CN113476497B - Prunella vulgaris extract with HSV (herpes Simplex Virus) resisting activity as well as preparation method and medical application thereof - Google Patents

Abstract

A Prunellae Spica extract with anti-HSV activity is prepared by collecting Prunellae Spica or its powder, adding 10-22 volume times of water, heating and reflux-extracting for 2-4 times, filtering, mixing filtrates, concentrating under reduced pressure, adding 0.2-1 volume times of ethanol to make ethanol concentration be 10-50% v/v, standing at 4 deg.C for 6-24 hr, centrifuging, washing obtained precipitate with 10-50% v/v ethanol for at least two times, collecting precipitate, and drying. The invention also provides a preparation method and application of the selfheal extract. The selfheal extract provided by the invention has obvious in-vivo and in-vitro anti-HSV effects, the in-vitro anti-HSV activity of the selfheal extract is doubled compared with that of an aqueous extract, and the selfheal extract also has stable inhibition effect on ACV drug-resistant HSV clinical strains and drug-resistant strains. Meanwhile, the medicine has good curative effect on treating herpes on skin and genitals caused by HSV-1 and HSV-2 infection.

Description

Prunella vulgaris extract with HSV (herpes Simplex Virus) resisting activity as well as preparation method and medical application thereof

Technical Field

The invention belongs to the technical field of medicines, relates to selfheal, and particularly relates to a selfheal extract with HSV (herpes simplex virus) resistance, and a preparation method and medical application thereof.

Background

Herpes Simplex Virus (HSV) is divided into type I (HSV-1) and type II (HSV-2), and the virus invades the body through oral cavity, genitals and the like, has long-term latency and repeated outbreak, has an increasing trend of morbidity, and can cause various diseases and serious complications. The clinical treatment medicine is mainly nucleoside analogues such as Acyclovir (ACV) and the like, and large-dose long-term administration is required for severe and frequently recurring cases, so that drug resistance is easily generated. Therefore, the development of anti-HSV drugs with different structures and mechanisms of action from acyclovir is urgently needed.

A traditional Chinese medicine antiviral screening platform is established in the early stage of an applicant team, traditional Chinese medicines with the effects of clearing heat and removing toxicity and antiviral activity reported in literatures are screened systematically, and the fact that the water extraction and alcohol precipitation part of the selfheal and part of polysaccharide components have remarkable HSV (herpes simplex virus) resisting effects is found.

The Prunellae Spica is dry cluster of Prunellae Spica Prunella vulgaris L. of Labiatae, and has effects of clearing pathogenic fire, improving eyesight, resolving hard mass, and relieving swelling. Can be used for treating conjunctival congestion, swelling and pain, eyeball nyctalgia, headache, giddiness, scrofula, goiter, mammary abscess, swelling and pain, etc., and has a long clinical history. For aphtha, fresh selfheal is added with salt and a little saliva, and the mixture is mashed and applied to an affected part, and the affected part is remained and not fully applied for three times, thus the aphtha is cured.

The Chinese invention patent application (201410561972.8) discloses a selfheal extract, which is prepared by the following steps: adding 10-22 times of water into Spica Prunellae, heating, reflux-extracting for 2-4 times, each for 0.5-2 hr, filtering, microfiltering the filtrate with 0.45 μm water film, collecting the filtrate after microfiltration with 0.45 μm water film, microfiltering the collected filtrate with 0.45 μm water film, passing through ultrafiltration membrane with molecular weight cutoff of 3-500kDa, collecting medicinal liquid not passing through the ultrafiltration membrane, concentrating, and drying. The Prunellae Spica water extract has anti-herpesvirus activity IC₅₀93.11 +/-23.46 mu g/mL; warp beamPolysaccharide extract IC after ultrafiltration purification₅₀77.56. + -. 4.19. mu.g/mL.

The Chinese invention application (201610066061.7) discloses a selfheal homogeneous polysaccharide, wherein the total sugar content of the homogeneous polysaccharide is 53-65%; the homogeneous polysaccharide has a molecular weight of 23.4kDa to 41.7 kDa; and said homopolysaccharide consists essentially of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose, the homopolysaccharide IC₅₀It was 84.84. mu.g/mL.

A research on ethanol fractional purification and physicochemical properties of Prunella vulgaris water-soluble polysaccharide (33, 4.1.1.2016, No.20,2012. Zhangxia, Neixiao, Li Chang, Xiongg) discloses a method for extracting Prunella vulgaris water-soluble polysaccharide: the method comprises the steps of obtaining prunella vulgaris polysaccharide from prunella vulgaris clusters by a water extraction and alcohol precipitation method, re-dissolving the obtained polysaccharide in water, and sequentially carrying out graded alcohol precipitation on the polysaccharide under the conditions that the volume fraction of ethanol is 20%, 30%, 40%, 50%, 60% and 70%, so as to obtain six polysaccharide components such as PVLP-1, PVLP-2, PVLP-3, PVLP-4, PVLP-5, PVLP-6 and the like. The basic physicochemical indexes of each component, such as sugar content, uronic acid content and protein content, are measured, the monosaccharide composition of each component is measured by gas chromatography, and the spectral properties of each component are inspected by scanning ultraviolet and infrared spectrums.

In view of the state of the prior art, the need to find the components of the anti-HSV of the selfheal, which have stronger anti-herpes virus activity, more concentrated active parts, safer and more efficient preparation process and are more suitable for industrial production, is needed.

Disclosure of Invention

The invention aims to provide a selfheal extract with centralized effective parts, simple and convenient extraction process and strong HSV (herpes Simplex Virus) resistance activity, a preparation method and medical application thereof.

