CN114848663A - Application of natural compound Solamargine and anti-HBV (hepatitis B virus) pharmaceutical composition - Google Patents
Application of natural compound Solamargine and anti-HBV (hepatitis B virus) pharmaceutical composition Download PDFInfo
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- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
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Abstract
An application of a natural compound Solamargine in preparing antiviral drugs relates to the field of virology, and the natural compound Solamargine can be effectively applied to the preparation of the antiviral drugs to achieve a better antiviral effect. In addition, the embodiment of the invention also provides an anti-HBV virus pharmaceutical composition, which comprises a natural compound Solamargine as an active ingredient and pharmaceutically acceptable auxiliary materials. The pharmaceutical composition has better anti-HBV activity and better curative effect on HBV infection.
Description
Technical Field
The invention relates to the field of virology, in particular to application of a natural compound Solamargine and an anti-HBV (hepatitis B virus) pharmaceutical composition.
Background
Hepatitis B Virus (HBV) is a member of the hepatitis b virus family, and consists of a unique DNA virus that initiates reverse transcription during replication. About 3.5 to 4 million people are chronically infected with HBV and HBV-related liver diseases such as cirrhosis, liver failure and hepatocellular carcinoma, resulting in 100 million deaths worldwide each year.
In the replication process of hepatitis b virus, viral DNA enters the nucleus of a host cell, and under the action of DNA polymerase, the nicks of both strands are filled up to form a supercoiled, covalently closed, circular DNA molecule (cccDNA). The cccDNA is an original template for replicating the pregenomic RNA of the hepatitis B virus, although the content of the cccDNA is low, only about 5-50 copies exist in each hepatocyte, the cccDNA has very important significance for replicating the hepatitis B virus and establishing the infection state, and the virus carrying state of a hepatitis B patient can be completely eliminated only by eliminating the cccDNA in a cell nucleus, so that the cccDNA is the target of antiviral treatment.
Currently, anti-HBV drugs used clinically mainly include two major classes of Interference (IFN) and Nucleoside (NAs) drugs, which act by regulating immunity and interfering with the replication process of hepatitis b virus, respectively. However, neither IFN nor NAs act directly on intrahepatic cccDNA, and thus complete cure of hepatitis b is almost impossible. In addition, the problems of drug resistance caused by NAs and the side effects of IFN have been faced by people. Therefore, the development of new anti-HBV viral drugs remains crucial for the treatment of clinical patients.
Disclosure of Invention
The invention aims to provide application of a natural compound Solamargine, which can be effectively applied to preparation of antiviral drugs to achieve a better antiviral effect.
Another objective of the present invention is to provide a pharmaceutical composition against HBV virus, which has better anti-HBV activity and better curative effect on HBV infection.
The embodiment of the invention is realized by the following steps:
an application of a natural compound Solamargine in preparing antiviral drugs.
A pharmaceutical composition for resisting HBV virus comprises a natural compound Solamargine as an active ingredient and pharmaceutically acceptable auxiliary materials.
The embodiment of the invention has the beneficial effects that:
the embodiment of the invention provides application of a natural compound Solamargine in preparation of an antiviral drug. It can be effectively applied to the preparation of antiviral drugs to achieve better antiviral effect. In addition, the embodiment of the invention also provides an anti-HBV virus pharmaceutical composition, which comprises a natural compound Solamargine as an active ingredient and pharmaceutically acceptable auxiliary materials. The pharmaceutical composition has better anti-HBV activity and better curative effect on HBV infection.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a graph showing the results of an assay of the inhibitory effect of various pharmaceutical compounds provided in test example 1 of the present invention on the expression level of HBV pgRNA;
FIG. 2 is a graph showing the results of the test of inhibitory effect of solargine, a natural compound, on the expression level of HBV DNA in cell culture supernatant, which is provided in test example 2 of the present invention;
FIG. 3 shows the results of northern blot analysis in test example 3 of the present invention;
FIG. 4 shows the results of a western blot assay provided in test example 4 of the present invention;
FIG. 5 is a schematic comparison of pGL4.74-HBpg-Ce and pGL4.74-HBenIIcp-Ce provided in Experimental example 5 of the present invention;
FIG. 6 is a schematic comparison of pGL4.74-HBenIIcp-Ce-del1 to pGL4.74-HBenIIcp-Ce-del10 provided in Experimental example 5 of the present invention;
FIG. 7 is a graph comparing the inhibitory effect of Solamargine, a natural compound provided in Experimental example 5 of the present invention, on pGL4.74-HBpg-Ce and pGL4.74-HBeniicp-Ce;
FIG. 8 is a graph comparing the inhibitory effect of Solamargine, a natural compound provided in Experimental example 5 of the present invention, on pGL4.74-HBpg-Ce and pGL4.74-HBeniicp-Ce.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The application of solaamargine, a natural compound, and the anti-HBV pharmaceutical composition according to the embodiments of the present invention are specifically described below.
