CN111437293B - Application of Bufonis venenum extract in preparing medicine for treating infectious diseases caused by SARS-CoV-2 virus - Google Patents

Application of Bufonis venenum extract in preparing medicine for treating infectious diseases caused by SARS-CoV-2 virus Download PDF

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CN111437293B
CN111437293B CN202010332639.5A CN202010332639A CN111437293B CN 111437293 B CN111437293 B CN 111437293B CN 202010332639 A CN202010332639 A CN 202010332639A CN 111437293 B CN111437293 B CN 111437293B
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CN111437293A (en
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曹鹏
陈姣
胡星星
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Jiangsu Provincial Insititute of Traditional Chinese Medicine
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    • A61K31/585—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin containing lactone rings, e.g. oxandrolone, bufalin
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Abstract

The invention discloses an application of a toad venom extract in preparing a medicament for treating infectious diseases caused by SARS-CoV-2 virus, which is found for the first time that the toad venom extract has a better inhibition effect on the SARS-CoV-2 which is currently pandemic worldwide, and clinical experiments show that the respiratory function improvement effect of a toad venom injection on a patient suffering from severe COVID-19 disease is remarkable. Clinical experiments and in-vitro antiviral experiments prove that the Bufonis venenum extract has the function of resisting the novel coronavirus SARS-CoV-2, has great significance in preventing, controlling and treating infection caused by the virus, particularly provides an effective candidate medicament for preventing and treating the COVID-19 caused by the SARS-CoV-2 which is currently in pandemic, and also provides a candidate medicament for preventing, controlling and treating the novel virus which possibly bursts in the future.

Description

Application of Bufonis venenum extract in preparing medicine for treating infectious diseases caused by SARS-CoV-2 virus
Technical Field
The invention belongs to the field of biological medicine, relates to a new application of a toad venom and an extract thereof, and in particular relates to an application of the toad venom extract in preparing a medicament for treating infectious diseases caused by SARS-CoV-2 virus.
Background
The host of highly pathogenic viruses is mainly wild animals, and as the ecological systems of humans and wild animals overlap more and more, the potential of new viruses to appear in humans is also increasing. The periodic cycling of viruses as heterogeneous members into and out of human and zoonotic hosts makes the development of specific antiviral therapies and vaccines exceptionally complex. Finding anti-coronavirus drugs with definite curative effects and broad spectrum can not only relieve the urgent need of current SARS-CoV-2 spreading, but also provide candidate drugs for prevention and treatment of new viruses which are likely to outbreak in the future.
The Bufonis venenum is dry secretion secreted by skin gland and ear gland of Bufo siccus or Bufo melanogaster belonging to Bufo siccus family. The toad venom mainly contains bufadienolide, non-cardiotonic sterols, indole derivatives and other chemical components of various types, and has pharmacological activities of cardiotonic, boosting, anesthetizing, analgesic, antibacterial, immunity enhancing, antitumor and the like. The traditional Chinese medicine preparation of the toad venom is widely applied to clinical practice of anti-tumor treatment, and has certain treatment effects on lung cancer, leukemia, liver cancer, gastric cancer, gynecological malignant tumors and the like. The chemical composition of the toad venom is complex and mainly comprises bufadienolide, 20, 21-epoxybufadienolide, cardiotonic sterene bufalin, indole derivatives, bufonin, sterols, peptides, amino acids and the like. Bufogenin, cinobufagin are the 3 major active monomers of bufonis.
Disclosure of Invention
The invention aims to solve the problem of lack of effective medicines for treating COVID-19 in clinic and provide medicines for safely and effectively inhibiting SARS-CoV-2 virus.
The technical scheme is as follows: the invention discovers that the toad venom extract can be used as an inhibitor of SARS-CoV-2 virus for the first time, inhibits the virus from infecting host cells, and verifies the treatment effect of the toad venom extract on COVID-19 through clinical tests. Thus, the application of the toad venom extract in preparing a medicament for treating infectious diseases caused by SARS-CoV-2 virus is proposed.
