Protein allergen marker in andrographis paniculata extract and detection method thereof

Abstract

The invention discloses a protein allergen marker in an andrographis paniculata extract and a detection method of the protein allergen marker in the andrographis paniculata extract, which are used for detecting the level of trace protein in the andrographis paniculata extract and related injections thereof by adopting an analysis method of ultrafiltration enrichment and proteomics combined with liquid chromatography-mass spectrometry. The invention provides a new method for evaluating the sensitizing substances of the andrographis paniculata extract and related injections thereof, and has important application value.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to a method for detecting residual protein components in an Andrographis paniculata extract. Background Art

[0002] Andrographis paniculata extract is a mixed substance extracted from the Chinese herbal medicine Andrographis paniculata with an appropriate solvent such as ethanol aqueous solution. Its main component, andrographolide, is one of the main active ingredients of Andrographis paniculata. It is a semi-floral-type diterpene lactone compound with a complex structure and difficult artificial synthesis. It mainly relies on extraction from the whole herb or leaves of Andrographis paniculata. Studies have found that andrographolide also has significant activity in antipyretic, antiviral, anti-inflammatory, and anti-cancer. Because andrographolide is almost insoluble in water, the bioavailability of oral preparations directly made is less than 10%. In order to improve bioavailability, the derivatives of Andrographis paniculata extract after sulfuric acid, sulfurous acid or succinic acid derivatization are used as raw materials to make Chinese medicine injections. The related injections with production approvals include Lianbizhi injection, Chuanhuning injection, Xiyanping injection, Yanhuning injection, etc. Literature reports that all four injections have some adverse reactions, among which allergic reactions account for a relatively large proportion. The cause of adverse allergic reactions may be related to macromolecules such as proteins. Compared with humans, plant proteins have a large difference in homology with humans and often have strong antigenicity. Trace amounts of protein will also increase the risk of adverse reactions during clinical use. Considering that when Andrographis paniculata extract is derivatized with acid, the protein will be hydrolyzed and broken into peptides, and the molecular weight will become smaller, which is more unfavorable for detection. Therefore, detecting the residual trace protein of the raw material Andrographis paniculata extract is more meaningful than detecting the protein of the injection, and can better guide the optimization of the process and improve the safety of the injection.

[0003] The "2400 Injection Related Substances Inspection Method" of the 2020 edition of the "Pharmacopoeia of the People's Republic of China" requires that Chinese medicine injections should be subject to a protein limit test. The inspection method is: unless otherwise specified, take 1 ml of injection solution, add 1 ml of newly prepared 30% sulfosalicylic acid solution, mix well, and let it stand for 5 minutes. No turbidity should appear; if the injection solution contains components that can produce precipitation when exposed to acid, 1 to 3 drops of tannic acid test solution can be added instead, and no turbidity should appear. Referring to this method, the injection solution was prepared according to the derivative of Andrographis paniculata extract. The inspection amount of Andrographis paniculata extract was about 100 mg. It was suspended with 5 ml of water and centrifuged. The supernatant was taken as the protein extract. 1 ml was taken for testing. As a result, no protein was detected. It is speculated that this is because the sensitivity of the inspection method is low, as described in the technical background of the authorized patent CN201010504574.4 (detection method of trace protein in traditional Chinese medicine injection): the protein limit of this method is about 37-110μg / ml. Proteins below this limit are difficult to detect by the pharmacopoeia method. Traditional Chinese medicine injections with protein content below 37μg / ml will be considered "qualified". Referring to the method of patent CN201010504574.4, using protein adsorption membrane, the minimum detection limit can be lower than 0.5μg / ml, and the injection test still did not detect it.

[0004] Therefore, developing a highly sensitive and highly specific detection method to qualitatively and quantitatively detect residual protein in Andrographis paniculata extract and its injection has become an urgent problem to be solved in the Chinese medicine injection industry. Referring to the authorized patent CN202010621272.9 (Detection method of trace plant protein components in Reduning injection and its intermediates), given that the detection target of the method described in the patent application is the water extract of compound Chinese medicine or the treated water extract, theoretically, the protein content in the water extract is relatively high; and Andrographis paniculata extract is extracted from the single medicinal material Andrographis paniculata with 90% to 95% ethanol, and the types of proteins contained are different and the residual amount is relatively much smaller, so the patented method is not universal, and whether it can be used successfully on the solid sample and related injections is unpredictable; in addition, the method described in the patent is relatively cumbersome, the operation process has large errors, takes a long time, uses more reagents, and has a greater risk of contamination. Summary of the invention

[0005] In order to overcome the shortcomings of the prior art, the present invention provides a method for detecting residual protein in Andrographis paniculata extract. The method adopts ultrafiltration, proteomics combined with liquid chromatography-mass spectrometry analysis to detect the residual protein content in Andrographis paniculata extract. The method has good specificity and high sensitivity and is suitable for application in quality control.