The embodiment of the invention provides application of a natural compound Solamargine in preparation of an antiviral drug.
Preferably, the virus is a DNA virus. Especially HBV virus among hepadnaviruses.
Solaargine is named Solamargine in Chinese, and has a structural formula shown in the specification,
solargine is a derivative of the steroid Solasodine derived from plants of the genus solanum, and exhibits anticancer activity in a variety of cancers. In the invention, the inventor finds that the natural compound solargine has certain antiviral activity, particularly shows a better inhibition effect on HBV virus, and can be applied to preparing antiviral drugs.
Further, the HBV viruses mentioned in the present invention include not only basic HBV viruses but also lamivudine-resistant HBV variants. Well solves the problem of drug resistance of the existing anti-HBV drugs.
Further, the inventors have found that the natural compound solargine has an antiviral effect by inhibiting transcription of covalently closed circular DNA in liver.
In addition, the natural compound solagargine also has antiviral effect by inhibiting the expression of pregenomic RNA of the HBV virus.
Through intensive research, the inventor further discovers that the natural compound Solamargine can play an antiviral effect by inhibiting the transcription of the core promoter of the HBV virus through targeting. The nucleic acid sequence of the core promoter is SEQ ID NO. 1.
In addition, the embodiment of the invention also provides an anti-HBV virus pharmaceutical composition which is characterized by comprising a natural compound Solamargine serving as an active ingredient and pharmaceutically acceptable auxiliary materials. The auxiliary materials can be solvents, propellants, solubilizers, solubilizing agents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integration agents, permeation promoters, pH regulators, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickeners, encapsulation agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants, and the like, depending on the dosage form of the medicament.
The features and properties of the present invention are described in further detail below with reference to examples.
Test example 1
This test example used various pharmaceutical compounds including the natural compound solagargine to test the inhibitory effect of each pharmaceutical compound on the expression level of HBV pgRNA. The information of the drug compounds involved is shown in table 1.
TABLE 1 pharmaceutical Compound information
The sample, reagent and instrument equipment used in this test example were configured as follows:
sample, reagent: the concentration of each drug compound solution was 5 uM; hepatoma cell lines HuH7 cells and HepG2 cells were purchased from a cell bank of Chinese academy of sciences; 10% fetal bovine serum, pancreatin purchased from Thermo Hyclone; the reverse transcription kit was purchased from vazyme; fluorescence quantification kits were purchased from MCE.
The instrument equipment comprises: 5% CO 2 Incubators were purchased from Bio-Red, USA; nanodrop one ultramicro uv spectrophotometer available from Thermo corporation; ABI StepOnePlus real-time fluorescent quantitative PCR instrument was purchased from Thermo Fisher.
The method comprises the following specific operation steps:
first, HBV stably expressing cell line is cultured (the specific culture steps can be referred toSun, Suofeng et al. “Establishment of stable cell lines in which the HBV genome replicates episomally for evaluation of antivirals.” Archives of medical science : AMS vol. 16,2 407-413. 20 Nov. 2018, doi:10.5114/aoms.2018.79712) And paving a 24-well plate when the cells grow full, replacing the solution with a medicinal compound solution after 1 day, extracting RNA after 3 days, measuring the concentration of the RNA by using a spectrophotometer, taking the same amount of RNA according to the concentration of the RNA, performing reverse transcription to obtain cDNA, and performing reverse transcription according to the kit instruction. Then, using cDNA as a template, SYBR Green qPCR Master Mix (2X) was used to prepare a reaction system of 10. mu.l SYBR Master Mix (2X), 0.4. mu.l ROX, 0.4. mu.l each of upstream and downstream primers, 2. mu.l cDNA, and 6.8. mu.l ddH2O 6.8. Beta-actin is used as an internal reference. The pg RNA level was detected using a fluorescent quantitative PCR instrument. The test results for each pharmaceutical compound are shown in figure 1.
