A fire mother extract and its application in the preparation of anti-type 1 herpes simplex virus drugs

Abstract

The present application relates to a kind of fire mother extract and its application in preparation anti-type 1 herpes simplex virus drug.The preparation method of the fire mother extract includes the following steps: fire mother is used as raw material, and ethanol extract is prepared by decocting alcohol deposition method, the ethanol extract is extracted with equal amount of petroleum ether, ethyl acetate in turn three times, respectively, and the water phase is collected and concentrated under reduced pressure, to obtain the ethyl acetate fraction, water phase fraction, the ethyl acetate fraction is chromatographed with silica gel column or the water phase fraction is chromatographed with macroporous resin, to obtain the fire mother extract.The fire mother extract provided in the present application mainly acts on HSV-1 virus infection early stage, can inhibit the expression of HSV-1 virus DNA, RNA, protein, can inhibit the production of virus particles, can be used for preparing anti-viral drug for type 1 herpes simplex virus, expand herpes virus infection disease treatment scheme, with wide application prospect and important clinical significance.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a Rubus corchorifolius L.f. extract and application thereof in preparation of an anti-type I herpes simplex virus drug. BACKGROUND

[0002] Herpes virus is a typical representative of the herpesviridae family. Currently, eight types of herpes virus that can infect humans have been discovered, including herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), human herpes virus type 6 (HHV-6), human herpes virus type 7 (HHV-7), and Kaposi's sarcoma-associated virus (KSHV). According to differences in genomic sequences and structures and physicochemical properties, human herpes viruses can be divided into three subfamilies: alpha herpesvirus subfamily, beta herpesvirus subfamily, and gamma herpesvirus subfamily. Alpha-herpes viruses include HSV-1, HSV-2, and VZV, which have relatively fast and short replication cycles in epithelial cells and fibroblasts, and are mainly latent in nervous system cells and reactivated when the individual's immunity is impaired. Beta-herpes viruses include HCMV, which usually establishes latency in non-neuronal cells and has a longer viral replication cycle. Gamma-herpes viruses establish latency in B lymphocytes and T lymphocytes, can transform latently infected cells, and induce tumors in infected hosts.

[0003] A mature HSV is composed of viral DNA, capsid, tegument, and envelope, and the outermost envelope contains at least 12 different glycoproteins, of which a small part exists as a heterodimer and the vast majority exists as a monomer. Once in contact with a suitable host, the viral glycoprotein will attach to the host cell surface receptor, and then induce fusion of the viral envelope with the host cell membrane, thereby delivering the viral contents to the host cell. HSV is divided into HSV-1 and HSV-2 according to differences in antigenicity. HSV-1 mainly infects mucosal epithelial cells and establishes lifelong infection in sensory neurons. The most common symptom of HSV-1 infection is the formation of painful vesicular lesions in the oral cavity and face. Symptoms include stinging, itching, or burning sensations, which can recur periodically and vary in frequency from person to person. In severe cases, it can lead to herpes labialis, corneal blindness, encephalitis, AIDS, Alzheimer's disease, and other diseases. Even if these diseases can be effectively treated, they still leave intractable sequelae, causing great suffering to humans.

[0004] At present, the main drugs for treating HSV-1 in clinic are nucleoside analogs represented by acyclovir (ACV) and ganciclovir (GCV), which target viral DNA polymerase to affect viral gene replication. These drugs all have problems such as low solubility, short half-life, low bioavailability and large side effects, and long-term use of these drugs to treat HSV-1 infection is easy to induce drug-resistant virus strains. Therefore, it is necessary to develop new drugs with high efficiency and low toxicity for HSV-1 infection. SUMMARY

[0005] Therefore, the purpose of the present application is to provide the use of a firethorn extract with a new mechanism of action, high efficiency and low toxicity in the preparation of an anti-HSV-1 drug, and to provide a new drug selection for the prevention and treatment of HSV-1 infection.

