryticatus decoction pieces, add 25 mL of water, use ultrasonic treatment with a frequency of 40 kHz for 30 minutes, heat in a 100°C water bath for 5 minutes, then cool to 20°C to 30°C, shake well, filter, and take the filtrate to obtain;

[0041] or

[0042] The preparation method of the first test solution is as follows: 0.2 g of Cantharis standard decoction or Cantharis formula granules is added into 25 mL of water, and ultrasonic treatment is performed for 30 min at a frequency of 40 kHz, and then the solution is cooled, shaken, filtered, and the filtrate is obtained.

[0043] As an improvement of the above technical solution, the preparation method of the second test solution is as follows: 0.8 g to 1.5 g of Cantharis fried decoction pieces is added into 20 mL to 40 mL of water or 10 vol% to 50 vol% methanol, and ultrasonic treatment is performed for 15 min to 45 min at a frequency of 20 kHz to 50 kHz, and then heating is performed for 2 min to 10 min at 95°C to 100°C, and then the solution is cooled to 20°C to 30°C, shaken, filtered, and the filtrate is obtained.

[0044] or

[0045] The preparation method of the second test solution is as follows: 0.1 g to 0.4 g of Cantharis fried standard decoction or Cantharis fried formula granules is added into 20 mL to 30 mL of water or 10 vol% to 50 vol% methanol, and ultrasonic treatment is performed for 20 min to 40 min at a frequency of 20 kHz to 50 kHz, and then the solution is cooled, shaken, filtered, and the filtrate is obtained.

[0046] As an improvement of the above technical solution, the preparation method of the second test solution is as follows: 1.0 g of Cantharis fried decoction pieces is added into 25 mL of water, and ultrasonic treatment is performed for 30 min at a frequency of 40 kHz, and then heating is performed for 5 min in a 100°C water bath, and then the solution is cooled to 20°C to 30°C, shaken, filtered, and the filtrate is obtained.

[0047] or

[0048] The preparation method of the second test solution is as follows: 0.2 g of Cantharis fried standard decoction or Cantharis fried formula granules is added into 25 mL of water, and ultrasonic treatment is performed for 30 min at a frequency of 40 kHz, and then the solution is cooled, shaken, filtered, and the filtrate is obtained.

[0049] As an improvement of the above technical solution, the characteristic spectrum has 9 peaks, wherein peak 1 is uric acid, peak 2 is hypoxanthine, peak 3 is xanthine, peak 4 is uridine, peak 5 is adenine, peak 7 is guanosine, and peak 9 is adenosine.

[0050] Peak 5 is the S peak, and the relative retention time of each characteristic peak is within ±10% of the specified value, the specified value of peak 1 is 0.41, the specified value of peak 2 is 0.51, the specified value of peak 3 is 0.61, the specified value of peak 4 is 0.72, the specified value of peak 6 is 1.08, the specified value of peak 7 is 1.52, and the specified value of peak 8 is 1.92.

[0051] Correspondingly, the present invention also discloses the application of the above-mentioned characteristic map construction method in the identification of Bombyx batryticatus and its counterfeits.

[0052] As an improvement of the above technical solution, the counterfeit products include silkworms, cornworms, mealworms, and beetle larvae that are not infected with Beauveria bassiana.

[0053] The implementation of the present invention has the following beneficial effects:

[0054] The present invention establishes a characteristic spectrum of Bombyx batryticatus, which has rich characteristic peak information and high resolution of each characteristic peak. Furthermore, the method is stable, accurate, and reliable, providing a reference for the identification of Bombyx batryticatus. Furthermore, the method can be applied to Bombyx batryticatus decoction pieces, Bombyx batryticatus standard decoction, Bombyx batryticatus formula granules, stir-fried Bombyx batryticatus decoction pieces, stir-fried Bombyx batryticatus standard decoction, and stir-fried Bombyx batryticatus formula granules, providing comprehensive quality control indicators. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 It is a survey map of the characteristics of Bombyx batryticatus;

[0056] Figure 2 This is the result of the intermediate precision inspection of the characteristic spectrum of Bombyx batryticatus;

[0057] Figure 3 This is a graph investigating the durability of Bombyx batryticatus's characteristic spectrum at different column temperatures;

[0058] Figure 4 This is a graph investigating the durability of Bombyx batryticatus at different flow rates;

[0059] Figure 5 This is a graph showing the durability of different chromatographic columns for the characteristic spectrum of Bombyx batryticatus;

[0060] Figure 6 This is a graph of the durability of Bombyx batryticatus at different pH values;

