Use of Tibetan medicinal material meconopsis racemosa maxim in anti-respiratory syncytial virus

Abstract

The application provides use of a Meconopsis racemosa extract in preparation of a respiratory syncytial virus resisting medicine. The experiment determines the medicine concentration of the Meconopsis racemosa extract in different ways through a CCK-8 experiment, explores the inhibiting effect of different Meconopsis racemosa on the RSV virus nucleic acid and the RSV virus by establishing a respiratory syncytial virus (RSV) fluorescence quantification method, so as to provide scientific and powerful support for preventing and controlling the RSV.

Description

TECHNICAL FIELD

[0001] The present application relates to a new use of Meconopsis horridula Hook.f. & Thomson. Specifically, the present application relates to a specific effective part of Meconopsis horridula Hook.f. & Thomson and its use in antiviral. BACKGROUND

[0002] Respiratory syncytial virus is one of the most important pathogens causing acute lower respiratory tract infection in infants and young children, and can also cause interstitial pneumonia and bronchiolitis, etc. It can also cause hospitalization and pneumonia death in the elderly and immunodeficient patients.

[58] Even though RSV respiratory syncytial virus has been concerned as early as 60 years ago, there is still a lack of effective vaccines and safe and efficient therapeutic drugs against the virus.

[0003] Currently, the treatment for the disease is limited to supportive treatment, and the clinically available therapeutic drugs are quite limited, and the therapeutic effect is poor. Therefore, it is necessary to explore new substances having inhibitory effect on respiratory syncytial virus. SUMMARY

[0004] Meconopsis horridula Hook.f. & Thomson is the flower or whole herb of Meconopsis horridula Hook.f. & Thomson in the Papaveraceae family, bitter and cold. It grows in the stone crevice of the mountain slope at an altitude of 4100-5400 meters. It has exact curative effects on clearing heat, relieving pain, promoting blood circulation and removing blood stasis, and has anti-tumor, anti-virus and heart maintenance effects. There is no report on its use in treating diseases related to respiratory syncytial virus.

[0005] Specifically, the present application provides a use of Meconopsis horridula Hook.f. & Thomson extract in the preparation of an anti-respiratory syncytial virus drug.

[0006] Further, through experimental screening, it is found that one extraction part of Meconopsis horridula Hook.f. & Thomson has excellent effect in resisting respiratory syncytial virus, which is significantly better than other extraction parts, and even better than ribavirin.

[0007] According to the above results, the Meconopsis horridula Hook.f. & Thomson extract in the present application is a 50% ethanol elution part of Meconopsis horridula Hook.f. & Thomson water extract in a macroporous resin.

[0008] In one specific embodiment of the present application, the Meconopsis horridula Hook.f. & Thomson extract is prepared by the following method:

[0009] The Meconopsis horridula Hook.f. & Thomson water extract is loaded onto a macroporous resin column, and water, 25% ethanol and 50% ethanol are used for elution in sequence, and the 50% ethanol eluate is collected to prepare the 50% ethanol elution part.

[0010] In another specific embodiment of the present application, the Meconopsis horridula Hook.f. & Thomson water extract refers to a liquid extracted by heating Meconopsis horridula Hook.f. & Thomson in water.

[0011] In order to prevent the macroporous resin from being blocked and improve the purity of the effective site, the water extract is subjected to alcohol precipitation before being subjected to the macroporous resin column, and the supernatant is subjected to the macroporous resin column again.

[0012] The drug is a drug for inhibiting respiratory syncytial virus nucleic acid.

[0013] The drug is a drug for treating diseases caused by respiratory syncytial virus.

[0014] Further, the diseases refer to lower respiratory tract infection, interstitial pneumonia, bronchiolitis.

[0015] The application further provides an anti-respiratory syncytial virus pharmaceutical composition comprising the extract of B. potaninii and ribavirin; wherein the extract of B. potaninii is a water extract of B. potaninii, and the 50% ethanol elution part in the macroporous resin.

