Application of pigeonpea in resisting respiratory syncytial virus infection

Abstract

The invention belongs to the technical field of plant pharmacy, and particularly relates to an effect of pigeonpea in resisting respiratory syncytial virus infection. Detecting that the pigeon pea protein extract is non-toxic to HEp-2 cells through a cell proliferation detection kit; a cell activity detection method and an indirect immunofluorescence method are adopted to determine that the pigeon pea protein extract has an obvious inhibition effect on the respiratory syncytial virus, the activity of inhibiting the respiratory syncytial virus after the pigeon pea protein is subjected to enzymolysis by alkaline protease is improved, and pigeon peas have relatively high biological safety and relatively strong respiratory syncytial virus infection resistance.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant pharmacy, and particularly relates to application of Cajanus cajan (Linn.) Millsp. in anti-respiratory syncytial virus infection. BACKGROUND

[0002] Respiratory syncytial virus (RSV) belongs to the family Paramyxoviridae, subfamily Pneumovirinae, and genus Pneumovirus, and is a non-segmented negative-sense single-stranded RNA virus with an envelope structure. As a highly infectious pathogen widely spread in the world, RSV can cause different degrees of symptoms from mild cold to severe respiratory disease, especially in infants, the elderly, and people with immune system damage or chronic respiratory disease. Currently, the commonly used drugs in clinic include ribavirin and palivizumab, etc. However, these antiviral drugs have problems such as poor efficacy, difficult administration, high cost, and drug resistance mutation. Therefore, developing new and efficient antiviral drugs has become an urgent need in the field of global public health.

[0003] In the world, medicinal plants have been used in traditional healthcare systems since ancient times, and are still the most important source of medicine for the vast majority of the world's population. Cajanus cajan (Linn.) Millsp. is a plant of the genus Cajanus in the family Fabaceae, and its seeds contain rich protein and high-quality oil. It is distributed in Yunnan, Sichuan, Guangxi and other places in China. Cajanus cajan has very high medicinal value, and has the effects of lowering blood sugar, lowering blood lipids, neuroprotection, anti-osteoporosis, treating cardiovascular and cerebrovascular diseases, and treating skin infections. The present application found that the protein of Cajanus cajan can inhibit the replication of RSV virus, which is expected to be applied to the development of drugs for preventing or treating RSV infection, and provides a new solution for the effective prevention and control of RSV. SUMMARY

[0004] The purpose of the present application is to provide the application of Cajanus cajan in the preparation of drugs or food-homologous health care products for resisting respiratory syncytial virus infection, and the Cajanus cajan has high biological safety and strong effect of resisting respiratory syncytial virus infection.

[0005] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: The application of Cajanus cajan in the preparation of drugs or food-homologous health care products for resisting respiratory syncytial virus infection.

[0006] Preferably, the application of the protein extract of Cajanus cajan in the preparation of drugs or food-homologous health care products for resisting respiratory syncytial virus infection.

[0007] Preferably, the application of the alkaline protease enzymatic hydrolysate of the protein of Cajanus cajan in the preparation of drugs or food-homologous health care products for resisting respiratory syncytial virus infection.

[0008] Preferably, the preparation method of the Cajanus cajan protein extract comprises: (1) after the Cajanus cajan seeds are crushed and defatted, the defatted Cajanus cajan is extracted under alkaline conditions, the supernatant is taken and the precipitate is collected under acidic conditions, the precipitate is dissolved and dialyzed, the dialysate is collected and dried; (2) after the Cajanus cajan seeds are crushed and defatted, the defatted Cajanus cajan is ultrasonically extracted in Tris-HCl buffer solution, ammonium sulfate is added to the supernatant for leaching, the precipitate is dissolved in water and dialyzed, the dialysate is collected and dried; (3) after the Cajanus cajan seeds are crushed and defatted, the defatted Cajanus cajan is leached with water, ammonium sulfate is added to the supernatant for leaching, the precipitate is dissolved in water and dialyzed, the dialysate is collected and dried; (4) the Cajanus cajan proteins obtained in the above steps (1) to (3) are mixed in equal amounts.

