Application of perfoliote knotweed herb aqueous extract in prevention and treatment of herpes virus or poxvirus infection

Abstract

The invention belongs to the field of medicines, and particularly discloses application of a perfoliote knotweed herb water extract in preventing and treating herpes virus and poxvirus infection. The extraction method of the perfoliote knotweed herb water extract comprises the following steps: S1, picking a perfoliote knotweed herb fresh product, cutting into sections, and crushing with a crusher; s2, performing ultrasonic extraction with deionized water at the normal temperature of 20-45 DEG C; s3, filtering with filter cloth to obtain a perfoliote knotweed herb water extract; and S4, standing the perfoliote knotweed herb water extract for 12-24 hours, and centrifuging to obtain water extract supernate. The perfoliote knotweed herb water extract obtained by the invention has inhibitory activity on poxviridae orthopoxvirus vaccinia virus infection, and can be used for treating pox, monkey pox and the like; the compound has inhibitory activity on herpes simplex virus type 1 and varicella-zoster virus infection of herpes viridae, and can be used for treating herpes simplex virus infection of oral cavity, periorbital and pudendum skin or mucosa and treating varicella and zoster. The method has application value in practice.

Description

Technical Field

[0001] The invention belongs to the field of medicines, and particularly relates to application of a water extract of Radix Codonopsis pilosulae in preventing and treating herpes virus and pox virus infections. Background Art

[0002] Polygonum perforatum Polygonum perfoliatum The whole herb of L. is also known as plowshare thorn, snake but not, snake retreat, river white grass, tiger thorn, triangular acid, cat claw thorn, and Miao medicine calls it Jiaouwan capsule. The medicine was first recorded in "Wanbing Huichun" and was used for snake bites. The 2020 edition of the Pharmacopoeia includes its dried above-ground part, and its effects are: clearing away heat and detoxifying, diuretic and detumescent, and cough; it can be used for sore throat, lung heat cough, children's cough, edema and oliguria, damp-heat diarrhea, eczema, furunculosis, snake and insect bites. "Jiangxi Folk Herbs" records: Fresh scutellaria baicalensis leaves, mashed and squeezed juice, adjusted with an appropriate amount of realgar powder, applied to the affected area to treat waist fire dan (herpes zoster). Fujian, Guangdong, Guangxi Zhuang Autonomous Region and other places also often use fresh scutellaria baicalensis to mash and apply externally to treat herpes zoster, eczema, carbuncle, furunculosis, and snake and insect bites.

[0003] The alcohol extract of Radix Angelicae Pubescentis slices showed anti-herpes virus HSV-1 activity at a toxic dose, which is different from the folk application of Radix Angelicae Pubescentis fresh products in the treatment of skin herpes. It has been reported that 80% ethanol was used as a solvent, 80℃ as an extraction temperature, a solid-liquid ratio of 1:10, and 2 times, each time for 2 hours, to extract flavonoid components in Radix Angelicae Pubescentis slices. The MTT method showed that the inhibition rate of the Radix Angelicae Pubescentis alcohol extract obtained by this method on HSV-1 was 40% at 780μg / ml, and there was a 20%-30% cytotoxic effect [Xu Yi. In vitro anti-herpes virus test of Radix Angelicae Pubescentis alcohol extract and research on the preparation process of gel, Hunan University of Traditional Chinese Medicine, 2017.].

[0004] However, there is no evidence that the fresh water extract of Radix Angelicae Pubescentis has a therapeutic effect on poxvirus and herpesvirus infections, nor has it had an inactivation effect on the above viruses. Summary of the invention

[0005] The invention is completed by studying the extraction method of the fresh water extract of Radix Corydalis and studying the therapeutic effect of the water extract on poxvirus and herpesvirus infection.