As can be seen from FIG. 1, each of the pharmaceutical compounds in Table 1 showed a certain inhibitory effect on the expression level of HBV pgRNA, compared with the blank (CTRL in the figure), wherein the inhibitory effect of the natural compound Solamargine (F2) was the most significant.
Test example 2
In the test example, a natural compound solagargine is adopted, and the inhibition effect of the solagargine on the expression level of HBV DNA in cell culture supernatant is detected by using a real-time fluorescent quantitative PCR method.
The sample, reagent and instrument configurations used in this test example were as follows:
sample, reagent: the solution of the natural compound solaamargine is 5 uM; lipo3000 transfection reagent was purchased from Thermo corporation, usa; a hepatitis B virus nucleic acid quantitative detection kit (PCR-fluorescent probe method) is purchased from Wuhanbaitai genetic engineering Co., Ltd.
The instrument equipment comprises: 5% CO 2 Incubators were purchased from Bio-Red, USA; ABI StepOnePlus real-time fluorescent quantitative PCR instrument was purchased from Thermo Fisher.
The method comprises the following specific operation steps:
collecting cell, collecting supernatant 300ul, centrifuging at 13000rpm for 5min, collecting supernatant 200ul, and testing in new EP tube. And taking out the negative quality control product, the HBV positive quality control product and the HBV working standard product in the reagent kit, dissolving at room temperature, and centrifuging at 5000rpm for several seconds. Preparing a reaction system (44.25 ul of PCR reaction liquid and 0.75ul of PCR enzyme system/hole), fully and uniformly mixing, centrifuging at 5000rpm for several seconds, respectively adding 45ul of reaction liquid into a PCR reaction tube, and respectively adding 5ul of processed samples (to-be-detected samples, negative quality control products, HBV positive quality control products and HBV working standard products). At 50 deg.C for 2 min; 3min at 95 ℃; reaction conditions are set for 40 cycles of reaction at 95 ℃ for 10s and 60 ℃ for 1 min. The test results are shown in FIG. 2.
As can be seen from FIG. 2, Solamargine was further verified and found to be effective in inhibiting the expression level of viral particles in the supernatant of HBV-infected cells by real-time fluorescent quantitative PCR.
Test example 3
In this test example, a natural compound solagargine was used, and its inhibitory effect on pgRNA was examined by northern blot.
The sample, reagent and instrument configurations used in this test example were as follows:
sample, reagent: the solution of the natural compound solaamargine is 5 uM; CDP-Star detection reagents are available from Japan GE Healthcare.
The instrument equipment comprises: 5% CO 2 Incubators were purchased from Bio-Red, USA; nanodrop one ultramicro uv spectrophotometer available from Thermo corporation; ChemiDocXRS + chemiluminescent gel imager was purchased from BIO-RAD, USA.
The method comprises the following specific operation steps:
after cell culture, HBV plasmid was transfected, and total RNA was extracted using TRI reagent. After treatment with DNase I and RNase inhibitor, at 1X 3- (N-morpholino) propanesulfonic acid (MOPS) buffer (20 mM MOPS, 5 mM sodium acetate and 2 mM EDTA). Samples were transferred to nylon membranes for 16 hours with 20 XSSC transfer buffer and then cross-linked to the membrane by UV light (120 mJ/cm 2). After washing, the blotting membrane was dried at room temperature. The blot was prehybridized at 68 ℃ with DIG Easy Hybridization buffer (Roche Diagnostics) and hybridized overnight at 68 ℃ with an appropriate DIG-labeled RNA probe labeled with DIG-11-UTP using the DIG Northern Starter Kit (Roche Diagnostics). The DIG labeled probe on the blot was detected using CDP-Star detection reagent. The test results are shown in FIG. 3.
As can be seen from FIG. 3, the expression of HBV pgRNA was significantly inhibited and the color development was weaker after the addition of the natural compound Solamargine compared to the blank. Thus, northern blot further demonstrated the inhibitory effect of solargine on pgRNA.