The invention provides a selfheal extract with HSV (herpes simplex virus) resisting activity, which is prepared by the following method: taking selfheal fruit clusters or powder thereof, adding 10-22 volume times of water, heating to 80-100 ℃, carrying out reflux extraction for 2-4 times, each time for 0.5-2h, filtering, combining filtrates, carrying out reduced pressure concentration, then adding 0.2-1 volume times of ethanol of the concentrated solution to ensure that the ethanol concentration is 10-50% v/v, standing at 4 ℃ for 6-24 h, centrifuging, washing the obtained precipitate with 10-50% v/v ethanol for at least two times, collecting the precipitate, and drying to obtain the selfheal extract with the anti-HSV activity.

Further, 0.2 to 1 volume of ethanol to an ethanol concentration of 30% v/v of the concentrated solution is added to precipitate, the precipitate is left to stand at 4 ℃ for 6 to 24 hours and then centrifuged, and the obtained precipitate is washed at least twice with 30% v/v ethanol.

Furthermore, the molecular weight of the selfheal extract with the anti-HSV activity is more than 2.0 kDa.

Furthermore, the selfheal extract with the anti-HSV activity contains 20-40% of total sugar, 5-25% of protein and 15-30% of polyphenol by mass.

The invention also provides a preparation method of the selfheal extract with the anti-HSV activity, which comprises the steps of taking selfheal fruit spikes or powder thereof, adding 10-22 volume times of water, heating to 80-100 ℃, carrying out reflux extraction for 2-4 times, each time for 0.5-2h, filtering, combining filtrate, carrying out reduced pressure concentration, then adding 0.2-1 volume time of ethanol into the concentrated solution to ensure that the ethanol concentration is 10-50% v/v, standing at 4 ℃ for 6-24 h, centrifuging, washing the obtained precipitate with 10-50% v/v ethanol for at least two times, collecting the precipitate, and drying to obtain the selfheal extract with the anti-HSV activity.

The invention also provides application of the selfheal extract in preparing a medicament or a health-care product for preventing or treating herpes simplex virus infection.

The invention also provides a pharmaceutical composition which is characterized by containing the selfheal extract with the HSV resistance activity and pharmaceutically acceptable auxiliary materials.

The invention also provides application of the selfheal extract with the anti-HSV activity in preparing a medicament for preventing or treating herpes simplex virus infection.

The anti-HSV in vitro activity of the selfheal extract with the anti-HSV activity is obviously improved compared with other extraction parts of the selfheal, and the anti-HSV in vitro activity of the selfheal extract with the anti-HSV activity is improved, and the anti-HSV in vitro activity of the selfheal extract has EC (effective fraction) of HSV-1 and HSV-2₅₀45.62. + -. 3.37. mu.g/mL and 57.14. + -. 2.37. mu.g/mL respectivelymL, selectivity index SI is greater than 10.96 and 8.75 respectively, and the anti-HSV activity of the compound is enhanced by about one time compared with that of a selfheal aqueous extract. The derivative has the same inhibiting effect on Acyclovir (ACV) resistant HSV strains as standard strains, has stable anti-HSV effect, and can be used for treating herpes virus infection caused by ACV resistant HSV strains. The extract has remarkable effect of inhibiting guinea pig skin lesion and hind limb paralysis caused by type I herpes, and the inhibiting activity of high and medium dosage groups is superior to ACV. Compared with a virus control group, the extract can obviously relieve the vagina infection symptom of mice caused by HSV-2, relieve lesion conditions of focus parts and prolong the service life of the mice. The extract has positive identification reaction of saccharide, protein and polyphenol components, and negative identification reaction of free amino acids, quinones, saponins and flavonoids components.

The in vitro CCK-8 colorimetric method experiment and the in vivo anti-HSV show that: the selfheal extract provided by the invention has obvious in-vivo and in-vitro anti-HSV effects, the in-vitro anti-HSV activity of the selfheal extract is doubled compared with that of an aqueous extract, and the selfheal extract also has stable inhibition effect on ACV drug-resistant HSV clinical strains and drug-resistant strains. Meanwhile, the medicine has good curative effect on treating herpes on skin and genitals caused by HSV-1 and HSV-2 infection.

Compared with the prior art, the invention has remarkable technical progress. The selfheal extract disclosed by the invention has more concentrated HSV-resistant active ingredients, stronger pertinence and higher drug forming property, and other ingredients are more definite in composition; in addition, in terms of process, the method has the advantages of less alcohol consumption, no toxic reagent involved in the process, economy and high efficiency, and is suitable for industrial production.

Compared with the preparation method disclosed in the research on ethanol fractional purification and physical and chemical properties of the selfheal water-soluble polysaccharide, the fractional alcohol precipitation operation related by the invention is simpler, ethanol soaking and dialysis treatment are not needed, and selfheal clusters are directly extracted by hot water and then added with ethanol with corresponding concentration to obtain the self-heal polysaccharide; the grading alcohol precipitation is more accurate in segmentation, ethanol with corresponding concentration is added for precipitation, and then the precipitate is washed by ethanol with the same concentration, so that the precipitates with other alcohol precipitation concentrations caused by incomplete alcohol precipitation can be reduced, and the gel chromatography shows that the molecular weight distribution of each grading precipitate obtained by the method is more concentrated; and the monosaccharide composition and the major component content of the two are different.

The details of various aspects of the invention are set forth in subsequent sections. The features, objects, and advantages of the invention will be apparent from the description and from the claims.

The present invention will be further described with reference to specific embodiments, but the scope of the invention as claimed is not limited to the following embodiments.

Drawings

FIG. 1 shows a process for preparing a Prunella vulgaris extract.

FIG. 2 shows the process of the fractional alcohol precipitation of the aqueous extract of Prunella vulgaris.

FIG. 3 shows HPGPC chromatograms of fractions of aqueous extract and fractions of alcohol precipitation.