[0009] Compared with the prior art, the present application has the beneficial effects of: The Cajanus cajan protein is extracted from the seeds of the food and medicine homologous plant Cajanus cajan, the raw material is easy to obtain, the cost is low, and the safety is high; it is verified that the Cajanus cajan protein with a concentration of 50-400 μg / mL has a strong ability to resist respiratory syncytial virus and has no cytotoxicity, the Cajanus cajan protein can be enzymolyzed by alkaline protease, and the product after alkaline protease enzymolysis has improved activity in inhibiting respiratory syncytial virus, and the enzymolysis liquid with a concentration of 100 μg / mL has the best inhibitory effect on respiratory syncytial virus. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 It is an SDS-PAGE diagram of the Cajanus cajan proteins extracted by different methods.

[0011] Figure 2 It is an SDS-PAGE diagram of the Cajanus cajan protein enzymolysis product.

[0012] Figure 3 It is the cytotoxicity of different concentrations of the Cajanus cajan protein and alkaline protease enzymolysis liquid.

[0013] Figure 4 It is the inhibitory effect of the Cajanus cajan protein enzymolysis liquid with different enzymolysis times on RSV.

[0014] Figure 5 It is a high-content fluorescence imaging diagram of the anti-RSV effect of different concentrations of the Cajanus cajan protein and alkaline protease enzymolysis liquid. DETAILED DESCRIPTION

[0015] Part of the raw materials used in the present application are as follows: the Cajanus cajan is purchased from Yunnan; the respiratory syncytial virus RSV-A2 strain is provided by the Jiangxia laboratory in Hubei.

[0016] Example 1 The present embodiment adopts seven methods of ultrasonic-assisted alkali dissolution and acid precipitation, Tris-HCl extraction, Tris-HCl extraction-ammonium sulfate precipitation, water extraction, ultrapure water extraction-ammonium sulfate precipitation, NaCl extraction and 70% ethanol extraction for the extraction of pigeon pea protein.

[0017] 1.1 Defatting treatment of pigeon pea material First, the pigeon pea seeds were crushed to 100 mesh and sieved, and the powder was weighed. Petroleum ether was added at a material to liquid ratio of 1:8, and ultrasonic extraction (400W) was performed for 60 min. The fume hood was then allowed to dry. After drying, the powder was sieved to 40 mesh and frozen at -20°C in a refrigerator for storage.

[0018] 1.2 Ultrasonic-assisted alkali dissolution and acid precipitation The defatted pigeon pea powder was dissolved in ultrapure water at a material to liquid ratio of 1:30, and the pH was adjusted to 9.0 with 1 M NaOH. Ultrasonic extraction (400W) was performed at 55°C for 2 h. The obtained suspension was centrifuged at 9000 rpm and 4°C to obtain the supernatant. The pH was adjusted to 4.5 with 1 M HCl, and the mixture was allowed to stand for 1 h. Then, the mixture was centrifuged at 9000 rpm and 4°C to obtain the precipitate. The precipitate was redissolved in ultrapure water, dialyzed overnight using a dialysis bag with a molecular weight cutoff of 5000 kDa, and vacuum freeze-dried for 48 h. The freeze-dried powder was stored at -20°C for future use.

[0019] 1.3 Tris-HCl extraction The defatted pigeon pea powder was dissolved in 1 M Tris-HCl buffer (pH 8.3) at a material to liquid ratio of 1:30, and extraction was performed at 50°C for 2 h. The obtained suspension was centrifuged at 9000 rpm and 4°C to obtain the supernatant. The supernatant was dialyzed overnight using a dialysis bag with a molecular weight cutoff of 5000 kDa, and vacuum freeze-dried for 48 h. The freeze-dried powder was stored at -20°C for future use.

