CN118526483B - Application of a biphenyl polyphenol compound derived from natural plants in the preparation of a drug for treating herpetic diseases - Google Patents
Application of a biphenyl polyphenol compound derived from natural plants in the preparation of a drug for treating herpetic diseases Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
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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
- A61P31/22—Antivirals for DNA viruses for herpes viruses
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Abstract
本发明提供了一种天然植物来源的联苯多酚化合物在制备治疗疱疹性疾病的药物中的应用，属于生物医药技术领域。所述联苯多酚化合物为Caesappanin C，它能显著抑制HSV在感染细胞中的增殖和病毒蛋白的表达。本发明通过建立细胞感染模型进行实验，结果表明Caesappanin C能够显著抑制HSV的体外增殖，降低HSV的mRNA和蛋白表达水平，抑制病毒空斑形成，且通过直接与病毒相互作用从而发挥抗病毒作用，并且有较高的活性和较低的细胞毒性，治疗指数较高。本发明通过实验表明植物来源的化合物Caesappanin C能够用于制备抗疱疹性疾病的药物。
The present invention provides an application of a natural plant-derived biphenyl polyphenol compound in the preparation of a drug for treating herpetic diseases, and belongs to the field of biomedical technology. The biphenyl polyphenol compound is Caesappanin C, which can significantly inhibit the proliferation of HSV in infected cells and the expression of viral proteins. The present invention conducts experiments by establishing a cell infection model, and the results show that Caesappanin C can significantly inhibit the in vitro proliferation of HSV, reduce the mRNA and protein expression levels of HSV, inhibit the formation of viral plaques, and exert an antiviral effect by directly interacting with the virus, and has higher activity and lower cytotoxicity, and a higher therapeutic index. The present invention shows through experiments that the plant-derived compound Caesappanin C can be used to prepare drugs for treating herpetic diseases.
Description
Technical Field
The invention belongs to the technical field of biology and medicine, and in particular relates to application of a biphenyl polyphenol compound from natural plants in preparation of a medicament for treating herpetic disease.
Background
Herpes simplex virus (Herpes Simplex Virus, HSV) is a member of the alpha subfamily of the herpesviridae, has a high infection rate in the population, and can cause stomatitis, keratitis, encephalitis and other diseases. HSV infection is neural latent and can remain latent for life in the host. HSV is classified into HSV-1 and HSV-2 according to serotypes, HSV-1 mainly causes herpes labialis and HSV-2 mainly causes herpes genitalis. The current drugs for treating herpes infection are all based on modified nucleosides and prodrugs thereof, such as acyclovir (Acyclovir, ACV), ganciclovir (GCV) and the like, but the drugs have low oral bioavailability or short blood half-life and cannot inhibit HSV reactivation, and can cause drug-resistant strains to appear after long-term administration.
CAESAPPANIN C is a biphenyl dimer, extracted from heartwood of indonesia CAESALPINIA SAPPAN l, having the potential to stimulate bone formation and regeneration. It shows potent proliferative stimulatory activity on primary osteoblasts in vitro. However, the anti-HSV effect of CAESAPPANIN C has not been reported.
Disclosure of Invention
The invention aims to provide an application of a biphenyl polyphenol compound from natural plants in preparing medicines for treating herpetic diseases. The biphenyl polyphenol compound is CAESAPPANIN C, and the CAS No. is 1913319-59-1. Experiments prove that the natural product CAESAPPANIN C from the plant source has obvious HSV resisting effect.
In order to achieve the aim of the invention, the invention is realized by adopting the following technical scheme:
The invention provides application of a biphenyl polyphenol compound from natural plants in preparing a medicament for treating herpetic diseases.
Further, the biphenyl polyphenol compound is CAESAPPANIN C, and the structural formula is as follows:
。
further, CAESAPPANIN C can significantly inhibit proliferation of HSV and expression of viral proteins in infected cells.
Furthermore, CAESAPPANIN C can obviously inhibit the virus key protein expression of HSV in infected cells in a dose-dependent manner in a dose range of 2.5 mu M-20 mu M.
Further, the virus key proteins are ICP5, ICP27 and gB.
Further, CAESAPPANIN C can significantly inhibit the mRNA transcription level of HSV very early protein ICP 0.
Furthermore, the action mechanism of CAESAPPANIN C anti-HSV virus is to directly inactivate virus and exert antiviral action by directly destroying virus structure.