FIG. 4 shows UPLC-QTOF-MS/MS chromatograms of Prunellae Spica water extract and Prunellae Spica alcohol extract.

Fig. 5 shows skin lesions of guinea pigs after using the prunella vulgaris extract.

Fig. 6 shows the evaluation of the prunella vulgaris extract on skin lesions of guinea pigs.

Fig. 7 shows the scoring of guinea pig hind limb paralysis by the extract of prunella vulgaris.

Fig. 8 shows the change in body weight of guinea pigs after using the prunella vulgaris extract.

FIG. 9 shows the pathological changes of mice treated with the Prunella vulgaris extract.

Figure 10 shows the visual appearance of a prunella vulgaris extract with anti-HSV activity.

FIG. 11 shows HPGPC chromatograms of a Prunella vulgaris extract having anti-HSV activity.

FIG. 12 shows an HPLC chromatogram of the monosaccharide composition of an extract of Prunella vulgaris with anti-HSV activity.

FIG. 13 shows a chromatogram of amino acids from an extract of Prunella vulgaris with anti-HSV activity.

FIG. 14 shows HPGPC chromatogram comparison of 30% ethanol precipitated fraction obtained by "ethanol fractional purification of water-soluble polysaccharides of Prunellae Spica and research on physicochemical properties thereof" (33, No.20,2012. Zhang Xia, Neixao, Li Chang, Xiongg, Navy.) in 2016).

FIG. 15 HPGPC chromatography of aqueous Prunella vulgaris extracts prepared in example 1 of the present invention compared with PVE 30.

FIG. 16PVE30 monosaccharide composition HPLC chromatogram.

Detailed Description

Example 1

The technical scheme of the invention is carried out according to the following steps:

the preparation method of the different extraction parts of the selfheal comprises the following steps:

(1) preparation of Prunellae Spica extract with different solvents (see figure 1)

Extracting prunella vulgaris with 90% ethanol: extracting 50g of selfheal with 90% ethanol 20 times by volume for 2 times and 2 hours, filtering, removing dregs of a decoction, concentrating the extract, performing rotary evaporation at 50 ℃ and reduced pressure concentration, and drying to obtain a 90% ethanol extract of selfheal, which is named as PV 90E.

Selfheal 70% ethanol extract: extracting 50g of selfheal with 20 times of ethanol with the mass percent concentration of 70% for 2 times and 2 hours, filtering, removing dregs of a decoction, concentrating the extracting solution, performing rotary evaporation at 50 ℃ and reduced pressure concentration, and drying to obtain a selfheal 70% ethanol extract, which is named as PV 70E.

(2) Fractional alcohol precipitation of selfheal aqueous extract

Taking prunella vulgaris clusters or powder thereof, adding water with the volume 10-22 times that of the prunella vulgaris clusters or powder thereof, heating to 80-100 ℃, carrying out reflux extraction for 2-4 times, each time for 0.5-2h, filtering, combining filtrates, concentrating under reduced pressure to a proper amount, adding ethanol with different concentrations, respectively collecting precipitates, namely 30% (PVE30) sample of prunella vulgaris, 50% (PVE50) sample of prunella vulgaris, 70% (PVE70) sample of prunella vulgaris, 85% (PVE85) sample of prunella vulgaris, and supernatant (PVES) of prunella vulgaris, as shown in figure 2.

The preparation method comprises the following steps:

preparing an alcohol precipitation mother solution: taking 50g of selfheal, adding 20 times of volume of purified water, extracting for 1 time and 2 hours, filtering, removing dregs of a decoction, performing rotary evaporation and reduced pressure concentration on an extracting solution at 50 ℃ until the ratio of material to liquid is 1: 1.04.

Preparation of the 30% alcohol precipitation fraction: taking 100mL of alcohol precipitation mother liquor, placing the liquor to room temperature, stirring the liquor by using a magnetic stirrer while slowly adding 46mL of ethanol to ensure that the concentration of the ethanol in the liquor reaches 30%, sealing and placing the liquor in a constant-temperature environment at 4 ℃ for 12 hours, taking out the liquor, filtering the liquor, washing the precipitate by using 30% ethanol by mass percent until the filtrate is colorless, collecting the precipitate, and drying the precipitate to obtain a 30% alcohol precipitation part (PVE 30); the filtrates are combined, and concentrated by rotary evaporation at 50 ℃ under reduced pressure until no alcohol smell exists, namely 30% supernatant.

Preparation of 50% alcohol precipitation fraction: accurately measuring the volume of 50% of supernatant, converting the ethanol addition amount according to the volume of 50% of supernatant, adding ethanol with corresponding volume while stirring by a magnetic stirrer until the ethanol concentration of the solution reaches 50%, sealing and placing in a refrigerator at 4 ℃ for 12 hours, taking out, placing to room temperature, filtering, washing the precipitate with ethanol with the mass percentage concentration of 50% until the filtrate is colorless, collecting the precipitate, and freeze-drying to obtain a 50% ethanol precipitation part (PVE 50); the filtrates are combined, and concentrated by rotary evaporation at 50 ℃ under reduced pressure until no alcohol smell exists, namely 50% supernatant.

Preparation of 70% alcohol precipitation fraction: accurately measuring the volume of 70% of supernatant, converting the ethanol addition amount according to the volume of 70% of supernatant, adding ethanol with a corresponding volume while stirring by a magnetic stirrer to enable the ethanol concentration of the solution to reach 70%, sealing and placing in an environment at 4 ℃, taking out after 12 hours, placing to room temperature, filtering, washing the precipitate with ethanol with the mass percentage concentration of 70% until the filtrate is colorless, collecting the precipitate, and drying to obtain a 70% ethanol precipitation part (PVE 70); the filtrates are combined, and concentrated by rotary evaporation at 50 ℃ under reduced pressure until no alcohol smell exists, namely 70% supernatant.