[0020] 1.4 Tris-HCl extraction and ammonium sulfate precipitation The defatted pigeon pea powder was dissolved in 1 M Tris-HCl buffer (pH 8.3) at a material to liquid ratio of 1:30, and ultrasonic extraction (400W) was performed at 45°C for 1 h. The obtained suspension was centrifuged at 9000 rpm and 4°C to obtain the supernatant. Ammonium sulfate powder was slowly added while stirring until the content reached 80%. The mixture was allowed to stand for 3 h at 4°C, and then centrifuged at 9000 rpm and 4°C to obtain the precipitate. The precipitate was redissolved in ultrapure water, dialyzed overnight using a dialysis bag with a molecular weight cutoff of 5000 kDa, and vacuum freeze-dried for 48 h. The freeze-dried powder was stored at -20°C for future use.

[0021] 1.5 Water extraction Defatted powder of C. cajan was dissolved in ultrapure water at a ratio of 1:30, and extracted at 40°C for 2 h on a shaker at 150 rpm. The obtained suspension was centrifuged at 9000 rpm and 4°C to obtain the supernatant. The supernatant was dialyzed overnight using a dialysis bag with a molecular weight cut-off of 5000 kDa, and then vacuum freeze-dried for 48 h. The freeze-dried powder was stored at -20°C for later use.

[0022] 1.6 Ultrpure water extraction and ammonium sulfate precipitation Defatted powder of C. cajan was dissolved in ultrapure water at a ratio of 1:30, and extracted at 4°C for 4 h. The obtained suspension was centrifuged at 9000 rpm and 4°C to obtain the supernatant. Ammonium sulfate powder was slowly added while stirring until the content reached 80%, and the mixture was allowed to stand at 4°C for 3 h. The precipitate was then obtained by centrifugation at 9000 rpm and 4°C. The precipitate was redissolved in ultrapure water, dialyzed overnight using a dialysis bag with a molecular weight cut-off of 5000 kDa, and then vacuum freeze-dried for 48 h. The freeze-dried powder was stored at -20°C for later use.

[0023] 1.7 NaCl extraction method Defatted powder of C. cajan was dissolved in 0.4 M NaCl at a ratio of 1:30, and extracted at 55°C for 2 h while stirring. The obtained suspension was centrifuged at 9000 rpm and 4°C to obtain the supernatant. The supernatant was dialyzed overnight using a dialysis bag with a molecular weight cut-off of 5000 kDa, and then vacuum freeze-dried for 48 h. The freeze-dried powder was stored at -20°C for later use.

[0024] 1.8 70% ethanol extraction Defatted powder of C. cajan was dissolved in 70% ethanol at a ratio of 1:30, and extracted for 1 h while oscillating. The obtained suspension was centrifuged at 9000 rpm and 4°C to obtain the supernatant. The supernatant was dialyzed overnight using a dialysis bag with a molecular weight cut-off of 5000 kDa, and then vacuum freeze-dried for 48 h. The freeze-dried powder was stored at -20°C for later use.

[0025] 1.9 Preparation of C. cajan protein enzymatic hydrolysate An equal amount of freeze-dried powder of C. cajan protein obtained in 1.2, 1.4, and 1.6 was mixed, and a solution with a concentration of 4 mg / mL was prepared using PBS. The pH was adjusted to 8.0, and alkaline protease (1.17 g / mL) was added at a mass ratio of 1:2. Enzymatic hydrolysis was performed at 50°C for 0 h, 0.1 h, 0.5 h, 1 h, 3 h, and 5 h on a shaker at 150 rpm, respectively. Then, the mixture was placed in a water bath at 100°C for 10 min. After cooling to room temperature, the supernatant was obtained by centrifugation at 8000 rpm and 4°C for 20 min, which was the C. cajan protein enzymatic hydrolysate.