Specifically, the infectious diseases caused by SARS-CoV-2 virus include, but are not limited to, diseases such as COVID-19, upper respiratory tract infection or gastroenteritis.
Specifically, the toad venom extract is prepared by the following steps:
(1) Cleaning Bufonis venenum, soaking with 8-10 times of water for injection for 0.5-24 hr, grinding, adding ethanol to make ethanol content in the extractive solution reach 50-90 vt%, and standing at 10-30deg.C for 1-6 days;
(2) Filtering the extraction solution obtained in the step (1), concentrating the filtrate until no alcohol smell exists, adding water for injection, stirring uniformly, refrigerating at a low temperature of between 0 and 5 ℃ and standing for 30 to 60 hours, filtering and separating out the filtrate, and concentrating to obtain the extract.
Specifically, the effective components in the Bufonis venenum extract at least comprise bufalin (molecular formula C) 24 H 34 O 4 ) Cinobufagin (molecular)C (C) 26 H 34 O 6 ) Ester bufogenin (Resibufogin, formula C) 24 H 32 O 4 ) Any one of the monomers or a combination of two or more monomers.
Preferably, the effective components in the toad venom extract are a composition of bufogenin, cinobufagin and ester bufogenin, and the total concentration of the bufogenin, the cinobufagin and the ester bufogenin in the toad venom extract is 10-53 mug/mL.
Most preferably, the mass ratio of the effective components bufogenin, cinobufagin and ester bufogenin in the bufogenin extract is (4.5-6) to (2.5-4).
The invention adopts dried toad venom to extract to obtain toad venom extract, and then can be matched with pharmaceutically acceptable carriers to prepare liquid injection, powder injection, tablets, capsules, soft capsules, powder, pills, oral liquid, ointment, nano preparation, microsphere preparation, gel preparation, granules or dressing and other dosage forms.
The beneficial effects are that:
(1) The invention discovers that the toad venom extract has better inhibition effect on SARS-CoV-2 which is currently pandemic worldwide for the first time, clinical experiments show that the toad venom injection has obvious effect of improving the respiratory function of patients with severe COVID-19, and clinical experience of using lopinavir and ritonavir is proved to have insignificant effect on patients with severe COVID-19;
(2) Clinical experiments and in-vitro antiviral experiments prove that the Bufonis venenum extract has the function of resisting the novel coronavirus SARS-CoV-2, has great significance in preventing, controlling and treating infection caused by the virus, particularly provides an effective candidate medicament for preventing and treating the COVID-19 caused by the SARS-CoV-2 which is currently in pandemic, and also provides a candidate medicament for preventing, controlling and treating the novel virus which possibly bursts in the future.
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The foregoing and/or other advantages of the invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings and detailed description.
FIG. 1 is a fingerprint of a Bufonis venenum injection prepared by the invention; wherein, the RT57.7 peak is bufalin; the RT67.9 peak is cinobufagin; the RT69.5 peak is the ester bufogenin.
Detailed Description
The invention will be better understood from the following examples.
Example 1: preparation of toad venom injection
Cleaning 2g of Bufonis venenum, soaking with 10 times of water for injection, grinding, adding ethanol to make ethanol content reach 75%, stirring, standing at 20deg.C for 5 days, refrigerating, filtering, concentrating the filtrate, recovering ethanol until no ethanol smell, adding 80mL water for injection, stirring, standing at 2deg.C for 40h, filtering, adding 8g of sodium chloride into the filtrate, stirring to dissolve, regulating pH to 4.5-6.5 with sodium hydroxide solution, adding water for injection to 1000mL, stirring, filtering, bottling, and sterilizing to obtain Bufonis venenum injection.