Preparation of 85% alcohol precipitation fraction: accurately measuring the volume of the supernatant of 85 percent, converting the adding amount of ethanol according to the volume of the supernatant of 85 percent, adding ethanol with corresponding volume while stirring by a magnetic stirrer to ensure that the concentration of the ethanol in the solution reaches 85 percent, sealing and placing the solution in an environment at 4 ℃, taking out the solution after 12 hours, placing the solution at room temperature, filtering the solution, washing the precipitate by using the ethanol with the mass percent concentration of 85 percent until the filtrate is colorless, collecting and drying the precipitate, namely an ethanol precipitation part of 85 percent (PVE 85); the filtrates were combined and concentrated by rotary evaporation at 50 ℃ under reduced pressure until no alcoholic smell was observed, i.e., 85% Supernatant (also total Supernatant, Supernatant No.: PVES).

Example 2

In the embodiment, identification reaction, HPGPC, and fractional alcohol precipitation parts (PVE30, PVE50, PVE70 and PVE85) are adopted for chemical characterization, and UPLC-DAD-QTOF-MS/MS analysis is carried out on alcohol extracts (PV90E and PV70E) of selfheal and supernatant liquid (PVES) obtained by water extraction and alcohol precipitation.

(1) Identification reaction of selfheal water extract, alcohol extract and classified alcohol precipitation parts

The method comprises the following steps: subjecting the above fractions to Molish reaction (saccharide), iodine solution reaction (starch), Coomassie brilliant blue reaction (protein), ninhydrin reaction (free amino acid), and FeCl₃The reaction (tannin), the acetic anhydride concentrated sulfuric acid reaction (saponin), the Feigl reaction (quinones), the hydrochloric acid magnesium powder reaction (flavone), the potassium iodide precipitation reaction (alkaloid) and the like.

As a result: the Molish reaction, the ferric trichloride reaction, the acetic anhydride concentrated sulfuric acid reaction and the Coomassie brilliant blue reaction of the selfheal water extract and the supernatant fluid obtained by water extraction and alcohol Precipitation (PVES) are positive, which shows that the selfheal water extract, the alcohol extract and the supernatant fluid obtained by water extraction and alcohol precipitation contain saccharides, polyphenols, tannins, saponins and protein components; ninhydrin reaction, Feigl reaction and potassium iodide precipitation reaction are all negative, which shows that the prunella vulgaris water extract, alcohol extract and supernatant fluid obtained by water extraction and alcohol precipitation does not contain free amino acids, quinones and alkaloids.

The Molishh reaction, ferric trichloride reaction and Coomassie brilliant blue staining of precipitation parts (PVE30, PVE50, PVE70 and PVE85) of selfheal in each grading alcohol precipitation are positive, which shows that the water extract, alcohol extract and supernatant of water extraction and alcohol precipitation of selfheal all contain saccharide, polyphenol, tannin and protein components; the acetic anhydride concentrated sulfuric acid reaction, the ninhydrin reaction, the Feigl reaction, the potassium iodide precipitation reaction and the magnesium hydrochloride powder reaction are all negative, which shows that the selfheal graded alcohol precipitation part does not contain saponins, free amino acids, quinone alkaloid and flavonoid components, and the table 1 shows.

TABLE 1 identification of various parts of Prunella vulgaris

(2) Fractional alcohol precipitation of each fraction by HPGPC analysis

The method comprises the following steps: preparation of standard solution:

accurately weighing 1mg of each of Pullulan (Pullulan) reference products D1, D2, D3, D4, D5, D6, D7 and D8 (the molecular weights are 6100, 9600, 21100, 47100, 107000, 194000, 337000 and 642000 in sequence), adding 1mL of 0.02mol/L sodium chloride aqueous solution filtered by a 0.22 mu m aqueous phase filter membrane to prepare 1mg/mL of standard solution, fully dissolving, centrifuging at 10000rpm for 10min, sucking supernatant of each centrifuged standard solution, and sequencing for sample injection.

Preparing a test solution:

precisely weighing 2mg of a sample, precisely weighing 1mL of ultrapure water for dissolving, adding 1mL of 0.02mol/L sodium chloride aqueous solution filtered by a 0.22 mu m water-phase filter membrane to prepare 1mg/mL of standard solution, centrifuging at 10000rpm for 10min after full dissolution, sucking the centrifuged supernatant of the sample, transferring the supernatant into a liquid-phase sample bottle, and sequencing for sample injection.

③ chromatographic conditions:

the Sugar KS-802 and the Sugar KS-804 chromatographic columns are connected in series; 0.02mol/L sodium chloride water solution is used as a mobile phase (0.22 mu m water phase filter membrane filtration); the flow rate is 0.8 mL/min; the injection volume is 20 mu L; the analysis time is 25 min; the detector is a differential detector (RID); the temperature of the detection cell is 40 +/-0.1 ℃.

Fourthly, drawing a standard curve:

taking Pullulan D1, D2, D3, D4, D5, D6, D7 and D8 standard solutions, filtering the standard solutions by a 0.45-micron pore filter, adopting the selected chromatographic conditions, collecting data by GPC software of Agilent company, drawing a standard curve, and carrying out linear equation fitting by taking the standard molecular weight as the ordinate and the retention time of corresponding chromatographic peaks as the abscissa.

Measuring the molecular weight of the sample to be measured:

the molecular weight distribution range of the sample was measured using Agilent GPC software under the above chromatographic conditions.