[0016] The application further provides the use of the extract of B. potaninii and ribavirin in the preparation of a combined drug for resisting respiratory syncytial virus. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Positive product is connected with PMD-19T to transform DH5a

[0018] Figure 2 Comparison of different parts of B. potaninii and RSV virus nucleic acid inhibition (incubation for 6 hours) DETAILED DESCRIPTION

[0019] Example 1

[0020] B. potaninii is taken, 12 times the amount of distilled water is used for heating reflux extraction, extraction is performed twice, the extract is combined and concentrated by filtration, and the supernatant is concentrated after precipitation by using ethanol.

[0021] The supernatant is subjected to macroporous resin, and is eluted with 6 times the column volume of distilled water and 25% and 50% ethanol in sequence, the 50% ethanol elution part is collected, and the extract of B. potaninii is obtained by freeze-drying after concentration under reduced pressure.

[0022] Example 2

[0023] B. potaninii is taken, 8 times the amount of distilled water is used for heating reflux extraction, extraction is performed three times, the extract is combined and concentrated by filtration, and the supernatant is concentrated after precipitation by using ethanol.

[0024] The supernatant is subjected to macroporous resin, and is eluted with 8 times the column volume of distilled water and 25% and 50% ethanol in sequence, the 50% ethanol elution part is collected, and the extract of B. potaninii is obtained by freeze-drying after concentration under reduced pressure.

[0025] Example 3

[0026] Take the multiple green wool, using 8 times the amount of distilled water heated reflux extraction, extraction 3 times, combined extract filtration and concentration, using ethanol precipitation to take supernatant concentration.

[0027] The supernatant was eluted with macroporous resin, and 8 times the column volume of distilled water and 50% ethanol were used in turn. The 50% ethanol elution fraction was collected, concentrated under reduced pressure, and freeze-dried to obtain the multiple green wool extract of the present application.

[0028] Example 4

[0029] 1. Materials and methods

[0030] 1.1 Sample

[0031] RSV: (ATCC-VR-26)

[0032] 1.2 Materials and instruments

[0033] RE-52A rotary evaporator (Shanghai Shen Sheng Technology Co., Ltd.); SterilGARD III Advance super clean bench (BAKER company); Optima L-100XP Ultracentrifuge (Beckman company); centrifuge 5427R low temperature refrigerated centrifuge (eppendorf company); PCR instrument (eppendorf company); pipette (eppendorf company); constant temperature water bath (Beijing Yongguangming Medical Instrument Co., Ltd.); protein electrophoresis instrument (Bio-Red company); transfer mold instrument (Bio-Red company); ChemiDoc MP Imaging Sys multifunctional imaging system (Bio-Red company); qPCR instrument (Bio-Red company); ZWY-100H constant temperature incubation oscillator box (Shanghai Zhicheng Co., Ltd.); MicroPure ST ultrapure water instrument (Thermo); U-HGLGPS fluorescence inverted microscope (OLYMPUS).

[0034] Vero cell line was preserved by the laboratory; CCK-8 reagent was purchased from Biyun Tian, ribavirin injection was preserved by the laboratory, RNAiso Plus (viral RNA extraction reagent), PrimeScript TM RT reagent kit (Perfect RealTime), TB Premix Ex Taq TM II (Tli RNaseH Plus), pMD TM19-T Vector Cloning Kit were purchased from TaKaRa Company; DNA purification kit, gel recovery kit and endotoxin-free plasmid extraction kit were purchased from OMEGA Company; competent cell DH5a was purchased from Beijing Tianzeyiin Company, and other reagents were analytical pure. M. horridula Hook. f. & Thoms was collected from Tibet and identified as Papaveraceae M. horridula Hook. f. & Thoms by Professor Liu Yuan of Southwest University for Nationalities.

[0035] Thomson.

[0036] 1.3 Extraction and separation of M. horridula Hook. f. & Thoms

[0037] 50.00 g of medicinal materials were precisely weighed, and the effective components of M. horridula Hook. f. & Thoms were extracted twice by heating reflux with ten times the amount of distilled water. The combined extract was filtered and concentrated, ethanol was added to make the alcohol content reach 70%, and then precipitated. The supernatant was concentrated. Macroporous resin was used to elute with 6 times the column volume of distilled water and 25%, 50%, and 95% ethanol of different concentrations, respectively. Each elution fraction was freeze-dried after being concentrated under reduced pressure. Four freeze-dried powders were taken and dissolved in an appropriate amount of cell maintenance solution to make the sample concentration 100 mg / mL. The samples were filtered through a 0.22 μm sterile microporous filter, and the four fractions were numbered as water, 25 alcohol, 50 alcohol, and 95 alcohol, respectively.