Test example 4
In this test example, a natural compound solagargine was used, and its inhibitory action on the expression of HBV core protein was detected using western blot.
The sample, reagent and instrument configurations used in this test example were as follows:
sample, reagent: the solution of the natural compound solaamargine is 5 uM; BCA kit was purchased from Thermo Fisher Scientific, USA; lipo3000 transfection reagent was purchased from Thermo corporation, usa; an SDS-PAGE gel preparation kit and an ECL chemiluminescence kit are purchased from Biyuntian corporation in China; goat anti-human HBsAg and rabbit anti-human HBsAg capsid protein antibody HBcAg were purchased from Bioss, mouse anti-human GAPDH antibody was purchased from ABClonal, and the corresponding secondary antibodies were purchased from Sericebio and Proteintech.
The instrument equipment comprises: microplate readers were purchased from USA BioTek Instruments; ChemiDocXRS + chemiluminescent gel imager was purchased from BIO-RAD, USA.
The method comprises the following specific operation steps:
the cells were lysed with RIPA lysate + PMSF (100: 1), collected in a 1.5ml EP tube, and centrifuged at 15000rpm for 20min at 4 ℃. Taking supernatant, determining protein concentration by adopting a BCA method, heating and denaturing the protein, performing gel electrophoresis on the protein through SDS-PAGE, transferring the protein to a PVDF membrane after electrophoresis, then sealing the protein for 1h by TBST containing 5% skimmed milk powder, incubating the primary antibody overnight, washing the membrane by the TBST, incubating the secondary antibody for 90min at room temperature, washing the membrane by the TBST, and finally performing exposure imaging by using a BIO-RAD ChemiDocXRS + chemiluminescence gel imaging instrument. The test results are shown in FIG. 4.
As can be seen from FIG. 4, the expression of HBV core protein can be inhibited by the natural compound Solamargine through strong and weak color development (right panel), but the surface protein is not significantly affected (left panel).
Test example 5
This experimental example was conducted to investigate the effect target of the natural compound solagargine.
The sample, reagent and instrument configurations used in this test example were as follows:
sample, reagent: a plasmid containing 1.24-fold HBV genome of HBV genotype pUC-HB-Ce is a gift from doctor Mizokami (Japan national center for Global health and medicine). pUC-HB-Ce contains 1.24-fold HBV genome from the consensus sequence of HBV genotype Ce, was artificially synthesized by Eurofins Genomics (Ebersberg, Germany) at each position according to the HBV genotype Ce clones (AB 014381, AB205124, AB033551, AB198081, AY596108, AB198080, AB222714 and AY 066028); the solaargine natural compound solution is 5 um; lipo3000 transfection reagent was purchased from Thermo corporation, usa; renilla luciferase assay kit was purchased from Promega, USA.
The instrument equipment comprises: 5% CO 2 Incubators were purchased from Bio-Red, USA; microplate reader purchased from USA BioTek Instruments; ABI StepOnePlus real-time fluorescent quantitative PCR instrument was purchased from Thermo Fisher.
The method comprises the following specific operation steps:
by searching the HBV sequence database, DNA fragments from HBV core promoters of HBV genotypes B and C were designed according to the most common nucleotides in HBV genotypes Bj and Ce, respectively, and then synthesized by Eurofins Genomics. The ENII/BCP fluorescent reporter vector (pGL4.74-HBenIIcp-Ce nt 1627-1817) is constructed by cloning to the upstream of luciferase gene in pGL4.74 fluorescent reporter vector through PCR amplification on the basis of HBV pre-genome core promoter fluorescent reporter vector (pGL4.74-HBpg-Ce, nt 900-1817) (as shown in FIG. 5). In the same way, a series of gene deletion mutants pGL4.74-HBenIIcp-Ce-del1 to pGL4.74-HBenIIcp-Ce-del10 (shown in FIG. 6) based on pGL4.74-HBenIIcp-Ce fluorescent reporter vectors are constructed.