[0006] The present invention provides a method for extracting a water extract of Radix Glehniae, which comprises the following steps: S1 collects fresh whole herb of Radix Glehniae, cuts it into segments, and then crushes it with a grinder; S2 was extracted by ultrasonication with deionized water at room temperature of 20-45°C; S3 is then filtered with filter cloth to obtain the water extract of the bar plate; After the S4 bark extract was allowed to stand for 12 to 24 hours, it was centrifuged to obtain the water extract supernatant 1; Optionally, the method further comprises: S5 decompressing and concentrating the supernatant, and then centrifuging to obtain a water extraction supernatant 2; Optionally, S6 freezes the supernatant 2 with liquid nitrogen and then freeze-dries it to obtain an aqueous extract of Radix Glehniae.

[0007] Preferably, The fresh whole herb of S. truncatum in S1 was collected in September; The amount of deionized water in S2 is 5-15 times, for example, 9-11 times, such as 10 times, that of the sample of the scutellaria baicalensis. The soaking time is 30 to 100 minutes, specifically 50 to 70 minutes, for example 60 minutes; The ultrasonic extraction time is 30 minutes to 100 minutes, specifically 50-70 minutes, for example 60 minutes.

[0008] Also preferably, In S2, ultrasonic extraction is performed at a frequency of 30-40 kHz, a power of 400-600 W, and a temperature of 36-42° C.; more preferably, ultrasonic extraction is performed at a frequency of 35 kHz, a power of 500 W, and a temperature of 40° C. for 1 hour.

[0009] More preferably, In S3, 300-mesh filter cloth was used for filtration; In S4, after standing at 4°C for 12 to 24 hours, the centrifugation condition is 2500-3500 rpm for 15-15 min, specifically 3000 rpm for 10 min; The reduced pressure concentration in S5 is carried out after reduced pressure concentration at 40° C., and the centrifugation condition is 2500-3500 rpm for 15-15 min, specifically 3000 rpm for 10 min.

[0010] Further comprising the steps of: S7 performs water-methanol gradient separation on the water extract of Radix Glehniae through a C8 extraction column, and collects the Radix Glehniae fractions collected from 60 to 80 minutes as the water extract of Radix Glehniae.

[0011] The method further comprises the following steps: S8 separating the water extract of Glehnia littoralis obtained in S7 through 1K and 3K concentration centrifuge tubes to obtain a GBGH-6 component group with a molecular weight less than 1000 and / or a GBGH-6 component group with a molecular weight greater than 3000 as the final water extract of Glehnia littoralis.

[0012] The invention also provides a water extract of Radix Codonopsis pilosulae obtained by the extraction method.

[0013] The invention discloses a pharmaceutical composition for preventing or treating herpes virus and / or pox virus infection, which comprises the water extract of Radix Codonopsis pilosulae as an active ingredient and a pharmaceutically acceptable auxiliary agent.

[0014] The present invention also provides the water extract of *Polygonum perfoliatum*, and the application of the pharmaceutical composition in the preparation of a drug for preventing or treating herpes virus and / or poxvirus infection.

[0015] Specifically, the herpes virus infection refers to herpes simplex virus infection of the oral cavity, periorbital area, genital skin or mucosa, chickenpox or herpes zoster; the poxvirus infection refers to a disease caused by an infection with a virus of the genus *Orthopoxvirus*, such as smallpox and monkeypox.

[0016] The advantage of the present invention is that for the first time, the therapeutic effects of extracts obtained by different extraction methods of *Polygonum perfoliatum* on poxvirus and herpes virus infections have been studied, and the therapeutic effects of the water extract of *Polygonum perfoliatum* on poxvirus and herpes virus infections have been obtained. In particular, through research, it has been found that the water extract of *Polygonum perfoliatum* is subjected to water-methanol gradient separation through a C8 extraction column, and the fraction of *Polygonum perfoliatum* collected in 60 to 80 minutes has a therapeutic effect on poxvirus and herpes virus infections. Specifically, the water extract of *Polygonum perfoliatum* of the present invention has inhibitory activity against the infection of vaccinia virus of the genus *Orthopoxvirus* in the family Poxviridae, and can be used to treat diseases caused by infections with viruses of the genus *Orthopoxvirus* such as smallpox and monkeypox. At the same time, it has inhibitory activity against the infections of herpes simplex virus type 1 and varicella-zoster virus in the family Herpesviridae, and can be used for the treatment of herpes simplex virus infections of the oral cavity, periorbital area, genital skin or mucosa, as well as chickenpox and herpes zoster. Through microscopic observation of cytopathic effects, the water extract of fresh *Polygonum perfoliatum* at 400 μg / ml can completely inhibit HSV-1, and no cytotoxicity is observed; the above results are verified again by immunofluorescence method. It has great application value in practice. Description of the Drawings

