A characteristic polypeptide of toad venom and its application in the quantitative determination of toad venom

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to a characteristic polypeptide of toad venom and its application in the quantitative determination of toad venom. The sequence of the characteristic polypeptide is: NNAYDINEER. The present invention is based on the precise identification and determination of characteristic polypeptides in toad venom, and aims to construct a toad venom quantitative system with stronger specificity and higher accuracy. Compared with traditional quantitative methods, this method focuses on highly representative and unique polypeptide components, which not only significantly improves the sensitivity and specificity of detection, but also effectively avoids the interference of non-target components, thereby ensuring the accuracy and reliability of quantitative results. This innovation not only provides strong technical support for the quality control and efficacy evaluation of toad venom, but also is an important supplement and improvement to the existing toad venom standards, which will help promote the process of standardization and scientific development of Chinese medicinal materials and ensure the safety and effectiveness of Chinese medicine products.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a toad venom characteristic polypeptide and application thereof in toad venom quantitative determination. Background Art

[0002] Toad venom is a kind of toad venom from the Chinese giant toad ( Bufo bufo gargarizans Cantor ) or the black-rimmed toad ( Bufo melanostictus The white serous fluid secreted by the parotid gland of Schneider is processed and dried to make the secretion. The active ingredients are steroid compounds, indole alkaloids, etc., which are often used in Chinese medicine prescriptions. Modern pharmacological research has confirmed that toad venom has the effects of strengthening the heart, increasing blood pressure, resisting shock, inhibiting platelet aggregation, respiratory excitement, anti-inflammatory, local anesthesia, resisting pathogenic microorganisms, improving the body's nonspecific immunity, anti-radiation, and anti-tumor.

[0003] In the prior art, methods such as ultraviolet spectrophotometry, thin layer chromatography, and high performance liquid chromatography are used to determine the contents of toad venom toxin base, bufalin, and resifobafox genin in toad venom. However, it is still impossible to detect adulteration of toad skin in toad venom. Summary of the invention

[0004] In view of the technical gaps and other problems existing in the prior art, the present invention provides a toad venom characteristic polypeptide.

[0005] The present invention also provides the application of the characteristic polypeptide of toad venom in the quantitative determination of toad venom.

[0006] The technical solution adopted by the present invention to achieve the above-mentioned purpose is:

[0007] The invention provides a toad venom characteristic polypeptide, the sequence of the characteristic polypeptide is shown in SEQ ID NO.1; the specific sequence is: NNAYDINEER.

[0008] The present invention also provides the use of the above-mentioned characteristic polypeptide in the quantitative determination of toad venom, comprising the following steps:

[0009] (1) Accurately weigh the toad venom sample and add appropriate amount of water for extraction. After cooling, obtain the crude extract;

[0010] (2) After the crude extract is diluted with water to a fixed volume, trypsin is added for enzymatic hydrolysis, and the sample to be tested is obtained after filtration;

[0011] (3) The samples to be tested were detected by triple quadrupole mass spectrometry.

[0012] Furthermore, in step (1), the ratio of the toad venom sample to water is: 1 g: 1-10 mL; the temperature of the added water is 20-50° C., preferably, the temperature is 50° C.

[0013] Furthermore, in step (1), the extraction method is ultrasound; the ultrasound time is 30-60 minutes.

[0014] Furthermore, in step (2), the volume ratio of the crude liquid to trypsin is 1:1; the concentration of the trypsin is 1 mg·mL -1 .

[0015] Furthermore, in step (2), the enzymatic hydrolysis is carried out at 37° C. for 5 hours.

[0016] Furthermore, in step (3), the parameters of the triple quadrupole mass spectrometry are: the mode is set to electrospray ionization (ESI) and positive ion MRM mode, Gas1 flow rate: 50 L∙h -1 , Gas2 flow rate: 50 L∙h -1 , spray voltage: 5.5 kV, source temperature: 550 °C.

[0017] The characteristic polypeptide provided by the present invention can also be further prepared into a kit and the like.

[0018] When the toad venom characteristic polypeptide provided by the present invention is used in the quantitative detection of toad venom, when the toad venom is adulterated with toad skin or other impurities, the content of the toad venom characteristic polypeptide will be reduced.