[0061] Figure 7 This is the spectrum diagram for identifying the characteristic peaks of the Bombyx batryticatus characteristic spectrum (peak 1 to peak 4);

[0062] Figure 8 This is the spectrum diagram for identifying the characteristic peaks of the characteristic spectrum of Bombyx batryticatus (peak 5, peak 7, peak 9);

[0063] Figure 9 This is an overlay of characteristic spectra of three batches of Bombyx Batryticatus Formula Granules;

[0064] Figure 10 It is an overlay of characteristic maps of 17 batches of Bombyx Batryticatus medicinal materials;

[0065] Figure 11 It is an overlay of characteristic spectra of 17 batches of standard decoction of Bombyx Batryticatus;

[0066] Figure 12is the superimposed chromatogram of 17 batches of fried silkworm chrysalis;

[0067] Figure 13 is the comparison chromatogram of silkworm chrysalis and its adulterants;

[0068] Figure 14 is the characteristic chromatogram of silkworm chrysalis obtained by using different chromatographic columns in Example 4;

[0069] Figure 15 is the characteristic chromatogram of silkworm chrysalis obtained by using different mobile phase B in Example 4;

[0070] Figure 16 is the characteristic chromatogram of silkworm chrysalis obtained by using different extraction solvents in Example 4;

[0071] Figure 17 is the characteristic chromatogram of silkworm chrysalis obtained by using different extraction methods in Example 4;

[0072] Figure 18 is the characteristic chromatogram of silkworm chrysalis obtained by using different extraction times in Example 4. DETAILED DESCRIPTION

[0073] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application will be further described in detail below with reference to the drawings and specific embodiments.

[0074] Establishment method of silkworm chrysalis characteristic chromatogram in Example 1

[0075] 1. Instruments, reagents and reagents

[0076] Instruments: SHIMADZU high performance liquid chromatograph (SHIMADZU LC40D-xs, Shimadzu Corporation); Thermo ultra-high performance liquid chromatograph (Thermo U3000, Thermo Fisher Scientific (China) Co., Ltd.); Waters Xselect HSS T3 chromatographic column (250mm×4.6mm, 5μm, No.: JS-280, JS-308, JS-322); Agilent ZORBAX SB-Aq C18 chromatographic column (250mm×4.6mm, 5μm); Waters Xbridge C18 chromatographic column (250mm×4.6mm, 5μm); one-hundredth balance (ME204E, Mettler-Toledo); millionth balance (XP26, Mettler-Toledo); ultrapure water system (Milli-Q Direct, Merck); digital controlled ultrasonic cleaner (KQ500D, Kunshan Ultrasonic Instrument Co., Ltd.); constant temperature water bath (HWS28 type, Shanghai Yiheng Scientific Instrument Technology Co., Ltd.).

[0077] Reagents: Methanol and 95% ethanol (Xilong Scientific Co., Ltd.) were of analytical grade; ammonium acetate (Guangzhou Chemical Plant); 36% acetic acid (Tianjin Komiou Chemical Reagent Co., Ltd.) were of HPLC grade; acetonitrile (Thermo Fisher Scientific (China) Co., Ltd.) was of HPLC grade; and water was ultrapure water (prepared in the laboratory).

[0078] Test drugs: uric acid reference substance (Shanghai Shidande Biological Service Technology Co., Ltd., batch number: 9602, content: 99.8%); uridine reference substance (China Food and Drug Inspection Institute, batch number: 110887-202104, content: 99.6%); hypoxanthine reference substance (China Food and Drug Inspection Institute, batch number: 140661-202005, content: 99.4%); xanthine reference substance (China Food and Drug Inspection Institute, batch number: 140662-200802, content: 99.1%); adenine reference substance (batch number: 110866-202203, content: 99. 8%, China Food and Drug Inspection Institute); guanosine reference substance (batch number: 111977-201501, content calculated as 93.6%, China Food and Drug Inspection Institute); adenosine reference substance (batch number: 110879-201703, content calculated as 99.7%, China Food and Drug Inspection Institute); Bombyx batryticatus reference medicinal materials (batch number: 125014-201202A, Guangdong Yifang Pharmaceutical Co., Ltd.); Bombyx batryticatus formula granules (batch numbers: KL01, KL02, KL03; source: Guangdong Yifang Pharmaceutical Co., Ltd.). The source information of Bombyx batryticatus medicinal materials, stir-fried Bombyx batryticatus decoction pieces, and Bombyx batryticatus standard decoction is shown in Table 1.