[0006] The above-mentioned object of the present application is achieved by the following technical solutions:

[0007] A preparation method of a firethorn extract with anti-HSV-1 activity, comprising the following steps:

[0008] S1: firethorn as raw material, 10-20 times by weight of water is added for decoction, the decoction time is 10-20 min, filtration, 5-15 times by weight of water is added for decoction of the residue, the decoction time is 10-20 min, filtration, the filtrates are combined, vacuum concentration, then ethanol is added to make the final concentration of ethanol 60% v / v, centrifugation after standing at 4℃ for 6-18 h, collection of supernatant, vacuum concentration, to obtain the ethanol extract;

[0009] S2: the ethanol extract is sequentially extracted with equal amount of petroleum ether and ethyl acetate three times, and the ethyl acetate phase and water phase are collected and vacuum concentrated to obtain the ethyl acetate fraction and water fraction;

[0010] S3: the ethyl acetate fraction is subjected to silica gel column chromatography, or the water fraction is subjected to macroporous resin chromatography to obtain the firethorn extract.

[0011] The firethorn extract provided by the present application mainly acts on the early stage of HSV-1 virus infection, can inhibit the expression of HSV-1 virus DNA, RNA and protein, and can inhibit the production of virus particles, which may inhibit the expression of downstream inflammatory factors by inhibiting the activation of virus-induced NF-κB, and can be used for preparing anti-HSV-1 virus drugs, expanding the treatment options for herpes virus infection diseases, and has broad application prospect and important clinical significance.

[0012] Further, the solid-liquid ratio of firethorn to water in step S1 is 1:15, the solid-liquid ratio of the residue to water is 1:10, the decoction time is 15 min, and the standing time is 12 h.

[0013] Further, the step S3 further comprises: taking the ethyl acetate fraction for silica gel column chromatography separation, gradient elution is carried out in a petroleum ether-ethyl acetate system with a volume ratio of 8:1-0:1, and the eluent of petroleum ether: ethyl acetate with a volume ratio of 0:1 is collected and concentrated to obtain the extract of Radix Rubi.

[0014] Further, the step S3 further comprises: taking the water phase fraction for D101 macroporous resin chromatography separation, elution is carried out with distilled water and 25% ethanol respectively, the elution speed is 3BV / h, and the 25% ethanol eluent is collected and concentrated to obtain the extract of Radix Rubi.

[0015] In some other embodiments, the extract of Radix Rubi as an active ingredient is applied to the preparation of a medicine for resisting type I herpes simplex virus.

[0016] Further, the medicine comprises a pharmaceutically acceptable excipient.

[0017] Further, the medicine is prepared into an injection, an oral liquid, granules, powder, tablets, capsules or a topical patch. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Results of detection of the cytotoxicity and anti-HSV-1 activity of the ethanol extract of Radix Rubi in Example 1 in Vero cells;

[0019] Figure 2 Results of detection of the cytotoxicity and anti-HSV-1 activity of the ethanol extract of Radix Rubi in Example 1 in Hela cells;

[0020] Figure 3 Results of detection of the cytotoxicity and anti-HSV-1 activity of the petroleum ether extract of Radix Rubi in Example 2 in Hela cells;

[0021] Figure 4 Results of detection of the cytotoxicity and anti-HSV-1 activity of the ethyl acetate extract of Radix Rubi in Example 2 in Hela cells;

[0022] Figure 5 Results of detection of the cytotoxicity and anti-HSV-1 activity of the water phase extract of Radix Rubi in Example 2 in Hela cells;

[0023] Figure 6 Results of detection of the cytotoxicity and anti-HSV-1 activity of the component Er1 in Example 3 in Hela cells;

[0024] Figure 7 Results of detection of the cytotoxicity and anti-HSV-1 activity of the component Er2 in Example 3 in Hela cells;

[0025] Figure 8 Results of testing of Example 3 component Er3 for cytotoxicity and anti-HSV-1 activity in Hela cells;

[0026] Figure 9 Results of testing of Example 4 component Fr1 for cytotoxicity and anti-HSV-1 activity in Hela cells;

[0027] Figure 10 Results of testing of Example 4 component Fr2 for cytotoxicity and anti-HSV-1 activity in Hela cells;

[0028] Figure 11 Results of testing of Example 4 component Fr3 for cytotoxicity and anti-HSV-1 activity in Hela cells;

[0029] Figure 12 Results of testing of Example 4 component Fr4 for cytotoxicity and anti-HSV-1 activity in Hela cells;

[0030] Figure 13 Fluorescence microscope images of HSV-GFP infected Hela cells at different concentrations of Example 5 component Er3;

[0031] Figure 14 Fluorescence microscope images of HSV-GFP infected Hela cells at different concentrations of Example 5 component Fr2;

[0032] Figure 15 Effect of Example 5 components Er3 and Fr2 on HSV-1 viral genome copy number in Hela cells;