[0006] In order to achieve the technical purpose of the present invention, the present invention provides the following technical solutions:

[0007] The method of patent CN202010621272.9 was improved from seven steps to four steps. The improvements include but are not limited to: step (1) pre-treatment with alkaline water to improve the protein extraction rate; step (2) ultrafiltration centrifugation, the acid used is controlled more accurately according to the isoelectric point of the protein, so that the protein precipitation is more complete; step (2) water is used for re-dissolution instead of denaturing solvent urea, and step (3) enzyme digestion is calculated by weight ratio to avoid the complex operation of protein content determination and desalination. The above improvements reduce the cost of expensive reagents, reduce pollution, and can improve efficiency and reduce errors.

[0008] The method for detecting and analyzing proteins in the puncture extract or injection specifically comprises the following steps:

[0009] (1) Preparation of sample solution: Take an appropriate amount of Andrographis paniculata extract or its related injection, add alkaline water to make a suspension, and take the supernatant after high-speed centrifugation;

[0010] (2) Enrichment and purification of trace proteins: The supernatant of step (1) is subjected to ultrafiltration centrifugation, the upper layer of the ultrafiltration centrifuge tube is taken and re-dissolved with water, and then acid is added to precipitate the protein, and the crude protein is washed and purified with acetone and evaporated;

[0011] (3) Protein enzymatic digestion: After the crude protein is re-dissolved in water, enzymes are added for enzymatic digestion;

[0012] (4) LC-MS analysis: The sample digested in step (3) was subjected to LC-MS analysis to identify the components of the crude protein composition of Andrographis paniculata. The data analysis process is as follows:

[0013] The LC-MS detection data were processed and analyzed using Raw data, and the data analysis software included Maxquant, Peaks and other software, preferably Peaks software. The raw mass spectrometry data were imported into PEAKS Studio 8.5 (Bioinformatics Solution Inc., Waterloo, Canada) for analysis. The error ranges for the precursor protein and fragment mass were 20 ppm and 0.2 Da, respectively, and carbamidomathylation was used as a fixed modification. The peptides analyzed by de novo had an average confidence (ALC) score of ≥50%. The resulting peptide data were exported as *.csv files and edited in Excel (Microsoft, Redmond, WA, ΜSA).

[0014] As a preferred embodiment, the method for detecting and analyzing proteins in the Andrographis paniculata extract or injection, step (1) the preparation method of the Andrographis paniculata extract is: take the Chinese medicinal material Andrographis paniculata with 90% to 95% ethanol, heat and reflux and extract 1 to 3 times. Each time for 0.5 to 2 hours, combine the extracts, concentrate, and recrystallize to obtain the Andrographis paniculata extract;

[0015] Take 15 g of Andrographis paniculata extract, add about 400 mL of 1 mol / L NaOH solution to adjust the pH value to 8.0-12.0; ultrasonicate for 20 min, let stand at 4°C overnight, filter and collect the supernatant.

[0016] As a preferred embodiment, the method for detecting and analyzing proteins in the extract or injection of the punctate, step (2) is to take the supernatant of step (1) and perform ultrafiltration centrifugation, the specification of the ultrafiltration centrifuge tube is 10kD, take the upper layer of the ultrafiltration centrifuge tube and add 350mL of water to dissolve it, add glacial acetic acid or perchloric acid or trichloroacetic acid to adjust its pH to 3.0-3.5 to precipitate protein, let it stand for 12 hours, pour out the supernatant, centrifuge at 12000r / min at low temperature for 6min to obtain protein precipitate, and evaporate the water; after obtaining the crude protein, wash and purify it with -20°C precooled acetone, centrifuge at 14000rpm at low temperature for 30min to obtain the precipitate, transfer it to a weighed vessel or centrifuge tube, and evaporate the acetone.