[0079] Table 1 Sample source information

[0080]

[0081] 2. Methods and Results

[0082] 2.1 Chromatographic conditions

[0083] A Waters Xselect HSS T3 column (4.6 mm × 250 mm, 5 μm) was used; acetonitrile was used as mobile phase A, and 5 mmol / L ammonium acetate solution (adjusted to pH 4.5 with 36% acetic acid solution) was used as mobile phase B. Gradient elution was performed according to the requirements in Table 2 below; the flow rate was 1.0 mL / min; the column temperature was 30°C; the detection wavelength was 260 nm; and the injection volume was 10 μL.

[0084] Table 2 Gradient elution table

[0085]

[0086] 2.2 Preparation of test solution

[0087] Cicada standard decoction, fried cicada standard decoction: take the appropriate amount of the product, grind, take about 0.2g, placed in a conical flask with a plug, add water 25mL, ultrasonic treatment (power 250W, frequency 40kHz) 30 minutes, cool, shake, filter, take the filtrate, get.

[0088] Cicada standard decoction, fried cicada standard decoction: take the appropriate amount of the product, grind, take about 0.2g, placed in a conical flask with a plug, add water 25mL, ultrasonic treatment (power 250W, frequency 40kHz) 30 minutes, cool, shake, filter, take the filtrate, get.

[0089] Cicada standard decoction, fried cicada standard decoction: take the appropriate amount of the product, grind, take about 0.2g, placed in a conical flask with a plug, add water 25mL, ultrasonic treatment (power 250W, frequency 40kHz) 30 minutes, cool, shake, filter, take the filtrate, get.

[0090] 2.3 Preparation of reference solution

[0091] Take 1g of the reference material of cicada, add water 25mL, ultrasonic treatment (power 250W, frequency 40kHz) 30 minutes, placed in a 100℃ water bath for 5 minutes, then cool to room temperature, shake, filter, take the filtrate, as the reference material reference solution. Take the appropriate amount of uric acid control, hypoxanthine control, xanthine control, uridine control, adenine control, guanosine control, adenosine control, add water to prepare 1mL of each containing uric acid 120μg, hypoxanthine 20μg, xanthine 30μg, uridine 15μg, adenine 20μg, guanosine 15μg, adenosine 25μg control solution, as the reference solution of the control.

[0092] 2.4 Determination method

[0093] Respectively, 10μL of the reference solution and the test solution were precisely taken and injected into the liquid chromatograph, and the determination was carried out, and the determination was carried out.

[0094] 3. Methodology

[0095] 3.1 Investigation of specificity

[0096] The water (blank) control reference solution and the test solution prepared from the cicada formula granules were taken respectively, and the chromatographic conditions of item 2.1 and the determination method of item 2.4 were used for determination. The results are shown in Figure 1 There are the same chromatographic peaks in the test solution chromatogram at the corresponding retention time of the reference solution chromatogram, and the negative has no interference, which shows that the method has good specificity.

[0097] 3.2 Precision investigation

[0098] Take the same batch of Bombyx batryticatus formula granules (KL01), prepare the test solution according to the method under 2.2 and the chromatographic conditions under 2.1, and inject it continuously for 6 times. Take peak 5 as the reference peak S, record the relative retention time and relative peak area of ​​each characteristic peak and peak S, and calculate the RSD value. The results show that the RSD values ​​of the relative retention time and relative peak area of ​​each characteristic peak are all less than 3%, indicating that the instrument precision is good.

[0099] 3.3 Repeatability study

[0100] Take 6 portions of sample powder from the same batch of Bombyx Batryticatus Formula Granules (KL01) and prepare 6 test solutions in parallel according to the method under 2.2. The samples were injected and measured according to the chromatographic conditions under 2.1. Peak 5 was used as the reference peak S. The relative retention time and relative peak area of ​​each characteristic peak to the S peak were recorded, and the RSD values ​​were calculated. The results showed that the RSD values ​​of the relative retention time and relative peak area of ​​each characteristic peak were all less than 3%, indicating that the method had good repeatability.

[0101] 3.4 Stability investigation

[0102] Take the same batch of silkworm formula granules (KL01) samples, prepare the test solution according to the method under 2.2, and inject the samples at 0, 2, 4, 6, 8, 10, and 18 hours according to the chromatographic conditions under 2.1. Take peak 5 as the reference peak S, record the relative retention time and relative peak area of ​​each characteristic peak and the S peak, and calculate the RSD value. The results show that the RSD values ​​of the relative retention time and relative peak area of ​​each characteristic peak are all less than 3%, indicating that the test solution has good stability within 18 hours.