[0033] Figure 16 Effect of Example 5 components Er3 and Fr2 on HSV-1 viral titer in Hela cells;

[0034] Figure 17 Effect of Example 5 components Er3 and Fr2 on HSV-1 a0 mRNA expression level in Hela cells;

[0035] Figure 18 Effect of Example 5 components Er3 and Fr2 on HSV-1 UL27 mRNA expression level in Hela cells;

[0036] Figure 19 Effect of Example 5 components Er3 and Fr2 on expression level of viral early protein ICP0 and late gB protein targeting in Hela cells;

[0037] Figure 20Effect of Example 5 components Er3 and Fr2 on the relative expression of ICP0 protein and gB protein in Hela cells;

[0038] Figure 21 Schematic diagram of the effect of Example 5 components Er3 and Fr2 on the HSV-1 action pathway;

[0039] Figure 22 Effect of Example 5 components Er3 and Fr2 on the HSV-1 viral genome copy number in Hela cells under different action pathways;

[0040] Figure 23 Effect of Example 5 components Er3 and Fr2 on the IL-1 β mRNA expression level in Hela cells;

[0041] Figure 24 Effect of Example 5 components Er3 and Fr2 on the IL-6 mRNA expression level in Hela cells;

[0042] Figure 25 Effect of Example 5 components Er3 and Fr2 on the TNF-α mRNA expression level in Hela cells;

[0043] Figure 26 Effect of Example 5 components Er3 and Fr2 on the NF-κΒ pathway p65 protein phosphorylation level in Hela cells;

[0044] Figure 27 Effect of Example 5 components Er3 and Fr2 on the wound healing of skin herpes virus infection model mice;

[0045] Figure 28 Effect of Example 5 components Er3 and Fr2 on the body weight of skin herpes virus infection model mice;

[0046] Figure 29 Effect of Example 5 components Er3 and Fr2 on the inflammation score of skin herpes virus infection model mice;

[0047] Figure 30 Effect of Example 5 components Er3 and Fr2 on the viral genome copy number of skin herpes virus infection model mice;

[0048] Figure 31 Effect of Example 5 components Er3 and Fr2 on the HSV-1 virus late gene gB mRNA expression level in skin herpes virus infection model mice. DETAILED DESCRIPTION

[0049] In recent years, monomer compounds extracted from natural Chinese medicine have become a hotspot in the research of anti-HSV-1, and these monomer compounds have low toxicity and multi-target functions. Therefore, the present application intends to screen compounds or compositions with anti-HSV-1 activity from natural Chinese medicine to develop new anti-HSV-1 drugs, especially high-efficiency and low-toxicity anti-HSV-1 drugs.

[0050] Polygonum chinense L. belongs to Polygonaceae and Polygonum, and is a kind of traditional medicine with a long history, which has the effects of clearing heat and dampness, cooling blood and detoxifying, soothing liver and eyesight, and activating blood and relaxing tendon. At present, Polygonum chinense L. has been reported to have the pharmacological effects of anti-inflammatory, anti-oxidation, anti-hepatitis B virus, and antibacterial, but there is no literature report on its therapeutic effect on herpes virus, especially on herpes simplex virus HSV-1.

[0051] In the previous research of the inventors, a variety of ethanol extracts of natural Chinese medicine were obtained by the method of decocting Chinese medicine in folk, and the in vitro anti-viral activity of the ethanol extracts of these natural Chinese medicine on HSV-1 was detected, and it was found that the ethanol extract of Polygonum chinense L. had in vitro anti-HSV-1 viral activity.

[0052] In order to find the effective anti-HSV-1 active ingredient in the ethanol extract of Polygonum chinense L., the inventors carried out the following screening process:

[0053] Firstly, the ethanol extract of Polygonum chinense L. was prepared by decocting and methanol precipitation method, and the obtained ethanol extract of Polygonum chinense L. was extracted with equal amount of petroleum ether, ethyl acetate three times in turn, to obtain petroleum ether fraction, ethyl acetate fraction and water phase fraction, respectively, and then the in vitro anti-HSV-1 activity of the three fractions was detected by using HSV-1 infected Hela cells, and it was found that the HSV-1 components were mainly concentrated in the ethyl acetate phase and the water phase. Then the ethyl acetate fraction and the water phase fraction were separated and purified respectively, to obtain 7 components Er1-Er3 and Fr1-Fr4, and their in vitro anti-HSV-1 activity was determined, the component with the strongest anti-HSV-1 activity was screened out and verified at the animal level. The components with the best anti-HSV-1 viral effect were Er3 component and Fr2 component, and their anti-viral mechanism was studied, and it was found that the Er3 component and the Fr2 component mainly acted on the early stage of HSV-1 virus infection, could inhibit the expression of HSV-1 virus DNA, RNA and protein, and could inhibit the production of virus particles.