[0017] Figure 1 . Anti-HSV-1 infection effect of the extract of fresh whole herb of *Polygonum perfoliatum*. Among them, the water extract of *Polygonum perfoliatum* at 400 μg / ml can effectively inhibit HSV-1 infection in Vero cells, but no inhibitory effect of the anhydrous ethanol extract on HSV-1 infection in Vero cells is observed.

[0018] Figure 2 . Anti-HSV-1 infection of the water extract of *Polygonum perfoliatum* in Vero cells.

[0019] Figure 3 . The water extracts of *Polygonum perfoliatum* harvested in different seasons all have the activity of anti-HSV-1 infection in Vero cells.

[0020] Figure 4 . Anti-HSV-1 infection activity of fractions of *Polygonum perfoliatum* (GBGH-6) with different molecular weights in Vero cells.

[0021] Figure 5 . Inhibitory effect of the water extract of fresh whole herb of *Polygonum perfoliatum* on VZV infection in MRC-5.

[0022] Figure 6 . Inhibitory effect of the aqueous extract of fresh whole herb of Polygonum perfoliatum L. on VACV infection in Vero cells. Detailed implementation manners

[0023] The present invention will be described below through specific implementation manners for better understanding, but it does not constitute a limitation to the present invention.

[0024] Example 1: Anti-herpes simplex virus activity of the aqueous extract of fresh Polygonum perfoliatum L. There are reports in the literature that the ethanol extract of dry Polygonum perfoliatum L. can reduce the herpes simplex virus infection rate, but it is quite different from the commonly used methods among the people. In this example, the inhibitory effects of the aqueous extract and ethanol extract of fresh Polygonum perfoliatum L. on HSV-1 infection were analyzed, and the harvesting time of the medicinal materials, thermal stability of the active ingredients, etc. were also analyzed. It was found that the aqueous extract of fresh Polygonum perfoliatum L. can more effectively inhibit HSV-1 infection in Vero cells. The aqueous extracts obtained by harvesting in summer and autumn both have anti-HSV-1 effects, and the components of Polygonum perfoliatum L. against HSV-1 have thermal stability.

[0025] Fresh whole herb of Polygonum perfoliatum L. was harvested in Meilin Town, Jieyang City, Guangdong Province in September. After being cut into small segments less than 5 cm, it was crushed with a pulverizer; one portion was taken respectively and soaked in 10-fold deionized water and absolute ethanol for 1 hour, and then ultrasonically extracted at a frequency of 35 kHz, a power of 500 W and 40 °C for 1 hour, and filtered through a 300-mesh filter cloth to obtain the aqueous extract and ethanol extract of Polygonum perfoliatum L.; after standing at 4 °C for 12 - 24 hours respectively, centrifuged at 3000 rpm for 10 min to obtain aqueous extract supernatant 1 and ethanol extract supernatant 1; the two supernatant 1 were concentrated under reduced pressure at 40 °C respectively, and then centrifuged at 3000 rpm for 10 min to obtain aqueous extract supernatant 2 and ethanol extract supernatant 2; the two supernatant 2 were rapidly frozen with liquid nitrogen and then freeze-dried to obtain the aqueous extract and ethanol extract of Polygonum perfoliatum L.