[0026] The experimental results are shown in Table 1. Figure 1As shown in Figure 1, lanes 1-7 are proteins obtained by different extraction methods, which are ultrasonic-assisted alkali-soluble acid precipitation extraction, Tris-HCl extraction, Tris-HCl extraction-ammonium sulfate precipitation, water extraction, ultrapure water extraction-ammonium sulfate precipitation, NaCl extraction method, and 70% ethanol extraction. The protein bands of the first six extraction methods have similarities, and the size distribution is 15-130 kDa. Two main proteins are detected in the regions of 55-72 kDa and 43-55 kDa, respectively. The protein band obtained by Tris-HCl extraction is thicker, and the protein content is higher. Tris-HCl extraction-ammonium sulfate precipitation and ultrapure water extraction-ammonium sulfate precipitation detect rich specific bands at about 130 kDa and 33-43 kDa. Therefore, the methods of Tris-HCl extraction, Tris-HCl extraction-ammonium sulfate precipitation, and ultrapure water extraction-ammonium sulfate precipitation are selected as the final protein extraction methods of Caesalpinia spinosa.

[0027] The SDS-PAGE diagram of the Caesalpinia spinosa protein enzymatic hydrolysate obtained by enzymolysis of the crude protein obtained by the three methods of Tris-HCl extraction, Tris-HCl extraction-ammonium sulfate precipitation, and ultrapure water extraction-ammonium sulfate precipitation mixed at a mass ratio of 1:1:1 is shown in Figure 2. Figure 2 As shown in Figure 2, lanes 1-6 represent different enzymolysis times, which are 0 h, 0.1 h, 0.5 h, 1 h, 3 h, and 5 h, respectively. The fragment size of the Caesalpinia spinosa protein after alkaline protease enzymolysis is concentrated at about 25 kDa and below 15 kDa.

[0028] Example 2 In this example, the cell proliferation detection kit (WST-1) is used to detect the effect of Caesalpinia spinosa protein and its enzymatic hydrolysate on HEp-2 cells, i.e., to quantify the cytotoxicity.

[0029] 2.1 Cell culture HEp-2 cells were inoculated in a 96-well plate at a density of 1×10 4 around, and cultured in DMEM medium containing 10% fetal bovine serum for 24 h.

[0030] 2.2 Preparation of different concentrations of Caesalpinia spinosa protein and its enzymatic hydrolysate The 4 mg / mL Caesalpinia spinosa protein prepared in 1.9 or the enzymatic hydrolysate obtained by alkaline protease for 0.5 h was mixed with DMEM medium containing 2% fetal bovine serum at a volume ratio of 1:10, 1:20, 1:40, and 1:80.

[0031] 2.3 Effect of different concentrations of Caesalpinia spinosa protein or protein enzymatic hydrolysate on HEp-2 cells Discard the culture medium in the 96-well plate in 2.1, add 100 μL of culture medium containing different concentrations of Caesalpinia diapenata protein or protein hydrolysate to each well, i.e. the treatment group, and set DMEM culture medium containing only 2% fetal bovine serum as the control group, and set three replicate wells for each group, and culture at 37°C in a 5% CO2 incubator for 72 h.

[0032] 2.4 Cell toxicity detection The cytotoxicity kit was used to evaluate the toxic effect of different concentrations of Caesalpinia diapenata protein or protein hydrolysate on cells. The specific operation is as follows: Discard the culture medium of the treatment group and the control group in the 96-well plate, wash the cells twice with PBS, and then add DMEM culture medium containing 10% WST-1, incubate at 37°C in a 5% CO2 incubator for 2 h, and then use an enzyme marker to measure the absorbance value at 450 nm.

[0033] The cell viability calculation formula is: The experimental results are shown in Figure 3 Different concentrations of Caesalpinia diapenata protein and alkaline protease hydrolysate do not show cytotoxicity to HEp-2 cells.

[0034] Example 3 In this example, the cell activity detection method was used to determine the inhibition of Caesalpinia diapenata protein hydrolysate on respiratory syncytial virus infected HEp-2 cells, i.e. to quantify the anti-respiratory syncytial virus activity.

[0035] 3.1 Cell culture HEp-2 cells were inoculated in a 96-well plate at a density of 1×10 4 around per well, and cultured in DMEM culture medium containing 10% fetal bovine serum for 24 h.