As a result: for the aqueous extract and fractionated alcohol precipitated fractions (PVE30, PVE50, PVE70, PVE85, PVEs), the sample molecular weight distribution range was determined using High Performance Gel Permeation Chromatography (HPGPC) and parallax Refractometer (RID) and the following results were obtained:

as shown in fig. 3, from the HPGPC chromatogram, the aqueous extract of prunella vulgaris mainly consists of 4 main peaks, wherein the HPGPC chromatographic peak (peak a) of PVE30 is mainly distributed at 2 peaks in the aqueous extract, the weight average molecular weight (Mw), the number average molecular weight (Mn) and the distribution coefficient (D ═ Mw/Mn) of each part of the aqueous extract of prunella vulgaris after fractional alcohol precipitation are statistically analyzed, the specific data are shown in table 2, and statistics show that the Mw of PVE30 is 42.54kDa, the Mn is 23.71kDa, which indicates that PVE30 does not contain micromolecule components with molecular weight below 2.0 kDa.

TABLE 2 molecular weight distribution of each fractionated alcohol-precipitated fraction of aqueous extract of Prunellae Spica

(4) UPLC-DAD-QTOF-MS/MS analysis of alcohol extract and supernatant obtained by water extraction and alcohol precipitation

The alcohol extract of Prunellae Spica and supernatant obtained by water extraction and alcohol precipitation are analyzed by Waters ACQUITY UPLC-QTOF-MS instrument, UPLC BEH Shield RP18 chromatographic column and ESI ion source are used to perform substance identification on the water extract and alcohol extract, and the components are quantitatively and qualitatively distinguished, and the chromatographic conditions are shown in Table 3.

TABLE 3UPLC chromatographic conditions

As a result: by measuring the polarity and molecular weight range of the extract, people can judge that the parts of prunella vulgaris 70% alcohol extract (PV70E), 90% alcohol extract (PV90E), supernatant (PVES) obtained by water extraction and alcohol precipitation and the like mainly comprise micromolecule components with the molecular weight below 2.0kDa, and carry out UPLC-DAD-QTOF-MS/MS analysis on the small molecule components, as shown in figure 4, the UPLC chromatographic peak similarity of each part is very high; according to the comparison of the ion fragment information of the components with the literature, the components comprise phenolic acid components, such as caffeic acid, rosmarinic acid and derivatives thereof; flavonoid components such as protocatechuic acid, rutin, tanshinol, isoquercitrin or its analogues; triterpenoid saponin components such as oleanolic acid or ursolic acid, Prunellae Spica saponin B, betulinic acid, corosolic acid, etc.; and small molecule mono-or oligosaccharide components such as gluconic acid and the like (see table 4).

TABLE 4 Prunella vulgaris extract ion fragment information and component estimation

Example 3

The extracted parts of the selfheal prepared in the embodiment 1 are subjected to activity tests of HSV-1/KOS and HSV-2/G resistant standard strains, and parts with optimal HSV resistant activity are screened out.

The method comprises the following steps: CCK-8 colorimetric method is used for detecting the in-vitro inhibition effect of the selfheal extract on standard virus strains HSV-1/KOS and HSV-2/G.

After counting Vero cells, inoculating the Vero cells to a 96-well cell culture plate at the density of twenty thousand cells per well, and placing the Vero cells at the constant temperature of 37 ℃ and CO₂In an incubator with 5% volume percentage concentration, after the cells grow to a monolayer, the upper layer culture solution is discarded, and 100 XTCID is added₅₀After one hour of adsorption, prunella vulgaris extract samples (100. mu.g/mL, 50. mu.g/mL, 25. mu.g/mL, 12.5. mu.g/mL) of different concentrations, each 3 wells, 200. mu.L/well, at 37 ℃ and 5% CO by volume, were added₂Culturing for 3 days, adding CCK-8 at 10 μ L/well after 3 days, measuring absorbance at 450nm with enzyme labeling instrument, and calculating the virus inhibition and cell survival rate of Prunellae Spica extract according to formula 1.

As a result: determining cytotoxicity and HSV-resistant drug effect of each part obtained by fractional alcohol precipitation by CCK-8 method, and calculating EC₅₀And SI values. The results are shown in Table 5, CC of fractions on Vero cells₅₀The values are all more than 500 mu g/mL, which shows that each part has no obvious cytotoxicity; wherein the EC of the 30% alcohol precipitated fraction (PVE30) of the aqueous extract₅₀The value was minimal, i.e. the activity was optimal, whereas the remaining fractionated alcohol precipitated fractions (PVE85, PVE70, PVE50) were less active against HSV, and the 90% alcohol extract (PV90E), 70% alcohol extract (PV70E) and the water and alcohol Precipitated Supernatant (PVEs) were inactive (see table 5).

TABLE 5 extraction yield and in vitro anti-HSV activity of Prunella vulgaris parts

Through the examples 1-3, the alcohol extract and supernatant fluid after water extraction and alcohol precipitation are obtained, the anti-HSV activity is not significant, and the parts mainly comprise small molecular compounds with Mw below 2.0kDa, wherein the small molecular compounds comprise monosaccharide oligosaccharides, flavonoids, phenolic acids, saponins and the like. The results show that the small molecular compound with the molecular weight less than 2.0kDa, including rosmarinic acid which is an index component of the selfheal in Chinese pharmacopoeia, is not the basis of the main anti-HSV substance of the small molecular compound, the small molecular compound plays the role of anti-HSV and is mainly and intensively distributed on the water extraction and alcohol precipitation part with larger molecular weight, and the anti-HSV activity of the 30 percent alcohol precipitation part (PVE30) of the selfheal water extract with the Mw of 42.54kDa and the Mn of 23.71kDa is optimal. Therefore, the molecular weight of the selfheal extract (PVE30) protected by the patent is more than 2.0 kDa.