[0019] The present invention is based on the precise identification and determination of characteristic polypeptides in toad venom, aiming to construct a toad venom quantitative system with stronger specificity and higher accuracy. Compared with traditional quantitative methods, this method focuses on highly representative and unique polypeptide components, which not only significantly improves the sensitivity and specificity of detection, but also effectively avoids the interference of non-target components, thereby ensuring the accuracy and reliability of the quantitative results. This innovation not only provides strong technical support for the quality control and efficacy evaluation of toad venom, but also is an important supplement and improvement to the existing toad venom standards, which will help promote the process of standardization and scientific development of Chinese medicinal materials and ensure the safety and effectiveness of Chinese medicine products.

[0020] The beneficial effects of the present invention are:

[0021] (1) The characteristic polypeptide provided by the present invention can effectively identify whether the toad venom sample is adulterated with toad skin, toad meat or toad bone, providing a solution for the detection of using toad skin, toad meat or toad bone to replace toad venom, filling the gap in this technology;

[0022] (2) The characteristic polypeptide provided by the present invention has excellent specificity and stability for toad skin, toad meat or toad bone, etc., has strong specificity and has good application prospects;

[0023] (3) The quantitative method of toad venom established by the present invention using characteristic peptides of toad venom has higher specificity and is an effective supplement to the current standard. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 To investigate the curve for the extraction method;

[0025] Figure 2 It is the investigation curve of material-liquid ratio;

[0026] Figure 3 To investigate the extraction temperature curve;

[0027] Figure 4 This is the enzymatic hydrolysis time investigation curve;

[0028] Figure 5 It is the result of specific test;

[0029] Figure 6 is the result of the linear relationship test;

[0030] Figure 7 is the result of the quantitation limit test;

[0031] Figure 8 The results of the durability test are shown in the figure. A: Agilent HSS C 18 Column, B: Waters BEH C 18 Chromatographic column, C: Thermo BDS C 18 Chromatographic columns;

[0032] Fig. 9 This is a graph of actual sample detection results of the method provided by the present invention. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is further explained and illustrated by means of specific embodiments below.

[0034] Example 1

[0035] 1 Experimental Materials

[0036] Table 1 Experimental instruments

[0037]

[0038] Table 2 Experimental materials

[0039]

[0040] Table 3 Information table of peptide biomarker reference substances

[0041]

[0042] 2 Method establishment

[0043] 2.1 Extraction method and detection

[0044] (1) Accurately weigh 10 mg of toad venom sample and add it to a 50 mL volumetric flask. Add appropriate amount of water, soak for 12 h, ultrasonicate for 30 min, ultrasonicate for 1 h, and reflux at 100 °C for 1 h. After cooling, adjust the volume to the scale line. Accurately measure 1 mL and add it to a 5 mL volumetric flask. Then accurately add trypsin (1 mg mL -1 ) 1 mL, dilute to the mark with water, hydrolyze at 37 °C for 5 h, filter and inject.

[0045] With the content as the ordinate and the extraction method as the abscissa, draw a curve, such as Figure 1 As shown: the content was the highest after ultrasound for 1 hour.

[0046] (2) The samples were detected by triple quadrupole mass spectrometry: the mode was set to electrospray ionization (ESI) and positive ion MRM mode, and the gas1 flow rate was 50 L∙h -1 , Gas2 flow rate: 50 L∙h -1 , spray voltage: 5.5 kV, source temperature: 550 °C.

[0047] 2.2 Investigation of material-liquid ratio

[0048] 100 mg, 50 mg, 10 mg and 5 mg of toad venom sample were accurately weighed and added to 20 mL, 50 mL, 50 mL and 50 mL volumetric flasks, respectively. Appropriate amount of water was added to make solutions with solid-liquid ratios of 5:1, 1:1, 1:5 and 1:10, respectively. Ultrasonication was performed for 1 h. After cooling, the volume was adjusted to the scale line. Then 1 mL was accurately measured and added to a 5 mL volumetric flask. Trypsin (1 mg mL -1 ) 1 mL, add water to the scale, incubate at 37 °C for 5 h, filter and inject.

[0049] With the content as the ordinate and the solid-liquid ratio as the abscissa, draw a curve, such as Figure 2 As shown: when the material-liquid ratio is 1:5, the content is the highest, and this material-liquid ratio is selected for subsequent experiments.