[0036] 3.2 Preparation of Caesalpinia diapenata protein hydrolysate with different hydrolysis times and virus mixture The 4 mg / mL Caesalpinia diapenata protein hydrolysate obtained by alkaline protease hydrolysis for 0 h, 0.1 h, 0.5 h, 1 h, 3 h and 5 h in 1.9 was mixed with DMEM culture medium containing 2% fetal bovine serum at a volume ratio of 1:5, and added to the 96-well plate at a volume of 50 μL per well, and 50 μL of respiratory syncytial virus (MOI = 1) was added, and three replicate wells were set for each group, and a cell well without adding virus and enzyme hydrolysate was set as a negative control group, and a cell well with only virus without enzyme hydrolysate was set as a positive control, and incubated at 35°C for 30 min.

[0037] 3.3 Infection of Hep-2 cells with different hydrolysis time enzyme hydrolysate and virus mixture Discard the culture medium in the 96-well plate in 3.1, add the enzyme solution mixed with virus prepared in 3.2 to the cells, and culture in a 35°C, 5% CO2 incubator for 72 h.

[0038] 3.4 Anti-virus detection The anti-virus effect of the enzyme solution at different time points was determined by chemiluminescence (CellTiter-Glo 2.0). The more cell survival indicates that the enzyme solution at the concentration has a stronger inhibitory effect on RSV. The specific operation is as follows: After the cells are cultured for 72 h, the enzyme solution mixed with virus in the 96-well plate is aspirated, the cells are washed twice with PBS, 50 μL of DMEM culture medium containing 2% fetal bovine serum is added, the CellTiter Glo and the culture medium are fully mixed at a volume ratio of 1:2, 50 μL of the mixed solution is added to each well, the cells are scraped off while being blown, and the light signal intensity is detected by using an enzyme-labeled instrument after the cells are fully lysed and mixed. The virus inhibition rate is calculated.

[0039] The experimental results are shown in Table 1. Figure 4 As shown in Table 1, the enzyme solution of alkaline protease at different time points has a significant inhibitory effect on respiratory syncytial virus without cytotoxicity. The Caesalpinia diapenata protein has a significant inhibitory effect on respiratory syncytial virus at 0 h of enzyme hydrolysis, and the inhibitory effect of the enzyme solution of alkaline protease on respiratory syncytial virus is enhanced after 0.1 h of enzyme hydrolysis. It can be known that the Caesalpinia diapenata protein exerts an inhibitory effect on respiratory syncytial virus, and the alkaline protease enzyme hydrolysis produces polypeptides with better anti-virus effect.

[0040] Example 4 In this example, the indirect immunofluorescence method is used to determine the inhibitory effect of Caesalpinia diapenata protein and enzyme solution of alkaline protease on respiratory syncytial virus, i.e., to quantify the anti-respiratory syncytial virus activity.

[0041] 4.1 Cell culture The cells are inoculated in the 96-well plate at a density of 1×10 4 The cells are inoculated in the 96-well plate at a density of 1×10

[0042] 4.2 Preparation of mixed solution of different concentrations of Caesalpinia diapenata protein or enzyme solution and virus 4.1 Preparation of virus mixed solution 4 mg / mL of Caesalpinia diapana protein or enzymatic hydrolysate was mixed with DMEM medium containing 2% fetal bovine serum at a ratio of 1:5, 1:10, 1:20, and 1:40, respectively, and added to a 96-well plate at a volume of 50 μL per well, and 50 μL of respiratory syncytial virus (MOI = 1) was added to each well, with 3 replicates for each group. Negative control wells were set up without the virus and Caesalpinia diapana protein or enzymatic hydrolysate, and positive control wells were set up without the Caesalpinia diapana protein or enzymatic hydrolysate. The mixture was incubated at 35°C for 30 min.

[0043] 4.3 Infection of Hep-2 cells with different concentrations of Caesalpinia diapana protein or enzymatic hydrolysate mixed with the virus The culture medium in the 96-well plate of 4.1 was discarded, and the virus mixed solution prepared in 4.2 was added to the cells, which were then cultured in a 35°C, 5% CO2 incubator for 72 h.