[0026] In this example, a herpes simplex virus HSV-1 infection model at the Vero cell level was used to analyze the anti-herpes simplex virus activity of Polygonum perfoliatum L. Vero cells in good growth state were seeded in 96-well plates. When they adhered and grew to 90% - 95% confluence, the HSV-1 virus solution diluted with 50 μL of serum-free DMEM medium (Gibco) was used at 100-fold TCID 50Vero cells were infected with the virus at a certain concentration, and 50 μL of serum-free DMEM medium was added to the normal control group. After preparing the aqueous extract dilution and ethanol extract dilution of Polygonum perfoliatum L. with DMEM containing 10% fetal bovine serum (Gibco), they were sterilized with a 0.22-μm sterile microporous filter membrane respectively. After 2 hours of infection, 50 μL of the aqueous extract dilution and ethanol extract dilution of Polygonum perfoliatum L. were added to the cell infection fluid in the corresponding wells, and gently tapped and mixed to make the final concentration of the aqueous extract of Polygonum perfoliatum L. reach 400 μg / ml and the final concentration of the ethanol extract of Polygonum perfoliatum L. reach 500 μg / ml, both equivalent to 20 mg of fresh Polygonum perfoliatum L. / ml; the normal control group and the HSV-1 virus infection model group were added with DMEM culture medium containing 10% fetal bovine serum. After the above cell culture plates were cultured in a 5% CO2 incubator at 37 °C for 72 hours, as shown under the microscope Figure 1 : All the cells in the HSV-1 infection model group showed lesions; the 400-μg / ml aqueous extract of Polygonum perfoliatum L. could inhibit the infection of HSV-1, and the cell state was good without lesions; no inhibitory effect of the drug on HSV-1 infection was seen in the 500-μg / ml ethanol extract group, and the cells showed lesions. Further, Vero cells infected with herpes simplex virus HSV-1 were intervened with the aqueous extract of Polygonum perfoliatum L. at 500, 400, 300, 250, 200, 100, 50, and 25 μg / ml. Immunofluorescence detection was performed 48 hours after HSV-1 infected Vero cells with the gD antibody of HSV-1+HSV-2 (abcam, AB6507) and goat anti-mouse fluorescent secondary antibody (abcam, AB150113), and DAPI was used to stain the cell nucleus as an internal reference control. As can be seen from Figure 2 : The 400-μg / ml aqueous extract of Polygonum perfoliatum L. could effectively inhibit the infection of herpes simplex virus HSV-1 in Vero cells.

[0027] Example 2: Anti-herpes simplex virus effect of the aqueous extract of fresh Polygonum perfoliatum L. harvested in different months The growth season of Polygonum perfoliatum L. mainly concentrates from May to October. By harvesting Polygonum perfoliatum L. in June in summer and September in autumn, according to the extraction conditions in Example 1: soaking in 10-fold deionized water for 1 hour, ultrasonic extraction at a frequency of 35 kHz, power of 500 W, and 40 °C for 1 hour, and filtering with a 300-mesh filter cloth to obtain the aqueous extract of Polygonum perfoliatum L. After standing at 4 °C for 12-24 hours, centrifuging at 3000 rpm for 10 min to obtain aqueous extract supernatant 1 and ethanol extract supernatant 1; after the two supernatant 1 were concentrated under reduced pressure at 40 °C and then centrifuged at 3000 rpm for 10 min again to obtain aqueous extract supernatant 2 and ethanol extract supernatant 2; the two supernatant 2 were quickly frozen in liquid nitrogen and then freeze-dried to obtain the aqueous extract of Polygonum perfoliatum L., and the aqueous extract of Polygonum perfoliatum L. harvested in different seasons was obtained by concentration and freeze-drying.