[0050] 2.3 Extraction temperature investigation

[0051] Weigh 10 mg of toad venom sample accurately and add it to a 50 mL volumetric flask. Add appropriate amount of water and sonicate for 1 h at 20 °C, 50 °C, and 100 °C. After cooling, dilute to the mark. Then accurately measure 1 mL and add it to a 5 mL volumetric flask. Add trypsin (1 mg mL -1 ) 1 mL, add water to the scale, incubate at 37 °C for 5 h, and inject the sample.

[0052] With the content as the ordinate and the extraction temperature as the abscissa, draw a curve, such as Figure 3 As shown: when the water temperature is 50 ℃, the extraction effect is best and the content is the highest.

[0053] 2.4 Investigation of enzymatic hydrolysis time

[0054] Weigh 10 mg of toad venom sample accurately and add it to a 50 mL volumetric flask. Add appropriate amount of water and sonicate at 50 °C for 1 h. After cooling, add water to the scale. Then accurately measure 1 mL and add it to a 5 mL volumetric flask. Add trypsin (1 mg mL -1 ) 1 mL, add water to the scale line, and inject samples continuously for 24 h to investigate the enzymatic hydrolysis time.

[0055] The curve was drawn with the peak area as the ordinate and the enzymatic hydrolysis time as the abscissa. The experimental results are shown in Figure 4 The results showed that the enzymatic hydrolysis became stable after 3.5 h. Considering all factors, 5 h was selected as the enzymatic hydrolysis time for subsequent experiments.

[0056] 3 Methodological investigation

[0057] Methodological investigations were conducted on the specificity, linearity, precision, repeatability, limit of quantification, accuracy, and durability of the established method to ensure the scientificity and rationality of the method.

[0058] 3.1 Exclusivity

[0059] Toad skin, toad meat and toad bones were processed by the same sample treatment method, and the mass-to-charge ratio and retention time were verified by the MRM mode of triple quadrupole mass spectrometry to verify the specificity of the toad venom characteristic peptide CS-Pep2 (NNAYDINEER).

[0060] The specificity test results are shown in Figure 5 The experimental results showed that the characteristic peptide CS-Pep2 in toad venom peaked at 8.01, while toad skin, toad meat and toad bone did not peak at this retention time, proving that the peptide marker has good specificity.

[0061] 3.2 Linearity

[0062] The reference solution was accurately measured to prepare standard curve solutions of 0.0012, 0.0186, 0.0593, 0.1186, 0.1483, 0.2372, 0.2965, 0.9488, and 1.8976 µg / mL, and the linear relationship was investigated using triple quadrupole mass spectrometry.

[0063] like Figure 6As shown, the ion pair (619.28→229.09) has a regression equation of Y = 7E+06X + 9561.3, R 2 The linear relationship is 0.9997.

[0064] 3.3 Precision

[0065] Take 2 µg / mL of the reference solution and inject 6 times continuously, and calculate the RSD%.

[0066] The experimental results show that the RSD% of the reference sample after 6 consecutive injections is 4.0969%, which is less than 5%, proving that the instrument has good precision.

[0067] Table 4 Precision test results

[0068]

[0069] 3.4 Repeatability

[0070] Six samples of toad venom were prepared in parallel, injected, and the RSD% was calculated.

[0071] The experimental results are shown in Table 5. The results show that the RSD% of the content of 6 parallel samples is 3.9421%, which is less than 5%, proving that the method has good repeatability.

[0072] Table 5 Repeatability test results

[0073]

[0074] 3.5 Limit of quantitation

[0075] Different concentrations of toad venom samples were injected, and the signal-to-noise ratio S / N=10 was determined as the quantification limit.

[0076] The experimental results are shown in Figure 7 The results showed that the signal-to-noise ratio (S / N) was about 10 at 0.001 µg / mL, so the limit of quantification was considered to be 0.001 µg / mL.

[0077] 3.6 Accuracy

[0078] The accuracy test results were expressed as recovery rate (%).

[0079] According to the concentration of the sample, the sample and the reference substance (characteristic polypeptide of toad venom) were mixed in a ratio of 1:1 and added to a 50 mL volumetric flask. Water was added and ultrasonicated for 1 h. After cooling, the volume was adjusted to the scale line. Then 1 mL was accurately measured and added to a 5 mL volumetric flask. Trypsin (1 mg mL -1 ) 1 mL, dilute to the mark, hydrolyze for 5 h, filter and inject.