[0054] Meanwhile, the inventors explore the influence of the Er3 component and the Fr2 component of the extract of the fire carambola on the activity of other herpes viruses through in vitro experiments, and find that the same extract has different antiviral activities on different herpes viruses. The Er3 component and the Fr2 component have good inhibitory effect on the HCMV virus of the beta-herpes virus genus, can affect the adsorption of the virus on the cell, and have the ability to inactivate the virus. However, the Er3 component and the Fr2 component have no inhibitory effect on the varicella-zoster virus (VZV), which may be related to the particularity of the virus transmission of the VZV. The progeny virus of the VZV cannot be released into the supernatant, so the infection mode of the VZV is different from that of other viruses, that is, the VZV completes virus infection through cell contact.

[0055] Therefore, the ethyl acetate fraction or the components Er1-Er3 can be used alone to prepare an anti-HSV-1 drug, and the components Er1-Er3 can also be mixed to prepare an anti-HSV-1 drug. Similarly, the aqueous phase fraction or the components Fr1-Fr4 can be used alone to prepare an anti-HSV-1 drug, and the components Fr1-Fr4 can also be mixed to prepare an anti-HSV-1 drug. Among them, the anti-HSV-1 drug prepared from the component Fr2 and the anti-HSV-1 drug prepared from the component Er3 have the best effect. In addition, the components Er3 and Fr2 can also be mixed to prepare an anti-HSV-1 drug.

[0056] Based on the above findings, the application of the Er3 component and the Fr2 component in the preparation of an anti-HSV-1 drug is provided, and the application includes at least one of the application in the preparation of a drug for inhibiting the replication of HSV-1 and the application in the preparation of a drug for preventing and / or treating HSV-1 infection.

[0057] The materials used in the embodiments of the application are as follows:

[0058] Human cervical cancer cells (Hela cells) and African green monkey kidney cells (Vero cells) are purchased from the American Type Culture Collection (ATCC) and are stored in liquid nitrogen for standby use.

[0059] The HSV-1-GFP virus is preserved by the laboratory.

[0060] The required experimental animals are BALB / c mice, which are purchased from the Southern Medical University and have a production license number of SCXK (Yue) 2021-0041.

[0061] DMEM medium, CCK-8, positive drug acyclovir (ACV) were purchased from GIBCO company; DAPI was purchased from Beijing Zhenxingjin company; Polygonum chinense dry product was purchased from Guangxi Yaoshan Wild Fresh Herbal Medicine Shop; virus genome extraction kit, total RNA extraction kit were purchased from Yixing company; β-actin antibody was purchased from Proteintech company; HSV-1 ICP0, gB antibody, p65, p-p65 antibody were purchased from Santa company.

[0062] The primers were synthesized by Shenguo Bioengineering Co., Ltd.

[0063] The application will be further described below in combination with specific examples. The experimental methods in the following examples not specified in the specific conditions are generally according to the conditions suggested by the manufacturer.

[0064] Detection of anti-HSV-1 activity of ethanol extract of Polygonum orientale

[0065] (1) Preparation of Polygonum chinense ethanol extract

[0066] 500g of Polygonum chinense raw medicinal material was weighed, 15 times water was added and heated to boiling and kept for 15min, and the filtrate 1 was collected. The filtrate 2 was obtained by adding 10 times water to the filtrate and heating to boiling and keeping for 15min, and then filtering. The filtrates obtained in two times were combined and concentrated to 1g / mL, anhydrous ethanol was added to the final ethanol concentration of 60%, 4℃ precipitation for 12h, the supernatant was collected, and concentrated by rotary evaporation, to obtain Polygonum chinense ethanol extract.