Example 4

Comparison of anti-HSV-1/KOS and anti-HSV-2/G standard strain activity of Prunellae Spica extract (PVE30) with Prunellae Spica water soluble polysaccharide

The method of the present application was used to prepare PVE 30.

According to the method of research on ethanol fractional purification and physicochemical properties of water-soluble polysaccharide of Prunella vulgaris (33 in 1/4/2016 (4/1/4), No.20,2012 Zhang Xia, Nei Shao Ping, Li Chang, Xiongying), the water-soluble polysaccharide of Prunella vulgaris is prepared, which comprises the following steps: the selfheal polysaccharide is obtained from selfheal fruit clusters by adopting a water extraction and alcohol precipitation method, the obtained polysaccharide is re-dissolved in water, and the polysaccharide is subjected to graded alcohol precipitation sequentially under the conditions that the volume fraction of ethanol is 20%, 30%, 40%, 50%, 60% and 70%, so that six polysaccharide components such as PVLP-1, PVLP-2, PVLP-3, PVLP-4, PVLP-5, PVLP-6 and the like are obtained. And selecting 30% alcohol precipitation part PVLP-2 for comparative analysis.

CCK-8 is the same as in example 3.

As a result: EC of PVE30 for HSV-1 and HSV-2₅₀45.62 +/-3.37 mu g/mL and 57.14 +/-2.37 mu g/mL respectively, the selectivity index SI is respectively more than 10.96 and 8.75, and the anti-HSV activity of the polysaccharide is doubled compared with that of common selfheal fruit-spike water-soluble polysaccharide (research on the alcohol classification and purification of common selfheal fruit-spike water-soluble polysaccharide and the physicochemical property thereof). EC between the two₅₀The values were statistically different (see table 6).

TABLE 6 comparison of the anti-HSV Activity of the Water-soluble polysaccharide of Prunella vulgaris with PVE30

T-test analysis was performed with statistical software, graphic prism, with P <0.05 indicating that the difference was statistically significant. (. P <0.05,. P <0.01,. P <0.001)

Accordingly, we propose a method for preparing a prunella vulgaris extract with stronger anti-HSV activity: taking selfheal fruit clusters or powder thereof, adding 10-22 times of water, heating to 80-100 ℃, reflux extracting for 2-4 times, each time for 0.5-2h, filtering, combining filtrates, concentrating under reduced pressure to a proper amount, adding 0.2-1 time of ethanol until the ethanol concentration is 10-50% v/v, standing for 6-24 h at 4 ℃, centrifuging, washing obtained precipitates with 10-50% v/v ethanol for at least two times, collecting the precipitates, and drying. To obtain Prunellae Spica extract PVE 30.

Example 5

Activity research of selfheal extract (PVE30) against HSV drug-resistant strains in vitro

The method comprises the following steps: CCK-8 colorimetric method is used for detecting the inhibition effect of the selfheal extract on drug-resistant strains HSV-1/Blue in vitro and clinical drug-resistant strains HSV-1/106 and HSV-1/153.

Vero cells were counted, inoculated into a 96-well plate at 2 ten thousand/well, allowed to stand in an incubator maintained at a constant temperature of 37 ℃ and a CO2 concentration of 5%, to allow Vero cells to grow into a monolayer, the supernatant culture solution was removed, and 0.085mL (containing 100 XTCID) of a dilution of each of the above virus stocks 1000-fold was inoculated thereto₅₀) After one hour of adsorption, the suspension is mixed with selfheal samples (100 mug/mL, 50 mug/mL, 25 mug/mL and 12.5 mug/mL) with different concentrations and added into Vero cells, 200 mug/well with 3 multiple wells of each concentration, and a normal Vero cell control group, an HSV virus control group and an ACV positive drug control group are arranged at the same time. Standing at constant temperature of 37 deg.C and CO₂Adding CCK-8 at a concentration of 5% into an incubator after 3 days, measuring absorbance at 450nm by using an enzyme-labeling instrument, and calculating the virus inhibition and cell survival rate of the selfheal extract according to the formula 1.

As a result: as shown in Table 7, the ACV administration concentration needs to be increased by about 300 times on two clinical strains, namely HSV-1/106, HSV-1/153 and a drug-resistant strain HSV-1/Blue, so that the inhibition effect similar to that of an anti-HSV-1/KOS standard strain can be exerted; therefore, compared with ACV, the selfheal extract has more stable effect of resisting ACV-resistant HSV-1 infection, and the activity of the drug-resistant strain is similar to that of a standard strain.

TABLE 7 anti-HSV-1/Blue, HSV-1/106 and HSV-1/153 activity of Prunella vulgaris extracts

Example 6

In vivo anti-HSV-1 activity study of Prunella vulgaris extract (PVE30)

The method comprises the following steps: (1) guinea pigs were anesthetized with isoflurane, depilated on the back, and 4 pieces of skin (1.5 cm. times.1.5 cm) were selected as the affected area. Sterilizing with 75% alcohol, puncturing into deep layer of skin with plum-blossom needle, adding dropwise 30 μ L HSV-1 stock solution, spreading with glass rod, friction infecting for 3min, and adding dropwise 30 μ L sterile physiological saline into normal control group. Randomly grouping after infection, wherein each experimental group comprises 2 guinea pigs, namely a normal control group; a virus control group; prunella vulgaris extract low dose group (20 mg/mL); a medium dose group (40mg/mL) of the selfheal extract; prunella vulgaris extract high dose group (80 mg/mL); positive drug ACV group (15 mg/mL).

(2) Administration was started 24 hours after infection, 50 μ L/mouse/dose, 2 times daily for 7 consecutive days for 2 weeks, and body weight and death were recorded, observed, and the degree of infected facial skin lesions and hind limb paralysis in each guinea pig were recorded according to the scoring criteria of table 8.