[0028] After Vero cells were infected with HSV-1 for 48 hours, immunofluorescence detection was performed using the gD antibody of HSV-1 + HSV-2 and goat anti-mouse fluorescent secondary antibody. From Figure 3 A of Figure 3 and B of 50 it can be seen that the half inhibitory dose (IC 50 ) of the aqueous extract of Polygonum perfoliatum L. harvested in September against HSV-1 infection in Vero cells was 137.4 μg / ml, while the IC 50 of the aqueous extract of Polygonum perfoliatum L. harvested in June against HSV-1 infection in Vero cells was 600.9 μg / ml. From the analysis of this result, Polygonum perfoliatum L. harvested in June and September both have the activity against HSV-1, and the anti-HSV-1 activity of the fresh herb aqueous extract of Polygonum perfoliatum L. harvested in September is better than that in June. It is speculated that this result is closely related to the rainfall and plant water content in different seasons.

[0029] Example 3: Anti-herpes simplex virus effects of fresh herb aqueous extracts by different methods The fresh herb of Polygonum perfoliatum L. harvested in September was soaked in 10-fold deionized water for 1 hour, and then the fresh herb extract of Polygonum perfoliatum L. was prepared by two methods: ultrasonic extraction in water at 40 °C for 1 hour and hot water decoction extraction for 30 minutes. After concentration and freeze-drying, the 40 °C ultrasonic aqueous extract of Polygonum perfoliatum L. and the hot water decoction extract of Polygonum perfoliatum L. were obtained. After Vero cells were infected with HSV-1 for 48 hours, immunofluorescence detection was performed using the gD antibody of HSV-1 + HSV-2 and goat anti-mouse fluorescent secondary antibody. From Figure 3 B of Figure 3 and C of

[0030] it can be seen that the aqueous extracts of Polygonum perfoliatum L. extracted at two temperature conditions of 40 °C and 100 °C both have the activity against HSV-1, indicating that the active components of Polygonum perfoliatum L. against herpes virus have thermal stability. After 500 mg of the 40 °C ultrasonic aqueous extract of Polygonum perfoliatum L. obtained in Example 1 was dissolved in 1 ml of 50% ethanol water, gradient separation was carried out through a C8 packing extraction column with a mass of 120 g. The gradient conditions were: 90% water - 10% methanol from 0 to 10 minutes, 90% water - 10% methanol → 0% water - 100% methanol from 10 to 110 minutes, and 100% methanol from 110 to 130 minutes. The flow rate was 10 ml / min. Through the water-methanol gradient separation by the C8 extraction column, the fraction of Polygonum perfoliatum L. collected from 60 to 80 minutes (GBGH-6) could inhibit the HSV-1 virus infection in Vero cells.

[0031] Specifically, the above-mentioned fraction of Polygonum perfoliatum L. was separated through 1K and 3K concentrative centrifugal tubes to obtain the GBGH-6 component group with a molecular weight less than 1000, the GBGH-6 component group with a molecular weight between 1000 and 3000, and the GBGH-6 component group with a molecular weight greater than 3000.

[0032] The specific method is as follows: For 3K and 1K concentrator centrifuge tubes, rinse: Rinse with 75% ethanol. Add 75% ethanol to the upper layer of the membrane, balance and centrifuge at 3000 rpm for 15 minutes. After removing the liquid in the upper layer of the membrane and the collection tube, rinse with deionized water. Add deionized water to the upper layer of the membrane, balance and centrifuge at 3000 rpm for 15 minutes, and then remove the liquid in the upper layer of the membrane and the collection tube.

[0033] GBGH-6 is concentrated by rotary evaporation at 40 °C and then freeze-dried to obtain a freeze-dried powder. Take 17.6 mg of the GBGH-6 freeze-dried powder, redissolve it with deionized water, centrifuge at 12000 rpm and take the supernatant, add it to the upper layer of a 3K concentrator centrifuge tube, centrifuge at 4000 rpm for 2 hours. After adding 2 ml of deionized water to the upper layer, centrifuge at 4 °C and 4000 rpm for 2 hours, and repeat this twice. Then, recover the liquid in the upper layer of the membrane and freeze-dry to obtain the fraction of GBGH-6 with a molecular weight greater than 3000; Transfer the collected liquid from the lower layer of the 3K concentrator centrifuge tube to the upper layer of the membrane of a pre-rinsed 1K concentrator centrifuge tube, centrifuge at 4000 rpm for 2 hours. After adding 2 ml of deionized water to the upper layer, centrifuge at 4 °C and 4000 rpm for 2 hours, and repeat this twice. Then, recover the liquid in the upper layer of the membrane and freeze-dry to obtain the fraction of GBGH-6 with a molecular weight of 1000 - 3000, and collect the lower layer liquid and freeze-dry to obtain the fraction of GBGH-6 with a molecular weight less than 1000.