Fingerprint spectrum: the fingerprint spectrum of the toad venom injection is measured by adopting a high performance liquid chromatography, and chromatographic conditions and system applicability tests are as follows: agilent Zorbax SB C18 (column length 250, inner diameter 4.6mm, particle size 5 μm) was used as a chromatographic column; acetonitrile is taken as a mobile phase A, and 0.1 percent ammonium acetate solution is taken as a mobile phase B for gradient elution; the detection wavelength is 296nm; the column temperature was 30℃and the flow rate was 1.0mL per minute. The number of theoretical plates is not less than 3000 based on bufogenin. The fingerprint of the obtained Bufonis venenum injection is shown in figure 1. Wherein, the RT57.7 peak is bufalin; the RT67.9 peak is cinobufagin; the RT69.5 peak is the ester bufogenin.
And (3) content measurement: taking bufogenin, cinobufagin and ester bufogenin reference substances, preparing into solutions containing bufogenin, cinobufagin and ester bufogenin 1.6 μg respectively per 1mL with 50% acetonitrile solution as reference, measuring Bufonis venenum injection content by high performance liquid chromatography, and preparing bufogenin-containing solution (C) per 1mL 24 H 34 O 4 ) About 1.16 μg, cinobufagin (C) 26 H 34 O 6 ) About 0.92g of ester bufogenin (C 24 H 32 O 4 ) About 0.92g, the total amount of the three monomers being 3.0. Mu.g.
Example 2: preparation of toad venom injection
Cleaning 2g of Bufonis venenum, soaking with 8 times of water for injection, grinding, adding ethanol to make the ethanol content reach 50%, stirring, standing at 10deg.C for 1 day, refrigerating, filtering, concentrating the filtrate, recovering ethanol until no ethanol smell, adding 80mL of water for injection, stirring, standing at 5deg.C for 30h, filtering, adding 8g of sodium chloride into the filtrate, stirring to dissolve, regulating pH to 4.5-6.5 with sodium hydroxide solution, adding water for injection to 1000mL, stirring, filtering, bottling, and sterilizing to obtain Bufonis venenum injection.
Fingerprint spectrum: the fingerprint spectrum of the toad venom injection is measured by adopting a high performance liquid chromatography, and chromatographic conditions and system applicability tests are as follows: agilent Zorbax SB C18 (column length 250, inner diameter 4.6mm, particle size 5 μm) was used as a chromatographic column; acetonitrile is taken as a mobile phase A, and 0.1 percent ammonium acetate solution is taken as a mobile phase B for gradient elution; the detection wavelength is 296nm; the column temperature was 30℃and the flow rate was 1.0mL per minute. The number of theoretical plates is not less than 3000 based on bufogenin. Fingerprint of Bufonis venenum injection is obtained.
And (3) content measurement: taking bufogenin, cinobufagin and ester bufogenin reference substances, preparing into solutions containing bufogenin, cinobufagin and ester bufogenin 1.6 μg respectively per 1mL with 50% acetonitrile solution as reference, measuring Bufonis venenum injection content by high performance liquid chromatography, and preparing bufogenin-containing solution (C) per 1mL 24 H 34 O 4 ) About 0.45 μg, cinobufagin (C) 26 H 34 O 6 ) About 0.3 μg of ester bufogenin (C 24 H 32 O 4 ) About 0.25. Mu.g, the total of the three monomers being 1.0. Mu.g.
Example 3: preparation of toad venom injection
Cleaning 2g of Bufonis venenum, soaking with 10 times of water for injection, grinding, adding ethanol to make the ethanol content reach 90%, stirring, standing at 30deg.C for 6 days, refrigerating, filtering, concentrating the filtrate to recover ethanol until no ethanol smell, adding 80mL water for injection, stirring, standing at 0deg.C for 60h, filtering, adding 8g of sodium chloride into the filtrate, stirring to dissolve, adjusting pH to 4.5-6.5 with sodium hydroxide solution, adding water for injection to 1000mL, stirring, filtering, bottling, and sterilizing to obtain Bufonis venenum injection.