[0038] 1.4 Primer

[0039] Table 1 Primers used in this study

[0040]

[0041] Primers were designed according to the sequences in Table 1 and synthesized by Shengong Bioengineering (Shanghai) Co., Ltd.

[0042] 1.5 Construction of standard plasmid

[0043] The total RNA in the sample was extracted by Trizol method, and then stored at -20℃ for standby. The above extracted RNA was used as a template to amplify the target gene using the primers in Table 1, and the obtained amplification fragments were subjected to gel recovery and linked with pMD19-T vector at 16℃ overnight; 5 μL of the linking product was added to 50 μL of competent cells, mixed, and subjected to ice bath for 60 min; the transformation tube was quickly transferred to an ice bath for 2-3 min; 800 μL of LB medium without antibiotics was added to each transformation tube, and incubated at 37℃ on a shaker at 150 rpm for 60 min; 100 μL was taken and inoculated on LB agar plates containing the corresponding resistance; the plates were inverted and cultured in a 37℃ incubator overnight; the transformation clones appeared in 12-16 h; the positive recombinants were identified by PCR, and sent to Shanghai Shengong Biotechnology Co., Ltd. for nucleic acid sequencing. The sequencing results were compared, the recombinant plasmid concentration was determined, and the copy number was calculated.

[0044] 1.6 Optimization of qPCR system and reaction conditions

[0045] The plasmid standard in Section 1.5 was used as a template, and the total reaction system was 25 μL according to the instructions of the fluorescent quantitative kit. The annealing temperature (58-60℃) and primer concentration (0.1-1 μL) were optimized to determine the optimal reaction system and conditions.

[0046] The plasmid standard in Section 1.5 was diluted by 10 times, and 1x10 7 ~ 1x10 2 copies / μL was selected for amplification. The standard curve was generated according to the fluorescent quantitative BIO-Rad CFX Maestro.

[0047] 1.8 CCK-8 experiment of A. potaninii and ribavirin

[0048] Vero cells were inoculated into a 96-well plate to form a single layer of cells. The A. potaninii extract and ribavirin injection were diluted with DMEM at 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, and 1:128, respectively. 100 μL of the DMEM and the diluted drug mixture was inoculated into the 96-well plate, and incubated for 6 h, 12 h, and 24 h, respectively, with 3 repeats per well. Negative wells and wells without cells were set. After 6 h, 12 h, and 24 h of incubation, 10 μL of CCK-8 reagent was added, and incubated for another 1 h. The absorbance value was read at OD 450 400 using an enzyme marker. The average OD 450 value of the repeated wells was taken, and the OD 450The values were compared and calculated using the cell viability calculation formula.

[0049] 1.9 Inhibition of RSV viral nucleic acid by B. viridis

[0050] 900 μL of pure water, 25% ethanol, 50% ethanol, and 95% ethanol B. viridis extract were mixed with 100 μL of RSV virus solution, respectively, and placed in a 37°C incubator for 6 h. The nucleic acid of the 6 h culture was extracted according to the Trizol method, and CT value and viral copy number analysis was performed according to the fluorescence quantitative method established in the laboratory. Ribavirin and a drug-free control group were also set up. GraphPad Prism software was used for difference analysis.

[0051] 1.10 Inhibition of RSV virus by B. viridis

[0052] Vero cells were transferred to a 24-well plate, and after the cells grew into a monolayer, 100 μL of RSV virus solution was added and incubated for 2 h. The incubated monolayer cells were added with 50% ethanol B. viridis extract diluted according to a 1:4 ratio, and DMEM PE medium was added to 2 mL. The mixture was incubated in a 37°C incubator for 12 h and 24 h, respectively, and a ribavirin and a drug-free control group were also set up. The culture of 12 h and 24 h was extracted according to the Trizol method, and CT value and viral copy number analysis was performed using the fluorescence quantitative method established in the laboratory. GraphPad Prism software was used for difference analysis.