[0067] (2) Cytotoxicity detection of Polygonum chinense ethanol extract

[0068] Hela cells or Vero cells were inoculated in a 96-well culture plate, each well containing 100μL of fresh complete culture medium, when the cell confluence reached more than 85%, the final concentration of 0.78mg / mL, 1.56mg / mL, 3.12mg / mL, 6.25mg / mL, 12.5mg / mL, 25mg / mL and 50mg / mL of Polygonum chinense ethanol extract (Polygonum chinense ethanol extract was dissolved in DMSO solution prepared by complete culture medium) was added, and then incubated at 37℃ in a 5% CO2 incubator for 48h, and then the cell activity was detected by CCK-8 method, and the half cytotoxicity concentration (CC 50 ) of Polygonum chinense ethanol extract was calculated.

[0069] (3) In vitro anti-viral activity of Polygonum chinense ethanol extract on HSV-1

[0070] Hela cells or Vero cells were inoculated in 96-well culture plates, and when the cell confluence reached more than 85%, HSV-1 virus was added to infect the cells, and then 0.78 mg / mL, 1.56 mg / mL, 3.12 mg / mL, 6.25 mg / mL, 12.5 mg / mL, 25 mg / mL and 50 mg / mL of the ethanol extract of Rubus corchorifolius L. (the ethanol extract of Rubus corchorifolius L. was dissolved in DMSO prepared with complete culture medium) were added, respectively, and the cells were further cultured at 37°C in a 5% CO2 incubator for 48 h. The GFP fluorescence produced by HSV-1 virus was detected, and the median effective concentration (EC 50 ) and the drug selection index (SI) of the ethanol extract of Rubus corchorifolius L. were calculated. 50 50 .

[0071] Referring to Figure 1 , Figure 2 and Table 1, the CC 50 , EC 50 and SI of the ethanol extract of Rubus corchorifolius L. in Vero cells were 26.34 mg / mL, 4.42 mg / mL and 6.19, respectively, and the CC 50 , EC 50 and SI of the ethanol extract of Rubus corchorifolius L. in Hela cells were 7.97 mg / mL, 1.03 mg / mL and 7.75, respectively. Therefore, the ethanol extract of Rubus corchorifolius L. had an inhibitory effect on HSV-1 in both Vero cells and Hela cells.

[0072] Table 1 Cytotoxicity and antiviral effect of the ethanol extract of Rubus corchorifolius L. in Hela and Vero cells

[0073]

[0074] Detection of anti-HSV-1 activity of each fraction of ethanol extract of Polygonum orientale

[0075] (1) Preparation of the extraction fraction of the fractionated extract of Rubus corchorifolius

[0076] ​The petroleum ether fraction, the ethyl acetate fraction and the water fraction of the Polygonum chinense ethanol extract obtained in Example 1 were collected and concentrated by rotary evaporation to obtain a petroleum ether fraction (Polygonum chinense PE extract), an ethyl acetate fraction (Polygonum chinense EtOAC extract) and a water fraction (Polygonum chinense extraction raffinate).

[0077] (2) Cytotoxicity test of the Polygonum chinense fractions

[0078] Hela cells were treated with three Polygonum chinense fractions at final concentrations of 15.6 mg / mL, 31.2 mg / mL, 62.5 mg / mL, 125 mg / mL, 250 mg / mL, 500 mg / mL and 1000 mg / mL, respectively, for 48 h, and then the cell viability was detected by CCK-8 method, and the CC 50 .

[0079] (3) In vitro anti-viral activity of the Polygonum chinense fractions against HSV-1

[0080] Hela cells were infected with HSV-1 virus, and the infected cells were treated with three Polygonum chinense fractions at final concentrations of 15.6 mg / mL, 31.2 mg / mL, 62.5 mg / mL, 125 mg / mL, 250 mg / mL, 500 mg / mL and 1000 mg / mL, respectively, for 48 h, and then the GFP fluorescence produced by HSV-1 virus was detected to calculate the EC 50 and SI of the drugs.

[0081] Referring to Figures 3 to 5 and Table 2, the petroleum ether fraction of Polygonum chinense had no significant effect on Hela cells and HSV-1 virus in Hela cells at concentrations ranging from 15.6 mg / mL to 1000 mg / mL, while the CC 50 of the ethyl acetate fraction and the water fraction of Polygonum chinense in Hela cells were 353.2 mg / mL and 980 mg / mL, respectively, the EC 50 were 41.61 mg / mL and 115.2 mg / mL, respectively, and the SI of HSV-1 virus in Hela cells were 8.488 and 8.507, respectively, indicating that the ethyl acetate fraction and the water fraction of Polygonum chinense had effective anti-HSV-1 activity.