TABLE 8 Guinea pig affected area skin lesion degree and hind limb paralysis condition score criteria

As a result: the Prunellae Spica extract has good in vivo anti-HSV-1 activity

(1) Inhibition of infected facial skin lesions

Establishing a guinea pig back skin infection HSV-1 virus model according to the method, and observing the focus state of each group of infected guinea pigs: on day 3 after infection of the skin of each guinea pig group, the skin damage due to the needle pricks in the normal control group had been substantially recovered, and redness of the skin at the infected site of each infected guinea pig was observed; the symptoms are obvious on the 4 th day, small herpes and red swelling begin to appear, the symptoms of HSV-1 infection rapidly develop on the 5 th to 6 th days, the number of small herpes eruptions on the skin of a part of infected face is increased to 5 or more than 5, and the serious patients can be combined into tablets; scabbing begins at the lesion infected by the virus on days 7-8, and healing is reached on days 9-10. The lesion scores for each group of infected facial skin are shown in fig. 6, and the specific lesion status is shown in fig. 5. On day 4, the skin lesions of each treatment group were significantly reduced compared to the virus control group, and the scores of ACV and the high and medium dose groups were significantly lower than those of the virus control group, wherein the high dose group was reduced by 83%, which was significantly better than 37% of ACV. On days 5-6, the high and medium dose groups continuously maintain significant inhibition, the lesion scores of the high dose group are respectively reduced by 67% and 75%, the lesion scores of the medium dose group are respectively reduced by 44% and 46%, but the low dose group does not show inhibition activity; whereas, the ACV group had only a 15% reduction in lesion score at day 5, and no difference from the virus control group at the time points thereafter. In general, the selfheal extract has a remarkable effect of inhibiting guinea pig skin lesions caused by type I herpes, and is dose-related, wherein the inhibiting activity of high-dose and medium-dose groups is superior to that of ACV groups.

(2) Inhibiting effect on hind limb paralysis

The model of the back skin infection of guinea pigs with HSV-1 virus was established according to the above method, administration of the guinea pigs of each group was started 24 hours after the infection with HSV-1 virus, and the action and presence of hind limb paralysis symptoms were observed in each group of guinea pigs, with the following results: each infected guinea pig showed symptoms of hind limb movement and paralysis on day 6, and peaked at day 8-9. The hind limb paralysis score for each group is shown in figure 7, with the paralysis lesions of each treated group being significantly lower than those of the virus control group on day 6. At day 7, the scores of the high, medium and low dose groups of the selfheal extract are still significantly lower than those of the virus control group, and are respectively reduced by 81%, 75% and 75%, while the score of the ACV group is only reduced by 25%. At the following time points, the scores of the high and medium dose groups continued to be maintained at 60-75% of significant inhibition, the score of the low dose group remained at 50% of significant inhibition, but the ACV group had completely lost inhibition and died guinea pigs at day 10, and the low dose group of the prunella spike extract also died 1 guinea pig at day 13 (resulting in a sudden drop in the score curve between days 13-14). Therefore, from the viewpoint of the inhibition effect on the hind limb paralysis, each dosage group of the test medicament has a remarkable inhibition effect on the hind limb paralysis caused by the herpes I, the activity is remarkably superior to that of the ACV group, but the dose correlation of the high and medium dosage groups is not obvious. Wherein, the hind limb of 1 guinea pig in the high dose group is recovered after mild paralysis of hind limb, the hind limb of 1 guinea pig in each of the medium and low dose groups is kept normal without paralysis, and 1 guinea pig in the low dose group dies.

(3) Weight change in guinea pigs

A model of dorsal skin infection of guinea pigs with HSV-1 virus was established according to [0061], and administration was started 24 hours after infection with HSV-1 virus in groups of guinea pigs, and changes in body weight within 14 days of administration were recorded in groups of guinea pigs, and the results are shown in FIG. 8: the weight of 6 groups of guinea pigs is increased within 0 to 3 days; the weights of the HSV-1 virus control group and the selfheal extract group slowly declined from the fourth day, and declined to the lowest on day 7, and then slowly increased again. It can be seen that the weight loss is essentially consistent with the viral pathogenesis. The weight loss in the ACV group was delayed by 2 days, but the weight loss was more rapid thereafter, and was closely related to severe hind limb paralysis. The weight of each dose of the selfheal extract is slightly higher than that of a virus control group in 4 to 8 days, which is related to the inhibition effect of the medicament on virus-induced skin and hindlimb lesions, but has no obvious dose correlation. On days 9 to 11, the weight of each dose group of the selfheal extract is basically consistent with that of a virus control group. Starting at day 12, the body weight of each experimental group increased to some extent, indicating that the guinea pigs began to adapt to the condition of hind limb paralysis, and in addition, the score of hind limb paralysis was decreased in the selfheal extract group. Therefore, the change of the weight of the guinea pig is positively correlated with the disease course, each dosage group of the selfheal extract has certain improvement effect on the weight average of the body, but the dispersion is larger, and no obvious dosage correlation exists.

Example 7

In vivo anti-HSV-2 activity study of Prunella vulgaris extract (PVE30)

The method comprises the following steps: (1) 40 mice were randomly divided into 5 groups of 8 mice each, namely a normal control group, a virus control group, a Prunella vulgaris extract low dose group (50mg/mL), a Prunella vulgaris extract high dose group (75mg/mL), and an ACV positive drug control group (100 mg/mL).