[0080] The experimental results showed that the average recovery rate of the three spiked samples was 98.9361%, which was in line with the recovery limit requirement of 85%-110% in the fourth part of the "Chinese Pharmacopoeia" analytical method validation guidelines.

[0081] Table 6 Recovery test results

[0082]

[0083] 3.7 Durability

[0084] The robustness of the column was investigated on three different brands, including Agilent HSS C 18 (100 mm × 2.1 mm, 1.8 µm) Column, Waters BEH C 18 (100 mm × 2.1 mm, 1.7 µm) column, Thermo BDS C 18 (100 mm × 2.1 mm, 2.2 µm) chromatographic column.

[0085] The experimental results are shown in Figure 8 ,The results showed that three different chromatographic columns all had single chromatographic peaks at different ,retention times, indicating that the method was robust.

[0086] 4. Sample determination

[0087] The content of 5 batches of toad venom collected from the medicinal material market was determined. 10 mg of toad venom sample was accurately weighed and added to a 50 mL volumetric flask to make a solution with a solid-liquid ratio of 1:5. The solution was ultrasonicated at 50 °C for 1 h, and then the volume was adjusted to the scale after cooling. Then 1 mL was accurately measured and added to a 5 mL volumetric flask. Trypsin (1 mg mL -1 ) 1 mL, add water to the scale, enzymatically hydrolyze at 37 °C for 5 h, filter and inject. The chromatography-mass spectrometry method is as follows:

[0088] Chromatographic method: The samples were separated by chromatography using an Agilent Eclipse C18 column (2.1×100 mm, 1.8 µm), with an injection volume of 5 µL and a flow rate of 0.3 mL min -1 , the column was kept at 43 °C. The mobile phase consisted of A (0.1% formic acid in water) and B (acetonitrile solution). Elution gradient: 0-20 min, 3%→25%B, 20-21 min, 25%→90%B, 21-24 min, 90%B, 24.1-30 min, 3%B.

[0089] Mass spectrometry method: The LC-MS / MS system was controlled using Analyst Software. The parameters were set as follows: the mode was set to electrospray ionization (ESI) and positive ion MRM mode, and the sheath gas flow rate was 46 L∙h -1 , Auxiliary gas flow rate: 850 L∙h -1 , Spray Voltage: 3.5 kV, Source temperature: 150 °C, Auxiliary gas temperature: 400 °C. Cone Voltage: 30 V, Collision voltage: 35 V.

[0090] The sample measurement results are shown in Table 7.

[0091] Table 7 Sample measurement results

[0092]

Claims

1. A toad venom characteristic polypeptide, characterized in that: The sequence of the characteristic polypeptide is shown in SEQ ID NO.1; the specific sequence is: NNAYDINEER.

2. Use of the characteristic polypeptide according to claim 1 in the quantitative determination of toad venom, characterized in that: The following steps are involved: (1) Accurately weigh the toad venom sample and add appropriate amount of water for extraction. After cooling, obtain the crude extract; (2) After the crude extract is diluted with water to a fixed volume, trypsin is added for enzymatic hydrolysis, and the sample to be tested is obtained after filtration; (3) The samples to be tested were detected by triple quadrupole mass spectrometry.

3. The use according to claim 2, characterized in that: In step (1), the ratio of the toad venom sample to water is: 1 g: 1-10 mL; and the temperature of the added water is 20-50°C.

4. The use according to claim 2 or 3, characterized in that: In step (1), the extraction is performed by ultrasound; the time of the ultrasound is 30-60 minutes.

5. The use according to claim 2, characterized in that: In step (2), the volume ratio of the crude extract to trypsin is 1:1; the concentration of trypsin is 1 mg·mL -1 .

6. The use according to claim 2 or 5, characterized in that: In step (2), the enzymatic hydrolysis is carried out at 37° C. for 5 h.

7. The use according to claim 2, characterized in that: In step (3), the parameters of the triple quadrupole mass spectrometry are: the mode is set to electrospray ionization and positive ion MRM mode, Gas1 flow rate: 50 L∙h -1 , Gas2 flow rate: 50 L∙h -1 , spray voltage: 5.5 kV, source temperature: 550 °C.

Images