[0082] Table 2 Cytotoxicity and anti-viral effect of Polygonum chinense fractions in Hela cells

[0083]

[0084] Detection of anti-HSV-1 activity of fractions Er1 to Er3 of Polygonum orientale

[0085] (1) Preparation of components Er1-Er3

[0086] The ethyl acetate extract of the fire charcoal mother liquor obtained in Example 2 was subjected to silica gel column chromatography, and a system of petroleum ether: ethyl acetate = 8:1-0:1 was selected. Each system was eluted at 3 column volumes, and 1 tube was collected every 20 mL. The obtained fractions were colored with 10% sulfuric acid ethanol, and components Er1, Er2 and Er3 were obtained according to TLC chromatography. The component in the section of petroleum ether: ethyl acetate = 2:1-1.5:1 was Er1, the component in the section of petroleum ether: ethyl acetate = 1:1-1:2 was Er2, and the component in the section of petroleum ether: ethyl acetate = 0:1 was Er3.

[0087] (2) Cytotoxicity detection of components Er1-Er3

[0088] Hela cells were treated with different concentrations of components Er1-Er3 for 48 h, and then the cell activity was detected by CCK-8 method, and the CC 50 was calculated.

[0089] (3) In vitro antiviral activity of components Er1-Er3 on HSV-1

[0090] HSV-1 virus infected cells were treated with different concentrations of components Er1-Er3 for 48 h, and the GFP fluorescence produced by HSV-1 virus was detected to calculate the drug EC 50 and SI.

[0091] Table 3 Cytotoxicity and antiviral effect of fire charcoal mother liquor extract in Hela cells

[0092]

[0093] Referring to Figures 6 to 8 and Table 3, the SI of components Er1-Er3 on HSV-1 virus in Hela cells was 8.86, 3.41 and 30.94 respectively, indicating that components Er1-Er3 of fire charcoal mother liquor all have effective anti-HSV-1 activity, and the selection index of component Er3 is the highest, which is used for subsequent pharmacodynamic test of antiviral activity.

[0094] Detection of anti-HSV-1 activity of fractions Fr1 to Fr4 of Polygonum orientale

[0095] (1) Preparation of components Fr1-Fr4

[0096] The macroporous resin chromatography was performed on the aqueous phase fraction of the fire charcoal mother liquor obtained in Example 2, and elution was performed with distilled water, 25% ethanol, 50% ethanol and 75% ethanol, respectively, at a speed of 3 BV / h, and four eluate fractions Fr1-Fr4 were collected.

[0097] (2) Cytotoxicity detection of components Fr1-Fr4

[0098] Hela cells were treated with different concentrations of components Fr1-Fr4 for 48 h, and then the cell activity was detected by CCK-8 method, and the CC 50 was calculated.

[0099] (3) In vitro antiviral activity of components Fr1-Fr4 against HSV-1

[0100] HSV-1 virus infected cells were treated with different concentrations of components Fr1-Fr4 for 48 h, and the GFP fluorescence produced by HSV-1 virus was detected to calculate the EC 50 and SI of the drugs.

[0101] Referring to Figures 9 to 12 and Table 4, the SI of components Fr1-Fr4 against HSV-1 virus in Hela cells were 25.51, 33.25, 11.19 and 3.45, respectively, indicating that the components Fr1-Fr4 of fire charcoal mother liquor all had effective anti-HSV-1 activity, and the selectivity index of component Fr2 was the highest, which was used for subsequent pharmacodynamic test of antiviral activity.

[0102] Table 4 Cytotoxicity and antiviral effect of fire charcoal mother liquor fraction in Hela cells

[0103]

[0104] Pharmacodynamic test of anti-viral activity of fractions Er3 and Fr2 of Polygonum orientale

[0105] (1) Inhibitory effect of components Er3 and Fr2 on HSV-1 virus

[0106] HSV-1 virus infected cells, and the infected cells were treated with component Er3 at a final concentration of 7.5 mg / mL, 15 mg / mL, 30 mg / mL, and component Fr2 at a final concentration of 25 mg / mL, 50 mg / mL, 100 mg / mL, with acyclovir (ACV) as a positive control group. After 12 h of treatment of the infected cells, the GFP fluorescence produced by HSV-1 virus was detected, total RNA and total protein of the cells were extracted, and the mRNA expression level and protein expression level of the immediate early gene a0 (early protein ICP0) and the late gene UL27 (targeting late protein gB protein) were detected by qPCR and Western blot, respectively. The primer sequences used in qPCR are shown in Table 5. Virions were harvested at 24 h and 48 h after treatment of the infected cells, and the viral genome copy number was calculated and the virus titer was detected by the PFU method.