[0103] 3.5 Intermediate precision study

[0104] Different analysts operated on different instruments on different days. Take an appropriate amount of Bombyx Batryticatus Formula Granules, grind them into powder, take about 1.0g, and weigh them accurately in 6 parallel portions. Prepare the test solution according to the test solution preparation method determined in 2.2. Inject and analyze according to the chromatographic conditions in 2.1. Use Peak 5 as the reference peak S. Record the relative retention time and relative peak area of ​​each characteristic peak and the S peak, and calculate the RSD value. The results are as follows. Figure 2 The results showed that the RSD values ​​of the six data of relative retention time, relative peak area and repeatability test were all less than 5.0%, indicating that the intermediate precision of this method was good.

[0105] 3.6 Durability inspection

[0106] 3.6.1 Investigation of different column temperatures

[0107] Take Jiegeng formula granules, grind finely, take about 1.0g, accurately weigh, prepare the test solution according to the preparation method of the test solution in item 2.2, except that the column temperature is 28℃, 30℃ and 32℃ respectively, other chromatographic conditions are the same as the provisions in item 2.1, sample analysis, take peak 5 as reference peak S, calculate the relative retention time and relative peak area of each characteristic peak and S peak, and calculate the RSD value. The results are as follows Figure 3 The RSD values of relative retention time and relative peak area are all less than 3.0%, which shows that the method has good robustness at different column temperatures.

[0108] 3.6.2 Investigation of different flow rates

[0109] Take Jiegeng formula granules, grind finely, take about 1.0g, accurately weigh, prepare the test solution according to the preparation method of the test solution in item 2.2, except that the flow rate is 0.9mL / min, 1.0mL / min and 1.1mL / min respectively, other chromatographic conditions are the same as the provisions in item 2.1, sample analysis, take peak 5 as reference peak S, calculate the relative retention time and relative peak area of each characteristic peak and S peak, and calculate the RSD value. The results are as follows Figure 4 The RSD values of relative retention time and relative peak area are all less than 5.0%, which shows that the method has good robustness at different flow rates.

[0110] 3.6.3 Investigation of different chromatographic columns

[0111] Take Jiegeng formula granules, grind finely, take about 1.0g, accurately weigh, prepare the test solution according to the preparation method of the test solution in item 2.2, except that the chromatographic column is different batch Waters Xselect HSS T3 chromatographic column (4.6mm×250mm, 5μm) chromatographic column, other chromatographic conditions are the same as the provisions in item 2.1, sample analysis, take peak 5 as reference peak S, calculate the relative retention time and relative peak area of each characteristic peak and S peak, and calculate the RSD value. The results are as follows Figure 5 The RSD values of relative retention time and relative peak area are all less than 5.0%, which shows that the method has good robustness to different chromatographic columns.

[0112] 3.6.4 Investigation of different pH values

[0113] Take Jiegeng formula granules, grind finely, take about 1.0g, accurately weigh, prepare the test solution according to the preparation method of the test solution in item 2.2, except that the pH value of mobile phase B is 4.4, 4.5 and 4.6 respectively, other chromatographic conditions are the same as the provisions in item 2.1, sample analysis, take peak 5 as reference peak S, calculate the relative retention time and relative peak area of each characteristic peak and S peak, and calculate the RSD value. The results are as follows Figure 6The RSD values of relative retention time and relative peak area are all less than 5.0%, which indicates that the method has good robustness for different chromatographic columns.

[0114] 3.7 Identification of characteristic peaks

[0115] Take the Heungcang Dispensing Granules, prepare the test sample solution according to the preparation method of the test sample solution in item 2.2, and separately take the test sample solution and the reference solution of the control drug into the sample for analysis according to the chromatographic conditions in item 2.1. Compare the retention time and spectrum of the characteristic peaks, as shown in Figure 7 and Figure 8 , determine that peak 1 is uric acid, peak 2 is hypoxanthine, peak 3 is xanthine, peak 4 is uridine, peak 5 is adenine, peak 7 is guanosine, and peak 9 is adenosine.

[0116] Example 2 Determination of Heungcang medicinal materials, standard decoction, dispensing granules, and fried Heungcang medicinal materials

[0117] 1. Determination of Heungcang dispensing granule samples

[0118] Take three batches of Heungcang dispensing granules (batch numbers: KL01, KL02, and KL03), prepare the test sample solution according to the preparation method of the test sample solution in item 2.2 of Example 1, and separately take the above-mentioned test sample solution and the reference solution of the Heungcang control drug into the sample for determination according to the chromatographic conditions in item 2.1 of Example 1. The superimposed chromatogram of the characteristic chromatograms of the three batches of samples is shown in Figure 9 Take peak 5 as the reference peak S, calculate the relative retention time and relative peak area of each characteristic peak and the peak S, and the calculation results are shown in Tables 3-4.