[0017] As a preferred embodiment, the method for detecting and analyzing proteins in the puncture extract or injection, the protein enzymatic cleavage method in step (3) is as follows: according to the weight of the protein obtained in step (2), all or part of it is dissolved by adding 100 to 500 μL of water, and pancreatic enzyme is added at a pancreatic enzyme: protein weight ratio of 1:20, and digested for 2 hours; for the second digestion, pancreatic enzyme is added at a pancreatic enzyme: protein weight ratio of 1:20, and digested overnight; after digestion is completed, 1% formic acid is added to terminate the digestion.

[0018] As a preferred embodiment, the method for detecting and analyzing proteins in the Andrographis paniculata extract or injection is characterized in that the data analysis of the LC-MS determination in step (4) identifies that the source protein is a composition containing multiple Andrographis paniculata proteins, including but not limited to T1UNP5_ANDPA, K9LXR9_ANDPA, T1UPW1_ANDPA, A0A5C0PVN4_ANDPA, T1UMK4_ANDPA, etc., and the molecular weight range covers the largest average molecular weight of 264,530 for T1UNP5_ANDPA to the smallest average molecular weight of 12,071 for T1UMK4_ANDPA.

[0019] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0020] (1) The present invention screens out ultrafiltration enrichment-proteomics and liquid chromatography-mass spectrometry analysis methods suitable for Andrographis paniculata extracts through a large number of experiments, and provides a new method for detecting trace proteins remaining in Andrographis paniculata extracts and related injections, which has high sensitivity and specificity. In the pre-treatment of step (1) of the method, alkaline water is first used to improve the protein extraction rate. In step (2), ultrafiltration centrifugation is used, and the acid used is more accurately controlled according to the isoelectric point of the protein, so that the protein precipitation is more complete. In step (2), water is used for re-dissolution instead of the denaturing solvent urea, and in step (3), the enzyme dosage calculated by weight ratio is used for enzymatic cleavage, avoiding protein content determination and desalting. The cost of expensive reagents is reduced, pollution is reduced, and efficiency can be improved.

[0021] (2) The Andrographis paniculata proteins T1UNP5_ANDPA, K9LXR9_ANDPA, T1UPW1_ANDPA, A0A5C0PVN4_ANDPA, and T1UMK4_ANDPA detected by the present invention can be used as allergen markers for Andrographis paniculata injection, which is of great significance for controlling the quality and safety of Andrographis paniculata injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the base pair arrangement diagram of the representative protein T1UNP5_ANDPA.

[0023] Figure 2 This is the base pair arrangement diagram of the representative protein K9LXR9_ANDPA.

[0024] Figure 3 This is the base pair arrangement diagram of the representative protein T1UPW1_ANDPA.

[0025] Figure 4 This is the base pair arrangement diagram of the representative protein A0A5C0PVN4_ANDPA.

[0026] Figure 5 This is the base pair arrangement diagram of the representative protein T1UMK4_ANDPA.

[0027] Figure 6 It is the total ion current chromatogram of the sample of the present invention. DETAILED DESCRIPTION

[0028] In order to better understand the content of the present invention, the following is further described in conjunction with specific examples, but the specific implementation methods are not intended to limit the content of the present invention. The experimental methods in the following examples that do not specify specific conditions are selected according to conventional methods and conditions, or according to the product specifications.

[0029] Unless otherwise specified, all reagents used in the examples of the present invention were commercially available.

[0030] The temperature in the embodiments of the present invention is room temperature unless otherwise specified.

[0031] The ratios of the components in the mixed solvents in the embodiments of the present invention are all volume ratios unless otherwise specified.

[0032] Example 1

[0033] 1. Preparation of Andrographis paniculata Extract

[0034] The feeding amount of the Andrographis paniculata extract is 90 kg, and 3 times the amount of 95% ethanol is added for the first time to soak for more than 30 hours, and the extract is filtered; 15% to 25% of the amount of medicinal charcoal is added to the extract, and the mixture is stirred evenly, heated to boiling, and then kept in a boiling state for decolorization for 2 hours. The decolorized filtrate is pumped into an alcohol double-effect vacuum concentrator to be concentrated under reduced pressure, and the ethanol is recovered until the medicinal solution is white and bubbly, and the concentrated solution is transferred to a crystallization tank through vacuum for crystallization, 95% ethanol is boiled to dissolve, and then cooled to precipitate crystals, and the filtered crystals are dried in a drying oven, and weighed to be 2.32 kg.