[0107] Referring to Figures 13 to 20 , components Er3 and Fr2 can significantly reduce the GFP fluorescence produced by HSV-1 virus, significantly reduce the viral genome copy number of HSV-1 at 24 h and 48 h after infection, and reduce the virus titer of HSV-1. In addition, components Er3 and Fr2 can significantly inhibit the mRNA level of a0 and UL27 and the protein level of ICP0 and gB, and show a dose-dependent manner.

[0108] Table 5 qPCR primers and sequences

[0109]

[0110] (2) Action mode of components Er3 and Fr2 in inhibiting HSV-1

[0111] Referring to Figure 21 , four groups of action modes were set: (a) pre-cell: the cells were pretreated with drugs for 1 h before infection with virus; (b) inactivation: the cells were infected after the drugs were incubated with virus for 2 h; (c) attachment: the cells were infected at 4°C after the drugs were mixed with virus; and (d) replication: the drugs were added after the virus infected the cells for 2 h. Virions were harvested after 24 h, and the viral genome copy number was detected.

[0112] Referring to Figure 22 , Er3 and Fr2 can significantly inhibit the adsorption of virus into cells and inhibit the growth of virus during the replication process.

[0113] (3) Other action pathways of components Er3 and Fr2 in inhibiting HSV-1

[0114] HSV-1 virus infected cells, the infected cells were treated with different concentrations of component Er3 and component Fr2, 12h later, total RNA of cells was extracted and qPCR was used to detect the mRNA expression of cytokines, primer sequences were shown in Table 6, at the same time, total protein of cells was extracted, and Western blot was used to detect the phosphorylation level of p65 protein in NF-κB pathway.

[0115] Table 6 qPCR primers and sequences

[0116]

[0117]

[0118] Referring to Figures 23 to 26 After HSV-1 infection, the expression of host cell related antiviral genes IL-1β, IL-6 and TNF-α was up-regulated, and the results showed that the mRNA expression level of cytokines was significantly inhibited after treatment with component Er3 and component Fr2; Western blot results showed that the phosphorylation modification of p65 was inhibited after treatment with component Er3 and component Fr2, and the activation of NF-κB was inhibited, therefore, it was speculated that component Er3 and component Fr2 could inhibit the activation of NF-κB induced by virus, and further inhibit the expression of downstream inflammatory factors.

[0119] (4) Therapeutic effect of component Er3 and component Fr2 in HSV-1 skin infection model mice

[0120] Before the experiment, BALB / c mice were adaptively fed for one week, and the animals could freely eat during the feeding period. The mice were randomly divided into 5 groups, 5 mice in each group, namely blank control group (Mock group), model group (HSV-1 group), Er3 treatment group, Fr2 treatment group and positive control group (ACV group). After the BALB / c mice were infected with HSV-1 after the depilation and scratching of the flank, a mouse skin herpes virus infection model was established, and the infected site was smeared with component Er3 or component Fr2 or positive drug for 10 consecutive days, twice a day, 10 μL each time. The skin virus infection site of the mice was observed and photographed every day, and the body weight of the mice was recorded at the same time. After 10 days, the mice were sacrificed, the pathological changes of the virus infection site were observed, and the inflammation score was performed. The total RNA of the infected skin tissue of the mice was extracted, and qPCR was used to detect the mRNA expression level of the late gene gB in the tissue, primer sequences were shown in Table 5, and PFU method was used to detect the HSV-1 virus titer of the infected skin tissue.

[0121] Referring to Figure 27, relative to the Mock group, the infected site of HSV-1 infection group appeared scab with the increase of infection time and the scab exceeded the HSV-1 inoculation area, reached the peak on the third day after infection, and then the wound of the infected site gradually healed with the extension of time, the wound of the infected site healed faster after Er3 or Fr2 was applied, and the phenomenon of scab exceeding the HSV-1 inoculation area did not appear. See Figure 28 and Figure 29 HSV-1 infection caused inflammation at the infected site but had no effect on the weight of the mice, and the inflammation scores of the Er3 group, the Fr2 group and the ACV group were significantly lower than that of the HSV-1 group, indicating that the extracts of firethorn (components Er3 and Fr2) can alleviate inflammation at the infected site and have no effect on the weight of the mice. See Figure 30 and Figure 31 Components Er3 and Fr2 significantly inhibited the replication of HSV-1 virus in the skin tissue of the mice.