[0034] As Figure 4 shown, through the gD immunofluorescence analysis of the HSV-1 infection model at the cellular level, it was found that: The fraction with a molecular weight greater than 3000 had an IC 50 of 23.09 μg / ml for HSV-1 infection in Vero; At a concentration of 500 μg / ml, the fraction with a molecular weight below 1000 showed activity in inhibiting HSV-1 infection in Vero; While within a concentration of 100 μg / ml, the fractions with molecular weights below 1000 and between 1000 - 3000 did not show activity in inhibiting HSV-1 infection in Vero. Thus, it was determined that the fraction of GBGH-6 with a molecular weight greater than 3000 could better inhibit HSV-1 infection in Vero cells at a lower dose. From this, it can be known that the active components against HSV-1 in the aqueous extract of Polygonum perfoliatum L. exist in the fractions with molecular weights less than 1000 and greater than 3000. The fraction of the aqueous extract of Polygonum perfoliatum L. with a molecular weight greater than 3000 has better anti-HSV-1 activity.

[0035] Example 5: Anti-varicella-zoster virus activity of the fresh Polygonum perfoliatum L. aqueous extract After the initial infection with herpes virus, it mostly turns into latent infection. When the body is stimulated by the outside world (chronic diseases, immune impairment, sun exposure, menstruation, emotional stress, etc.), it recurs, seriously affecting the health of humans and other animals. Although there are historical literature records of using the juice of Polygonum perfoliatum to treat "shingles" and clinical experience of using its fresh product to treat herpes zoster, there are currently no relevant reports on the inhibition of VZV infection by the water extract of Polygonum perfoliatum. In this example, the inhibitory effect of Polygonum perfoliatum on VZV infection was analyzed, and it was found that the water extract of fresh Polygonum perfoliatum could more effectively inhibit HSV-1 infection in MRC-5.

[0036] The MRC-5 cell line and EMEM culture medium were purchased from ATCC, and the varicella-zoster virus VZV with GFP fluorescence label was gifted by the Chinese Center for Disease Control and Prevention. MRC-5 cells in good growth state were seeded in 96-well plates. When they adhered and grew to 90%-95% confluence, the VZV virus solution diluted with 50 μL of serum-free EMEM medium (HyClone) was used to infect MRC-5 cells at a concentration of 100-fold TCID 50 The concentration was used to infect MRC-5 cells, and 50 μL of serum-free DMEM medium was added to the normal control group. The water extract of Polygonum perfoliatum by ultrasonic extraction at 40 °C in Example 1 was used, that is, Polygonum perfoliatum was soaked in 10-fold deionized water for 1 hour and then extracted by ultrasonic extraction at 40 °C for 1 hour to prepare the water extract of Polygonum perfoliatum. A 500 μg / ml dilution of the water extract of Polygonum perfoliatum was prepared with EMEM containing 10% fetal bovine serum (Gibco), sterilized with a 0.22 μm sterile microporous filter membrane, and serially diluted to 7.8 μg / ml. After 2 hours of infection, 50 μL of the dilution of the water extract of Polygonum perfoliatum and the dilution of the ethanol extract of Polygonum perfoliatum were added to the cell infection solution in the corresponding wells, and gently tapped and mixed to make the final concentration of the water extract of Polygonum perfoliatum reach 250, 125, 62.5, 31.2, 15.6, 7.8 μg / ml. The above cell culture plates were placed in a 5% CO2 incubator and cultured at 37 °C for 4 days. As Figure 5 seen in A in it: In the VZV infection model group, some cells showed lesions, and no lesions were seen in the cells in the intervention groups of 100 μg / ml of Polygonum perfoliatum and acyclovir; continued to culture until the 9th day after infection, under the microscope, large areas of cells in the VZV infection model group showed lesions, and lesions also appeared in the positive drug acyclovir intervention group of 100 μg / ml, while no obvious cell lesions were seen in the intervention group of 100 μg / ml of the water extract of Polygonum perfoliatum. The VZV infection rate was calculated by analyzing the number of MRC-5 cells with GFP fluorescence using the CQ1 high-content cell imaging system. VZV infection rate = (number of GFP fluorescence in the detection well - average number of GFP fluorescence in the normal control group) / (average number of GFP fluorescence in the infection group - average number of GFP fluorescence in the normal control group) * 100%. As Figure 5 seen in B in it, the IC 50 of the water extract of Polygonum perfoliatum in inhibiting varicella-zoster virus VZV infection in MRC-5 cells was 21.86 μg / ml.