[0035] 2. Preparation of sample solution

[0036] (1) Preparation of sample solution: 15 g of Andrographis paniculata extract was added with about 400 mL of 1 mol / L NaOH solution to adjust the pH value to 10.0; ultrasonication was performed for 20 min, and the solution was allowed to stand at 4°C overnight, then filtered and the supernatant was collected;

[0037] (2) Enrichment and purification of trace proteins: The supernatant of step (1) is subjected to ultrafiltration centrifugation. The specification of the ultrafiltration centrifuge tube is 10 kD. The upper layer of the ultrafiltration centrifuge tube is taken and 350 mL of water is added to dissolve it. Glacial acetic acid is added to adjust its pH to 3.2 to precipitate the protein. The mixture is allowed to stand for 12 hours. The supernatant is poured out and the protein precipitate is obtained by centrifugation at 12000 r / min at low temperature for 6 minutes. The water is evaporated. After the crude protein is obtained, it is washed and purified with -20°C precooled acetone. The precipitate is obtained by high-speed centrifugation at 14000 rpm at low temperature for 30 minutes. The precipitate is transferred to a weighed vessel or centrifuge tube and the acetone is evaporated.

[0038] (3) Enzymatic digestion of protein: Take the protein from step (2), add 500 μL of water, add trypsin at a ratio of 1:20 by weight of trypsin:protein, and digest for 2 h; for the second digestion, add trypsin at a ratio of 1:20 by weight of trypsin:protein, and digest overnight; after digestion, add 1% formic acid to terminate digestion.

[0039] 3. LC-MS analysis

[0040] The solution after protein digestion in step 2 above was taken for LC-MS analysis.

[0041] The specific conditions for LC-MS analysis are:

[0042] A Q Exactive PLUS quadrupole-Orbitrap combined mass spectrometer was used, equipped with nanoLC; a capillary column of 75 μm × 100 mm, 1.8 μm C18 beads; an 80-min gradient elution (0-5 min, 5% B; 5-60 min, 5-50% B; 60-70 min, 50-100% B; 70-80 min, 100% B), mobile phase A was water containing 1% glacial acetic acid, mobile phase B was acetonitrile containing 1% glacial acetic acid, flow rate was 200 nL / min; injection volume was 1 μL.

[0043] Mass spectrometry conditions: positive ion scanning mode, primary mass spectrometry scanning range m / z 300~2000Da, resolution 50,000, automatic gain control target 4×e 6 , maximum dwell time 50ms; secondary mass spectrometry resolution 5,000, automatic gain control target 2×e 5 , maximum dwell time: 50ms, parent ion isolation width; 2m / z, dynamic exclusion on, time 30s, CID collision induced dissociation mode. The total ion current chromatogram of the sample was obtained by LC-MS analysis ( Figure 6 ). The peptide components were separated by a reverse phase C18 column, and the peaks appeared one after another according to the differences in the polarity of the peptides. The same chromatographic peak was composed of many primary scanning and secondary fragmentation substances with different mass-to-charge ratios.

[0044] The LC-MS / MS data were processed and analyzed using Raw data, and the data analysis software used was Peaks software.

[0045] The mass spectrometry raw data were imported into PEAKS Studio 8.5 (Bioinformatics Solution Inc., Waterloo, Canada) for analysis. The error ranges for the precursor protein and fragment mass were 20 ppm and 0.2 Da, respectively, and carbamidomathylation was used as a fixed modification. The peptides analyzed de novo had an average confidence level (ALC) score of ≥ 50%. The resulting peptide data were exported as *.csv files and edited in Excel (Microsoft, Redmond, WA, ΜSA).

[0046] De novo comparison results of plant-derived peptide sequences of Andrographis paniculata extract. The de novo sequencing analysis in Peaks software is de novo sequencing. Sequencing can be performed without using any reference sequence data. It only needs to use bioinformatics methods to analyze the peptide secondary mass spectrometry and perform splicing and assembly to obtain the peptide sequence. Each peptide obtained by Peaks search can be determined by the secondary mass spectrum. Usually, the fragment ions produced by peptide fragmentation can be divided into two types, one of which contains the amino terminal (a, b, c) and the other contains the carboxyl terminal (x, y, z). Among the ions produced in low-energy collision-induced fragmentation and high-energy collision-induced fragmentation, b ions and y ions are the main ions, and the by ion is at the peptide bond position. There is a difference of one amino acid residue between two adjacent b-ions and y-ions, while glutamine, arginine and lysine residues may usually lose an amino group and lose 17D mass number, and serine, threonine and glutamic acid residues may lose a water molecule and lose 18D mass number.