[0122] The present application detected the toxicity of the ethanol extract of firethorn on cells and its inhibitory effect on herpes simplex virus type 1 (HSV-1) in cells on African green monkey kidney Vero cells and human cervical cancer Hela cells, and the results showed that the ethanol extract of firethorn had dose-dependent anti-HSV-1 activity in vitro. The ethanol extract of firethorn was further separated and purified, and the anti-HSV-1 activity of the separated components was detected, and the components with the best antiviral effect were screened by selecting the index, i.e. the components Er3 separated from the ethyl acetate fraction and the components Fr2 separated from the water fraction, and the pharmacodynamics of the antiviral activity of the two components was further studied. The results of the cell experiment showed that the components Er3 and Fr2 mainly acted on the early stage of HSV-1 virus infection, and could inhibit the expression of HSV-1 virus DNA, RNA and protein, and could inhibit the production of virus particles, and had obvious anti-herpes simplex virus effect, and the experimental results showed that the components Er3 and Fr2 might inhibit the expression of downstream inflammatory factors by inhibiting the activation of virus-induced NF-κB, and the in vivo experiment further showed that the components Er3 and Fr2 improved the skin herpes caused by HSV-1 infection in mice.

[0123] In summary, the extracts of firethorn, i.e. the components Er3 and Fr2 provided by the present application, can inhibit HSV-1 infection through a new action pathway, have the advantages of good safety and high selection index, and can be used for preparing antiviral drugs against herpes simplex virus type 1 (HSV-1), expanding the treatment scheme of herpes virus infection diseases, and have wide application prospect and important clinical significance.

[0124] The above-described embodiments are merely preferred embodiments of the present application, and the protection scope of the present application is not limited thereto. Any equivalent substitutions or transformations made by those skilled in the art based on the present application are within the protection scope of the present application. The protection scope of the present application is subject to the claims.

Claims

1. A firethorn extract, characterized in that, The application relates to a preparation method of a firethorn extract for preparing an anti-HSV-I drug, and the preparation method comprises the following steps: S1: firethorn is used as raw material, 10-20 times of water by weight is added for decocting, the decocting time is 10-20 min, filtration is conducted, 5-15 times of water by weight is added for decocting the filter residue, the decocting time is 10-20 min, filtration is conducted, the filtrates are combined, vacuum concentration is conducted, then ethanol is added to make the final concentration of ethanol 60% v / v, centrifugation is conducted after standing at 4 DEG C for 6-18 h, the supernatant is collected, and vacuum concentration is conducted, so as to obtain an ethanol extract; S2: the ethanol extract is sequentially extracted with equal amounts of petroleum ether and ethyl acetate for three times, the ethyl acetate phase and the water phase are collected and vacuum concentrated, so as to obtain an ethyl acetate fraction and a water phase fraction; S3: the ethyl acetate fraction is subjected to silica gel column chromatography, or the water phase fraction is subjected to macroporous resin chromatography, so as to obtain the firethorn extract; The silica gel column chromatography of the ethyl acetate fraction comprises the following steps: the ethyl acetate fraction is subjected to silica gel column chromatography separation, a petroleum ether-ethyl acetate system with a volume ratio of 8:1-0:1 is used for gradient elution, the eluent with a volume ratio of 0:1 of petroleum ether: ethyl acetate is collected and concentrated, and the firethorn extract is obtained; The macroporous resin chromatography of the water phase fraction comprises the following steps: the water phase fraction is subjected to macroporous resin chromatography separation, distilled water and 25% ethanol are used for elution, the elution speed is 3 BV / h, the 25% ethanol eluent is collected and concentrated, and the firethorn extract is obtained.

2. The extract of Combretum lognium of claim 1, wherein, In the step S1, the material-liquid ratio of the firethorn to water is 1:15, the material-liquid ratio of the filter residue to water is 1:10, the decocting time is 15 min, and the standing time is 12 h.

3. The firethorn extract according to any one of claims 1-2 is used as an active ingredient in the preparation of an anti-HSV-I drug.

4. Use according to claim 3, characterized in that, The drug comprises pharmaceutically acceptable adjuvants.

5. Use according to claim 4, characterized in that, The drug is prepared into injections, oral liquids, granules, powders, tablets, capsules or external use patches.

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