[0053] 2. Results and analysis

[0054] 2.1 Optimization of qPCR reaction system and parameters

[0055] The optimized 25 μL reaction system: TB Green Premix Ex Taq II 12.5 μL; 1 μL of each of type 2 upstream and downstream primers; 2 μL of plasmid cDNA template; and 8.5 μL of ddH2O. The reaction program: 95°C pre-denaturation for 30 s; 95°C for 5 s, 60°C annealing and extension for 10 s, collect fluorescence signal, 45 cycles. After amplification, 65-95°C with 0.5°C increment, each temperature treatment for 5 s to read the results.

[0056] 2.2 Preparation of standard

[0057] The positive cDNA was used as a template for routine PCR amplification to obtain the RSV-N gene fragment (127 bp), which was consistent in size with the target fragment. Figure 1 The size of the band obtained after gel recovery, ligation, transformation, positive clone amplification, and plasmid extraction and verification using M13 primers was about 714 bp, and the positive bacterial sequencing result was consistent with the expected result, indicating that the recombinant plasmid standard was successfully established.

[0058] 2.3 Establishment of qPCR standard curve

[0059] Dilute 1x10 by 10 times 7 ~1x10 2 The copies / µL plasmid was amplified according to the optimized reaction system and parameters. The software automatically generated a standard curve, and the correlation coefficient R of the RSV standard curve was calculated. 2 =0.9922, the standard equation is y = -2.6811x + 38.305, and the amplification efficiency of the target gene is 99.523%. The results show that the RSV real-time PCR method established in this study has good linearity.

[0060] 2.4 CCK-8 Experimental Results

[0061] 2.4.1 Cell viability of *Meconopsis lanceolata* at different dilutions in pure water

[0062] Table 2. Cell viability of *Meconopsis lanceolata* at different dilutions after 6h, 12h, and 24h of incubation in water.

[0063]

[0064] The results showed that during the 6-hour incubation period, the aqueous extract of *Meconopsis lanceolata* had a significant impact on Vero cells. After a 1:128 dilution, the impact on Vero cells gradually decreased, and cell viability returned to normal, with the positive and negative OD values ​​being closest. During the 12-hour incubation period, after a 1:64 dilution, the impact of the aqueous extract on Vero cell viability decreased, and Vero cell viability began to return to normal. During the 24-hour incubation period, the 1:8 dilution had the least impact on Vero cell viability. In conclusion, incubation of *Meconopsis lanceolata* with Vero cells at dilutions below 1:128 for 6 hours has minimal impact on cell viability; incubation at dilutions of 1:64 or lower for 12 hours has minimal impact on Vero cell viability; and incubation at a 1:8 dilution for 24 hours has minimal impact on Vero cell viability.

[0065] 2.4.2 Cell viability of *Meconopsis thornata* at different dilutions in 25% ethanol fraction

[0066] Table 3. Cell viability of *Meconopsis lanceolata* at different dilutions after incubation with 25% ethanol for 6 h, 12 h, and 24 h.

[0067]

[0068] Results show that: in the 6 hours of incubation results, 1:4 dilution of 25% ethanol parts of Meconopsis racemosa have no significant effect on the Vero cell, in the 12 hours of incubation results, 1:128 dilution of 25% ethanol parts of Meconopsis racemosa have less effect on the Vero cell viability. In the 24 hours of incubation results, 25% ethanol parts of Meconopsis racemosa have effect on the Vero cell viability in the dilution ratio of 1:1 to 1:16, but the effect gradually decreases after 1:32 dilution, and the Vero cell viability tends to be stable in the dilution ratio of 1:32 to 1:128. In summary, 25% ethanol parts of Meconopsis racemosa have no significant effect on the Vero cell viability after incubation with the cell for 6 hours at the dilution ratio of 1:4 or less, have less effect on the Vero cell viability after incubation with the cell for 12 hours at the dilution ratio of 1:128 or less, and have less effect on the Vero cell viability after incubation with the cell for 24 hours at the dilution ratio of 1:32 or less.