Further, the CAESAPPANIN C working stage is mainly in the process of compound pretreatment virus.
Further, the herpetic disease includes herpetic encephalitis pneumonia, genital herpes and cornea diseases.
The invention also provides a medicine for treating diseases caused by herpes simplex virus infection, which contains CAESAPPANIN C as an active ingredient and pharmaceutically acceptable auxiliary materials.
Compared with the prior art, the invention has the following beneficial technical effects:
Aiming at the defects of the existing anti-HSV drugs, the invention selects natural products CAESAPPANIN C derived from plants as research objects, and reveals the effect of inhibiting HSV-induced reaction at in-vivo and in-vitro levels. CAESAPPANIN C has better inhibition effect on cytopathy induced by HSV, can inhibit cytopathy caused by HSV infection, inhibit the expression of HSV key viral proteins and inhibit the mRNA level of some viral proteins, thereby having strong inhibition effect on cytopathy reaction caused by HSV and improving the symptom and tissue injury of mice caused by HSV infection.
The invention proves that CAESAPPANIN C has the effects of inhibiting HSV-induced cytopathic reaction and relieving the tissue injury, survival rate and weight loss of mice caused by HSV infection for the first time. Therefore, CAESAPPANIN C can be developed into a novel medicine for preventing and/or treating HSV infectious diseases. Has wide market application prospect.
Other features and advantages of the present invention will become apparent upon review of the detailed description of the invention in conjunction with the drawings.
Drawings
FIG. 1 is a graph showing the half-maximal inhibitory concentrations of CAESAPPANIN C in Vero cells and HaCaT cells, respectively, on HSV-1 and HSV-2, and the cytotoxicity assays on these two cells, in example 1.
FIG. 2 shows that CAESAPPANIN C of example 1 can significantly inhibit the expression levels of viral proteins in Vero cells after infection with HSV-1 and HSV-2 by pretreatment of the virus, and the detected proteins are viral early protein ICP27 and late protein ICP5. The internal reference is GAPDH.
FIG. 3 shows that CAESAPPANIN C of example 1 was able to significantly inhibit the expression of viral proteins in Vero cells after infection with HSV-1 and HSV-2 in a dose dependent manner, and the proteins detected were viral early proteins ICP27 and gB. The internal reference is beta-actin.
FIG. 4 shows the detection of mRNA expression levels of HSV early protein ICP0 and late protein ICP5 by RT-PCR.
FIG. 5 is a direct inactivation of HSV-1 by CAESAPPANIN C of example 1, validated using a plaque reduction assay.
FIG. 6 is a graph of the direct inactivation of HSV-1 by CAESAPPANIN C of example 1 as further validated by the Virucidal assay.
FIG. 7 is a membrane fusion inhibition assay of CAESAPPANIN C of example 1 following infection of Vero cells with HSV-2.
Wherein the biphenylpolyphenol compounds CAESAPPANIN C of natural plant origin are designated CAESAPPANIN C throughout the drawings. Acyclovir Wei Zaisuo, a positive drug, is referred to as ACV in the drawings.
Detailed Description
The technical scheme of the present invention will be described in further detail with reference to the accompanying drawings and the detailed description, but should not be construed as limiting the invention. Unless otherwise indicated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise indicated.
According to the internationally accepted method, the inhibition of CAESAPPANIN C on herpes simplex virus infection in vitro was evaluated. CAESAPPANIN C adopted by the invention is a commercially available product, and the chemical structural formula of CAESAPPANIN C is shown as follows:
。
example 1: CAESAPPANIN C in vitro HSV Activity inhibition assay
1. CAESAPPANIN C cytotoxicity test and CPE inhibition test
Two kinds of Vero and HaCaT cells were spread in 96-well plates, placed in an incubator at 37℃and cultured with 5% CO 2, and cytotoxicity of CAESAPPANIN C on three kinds of different cells was measured. After the cells grow to 100%, the original culture solution is sucked and removed, CAESAPPANIN C (final concentration of 500 mu M, 250 mu M, 125 mu M, 62.5 mu M, 31.25 mu M, 15.625 mu M, 7.8125 mu M and 3.90625 mu M) subjected to gradient dilution is added into a 96-well plate, three compound wells are arranged in each concentration, and a blank control group is arranged at the same time. 37. After 24 h ℃culture, the solution was pipetted, 4% paraformaldehyde was added, the solution was fixed at room temperature for 15: 15 min, 0.1% (w/v) crystal violet was added after pipetting, the solution was stained at room temperature for 15: 15 min, washed with running water, dried at room temperature, and the A540nm value was measured. The concentration of compound that decreased cell viability by 50% (CC 50) was calculated using GRAPHPAD PRISM 8.0.0 and the cell viability was plotted.