[0047] like Figure 1 to Figure 3 The proteins identified by data analysis are T1UNP5_ANDPA, K9LXR9_ANDPA, T1UPW1_ANDPA, A0A5C0PVN4_ANDPA, and T1UMK4_ANDPA. The largest molecular weight is T1UNP5_ANDPA with an average molecular weight of 264,530, and the smallest molecular weight is T1UMK4_ANDPA with an average molecular weight of 12,071. These trace proteins can be used as markers of allergens in Andrographis injection.

[0048] The above is only a preferred embodiment of the present invention. It should be pointed out that technicians in this technical field can make several improvements and modifications without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of proteins T1UNP5_ANDPA, K9LXR9_ANDPA, T1UPW1_ANDPA, A0A5C0PVN4_ANDPA, and T1UMK4_ANDPA as allergen markers of Andrographis paniculata extract or its related injections.

2. The method for detecting protein according to claim 1, characterized in that: The following steps are involved: (1) Preparation of sample solution: Take an appropriate amount of Andrographis paniculata extract or its related injection, add alkaline water to make a suspension, and collect the supernatant after high-speed centrifugation; (2) Enrichment and purification of trace proteins: The supernatant of step (1) is subjected to ultrafiltration centrifugation, the upper layer of the ultrafiltration centrifuge tube is taken and re-dissolved with water, and then acid is added to precipitate the protein, and the crude protein is washed and purified with acetone and evaporated; (3) Protein enzymatic digestion: After the crude protein is re-dissolved in water, enzymes are added for enzymatic digestion; (4) LC-MS analysis: The sample digested in step (3) was subjected to LC-MS analysis to identify the components of the crude protein composition of Andrographis paniculata and determine the molecular weight range.

3. The method for detecting protein according to claim 2, characterized in that: The Andrographis paniculata extract in step (1) is prepared by extracting the Andrographis paniculata from the Chinese medicinal material with ethanol having a volume concentration of 90% to 95%, heating and refluxing, combining the extracts, concentrating, and recrystallizing to obtain the Andrographis paniculata extract.

4. The method for detecting protein according to claim 2, characterized in that: The alkaline water in step (1) is an aqueous solution of an alkaline reagent such as sodium hydroxide, potassium hydroxide or ammonia water, and the pH value after adding the alkali is 8.0-12.

0.

5. The method for detecting protein according to claim 2, characterized in that: The pore size of the ultrafiltration centrifuge tube in step (2) is 5-20KD, the acid used for acid addition is glacial acetic acid, trifluoroacetic acid, or perchloric acid, and the pH value after acid addition is 2.5-4.

0.

6. The method for detecting protein according to claim 2, characterized in that: The enzyme used in step (3) is pancreatin or amylase.

7. The method for detecting protein according to claim 6, characterized in that: Trypsin was added at a ratio of 1:20 for protein weight and digested for 2 hours. For the second digestion, trypsin was added at a ratio of 1:20 for protein weight and digested overnight. After digestion was completed, 1% formic acid was added to terminate the digestion.

8. The method for detecting protein according to claim 2, characterized in that: The specific conditions for LC-MS analysis are: Q Exactive PLUS quadrupole-Orbitrap combined mass spectrometer equipped with nanoLC; 75μm×100mm, 1.8μm C18 beads capillary column; mobile phase A is water containing 1% glacial acetic acid, mobile phase B is acetonitrile containing 1% glacial acetic acid, gradient elution: 0-5 minutes, 5% B; 5-60 minutes, 5-50% B; 60-70 minutes, 50-100% B; 70-80 minutes, 100% B; flow rate 200nL / min; injection volume 1μL; Mass spectrometry conditions: positive ion scanning mode, primary mass spectrometry scanning range m / z 300~2000Da, resolution 50,000, automatic gain control target 4×e 6 , maximum dwell time 50ms; secondary mass spectrometry resolution 5,000, automatic gain control target 2×e 5 , maximum dwell time: 50 ms, parent ion isolation width; 2 m / z, dynamic exclusion on, time 30 s, CID collision induced dissociation mode.

9. The method for detecting residual protein in Andrographis paniculata extract according to claim 1, characterized in that: The data analysis of step (4) by LC-MS identified that the source protein was a composition containing multiple Andrographis paniculata proteins, including T1UNP5_ANDPA, K9LXR9_ANDPA, T1UPW1_ANDPA, A0A5C0PVN4_ANDPA, and T1UMK4_ANDPA, with a molecular weight range covering the largest average molecular weight of 264,530 for T1UNP5_ANDPA and the smallest average molecular weight of 12,071 for T1UMK4_ANDPA.

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