[0119] Table 3 Characteristic chromatogram of three batches of Heungcang dispensing granules (relative retention time)

[0120]

[0121] Table 4 Characteristic chromatogram of three batches of Heungcang dispensing granules (relative peak area)

[0122]

[0123] 2. Determination of Heungcang medicinal material samples

[0124] Take Heungcang medicinal material powder (pass through a No. 3 sieve), prepare the test sample solution according to the method in item 2.2 of Example 1, and separately take the above-mentioned test sample solution and the reference solution of the Heungcang control drug into the sample for determination according to the chromatographic conditions in item 2.1 of Example 1. The superimposed chromatogram of the characteristic chromatograms of the 17 batches of samples is shown in Figure 10 Take peak 5 as the reference peak S, calculate the relative retention time, relative peak area, and RSD value of each characteristic peak and the peak S, and the calculation results are shown in Tables 5-6.

[0125] Table 5 Characteristic spectra of 17 batches of Bombyx batryticatus medicinal materials (relative retention time)

[0126]

[0127]

[0128] Table 6 Characteristic spectra of 17 batches of Bombyx batryticatus medicinal materials (relative peak area)

[0129]

[0130] 3. Determination of Bombyx Batryticatus Standard Decoction Samples

[0131] Take the standard decoction of silkworm respectively, and prepare the test solution according to the method under 2.2 in Example 1. Accurately aspirate the above test solution and the reference solution of silkworm control medicinal material respectively, and inject and measure the sample according to the chromatographic conditions under 2.1 in Example 1. The characteristic spectrum overlay of 17 batches of samples is shown in the figure below. Figure 11 Taking peak 5 as the reference peak S, the relative retention time, relative peak area and RSD value of each characteristic peak and peak S were calculated. The calculation results are shown in Tables 7 and 8.

[0132] Table 7 Characteristic spectra of 17 batches of standard decoction of Bombyx batryticatus (relative retention time)

[0133]

[0134]

[0135] Table 8 Characteristic spectra (relative peak areas) of 17 batches of standard decoctions of Bombyx batryticatus

[0136]

[0137]

[0138] 4. Determination of fried silkworm slices

[0139] Take the fried silkworm slices separately, prepare the test solution according to the method under 2.2 in Example 1, accurately draw the above test solution and silkworm reference medicinal material reference solution respectively, refer to the chromatographic conditions under 2.1 in Example 1 for sample injection and determination, and the characteristic spectrum overlay of 17 batches of samples is as shown in the figure below. Figure 12 Taking peak 5 as the reference peak S, the relative retention time, relative peak area and RSD value of each characteristic peak and peak S were calculated. The calculation results are shown in Tables 9 and 10.

[0140] Table 9 Characteristic spectra of 17 batches of fried silkworm slices (relative retention time)

[0141]

[0142] Table 10 Characteristic spectra of 17 batches of fried silkworm slices (relative peak area)

[0143]

[0144] Example 3: Comparative study of Bombyx batryticatus and other species.

[0145] This example uses the Bombyx batryticatus characteristic spectrum established in Example 1 to detect common counterfeit Bombyx batryticatus products. Specifically, the counterfeit products include silkworms, silkworm pupae, beetle larvae, mealworms, cornworms, etc. The specific sample sources are shown in Table 11.

[0146] Table 11 Sample source information

[0147]

[0148] See also Figure 13 The test results showed that compared with the silkworm, other counterfeit products only had peaks 1, 2 and 7 as common peaks, and the rest of the chromatographic peaks were missing, specifically: silkworm lacked peaks 3, 6, 8 and 9; silkworm pupae lacked peaks 6, 8 and 9; cornworm lacked peaks 6 and 8; mealworm lacked peaks 5, 6, 8 and 9; and beetle larvae lacked peaks 4, 5, 6 and 8.

[0149] Example 4 Investigation of chromatographic conditions and preparation of test solution

[0150] 4.1 Investigation and determination of chromatographic conditions

[0151] (1) Investigation of different chromatographic columns

[0152] A Waters Xselect HSS T3 column (250 mm × 4.6 mm, 5 μm); an Agilent ZORBAXSB-Aq C18 column (250 mm × 4.6 mm, 5 μm); and a Waters Xbridge C18 column (250 mm × 4.6 mm, 5 μm) were used respectively. The remaining chromatographic conditions were the same as those under 2.1 in Example 1. The above-mentioned Bombyx Batryticatus Formula Granule test solution was accurately drawn and injected for determination. The results are shown in FIG. Figure 14 As shown in the figure, it can be seen that the Waters Xselect HSS T3 column has the best separation effect.