[0037] Example 6: Water extract of fresh Polygonum perfoliatum inhibits poxvirus infection In this example, a vaccinia virus VACV infection model at the Vero cell level was used to analyze the anti-poxvirus effect of Polygonum perfoliatum. Vero cells in good growth state were evenly seeded into a 12-well plate and cultured in a 5% CO2 incubator at 37 °C for 24 hours until the cells grew into a monolayer. The VACV virus solution diluted with DMEM was used. Through preliminary experiments, the dilution factor at which the plaque count was about 100 after adding 400 μL of the virus solution and 400 μL of DMEM culture medium for 48 hours was determined as the working solution concentration of the VACV virus.

[0038] The supernatant of Vero cells in the 12-well plate was removed. 400 μL of DMEM culture medium was added to the negative control wells, and 400 μL of the working solution of the VACV virus solution was added to the infection wells. After standing in a 5% CO2 incubator at 37 °C for 1 hour, 400 μL of the diluted solution of the water extract of Polygonum perfoliatum obtained by ultrasonic extraction of Polygonum perfoliatum at 40 °C in Example 1 at the corresponding concentration was added to the wells in the drug intervention group, and gently shaken and mixed to make the final concentration of the water extract of Polygonum perfoliatum in the corresponding wells reach 400, 200, 100, 50 μg / ml. Another 400 μl of DMEM culture medium was added to the negative control wells and the VACV infection model wells. After standing in a 5% CO2 incubator at 37 °C for another 1 hour, 1 ml of the covering solution pre-warmed to 37 °C, that is, 2% carboxymethyl cellulose sterilized and an equal volume of 2×DMEM culture medium containing double antibiotics, was added to each well of the 12-well plate. After incubating in a 5% CO2 incubator at 37 °C for 48 h, 1 mL of 4% paraformaldehyde fixative was added to each well. After standing at room temperature for 1 hour, the liquid in the well plate was removed, and the residues on the surface were washed with water. Then, each well was stained with 1 ml of 0.2% crystal violet (2.5 L of 75% alcohol + 7.5 g of crystal violet) solution for 1 hour. After pouring out the staining solution, it was gently rinsed with water and inverted to dry. Photographs were taken and counted to analyze the inhibition rate. The results are as Figure 6 shown.

[0039] As Figure 6 can be seen, the water extract of fresh whole herb of Polygonum perfoliatum can completely inhibit the infection of vaccinia virus VACV in Vero cells at 200 μg / ml; in addition, after VACV was co-incubated with the water extract of Polygonum perfoliatum at 50 μg / ml and then acted on Vero cells, there were no VACV infection plaques on Vero cells, which indicates that the water extract of Polygonum perfoliatum at 50 μg / ml can inactivate herpes virus, and wet compresses, hydrogels or other preparations acting on the infected skin surface can kill the live virus on the skin surface and reduce the risk of poxvirus transmission.