Cell viability = (dosing group-blank)/(control group-blank) ×100%;
As shown in FIG. 1 and Table 1, CAESAPPANIN C was safe and nontoxic to Vero and HaCaT cells at 250. Mu.M and below, and was cytotoxic to HaCaT cells at 500. Mu.M while cell viability remained above 60%. As can be seen, CAESAPPANIN C is less cytotoxic to Vero, haCaT cells. CPE inhibition assay CAESAPPANIN C activity against HSV-1 (strain F) and HSV-2 (type 333) on both Vero and HaCaT cells was evaluated, IC 50 was calculated by whole-process dosing, and therapeutic index (si=cc50/IC 50) was calculated. As shown in FIG. 1 and Table 1, CAESAPPANIN C has excellent anti-HSV effect on all three cells, and has a certain dose dependence with IC 50 value of less than 15 μm. CAESAPPANIN C has better anti-HSV-2 activity than HSV-1 in Vero and HaCaT, and has better anti-HSV activity than HaCaT in Vero cells, and has certain cell dependence.
Cytotoxicity and antiviral Effect of the compounds of Table 1 in different cells
2. Influence of Western blot detection CAESAPPANIN C on expression of HSV viral proteins
(1) Effect of different concentrations CAESAPPANIN C on HSV viral protein expression: vero cells were inoculated in 6-well plates, after the cells had grown one layer, CAESAPPANIN C were diluted in a gradient and the whole procedure was performed on HSV-1 and HSV-2 infected Vero cells (pretreatment of cells, pretreatment of virus, administration at the time of adsorption, and administration after adsorption). At the same time, virus and blank control groups are provided, and cell lysate is collected after 12 h. Preparing cell lysate, sucking and removing the original culture solution, adding 80 μl of lysate into each well, lysing 20 min at 4deg.C, and decocting 20 min at 100deg.C. The extracted protein concentration was measured by BCA kit, separated by SDS-PAGE and transferred to NC membrane. After blocking overnight at 4℃in 5% nonfat milk powder, NC membranes were washed and incubated with anti-HSV-1/2 ICP27 antibody (1:1000 dilution) and anti-HSV-1/2 gB antibody (1:1000 dilution) at 37℃for 2 h. After three washes of 1 XTBST, the AP-labeled secondary antibody (1:5000 dilution) was incubated at 37℃for 2 h. After washing three times with 1 XTBST, the protein bands were observed and analyzed by alkaline phosphatase assay kit, which developed at room temperature.
(2) Inoculating Vero cells in a 6-well plate, and after the cells grow to form a layer, respectively treating the cells by different action modes: pretreatment cells (pre+cell): CAESAPPANIN C (20 μm) acts on cell 1 h at 37 ℃, aspirates, adsorbs HSV (moi=0.1) 1.5 h at 4 ℃, and changes to maintenance fluid; pretreatment Virus (pre+virus): CAESAPPANIN C (20. Mu.M) and HSV were mixed and incubated at 37℃for 1:1 h, transferred to a 6-well plate from which the original medium was aspirated, and allowed to act at 4℃for 1.5: 1.5 h, and changed to a maintenance solution; at Adsorption (Adsorption) administration: CAESAPPANIN C (20 μm) and HSV (moi=0.1) were mixed and then added to a 6-well plate to act at 4 ℃ for 1.5: 1.5 h, and the mixture was removed by suction and changed into a maintenance solution; post-adsorption (Post-adsorption) dosing: HSV adsorbed cells 1.5 h at 4deg.C, the original virus solution was discarded, changed to maintenance solution and CAESAPPANIN C (20 μm) was added, with virus and blank. 12 After h, cell lysates were collected. Preparing cell lysate, sucking and removing the original culture solution, adding 80 μl of lysate into each well, lysing 20 min at 4deg.C, and decocting 20 min at 100deg.C. The extracted protein concentration was measured by BCA kit, separated by SDS-PAGE and transferred to NC membrane. After blocking overnight at 4℃in 5% nonfat milk powder, NC membranes were washed and incubated with anti-HSV-1/2 ICP27 antibody (1:1000 dilution) and anti-HSV-1/2 ICP5 antibody (1:1000 dilution) at 37℃for 2 h. After three washes of 1 XTBST, the AP-labeled secondary antibody (1:5000 dilution) was incubated at 37℃for 2 h. After washing three times with 1 XTBST, the protein bands were observed and analyzed by developing color at room temperature using an alkaline phosphatase test kit.