[0153] (2) Investigation of ammonium acetate solutions with different concentrations

[0154] The chromatographic conditions are the same as those in Example 1, 2.1, except that the mobile phase B is 3 mmol / L, 5 mmol / L, 10 mmol / L ammonium acetate solution (pH value is adjusted to 4.5 with 36% acetic acid solution) respectively, and the above Fomula Granules of Fleeceflower Root test sample solutions are precisely taken for injection determination, and the results are shown in Table 2. Figure 15 As shown in Table 2, it can be seen that the separation effect is the best when the mobile phase B is 5 mmol / L ammonium acetate solution (pH value is adjusted to 4.5 with 36% acetic acid solution).

[0155] 4.2 Preparation of test sample solution

[0156] (1) Investigation of extraction solvent

[0157] A certain amount of Fomula Granules of Fleeceflower Root (KL01) is finely ground, about 0.2 g is precisely weighed, 7 groups in parallel, 2 portions in each group, and is placed in a conical flask with a plug. 10% methanol, 50% methanol, 70% methanol, 10% ethanol, 50% ethanol, and 70% ethanol are precisely added to the flask respectively, the weight is determined, and the sample is ultrasonically treated (power 250 W, frequency 40 kHz) for 30 minutes. After cooling, the weight is determined again, the lost weight is made up with the corresponding solvent, shaken well, filtered, and the filtrate is taken. The injection is analyzed according to the chromatographic conditions in Example 1, 2.1, and the results are shown in Table 12, Figure 16 . The results show that when different solvents are used for extraction, the "total peak area / sample weight" is larger and the chromatographic peak has a better peak shape when water is used as the extraction solvent, so water is selected as the extraction solvent.

[0158] Table 12 Investigation results of extraction solvent of characteristic chromatogram of Fomula Granules of Fleeceflower Root

[0159]

[0160]

[0161] (2) Investigation of extraction method

[0162] A certain amount of Fomula Granules of Fleeceflower Root (KL01) is finely ground, about 0.2 g is precisely weighed, 2 groups in parallel, 2 portions in each group, and is placed in a conical flask with a plug. Water is precisely added to the flask, the weight is determined, and the sample is ultrasonically treated (power 250 W, frequency 40 kHz) for 30 minutes and heated to reflux for 30 minutes, respectively. After cooling, the weight is determined again, the lost weight is made up with water, shaken well, filtered, and the filtrate is taken. The injection is analyzed according to the chromatographic conditions in Example 1, 2.1, and the results are shown in Table 13, Figure 17 . The results show that when different extraction methods are used, there is no obvious difference in the peak shape, separation effect, and "total peak area / sample weight" of each characteristic peak. Considering the convenience of operation, ultrasonic extraction is selected.

[0163] Table 13 Investigation results of extraction method of characteristic chromatogram of Fomula Granules of Fleeceflower Root

[0164]

[0165] (3) Extraction time investigation

[0166] Take appropriate amount of Cantharis formula granules (KL01), grind finely, take about 0.2g, parallel 3 groups, 2 portions in each group, accurately weigh, place in a conical flask with a stopper, accurately add water 25ml, weigh, ultrasonic treatment (power 250W, frequency 40kHz) for 15 minutes, 30 minutes and 45 minutes respectively, cool, weigh again, make up the weight loss with water, shake well, filter, take the filtrate. Analyze by the chromatographic conditions in item 2.1 of Example 1, the results are shown in Table 14, Figure 18 . The results show that there is no significant difference in the "total peak area / sample weight" of the 9 characteristic peaks of different extraction times, indicating that ultrasonic extraction for 30 minutes can completely extract, considering the influence of experimental environment, in order to ensure the robustness of the method, the ultrasonic extraction time is selected as 30 minutes.

[0167] Table 14 Investigation results of characteristic chromatogram of Cantharis formula granules

[0168]

[0169] The above is the preferred embodiment of the application. It should be noted that for those skilled in the art, without departing from the principles of the application, a number of improvements and refinements can be made, which are also considered within the scope of protection of the application.