[0069] 2.4.3 Cell viability of 50% ethanol extracted Meconopsis racemosa at different dilution ratios

[0070] Table 4 Cell viability of 50% ethanol parts of Meconopsis racemosa incubated for 6h, 12h, 24h at different dilution ratios

[0071]

[0072] Results show that: in the 6 hours of incubation results, 1:1 to 1:8 dilution of 50% ethanol Meconopsis racemosa have greater effect on the Vero cell, and the effect gradually decreases after 1:16 dilution, and the cell viability tends to be normal. In the 12 hours of incubation results, 1:32 dilution of 50% ethanol Meconopsis racemosa have no effect on the Vero cell viability. In the 24 hours of incubation results, 1:128 dilution of 50% ethanol Meconopsis racemosa have no effect on the Vero cell viability. In summary, 50% ethanol Meconopsis racemosa have less effect on the Vero cell viability after incubation with the cell for 6 hours at the dilution ratio of 1:16 or less, have less effect on the Vero cell viability after incubation with the cell for 12 hours at the dilution ratio of 1:32 or less, and have no effect on the Vero cell viability after incubation with the cell for 24 hours at the dilution ratio of 1:128 or less.

[0073] 2.4.4 Cell viability of 95% ethanol extracted Meconopsis racemosa at different dilution ratios

[0074] Table 5 Cell viability of 95% ethanol parts of Meconopsis racemosa incubated for 6h, 12h, 24h at different dilution ratios

[0075]

[0076]

[0077] Results showed that in the 6 hours of incubation results, 95% ethanol parts of Meconopsis torquifolia had less impact on the viability of vero cells at a dilution of 1:8. In the 12 hours of incubation results, after dilution of 1:32, 95% ethanol parts of Meconopsis torquifolia gradually reduced the impact on vero cells, and the cell viability tended to be normal. In the 24 hours of incubation results, 95% ethanol parts of Meconopsis torquifolia had an impact on the viability of vero cells at a dilution ratio of 1:1 to 1:16, but after dilution of 1:32, the impact gradually decreased, and at a dilution ratio of 1:32 to 1:128, the viability of vero cells tended to be stable. In summary, 95% ethanol parts of Meconopsis torquifolia had less impact on the viability of vero cells after incubation for 12 hours and 24 hours at a ratio of 1:32 or less, and had no significant impact on the viability of vero cells after incubation for 6 hours at a dilution ratio of 1:8.

[0078] 2.4.5 Cell viability at different dilutions of ribavirin

[0079] Table 6 Cell viability at different dilutions of ribavirin incubated for 6h, 12h, 24h

[0080]

[0081]

[0082] Results showed that in the 6 hours of incubation results, ribavirin had a greater impact on vero cells, and after dilution of 1:32, the impact of ribavirin on vero cells gradually decreased, the cell viability tended to be normal, and the positive OD value was closest to the negative OD value. In the 12 hours of incubation results, after dilution of 1:128, the impact of ribavirin on vero gradually decreased, and the cell viability tended to be normal. In the 24 hours of incubation results, ribavirin had an impact on the viability of vero cells at a dilution ratio of 1:1 to 1:4, but after dilution of 1:8, the impact gradually decreased, and at a dilution ratio of 1:8 to 1:128, the viability of vero cells tended to be stable. In summary, ribavirin had less impact on the viability of vero cells after incubation for 6 hours at a ratio of 1:32 or less, and for 12 hours at a ratio of 1:128 or less, and for 24 hours at a ratio of 1:8 or less.