As shown in fig. 2 and 3, CAESAPPANIN C can significantly inhibit the expression of key proteins of HSV-1 and HSV-2 in a dose of 10 μm and 20 μm in a dose-dependent manner, and CAESAPPANIN C is mainly used for resisting HSV by pre-treating viruses and post-adsorption administration, wherein the effect of the pre-treating viruses is most significant, the post-adsorption administration also has a certain inhibition effect, and the pre-treated cells and the post-adsorption administration have little influence on inhibiting the expression of viral proteins. The experimental results show that CAESAPPANIN C can inhibit the expression of HSV virus protein in a dose-dependent manner, and the acting stage mainly comprises the steps of pretreatment of the virus by the compound so as to inhibit inflammatory response caused by virus infection.
3. Real-time fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) further verifies CAESAPPANIN C action mode for inhibiting HSV (HSV)
(1) Inoculating Vero cells in a 6-well plate, and after the cells grow to form a layer, respectively treating the cells by different action modes: pretreatment cells (pre+cell): CAESAPPANIN C (20 μm) acts on cell 1 h at 37 ℃, aspirates, adsorbs HSV (moi=0.1) 1.5 h at 4 ℃, and changes to maintenance fluid; pretreatment Virus (pre+virus): CAESAPPANIN C (20. Mu.M) and HSV were mixed and incubated at 37℃for 1:1 h, transferred to a 6-well plate from which the original medium was aspirated, and allowed to act at 4℃for 1.5: 1.5 h, and changed to a maintenance solution; at Adsorption (Adsorption) administration: CAESAPPANIN C (20 μm) and HSV (moi=0.1) were mixed and then added to a 6-well plate to act at 4 ℃ for 1.5: 1.5 h, and the mixture was removed by suction and changed into a maintenance solution; post-adsorption (Post-adsorption) dosing: HSV adsorbed cells 1.5 h at 4deg.C, the original virus solution was discarded, changed to maintenance solution and CAESAPPANIN C (20 μm) was added, with virus and blank.
(2) After 8h of viral infection, total RNA of Vero cells was extracted according to the instructions using RNA extraction kit.
(3) After concentration detection of the extracted RNA by Nanodrop, two-step RT-PCR was performed using Applied Biosystems 7500: 7500 Fast Real Time PCR System according to the instructions of Evo M-MLV reverse transcription kit and SYBR block Green Premix Pro TAQ HS QPCR KIT II.
TABLE 2 RT primer sequences for PCR
As shown in fig. 4, the RT-PCR detected the mRNA expression levels of the HSV early protein ICP0 and the late protein ICP5, and the mRNA expression level of ICP0 was greatly reduced after CAESAPPANIN C pretreatment of cells and pretreatment of viruses, especially the pretreatment of viruses was most remarkable, almost reducing the mRNA expression level of ICP0 to 0; in contrast, ICP5 significantly decreased mRNA levels after CAESAPPANIN pretreatment of the virus, which was also confirmed by the previous Western blot results.
4. Plaque assay
(1) Plaque reduction assay: spreading Vero cells in a 12-hole plate, after the cells grow into one layer, carrying out mixing treatment on HSV-1 and CAESAPPANIN C of gradient dilution (final concentration of 20 mu M, 10 mu M, 5 mu M, 2.5 mu M and 1.25 mu M), carrying out mixing treatment on HSV-2 and CAESAPPANIN C of gradient dilution (final concentration of 10 mu M, 5 mu M, 2.5 mu M, 1.25 mu M and 0.625 mu M), and setting a virus control group. 37. After pretreatment at 1 h ℃, the pretreatment solution is added into a 12-well plate, 1 mL of the pretreatment solution is added into each well, 1 h is incubated at 37 ℃, the pretreatment solution is absorbed and removed, the prepared Overlay medium 1 mL is added, after cooling at room temperature for 10 min, the pretreatment solution is inverted into a 37 ℃ incubator, and after plaque grows out, the pretreatment solution is fixedly dyed and counted.