Claims

1. A method for constructing a characteristic spectrum of Bombyx batryticatus medicinal materials, decoction pieces, standard decoctions, and formula granules, characterized in that: include: Extracting the first Bombyx batryticatus sample with water or 10 vol% to 50 vol% methanol to obtain a first test solution; the first Bombyx batryticatus sample includes Bombyx batryticatus medicinal materials, Bombyx batryticatus decoction pieces, Bombyx batryticatus standard decoction or Bombyx batryticatus formula granules; Extracting a Bombyx batryticatus reference medicinal material and a reference substance with water to obtain a reference solution; wherein the reference substances include a uric acid reference substance, a hypoxanthine reference substance, a xanthine reference substance, a uridine reference substance, adenine reference substance, a guanosine reference substance, and an adenosine reference substance; The first test solution and the reference solution are measured by liquid chromatography to obtain; The liquid chromatograph uses a Waters Xselect HSS T3 chromatographic column with a column length of 250 mm, a column diameter of 4.6 mm, a particle size of the stationary phase of 5 μm, a detection wavelength of 260 nm, and acetonitrile as mobile phase A and ammonium acetate solution with a concentration of 3 mmol / L to 10 mmol / L as mobile phase B for gradient elution. The gradient elution curve is: 0 min to 16 min, mobile phase A is 0%, mobile phase B is 100%; 16 min to 40 min, mobile phase A from 0% to 3%, mobile phase B from 100% to 97%; 40-47 min, mobile phase A from 3% to 6%, mobile phase B from 97% to 94%; From 47 to 55 minutes, mobile phase A increased from 6% to 15%, and mobile phase B increased from 94% to 85%; Wherein, the pH of the mobile phase B is 4.4-4.

6.

2. A method for constructing a characteristic spectrum of fried silkworm slices, standard decoctions, and formula granules, characterized in that: include: Extracting the second Bombyx batryticatus sample with water or 10 vol% to 50 vol% methanol to obtain a second test solution; the second Bombyx batryticatus sample includes fried Bombyx batryticatus decoction pieces, fried Bombyx batryticatus standard decoction or fried Bombyx batryticatus formula granules; Extracting a Bombyx batryticatus reference medicinal material and a reference substance with water to obtain a reference solution; wherein the reference substances include a uric acid reference substance, a hypoxanthine reference substance, a xanthine reference substance, a uridine reference substance, adenine reference substance, a guanosine reference substance, and an adenosine reference substance; The second test solution and the reference solution are measured by liquid chromatography to obtain; The liquid chromatograph uses a Waters Xselect HSS T3 chromatographic column with a column length of 250 mm, a column diameter of 4.6 mm, a particle size of the stationary phase of 5 μm, a detection wavelength of 260 nm, and acetonitrile as mobile phase A and ammonium acetate solution with a concentration of 3 mmol / L to 10 mmol / L as mobile phase B for gradient elution. The gradient elution curve is: 0 min to 16 min, mobile phase A is 0%, mobile phase B is 100%; 16 min to 40 min, mobile phase A from 0% to 3%, mobile phase B from 100% to 97%; 40-47 min, mobile phase A from 3% to 6%, mobile phase B from 97% to 94%; From 47 to 55 minutes, mobile phase A increased from 6% to 15%, and mobile phase B increased from 94% to 85%; Wherein, the pH of the mobile phase B is 4.4-4.

6.

3. The method for constructing a characteristic map according to claim 1 or 2, wherein: The pH of the mobile phase B is adjusted to 4.4-4.6 by a pH adjusting solution, wherein the pH adjusting solution is a 30 vol%-98 vol% acetic acid solution; The column temperature of the chromatographic column of the liquid chromatograph is 28° C. to 32° C., and the flow rate is 0.9 mL / min to 1.1 mL / min.

4. The method for constructing a characteristic map according to claim 3, wherein: The concentration of the ammonium acetate solution is 5 mmol / L and the pH is 4.5; The column temperature of the chromatographic column of the liquid chromatograph was 30° C., and the flow rate was 1 mL / min.

5. The method for constructing a characteristic map according to claim 1 or 2, wherein: The reference solution includes a reference medicinal material reference solution and a reference product reference solution; The preparation method of the control medicinal material reference solution is as follows: take 0.5g~1.2g of Bombyx batryticatus control medicinal material, add 20mL~30mL of water, use ultrasonic treatment with a frequency of 20kHz~50kHz for 20min~40min, heat at 95℃~100℃ for 2min~10min, cool to 20℃~30℃, filter, and take the filtrate to obtain; The preparation method of the reference substance solution is as follows: take appropriate amounts of uric acid reference substance, hypoxanthine reference substance, xanthine reference substance, uridine reference substance, adenine reference substance, guanosine reference substance, and adenosine reference substance, add water to make a mixed solution containing 110μg~130μg of uric acid, 10μg~30μg of hypoxanthine, 20μg~40μg of xanthine, 10μg~20μg of uridine, 10μg~30μg of adenine, 10μg~20μg of guanosine, and 20μg~30μg of adenosine per 1mL, to obtain the result.