Fingerprint spectrum: the fingerprint spectrum of the toad venom injection is measured by adopting a high performance liquid chromatography, and chromatographic conditions and system applicability tests are as follows: agilent Zorbax SB C18 (column length 250, inner diameter 4.6mm, particle size 5 μm) was used as a chromatographic column; acetonitrile is taken as a mobile phase A, and 0.1 percent ammonium acetate solution is taken as a mobile phase B for gradient elution; the detection wavelength is 296nm; the column temperature was 30℃and the flow rate was 1.0mL per minute. The number of theoretical plates is not less than 3000 based on bufogenin. Obtaining fingerprint diagram spectrogram of Bufonis venenum injection.
And (3) content measurement: taking bufogenin, cinobufagin and ester bufogenin reference substances, preparing into solutions containing bufogenin, cinobufagin and ester bufogenin 1.6 μg respectively per 1mL with 50% acetonitrile solution as reference, measuring Bufonis venenum injection content by high performance liquid chromatography, and preparing bufogenin-containing solution (C) per 1mL 24 H 34 O 4 ) About 2.5 μg, cinobufagin (C) 26 H 34 O 6 ) About 1.3 μg of ester bufogenin (C 24 H 32 O 4 ) About 1.2. Mu.g, the total of the three monomers being 5.0. Mu.g.
Example 4: clinical test effect of Bufonis venenum injection on SARS-CoV-2
By adopting a prospective random control study method, 50 cases of heavy/critical patients, which are in line with the clinical diagnosis of COVID-19 or the laboratory diagnosis, of the medical subjects of critical illness in the medical subjects of Xia Oudi people in Wuhan City, hubei province, 2 months 05 days 2020 to 3 months 05 days, are selected, and the patients are divided into a treatment group (standard treatment+Bufonis venenum injection) and a control group (standard treatment only) according to a ratio of 1:1 by adopting a random digital method. Both groups received indifferent standard treatment for covd-19, including peramivir, an empirical antiviral treatment for interferon alpha; empirical antibiotics cover possible pathogenic bacteria; nutritional support; normative hormone treatment; acid inhibiting and stomach protecting etc. The treatment group was given a Bufonis venenum injection (Jiangsu Pu Jin Yaoye Co., ltd., 10 ml/min., product lot number: 191002; approval document number: national drug standard character Z32020694) 20ml, 0.9% physiological saline 250ml was added for intravenous infusion at a rate of 125ml/h, and the control group was given a physiological saline 250ml 0.9% for intravenous infusion at a rate of 125ml/h for 7 days.
And (3) observing the indexes:
baseline: gender, age, body Mass Index (BMI), time of onset to visit, pulse oxygen saturation, underlying disease, vital signs at time of group entry (worst day Index), initial respiratory support, drug use, and the like.
The main observation indexes are as follows: PO for treatment of day 1, 7 2 /FiO 2 And ROX index.
Other observations: day 1, 7 white blood cell count, lymphocyte count, respiratory support decreases to guaiac step time. Because the noninvasive mechanical ventilation, high-flow oxygen therapy, mask oxygen inhalation and nasal catheter oxygen inhalation support the force of the force to the patient gradually weakens, the respiratory support is reduced to a healing step time is defined as: after the group is put into the group, the high-grade support is switched to the low-grade support, or the oxygen absorption concentration is reduced by 50% than the previous one. The patient maintains pulse oxygen saturation above 94% after the support mode conversion, and the respiratory rate is not increased by more than 20% as the conversion is successful.
Safety index: (1) liver function index (AST, ALT, TB); (2) renal function index (Cr); (3) systemic or local rashes cannot be explained by other causes; (4) digestive tract symptoms (nausea, vomiting, abdominal pain, diarrhea); (5) new-onset arrhythmia.
Statistical analysis: data processing was performed using SPSS 22.0 software, and the normal distribution metrology data was expressed as mean.+ -. Standard deviation (_x_s), with independent sample t-test for group comparisons. Count data is expressed as a rate (%), and comparisons between groups are tested using χ2. Multiple-factor regression analysis is carried out firstly, single-factor linear regression analysis is carried out, each index with statistical significance on regression coefficients is screened out, and then all the screened indexes are incorporated into the system for multi-factor linear regression analysis.