(2) Vero cells were plated in 12-well plates, after the cells had grown one layer, HSV-1/HSV-2 was mixed with CAESAPPANIN C (final concentration 20 μm) and a virus control group without CAESAPPANIN C was set. 37. Pretreating at the temperature of 1 h ℃, diluting the pretreatment liquid according to the multiple of 10-106, then adding the diluted pretreatment liquid into a 12-hole plate, incubating at the temperature of 37 ℃ for 1 h, absorbing, adding an Overlay culture medium, cooling, inverting the mixture into a 37 ℃ incubator, standing for dyeing after plaque grows out, and counting.
As shown in FIG. 5, the inactivation rate of HSV-1 was about 50% at CAESAPPANIN C at 1.25. Mu.M, the HSV-1 virus particles were almost completely inactivated at 10. Mu.M, and the inactivation effect on the virus was dose-dependent. For HSV-2, the inactivation rate was about 70% at CAESAPPANIN C concentrations of 1.25, and almost complete inactivation of the viral particles was achieved at concentrations up to 5. Mu.M. CAESAPPANIN C was shown to exert antiviral effects by binding directly to HSV particles.
The plaque reduction test shows that CAESAPPANIN C has dose dependency on the direct inactivation of HSV, but the CAESAPPANIN C concentration of the pretreated virus in adsorption is still within the effective range, so that the effect of the pretreated virus in the adsorption and post-adsorption stage cannot be excluded, and the direct inactivation is more accurately verified outside the effective concentration range of CAESAPPANIN C by using the Virucidal test. HSV-1 was previously treated with CAESAPPANIN C or not, and then diluted in a 10-fold gradient, so that the CAESAPPANIN C concentration had reached 1/1000 to 1/10000 of the normal effective concentration, and the adsorption and subsequent action phases were hardly affected. As shown in fig. 6, it is evident that the group treated with CAESAPPANIN C significantly reduced the viral titer of HSV-1, indicating that CAESAPPANIN C is indeed capable of exerting an antiviral effect by acting on HSV particles.
5. Membrane fusion inhibition assay
Vero cells were plated in 6-well plates, and after incubation of gradient diluted CAESAPPANIN C with HSV-2 (moi=3.0) at 37 ℃ for 1h, 4 ℃ to infect cells for 1.5 h, compound-free medium was added, after incubation at 37 ℃ for 6h, CAESAPPANIN C (final concentrations 40 μm, 20 μm, 10 μm, 5 μm) was added, two duplicate wells per group, and incubation at 37 ℃ was continued for 2 h. Blank and virus control groups were set simultaneously. After 4% paraformaldehyde is used for fixing cells 10min and double distilled water is used for washing twice, 0.25% (v/v) Triton X-100 is penetrated for 10min, the cells are washed again, hematoxylin dye solution for removing sediment by centrifugation is used for dyeing 10min, the cells are washed once rapidly, after 2 min of the cells are washed again, 95% ethanol is added for treatment 10 s, the double distilled water is used for washing for 10min, eosin dye solution is used for dyeing 2 min, 70% ethanol is used for washing twice, and an optical microscope is used for observing the inhibition effect on the formation of the cells, and photographing is carried out for analysis.
As shown in fig. 7, the experimental result shows that CAESAPPANIN C cannot obviously inhibit the membrane fusion process after the HSV-2 is infected for 8h, CAESAPPANIN C (final concentration 40 mu M, 20 mu M, 10 mu M and 5 mu M) treatment has only slight inhibition effect on the membrane fusion process after the Vero is infected with the HSV-2 virus, the membrane fusion degree of the compound group is not obviously different from that of the virus control group, the CAESAPPANIN C does not inhibit the process of the HSV infection cell by inhibiting the membrane fusion, and the CAESAPPANIN C anti-HSV virus action mechanism is to directly inactivate the virus and mainly acts by directly destroying the virus structure.
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting thereof; although the invention has been described in detail with reference to the foregoing embodiments, it will be apparent to one skilled in the art that modifications may be made to the technical solutions described in the foregoing embodiments, or equivalents may be substituted for some of the technical features thereof; such modifications and substitutions do not depart from the spirit and scope of the corresponding technical solutions.
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