Claims

1. A method for extracting the aqueous extract of Polygonum perfoliatum L., characterized in that, It includes the following steps: S1 Collect fresh whole plants of Polygonum perfoliatum L., cut them into sections, and then crush them with a pulverizer. S2 Use deionized water to perform ultrasonic extraction at room temperature of 20 - 45°C. S3 Then filter with a filter cloth to obtain the water extract of Polygonum perfoliatum L. S4 After the water extract of Polygonum perfoliatum L. stands for 12 - 24 hours, centrifuge to obtain the supernatant 1 of the water extract. Optionally, it further includes: S5 After decompression and concentration of the supernatant, centrifuge again to obtain the supernatant 2 of the water extract. Further optionally, S6 After rapid freezing of the supernatant 2 with liquid nitrogen, perform freeze-drying to obtain the water extract of Polygonum perfoliatum L.

2. The extraction method according to claim 1, wherein In S1, the fresh whole plants of Polygonum perfoliatum L. are collected in September. In S2, the amount of deionized water used is 5 - 15 times that of the Polygonum perfoliatum L. sample, for example, 9 - 11 times, such as 10 times. The soaking time is 30 minutes to 100 minutes, specifically 50 - 70 minutes, for example, 60 minutes. The ultrasonic extraction time is 30 minutes to 100 minutes, specifically 50 - 70 minutes, for example, 60 minutes.

3. The extraction method according to claim 2, wherein In S2, perform ultrasonic extraction at a frequency of 30 - 40 kHz, a power of 400 - 600 W, and a temperature of 36 - 42°C; more preferably, perform ultrasonic extraction at a frequency of 35 kHz, a power of 500 W, and a temperature of 40°C for 1 hour.

4. The extraction method according to claim 1, wherein In S3, filter with a 300-mesh filter cloth. In S4, after standing at 4°C for 12 - 24 hours, the centrifugation conditions are centrifugation at 2500 - 3500 revolutions per minute for 15 - 15 minutes, specifically centrifugation at 3000 revolutions per minute for 10 minutes. In S5, after decompression and concentration at 40°C, the centrifugation conditions are centrifugation at 2500 - 3500 revolutions per minute for 15 - 15 minutes, specifically centrifugation at 3000 revolutions per minute for 10 minutes.

5. The extraction method according to claim 1, wherein It further includes the following steps: S7 Perform water - methanol gradient separation on the water extract of Polygonum perfoliatum L. through a C8 extraction column, and collect the fraction of Polygonum perfoliatum L. collected in 60 to 80 minutes as the water extract of Polygonum perfoliatum L.

6. The extraction method according to claim 5, characterized in that It further includes the following steps: S8 Separate the water extract of Polygonum perfoliatum L. obtained in S7 through 1K and 3K concentrator centrifugal tubes to obtain the GBGH-6 component group with a molecular weight less than 1000 and / or the GBGH-6 component group with a molecular weight greater than 3000 as the final water extract of Polygonum perfoliatum L.

7. The water extract of Polygonum perfoliatum L. obtained by the extraction method according to any one of claims 1 to 6.

8. A pharmaceutical composition for preventing or treating herpesvirus and / or poxvirus infections, characterized in that, It uses the water extract of Polygonum perfoliatum L. according to claim 7 as an active ingredient, and pharmaceutically acceptable adjuvants.

9. The application of the water extract of Polygonum perfoliatum L. according to claim 7 and the pharmaceutical composition according to claim 8 in the preparation of a drug for preventing or treating herpes virus and / or poxvirus infection.

10. The application according to claim 9, wherein The herpes virus infection refers to herpes simplex virus infection of the oral cavity, periorbital area, genital skin or mucosa, chickenpox or herpes zoster; the poxvirus infection refers to diseases caused by orthopoxvirus infection, such as smallpox and monkeypox.

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