Results:
general conditions: the study was included in a total of 50 patients, 25 in each of the treatment and control groups. 3 patients in the treatment group who are not completed in the treatment course and transferred to the hospital are 1 patient without checking indexes on time; 6 patients in the control group, whose treatment course was not completed, were removed. There were 40 patients who were eventually included in the statistics, 21 of the treatment groups and 19 of the control groups. The differences in sex, age, BMI, time of onset to visit, basal disease, basal vital signs in the group, etc. were not statistically significant in the two groups of patients, as shown in table 1.
Table 1 comparison of general data for two groups of patients
And (3) observing the indexes: initial oxygenation index PO for two groups of patients 2 /FiO 2 The differences were statistically not significant, and PO was applied to 20 (95.2%) patients in treatment group during treatment 2 /FiO 2 Improved PO for control group 13 (68.4%) patients 2 /FiO 2 Improved, there was a statistical difference between groups (p=0.026); treatment group oxygenation index PO at day 7 2 /FiO 2 Significantly higher than the control, the differences were statistically significant (P < 0.001). The same trend was shown by the ROX index, which was 14.01±3.99 in the treatment group on day 7, and a clear increase was observed in the treatment group 9.64±5.54 compared with the control group, and the difference was statistically significant (p=0.006). The difference between the peripheral blood leucocyte level treatment of the two groups of patients before and after the treatment has no statistical significance; however lymphocyte levels increased on day 7 in the treatment group compared to pre-treatment, but there was no statistical difference in the differences (P>0.05 But the difference was statistically significant (p=0.018) compared to control group day 7. The treatment group had a step down time of 5 days, which was 1 day shorter than the control group, and the difference was statistically different (p=0.016), as shown in table 2.
Table 2 comparison of two sets of intervention situations
* Statistical differences between Day 1 and Day 7 in the group, P <0.05
The statistical difference among groups is that P is less than 0.05
And + -represents the average + -SD
Multifactor regression analysis: at day 7 oxygenation index PO 2 /FiO 2 As a dependent variable, the single factor regression analysis is carried out item by item on the basic condition of the patient, various indexes during the group entering and the therapeutic intervention measures, so that the following is found: the four index regression coefficients of the oxygenation index on day 1, the peripheral blood lymphocyte count on day 1, the initial respiratory support mode and the toad venom injection have statistical significance, and then the multi-factor regression analysis is carried out again to find that the three index regression coefficients of the toad venom injection intervention, the oxygenation index on day 1 and the initial respiratory support mode have statistical significance (P < 0.05), the peripheral blood lymphocyte count regression coefficient on day 1 has no statistical significance (P > 0.05), wherein only the toad venom injection intervention has statistical significance (P < 0.001) in the treatment intervention measures and has the largest contribution to the regression model (beta=0.486), as shown in table 3.
TABLE 3 multifactor regression analysis
Safety index: the treatment group did not find common adverse reactions such as rash, vomiting, diarrhea, etc. during the treatment period. The differences of the safety indexes of the two groups of patients before and after treatment have no statistical significance (P is more than 0.05), and the synchronous comparison differences among the groups have no statistical significance (P is more than 0.05) as shown in the table 4.