[0044] 4.4 Indirect immunofluorescence After 72 h of cell culture, 8% cell tissue fixative was added at a volume ratio of 1:1, and the mixture was allowed to stand at room temperature for 1 h. After the supernatant was removed, the cells were washed with PBS 3 times. 0.5% Triton X-100 was added for permeation, and the mixture was allowed to stand at room temperature for 30 min. After the supernatant was removed, the cells were washed with PBS 3 times. 0.1% Triton X-100 containing 0.2% BSA was added for blocking at room temperature for 30 min. After the supernatant was removed, the cells were washed with PBS 3 times. Human anti-RSV neutralizing antibody (primary antibody) diluted in 0.1% Triton X-100 was added, and the mixture was incubated at 4°C overnight. After the primary antibody was removed, the cells were washed with PBS 6 times. Anti-human IgG (H+L) diluted in PBS (1:1000 dilution) was added, and the mixture was incubated at room temperature for 2 h in the dark. The cells were washed with PBS 6 times. PBS-diluted Hoechst 33258 (1:1000 dilution) was then added, and the mixture was incubated at room temperature in the dark for 10 min. The cells were washed with PBS 3 times. Finally, a high-content cell analyzer was used to take photographs and perform statistical analysis.

[0045] The results are shown in Table 1. Figure 5 As shown in Table 1, the virus fluorescence signal was reduced in the Caesalpinia diapana protein treatment groups at different concentrations, without cytotoxicity. Among them, 200 μg / mL of Caesalpinia diapana protein had the best inhibitory effect on respiratory syncytial virus, with a reduction of about 50% in the fluorescence signal. The enzymatic hydrolysate of Caesalpinia diapana protein had an inhibitory effect on respiratory syncytial virus at a concentration of 100-400 μg / mL. Among them, 100 μg / mL of the enzymatic hydrolysate had the strongest inhibitory effect on respiratory syncytial virus, with a reduction of about 50% in the fluorescence signal. However, at a concentration of 50 μg / mL, the enzymatic hydrolysate did not show an inhibitory effect on respiratory syncytial virus.

Claims

1. Use of Caesalpinia diapora in the preparation of a medicine or a health product for preventing respiratory syncytial virus infection.

2. Use of a protein extract of Caesalpinia diapora in the preparation of a medicine or a health product for preventing respiratory syncytial virus infection.

3. Use of an alkaline protease enzymatic hydrolysate of a protein extract of Caesalpinia diapora in the preparation of a medicine or a health product for preventing respiratory syncytial virus infection.

4. Use according to claim 2, characterized in that, The method for preparing the protein extract of Caesalpinia diapora comprises: (1) grinding Caesalpinia diapora seeds, defatting the ground seeds, extracting the defatted seeds under alkaline conditions, collecting the supernatant, collecting the precipitate under acidic conditions, dissolving the precipitate, dialyzing the solution, collecting the dialysate, and drying the dialysate; (2) grinding Caesalpinia diapora seeds, defatting the ground seeds, extracting the defatted seeds in Tris-HCl buffer, adding ammonium sulfate to the supernatant, dissolving the precipitate in water, dialyzing the solution, collecting the dialysate, and drying the dialysate; (3) grinding Caesalpinia diapora seeds, defatting the ground seeds, extracting the defatted seeds in water, adding ammonium sulfate to the supernatant, dissolving the precipitate in water, dialyzing the solution, collecting the dialysate, and drying the dialysate; (4) mixing equal amounts of the protein extracts obtained in steps (1) to (3).

5. Use according to claim 3, characterized in that, The enzymatic hydrolysate is obtained by subjecting the protein extract of Caesalpinia diapora of claim 4 to alkaline protease enzymatic hydrolysis.

6. Use according to claim 4, characterized in that, The amount of ammonium sulfate added in steps (2) and (3) is 80% of the extract.

7. Use according to claim 4, characterized in that, The molecular weight cut-off in the dialysis is 5000 kDa.

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