CN109528703B - Application of 3-acetate-emodin in the preparation of anti-herpes simplex virus type I drugs - Google Patents
Application of 3-acetate-emodin in the preparation of anti-herpes simplex virus type I drugs Download PDFInfo
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid
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- 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3‑乙酸大黄素在制备抗单纯疱疹病毒I型药物中的应用，属于抗病毒药物技术领域。本发明提供的3‑乙酸大黄素，将大黄素中3位C上的羟基替换为乙酸基，大大提高了溶解性，具有更好的成药性。实验证明：3‑乙酸大黄素对HSV‑1毒株感染Hep‑2细胞的毒性很小，3‑乙酸大黄素可显著降低Hep‑2细胞中HSV‑1 gD基因表达同时抑制HSV‑1在Hep‑2细胞中的复制与感染，且呈浓度剂量效应。本发明制备的3‑乙酸大黄素具有显著抗HSV‑1病毒的特性，成为临床治疗HSV‑1病毒感染的候选药物。
The invention provides the application of 3-acetate-emodin in the preparation of anti-herpes simplex virus type I medicine, and belongs to the technical field of antiviral medicines. The 3-acetate-emodin provided by the invention replaces the hydroxyl group on the 3-position C in the emodin with an acetate group, which greatly improves the solubility and has better druggability. Experiments have shown that: 3-emodin acetate has little toxicity to Hep-2 cells infected with HSV-1 strain, and 3-emodin acetate can significantly reduce the expression of HSV-1 gD gene in Hep-2 cells and inhibit the expression of HSV-1 in Hep-2 cells. Replication and infection in ‑2 cells in a concentration-dose effect. The 3-acetate-emodin prepared by the invention has the characteristic of remarkably anti-HSV-1 virus, and becomes a candidate medicine for clinical treatment of HSV-1 virus infection.
Description
Technical Field
The invention belongs to the technical field of antiviral drugs, and particularly relates to application of 3-emodin acetate in preparation of anti-herpes simplex virus I drugs.
Background
Herpes simplex virus type I (HSV-1) is the most common pathogen in human, and human is the only natural host, and can cause latent or cause oral and genital herpes, conjunctivitis, eczema herpeticum and even herpetic encephalitis with extremely high mortality rate. AIDS associated with HSV-1 infection complicates AIDS treatment. Most oral and genital herpes infections are asymptomatic, and symptoms of herpes include painful blisters or ulcers at the site of infection. Herpes infections are most contagious once the symptoms appear, but can still be transmitted to others without symptoms, and are extremely contagious.
Most current therapies for HSV-1 viral infection use nucleotide, nucleoside or pyrophosphate analogs such as acyclovir, valacyclovir, penciclovir, famciclovir, and the like. After the medicines are taken up by cells infected with viruses, the thymidine kinase coded by the viruses and intracellular kinase are phosphorylated and compete with nucleotides to inhibit virus DNA polymerase, so that the synthesis of virus DNA chains is stopped, and the aim of resisting viruses is fulfilled. Although these drugs have significant efficacy in the treatment of many acute infections, helping patients reduce the severity and frequency of symptoms, they do not completely cure the infection and long-term use can lead to the emergence of resistant viral strains, which is more likely to occur especially in immunocompromised patients, and it is therefore essential to find alternative approaches to combat HSV-1 infection and replication.
Emodin is dried rhizome and root of Polygonum cuspidatum of Polygonaceae, is an orange long needle crystal, and is orange in acetone and yellow in methanol, and belongs to plant type medicine. The chemical name of the anthraquinone is 1'3' 8-trihydroxy-6-methylanthraquinone, the molecular formula is C15H10O5, the molecular weight is 270.23, the melting point is 256-257 ℃, the anthraquinone has special reaction, and the anthraquinone is hardly dissolved in water and is dissolved in ethanol and alkali solution. Modern pharmacological research finds that emodin has various effects of anti-tumor activity, antimicrobial growth (such as staphylococcus, hemolytic streptococcus and the like), spasmolysis, cough relieving, immunosuppression, diuresis, purgation and the like; meanwhile, the medicament also has the effects of resisting influenza virus, hepatitis B virus, coxsackie virus, rubella virus and the like, but the treatment condition of the medicament on herpes simplex virus I is not reported. Emodin is insoluble in water and only soluble in alkali and some organic solvents such as ethanol, dimethyl sulfoxide (DMSO), which hinders the application of emodin in clinical development.
Disclosure of Invention
In view of the above, the invention aims to provide 3-emodin acetate, a preparation method thereof and application thereof in preparation of anti-herpes simplex virus I type drugs, wherein the 3-emodin acetate is increased in water solubility or lipid solubility, has better drug forming property, and becomes a candidate drug for clinical treatment of HSV-1 virus infection.
In order to achieve the above object, the present invention provides the following technical solutions:
an application of 3-acetic acid emodin with a structure shown in a formula a in preparing anti-herpes simplex virus I type medicine,
preferably, the concentration of the 3-emodin acetate is 10-30 mu mol/L, more preferably 15-28 mu mol/L, and most preferably 20 mu mol/L. The administration time is preferably before or as soon as possible after infection with HSV-1.