6. The method for constructing a characteristic map according to claim 5, wherein: The preparation method of the control medicinal material reference solution is as follows: take 1.0 g of Bombyx batryticatus control medicinal material, add 25 mL of water, use ultrasonic treatment with a frequency of 40 kHz for 30 minutes, heat in a water bath at 100° C. for 5 minutes, cool to 20° C.-30° C., filter, and take the filtrate to obtain; The preparation method of the reference substance solution is as follows: take appropriate amounts of uric acid reference substance, hypoxanthine reference substance, xanthine reference substance, uridine reference substance, adenine reference substance, guanosine reference substance, and adenosine reference substance, add water to make a mixed solution containing 120 μg of uric acid, 20 μg of hypoxanthine, 30 μg of xanthine, 15 μg of uridine, 20 μg of adenine, 15 μg of guanosine, and 25 μg of adenosine per 1 mL.

7. The method for constructing a characteristic spectrum according to claim 1, wherein: The preparation method of the first test solution is as follows: take 0.8g~1.5g of Bombyx batryticatus medicinal materials or Bombyx batryticatus decoction pieces, add 20mL~40mL of water or 10vol%~50vol% methanol, use ultrasonic treatment with a frequency of 20kHz~50kHz for 15min~45min, heat at 95℃~100℃ for 2min~10min, cool to 20℃~30℃, filter, and take the filtrate to obtain; or The preparation method of the first test solution is as follows: take 0.1g~0.4g of Bombyx batryticatus standard decoction or Bombyx batryticatus formula granules, add 20mL~30mL of water or 10vol%~50vol% methanol, use ultrasonic treatment with a frequency of 20kHz~50kHz for 20min~40min, cool, shake well, filter, and take the filtrate to obtain the first test solution.

8. The method for constructing a characteristic spectrum according to claim 7, wherein: The preparation method of the first test solution is as follows: take 1.0 g of Bombyx batryticatus medicinal materials or Bombyx batryticatus decoction pieces, add 25 mL of water, use ultrasonic treatment with a frequency of 40 kHz for 30 minutes, heat in a 100°C water bath for 5 minutes, then cool to 20°C~30°C, shake well, filter, and take the filtrate to obtain; or The preparation method of the first test solution is as follows: take 0.2 g of Bombyx batryticatus standard decoction or Bombyx batryticatus formula granules, add 25 mL of water, use ultrasonic treatment with a frequency of 40 kHz for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the first test solution.

9. The method for constructing a characteristic spectrum according to claim 2, wherein: The preparation method of the second test solution is as follows: take 0.8g~1.5g of fried silkworm slices, add 20mL~40mL of water or 10vol%~50vol% methanol, use ultrasonic treatment with a frequency of 20kHz~50kHz for 15min~45min, heat at 95℃~100℃ for 2min~10min, cool to 20℃~30℃, filter, and take the filtrate to obtain; or The preparation method of the second test solution is as follows: take 0.1g~0.4g of stir-fried silkworm standard decoction or stir-fried silkworm formula granules, add 20mL~30mL of water or 10vol%~50vol% methanol, use ultrasonic treatment with a frequency of 20kHz~50kHz for 20min~40min, cool, shake well, filter, and take the filtrate to obtain the second test solution.

10. The method for constructing a characteristic spectrum according to claim 9, wherein: The preparation method of the second test solution is as follows: take 1.0 g of fried silkworm slices, add 25 mL of water, use ultrasonic treatment with a frequency of 40 kHz for 30 minutes, heat in a 100°C water bath for 5 minutes, then cool to 20°C~30°C, shake well, filter, and take the filtrate to obtain; or The preparation method of the second test solution is as follows: take 0.2g of stir-fried silkworm standard decoction or stir-fried silkworm formula granules, add 25mL of water, use ultrasonic treatment with a frequency of 40kHz for 30min, cool, shake well, filter, and take the filtrate to obtain the second test solution.

11. The method for constructing a characteristic map according to claim 1 or 2, wherein: The characteristic spectrum has a total of 9 peaks, among which peak 1 is uric acid, peak 2 is hypoxanthine, peak 3 is xanthine, peak 4 is uridine, peak 5 is adenine, peak 7 is guanosine, and peak 9 is adenosine; Taking peak 5 as the S peak, the relative retention times of the remaining characteristic peaks are within ±10% of the specified value. The specified value of peak 1 is 0.41, the specified value of peak 2 is 0.51, the specified value of peak 3 is 0.61, the specified value of peak 4 is 0.72, the specified value of peak 6 is 1.08, the specified value of peak 7 is 1.52, and the specified value of peak 8 is 1.92.

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