Table 4 comparison of patient safety metrics for two groups
Example 5: in vitro anti-SARS-CoV-2 virus experiment of Bufonis venenum extract
The method comprises the following steps: vero E6 cells were grown at 5X 10 4 Individual cells/well were inoculated into 96-well plates and cultured in DMEM medium supplemented with 10% fbs for 16 hours under 5% co 2 37 ℃. The medium was then decanted, the cells were washed with PBS buffer, and a concentration of drug was added to 50. Mu.L/well of cell fixative and incubated with the cells for 1h. As a control, 50. Mu.L/Kong Peiyang base was used. The dosed or non-dosed cells were then infected with SARS-CoV-2 virus for 2 hours with a multiplicity of infection MOI of 0.05.The virus-drug mixture was decanted off and fresh drug-containing medium was used at 5% CO 2 Cells were further cultured at 37℃for 48 hours. The culture supernatant was collected, left at 56℃for 30min, and viral RNA was extracted, and the effect of the drug on viral RNA replication was examined by qRT-PCR. Extracting viral RNA with HP RNA extraction kit (Roche), performing PCR reaction with viral nucleic acid detection kit according to the instruction, and calculating 2 by CT value displayed by PCR instrument -ΔCT The value, namely the relative viral replication rate of the drug group and the control group, and the calculation formula of the viral replication inhibition rate is as follows: (1-2 -ΔCT ) 100% and the results are shown in Table 5. Wherein, the toad venom injection is prepared in the example 1, and diluted to a final concentration of 20ng/mL; bufogenin, cinobufagin and ester bufogenin are all monomer pure products, and are prepared into mother liquor with the concentration of 20 mug/mL, and then diluted into the required final concentration to measure the activity of SARS-CoV-2.
TABLE 5 inhibition of SARS-CoV-2 RNA replication by different drugs
As is clear from Table 5, the inhibition ratio of the bufonin injection to the RNA replication of SARS-CoV-2 virus at 20ng/mL was 73.4%, and the three main monomers bufogenin, cinobufagin and ester bufogenin all had the inhibition effect to the RNA replication of SARS-CoV-2 virus, among them, the inhibition effect of bufogenin monomer was the best. When the three monomers are combined or two by two, the three monomers of bufalin, cinobufagin and ester bufogenin are found to have the best inhibition effect on viral RNA replication, but the inhibition effect is lower than that of a bufalin injection, which shows that other components except bufalin, cinobufagin and ester bufagin in the bufalin injection can have inhibition effect on SARS-CoV-2 virus.
The invention provides a method and a method for applying a toad venom extract in preparing a medicine for treating infectious diseases caused by SARS-CoV-2 virus, and the method and the way for realizing the technical scheme are numerous, the above is only a preferred embodiment of the invention, and it should be pointed out that a plurality of improvements and modifications can be made to a person of ordinary skill in the art without departing from the principle of the invention, and the improvements and the modifications are also considered as the protection scope of the invention. The components not explicitly described in this embodiment can be implemented by using the prior art.

Claims (4)

1. Application of Bufonis venenum extract or Bufonis venenum injection in preparing medicine for treating infectious diseases caused by SARS-CoV-2 virus; the toad venom extract is characterized in that the effective components in the toad venom extract are any one of the following components:
bufalin, or
Bufogenin, cinobufagin, or ester bufogenin, or a combination thereof
A combination of bufalin and cinobufagin, or
A combination of bufalin and a ester bufogenin;
the infectious diseases caused by SARS-CoV-2 virus include COVID-19, upper respiratory tract infection or gastroenteritis.
2. The use according to claim 1, wherein the venenum bufonis extract is prepared by:
(1) Cleaning a dried toad venom product, soaking the dried toad venom product in 8-10 times of injection water for 0.5-24 hours, grinding, adding ethanol to enable the ethanol content in an extracting solution to reach 50-90 vt%, and standing and extracting at 10-30 ℃ for 1-6 days;
(2) Filtering the extraction solution obtained in the step (1), concentrating the filtrate until no alcohol smell exists, adding water for injection, stirring uniformly, refrigerating at a low temperature of 0-5 ℃ and standing for 30-60 hours, filtering and separating out the filtrate, and concentrating to obtain the product.
3. The use according to claim 1, wherein the effective component in the bufonis extract is a composition of bufogenin, cinobufagin and ester bufogenin, and the total concentration of the three components in the bufonis extract is 10-53 μg/mL.
4. The use according to claim 1, wherein the mass ratio of the bufogenin, cinobufagin and bufogenin is (4.5-6): 2.5-4.
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