The dosage form of the drug is not particularly limited, and the drug can be prepared into dosage forms of drugs well known in the art, such as injection, spray, powder, injection and the like. The preparation method of the dosage form is not particularly limited in the present invention, and a preparation method of a pharmaceutical dosage form well known in the art may be employed.
The 3-acetic acid emodin provided by the invention has the advantages that the hydroxyl on the C position in the emodin is replaced by the acetic acid group, so that the solubility is greatly improved, the emodin has better drug forming property, and the antiviral function is kept. Experiments prove that: the toxicity of the 3-emodin acetate to the Hep-2 cells infected by HSV-1 strain is very small, the survival rate of the 3-emodin acetate to the Hep-2 cells is close to 100 percent at 10 mu mol/L, and the half lethal dose (CC) of the 3-emodin acetate to the Hep-2 cells50) The concentration was 31.33. mu. mol/L. The expression of HSV-1gD mRNA in cells is detected through fluorescent real-time quantitative PCR, and the 3-acetic acid emodin can reduce the expression of HSV-1gD gene in Hep-2 cells after infection at 48h and has concentration dose effect. At the same time, the user can select the desired position,the result of the plaque experiment shows that: the 3-emodin acetate can obviously inhibit the replication and infection of HSV-1 in Hep-2 cells and has concentration dose effect. Therefore, the 3-emodin acetate prepared by the invention has the characteristic of obviously resisting HSV-1 virus and can be used as a candidate medicament for clinically treating HSV-1 virus infection.
Drawings
FIG. 1 shows the reaction formula for the preparation of 3-emodin acetate;
FIG. 2 is a nuclear magnetic hydrogen spectrum of emodin-3-acetate;
FIG. 3 is the in vitro anti-HSV-1 effect of 3-emodin with different concentrations, wherein FIG. 3-A is a bar graph of HSV-1gD gene expression level in Hep-2 cells infected with 3-emodin with different concentrations detected by fluorescent real-time quantitative PCR; FIG. 3-B is a graph showing the plaque formation of virus in different concentrations of emodin-3-acetate treated HSV-1 infected Hep-2 cells detected by plaque assay;
FIG. 4 is a graph of the inhibition of HSV-1gD gene expression by 3-acetorhein versus time.
Detailed Description
The features and advantages of the present invention will be further understood from the following detailed description taken in conjunction with the accompanying drawings. The examples provided are merely illustrative of the method of the present invention and do not limit the remainder of the disclosure in any way.
Example 1
The preparation method of the 3-emodin acetate refers to the specific implementation mode of ZL201510974947.7 in the specification [ example 1 ]. The preparation reaction formula is shown in figure 1.
125mg of emodin and 138mg of potassium carbonate are added into 5mL of acetone, mixed evenly, then 71mg of alpha-bromomethyl acetate is added dropwise, and reflux reaction is carried out at 50 ℃. After the reaction is finished, adding hydrochloric acid to adjust the pH value to 1-2, filtering to obtain a red solid, and drying in a vacuum drying oven at 40 ℃. Adding 74.3mg of the product into a round-bottom flask, adding 34.7mg of sodium hydroxide and 15mL of ethanol, stirring at 30 ℃ for 4h, placing the reaction solution until no liquid is separated out, adding 10mL of water for dilution, adjusting the pH value to 1-2 by using hydrochloric acid, extracting an aqueous phase by using ethyl acetate, combining organic phases, drying by using sodium sulfate, and drying to obtain a red solid. The product was identified as emodin-3-acetic acid by nuclear magnetic hydrogen spectroscopy (see FIG. 2).
Example 2
MTT method for detecting cytotoxicity of 3-emodin acetate
mu.L of 3-emodin acetate solution diluted with DMEM (containing 2% fetal calf serum, V/V) to different concentrations (0.1. mu.M, 1. mu.M, 10. mu.M, 100. mu.M, 1000. mu.M) was added to the growing monolayer of Hep-2 cells, and cultured for 48 h. Cells not treated with emodin 3-acetate (100. mu.L of DMEM containing 2% fetal bovine serum) were used as normal control. The cytotoxicity effect of emodin-3-acetate was evaluated by detecting the absorbance values of the normal control group and the drug group by the MTT method, and calculating the cell survival rate (cell survival rate ═ average absorbance value of drug group/average absorbance value of normal control group × 100%).
The results show that the 3-emodin acetate has the following toxic effect on Hep-2 cells: cell adhesion, rounding, breaking and shedding, increasing intracytoplasmic granules, enhancing refractivity and obviously reducing light absorption value. The toxicity of 3-emodin acetate to Hep-2 cells is very small, the survival rate of the 3-emodin acetate to Hep-2 cells is close to 100% at 10 mu M, and the median lethal dose (CC) of the 3-emodin acetate to Hep-2 cells is calculated50) 31.33 μ M, and therefore the concentration of emodin-3-acetate was chosen to be less than CC 5020 μ M for subsequent antiviral studies.