After the cells are sampled in a 24-well plate, the cells are lysed, 120 muL of Lysis solution is added into the cell Lysis solution, which is 1 XLysis Buffer (the component A is diluted by 4:1 by sterile water), and then the culture plate is placed on a micro-oscillator to shake for 15 min at room temperature, so that the cells are fully lysed. The lysate was collected in an EP tube and centrifuged at 13000rpm for 5 min. After centrifugation the supernatant was transferred to a new EP tube. And diluting the C component (1 mu L C component: 100 mu L B component) into renilla luciferase working solution by the B component. And adding the cell lysate into the measuring tube according to the volume of 20 muL, adding 100 muL of Renilla luciferase working solution, and detecting the fluorescence expression amount after fully and uniformly mixing. The test results are shown in fig. 7 and 8.
In FIG. 7, the expression of the white promoter (pGL4.74-HBpg-Ce), ENII/BCP (pGL4.74-HBeniicp-Ce) is shown compared without and with the addition of the natural compound Solamargine, respectively. As can be seen in FIG. 7, similar inhibitory effects were exhibited on pGL4.74-HBpg-Ce and pGL4.74-HBeniicp-Ce after adding the natural compound Solamargine, indicating that the ENII/BCP fragment is the core promoter with the nucleic acid sequence of SEQ ID NO: 1.
Furthermore, a comparison of the expression of pGL4.74-HBenIIcp-Ce-del1 to pGL4.74-HBenIIcp-Ce-del10 without and with the addition of the natural compound Solamargine is given in FIG. 8, respectively. As can be seen from FIG. 8, after the natural compound Solamargine is added, the inhibition effect on pGL4.74-HBeniicp-Ce-del2 is the worst, which indicates that the deleted gene fragment in pGL4.74-HBeniicp-Ce-del2 has the most significant effect on the effect of the natural compound Solamargine, the deleted gene fragment is the target of the natural compound Solamargine, and the nucleic acid sequence of the deleted gene fragment is SEQ ID NO: 2.
In conclusion, the embodiment of the invention provides an application of a natural compound Solamargine in preparation of an antiviral drug. It can be effectively applied to the preparation of antiviral drugs to achieve better antiviral effect. In addition, the embodiment of the invention also provides an anti-HBV virus pharmaceutical composition, which comprises a natural compound Solamargine as an active ingredient and pharmaceutically acceptable auxiliary materials. The pharmaceutical composition has better anti-HBV activity and better curative effect on HBV infection.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
<110> hospital for province people in Henan
Application of natural compound Solamargine in preparation of antiviral drugs
<160> 2
<210> 1
<211>191
<212> DNA
<213> Artificial sequence
<400> 1
GCCCACCAGG TCTTGCCCAA GGTCTTACAT AAGAGGACTC TTGGACTCTC AGCAATGTCA 60
ACGACCGACC TTGAGGCATA CTTCAAAGAC TGTTTGTTTA AAGACTGGGA GGAGTTGGGG 120
GAGGAGATTA GGTTAAAGGT CTTTGTACTA GGAGGCTGTA GGCATAAATT GGTCTGTTCA 180
CCAGCACCAT G 191
<210> 2
<211> 35
<212> DNA
<213> Artificial sequence
<400> 2
GCCCAAGGTC TTACATAAGA GGACTCTTGG ACTCT 35
Claims (10)
1. An application of a natural compound Solamargine in preparing antiviral drugs.
2. The use of claim 1, wherein the virus is a DNA virus.
3. The use according to claim 2, wherein the virus is a hepadnavirus.
4. The use according to claim 3, wherein the hepadnavirus is an HBV virus.
5. The use according to claim 4, wherein the HBV virus comprises a lamivudine-resistant HBV variant.
6. The use according to any one of claims 1 to 5, wherein the natural compound Solamargine has a structural formula of,
the use according to claim 3 or 4, wherein the natural compound Solamargine has an antiviral effect by inhibiting the transcription of covalently closed circular DNA in the liver.
7. The use according to claim 3 or 4, wherein the natural compound solargine has an antiviral effect by inhibiting the expression of the pregenomic RNA of the HBV virus.
8. The use according to claim 3 or 4, wherein the natural compound solamgine has an antiviral effect by targeting the core promoter of the HBV virus to inhibit its transcription.
9. The use of claim 8, wherein the nucleic acid sequence of the core promoter is SEQ ID NO 1.
10. A pharmaceutical composition for resisting HBV virus is characterized by comprising a natural compound Solamargine serving as an active ingredient and pharmaceutically acceptable auxiliary materials.
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