[0083] 2.5 Inhibition of RSV viral nucleic acid by Meconopsis torquifolia

[0084] The results show that the nucleic acid CT value after incubation with 50% ethanol extract of M. potaninii and RSV virus has significant difference with pure water extract of M. potaninii, 25% ethanol extract of M. potaninii incubated with RSV for 6h, 95% ethanol extract of M. potaninii incubated with RSV for 6h, no drug control (P=0.002, P=0.0011, P=0.008, P<0.0001), and no difference with ribavirin injection incubated with RSV for 6h (P=0.9988)

[0085] Table 7

[0086]

[0087] Note: A: positive control

[0088] B: pure water extract of M. potaninii incubated with RSV for 6h

[0089] C: 25% ethanol extract of M. potaninii incubated with RSV for 6h

[0090] D: 50% ethanol extract of M. potaninii incubated with RSV for 6h

[0091] E: 95% ethanol extract of M. potaninii incubated with RSV for 6h

[0092] F: ribavirin injection incubated with RSV for 6h

[0093] 2.6 Inhibition of RSV virus by M. potaninii

[0094] The results show that M. potaninii has obvious inhibitory effect on RSV virus, and the CT value of no drug control has significant difference with ribavirin incubated with RSV for 24h, ribavirin incubated with RSV for 12h, 50% ethanol extract of traditional Chinese medicine incubated with RSV for 24h, and 50% ethanol extract of traditional Chinese medicine incubated with RSV for 12h (P value is less than 0.0001)

[0095] Table 8

[0096]

[0097] Note: A: no drug control

[0098] B: ribavirin incubated with RSV for 24h

[0099] C: ribavirin incubated with RSV for 12h

[0100] D: 50% ethanol extract of traditional Chinese medicine incubated with RSV for 24h

[0101] E: 50% ethanol extract of traditional Chinese medicine incubated with RSV for 12h

[0102] 3. Summary:

[0103] Finally, through CCK-8 experiment, it was determined that the extract of Meconopsis racemosa had less effect on cells at a dilution of 1:4, and the dilution of 1:4 was selected for subsequent experiments. In the experiment of Meconopsis racemosa on RSV viral nucleic acid inhibition, it was found that the 50% ethanol extract of Meconopsis racemosa had the best inhibitory effect on RSV viral nucleic acid. After incubating the 50% ethanol extract of Meconopsis racemosa with RSV virus at the cellular level, it was found that Meconopsis racemosa had an inhibitory effect on RSV.

Claims

1. The use of Artemisia scoparia extract in the preparation of a drug for resisting respiratory syncytial virus; the Artemisia scoparia extract is 50% ethanol elution fraction of Artemisia scoparia water extract on macroporous resin; before being loaded on the macroporous resin column, the water extract is alcohol precipitated, and the supernatant is loaded on the macroporous resin column again; during the alcohol precipitation, the concentration of ethanol in the water extract reaches 70%.

2. Use according to claim 1, characterized in that, The Artemisia scoparia extract is prepared by the following method: Artemisia scoparia water extract is loaded on a macroporous resin column, and eluted with water, 25% ethanol and 50% ethanol in sequence, and the 50% ethanol eluate is collected to prepare the 50% ethanol elution fraction.

3. Use according to claim 1, characterized in that, The Artemisia scoparia water extract refers to a liquid obtained by heating Artemisia scoparia in water.

4. Use according to claim 1, characterized in that, The drug is a drug for inhibiting respiratory syncytial virus nucleic acid.

5. Use according to claim 1, characterized in that, The drug is a drug for treating diseases caused by respiratory syncytial virus.

6. Use according to claim 5, characterized in that, The diseases refer to lower respiratory tract infection, interstitial pneumonia and bronchiolitis.

7. A pharmaceutical composition against respiratory syncytial virus, characterized by, It comprises Artemisia scoparia extract and ribavirin; the Artemisia scoparia extract is 50% ethanol elution fraction of Artemisia scoparia water extract on macroporous resin; before being loaded on the macroporous resin column, the water extract is alcohol precipitated, and the supernatant is loaded on the macroporous resin column again; during the alcohol precipitation, the concentration of ethanol in the water extract reaches 70%.

8. The use of Artemisia scoparia extract and ribavirin in the preparation of a combined drug for resisting respiratory syncytial virus; the Artemisia scoparia extract is 50% ethanol elution fraction of Artemisia scoparia water extract on macroporous resin; before being loaded on the macroporous resin column, the water extract is alcohol precipitated, and the supernatant is loaded on the macroporous resin column again; during the alcohol precipitation, the concentration of ethanol in the water extract reaches 70%.

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