Example 3
3-emodin acetate has in vitro anti-HSV-1 effect and concentration dose effect
After 2h of infecting Hep-2 cells with HSV-1 standard strain KOS strain (100PFU/ml) (establishing HSV-1 infected cell model by respectively applying fluorescence real-time quantitative PCR and plaque assay), 3-emodin solution with different concentrations (2.5 mu mol/L, 5 mu mol/L, 10 mu mol/L and 20 mu mol/L) is used for treating the cells for 48h, then the cells are collected, 20 mu M Acyclovir (ACV) is used as a positive control, and water is used as a blank control (Con). The fluorescent real-time quantitative PCR method is used for detecting the plaque forming condition in the cell plate by HSV-1gD mRNA expression he plaque assay, and the antiviral effect of the 3-rhein acetate on HSV-1 is determined from two angles of virus nucleic acid and virus titer. The method comprises the following specific steps:
(1) the HSV-1gD gene expression is detected by using fluorescent real-time quantitative PCR, GAPDH is used as an internal reference, and a 3-emodin acetate treatment group is not added and is used as a control group. The nucleotide sequences of the primers are as follows:
gD gene upstream primer: 5'-gccccgctggaactactatg-3' (SEQ ID No. 1);
gD gene downstream primer: 5'-ttatcttcacgagccgcagg-3' (SEQ ID No. 2);
GAPDH upstream primer: 5'-ggtggtctcctctgacttcaaca-3' (SEQ ID No. 3);
GAPDH downstream primer: 5'-gttgctgtagccaaattcgttgt-3' (SEQ ID No. 4).
1 μ L of total RNA sample was subjected to reverse transcription using RT system (Promega), and the reaction was terminated with random primers at 37 ℃ for 1h and 94 ℃ for 5min, and the product was stored at 4 ℃. The reverse transcription product cDNA was used as a reaction template for real-time quantitative RT-PCR, and 1.5. mu.L of RNA reverse transcription product cDNA, 0.3. mu.L of upstream and downstream primers (20pmol), and 7.5. mu.L of LSYBR green mixed solution were taken, and water was supplemented to a total volume of 15. mu.L, and detection was performed on a fluorescence quantitative PCR instrument (BioRad). The reaction procedure is as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 10s, annealing at 60 ℃ for 10s, and extension at 72 ℃ for 15s, for 40 cycles.
The results are shown in FIG. 3-A, and compared with the blank control group, emodin 3-acetate at 48h after infection can reduce HSV-1gD gene expression in Hep-2 cells, and shows concentration dose effect (FIG. 3-A, P < 0.01). When 20 mu M acyclovir is used as a positive control group, compared with a CON group, the gD expression level is very low, and the relative value approaches to 0, so that the anti-HSV-1 effect is better. Meanwhile, the expression level of the 20 mu M3-emodin acetate is lower, although the expression of gD gene can not be completely inhibited, which shows that the invention has similar antiviral effect compared with the known aciclovir.
(2) Plaque assay calculate virus titer: after 2h of infecting Hep-2 cells with HSV-1 strain, discarding virus liquid, adding methyl cellulose culture solution containing 3-emodin acetate with different concentrations, continuing culturing for 48h, fixing with methanol for 10min, adding crystal violet for dyeing, and removing unfixed parts in the cell plate with running water. The cell plates were observed for the formation of hollow spots and counted.
The results are shown in figure 3-B, which shows that the 3-emodin acetate can obviously inhibit the replication and infection of HSV-1 in Hep-2 cells, and has concentration dose effect.
Example 4
The 3-emodin acetate has time effect in vitro anti-HSV-1 effect
After 2h of infecting Hep-2 cells by HSV-1 strain, 3-emodin acetate (20 mu mol/L) is respectively treated for 3h, 6h, 12h, 24h and 48h, and then the cells are collected. HSV-1gD gene expression in the cells was detected using fluorescent real-time quantitative PCR as in example 3 (1).
The results are shown in FIG. 4, and the effect of 3-acetic acid emodin anti-HSV-1 in Hep-2 cells shows a time effect.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Wuhan university
Application of <120> 3-emodin acetate in preparation of anti-herpes simplex virus I-type drugs
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
<210> 3
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ggtggtctcc tctgacttca aca 23
<210> 4
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
gttgctgtag ccaaattcgt tgt 23
Claims (4)
1. An application of 3-acetic acid emodin with a structure shown in a formula a in preparing anti-herpes simplex virus I type medicine,
2. the use according to claim 1, wherein the concentration of emodin-3-acetate is 10-30 μmol/L and the administration time is before or after HSV-1 infection.
3. The use according to claim 2, wherein the concentration of emodin-3-acetate is 15 to 28 μmol/L.
4. The use according to claim 3, wherein the concentration of emodin-3-acetate is 20 μmol/L.
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| Title |
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| The effect of emodin, an anthraquinone derivative extracted from the roots of Rheum tanguticum, against herpes simplex virus in vitro and in vivo;Hai-Rong Xiong等;《Journal of Ethnopharmacology》;20101102;第133卷;第718–723 页 * |
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