(2) Disinfecting the pudendum of each group of mice by medical alcohol, and repeatedly rubbing the vaginal mucosa of the mice by using a rough and thin glass rod; knocking and hitting the mouse vulva surface with a medical seven-star needle for several times to enable the skin to ooze small blood beads, then extending the gastric lavage needle into the vagina of the mouse for 3cm, and injecting 100 mu L (1 multiplied by 107PFU/mL) of HSV-2 virus solution to enable the HSV-2 to permeate into vulval mucosal tissues. The sample preparation group is given 30 mu L of selfheal extract aqueous solution every day, and the positive drug control group is given equal volume of ACV aqueous solution, and continuous intervention is carried out for 7 days. Recording the weight change, the survival rate, the average survival days and the virus infection rate of the mice during the establishment of the vaginal infection HSV-2 model-making administration of the mice, and scoring and summarizing the skin injuries and the pathological changes of the infected mice according to the scoring standards in the table 9.

TABLE 9 skin injury and lesion scoring criteria for infected mice

(3) The drug intervention was continued for 7 days, and after another observation for 7 days, the experimental animals were sacrificed. The change in body weight of the mice was recorded daily and data analysis was performed on the survival rate, infection rate and lesion score of the mice.

As a result: the Prunellae Spica extract (PVE30) has good in vivo anti-HSV-2 activity

(1) Body weight changes in mice

According to the method, a mouse vagina infection HSV-2 virus model is established, the medicine is continuously intervened for 7 days, the observation is carried out for 7 days after the medicine is stopped, the weight change of each group of mice is recorded, and the results are shown in tables 10 and 11: the weight of the mice in the selfheal extract high-dose group is close to that of the ACV positive drug control group, the mice are continuously observed for 7 days after the drug is stopped, and the weight of the mice in the selfheal extract high-dose group is close to that of the normal control group and is heavier than that of the mice in other groups.

TABLE 10 continuous intervention 7 days (pre-dose-7 days) mice daily weight change (g/mouse)

TABLE 11 daily weight change (g/mouse) of mice 7 days after drug withdrawal (days 8-14)

(2) Survival rate and infection rate of mice

According to the method, a mouse vaginal infection HSV-2 model is established, external administration intervention is continuously carried out on each group of mice for 7 days, observation is carried out for 7 days after the administration is stopped, the survival rate and the infection rate of the mice are analyzed and recorded, and the results are as follows: compared with the virus control group, the onset time and death time of the selfheal extract high-dose group are delayed by 1-2 days, the survival rate of mice in the selfheal extract dried group is higher than that of the virus control group, and the average life of the mice in the high-dose group is prolonged by about 1 day (see table 12). The average survival days and the virus infection rate of the animals are reduced by 25 percent (see table 13).

Table 12 mouse survival (%)

TABLE 13 average survival days (days) and viral infection Rate (%)

(3) Mouse lesion scoring

According to the method, a mouse vagina infection HSV-2 virus model is established, selfheal extracts of all groups of mice are externally coated and continuously intervened for 7 days, the pathological changes of the mice are scored from the 4 th day of inoculation of HSV-2/G virus, and the results are shown in a table 14 and a table 9: the mice of the virus control group have the phenomena of red and swollen vaginal orifice, red and swollen vaginal periphery, red and swollen spread to rat tail and two sides, large-area lesion ulceration of lesions, lower limb paralysis and the like, and the total lesion score of the mice of the selfheal extract intervention group is obviously lower than that of the virus control group.

TABLE 14 summary of mouse lesion scores

Example 8

Chemical characterization of Prunella vulgaris extract (PVE30) with anti-HSV activity

1. A Prunella vulgaris extract (PVE30) with anti-HSV activity has physical and chemical properties determined as follows:

the method comprises the following steps: a Prunella vulgaris extract (PVE30) with anti-HSV activity has color observation and solubility determination:

as a result: an anti-HSV activity of Prunellae Spica extract (PVE30) is brown powder (shown in figure 10), is easily soluble in water, and is insoluble in organic solvent such as high-concentration methanol, ethanol, acetone, ethyl acetate, chloroform, n-butanol, etc.

2. Identification reaction of Prunellae Spica extract (PVE30) with HSV resisting activity

The method comprises the following steps: subjecting the above components to Molish reaction (saccharide), iodine solution reaction (starch), Coomassie brilliant blue reaction (protein), ninhydrin reaction (free amino acid), and FeCl₃The reaction (tannin), the acetic anhydride concentrated sulfuric acid reaction (saponin), the Feigl reaction (quinones), the hydrochloric acid magnesium powder reaction (flavone), the potassium iodide precipitation reaction (alkaloid) and the like.

As a result: the identification reaction results of various major components in the selfheal extract (PVE30) with the anti-HSV activity are shown in Table 15, and the Molish reaction, the ferric trichloride reaction and the Coomassie brilliant blue staining are positive, which shows that the PVE30 contains saccharide, polyphenol, tannin and protein components; the acetic anhydride concentrated sulfuric acid reaction, the ninhydrin reaction, the Feigl reaction, the potassium iodide precipitation reaction and the hydrochloric acid cent reaction are all negative, which shows that the selfheal graded alcohol precipitation part does not contain saponins, free amino acids, quinones, alkaloids and flavonoids.

TABLE 15 identification of chemical composition of a Prunella vulgaris extract (PVE30) with anti-HSV activity

3. A Prunellae Spica extract (PVE30) with HSV resisting activity and having molecular weight range

The method comprises the following steps: preparation of standard solution: accurately weighing 1mg of each of Pullulan (Pullulan) reference products D1, D2, D3, D4, D5, D6, D7 and D8 (the molecular weights are 6100, 9600, 21100, 47100, 107000, 194000, 337000 and 642000 in sequence), adding 1mL of 0.02mol/L sodium chloride aqueous solution filtered by a 0.22 mu m aqueous phase filter membrane to prepare 1mg/mL of standard solution, fully dissolving, centrifuging at 10000rpm for 10min, sucking supernatant of each centrifuged standard solution, and sequencing for sample injection.

Preparing a test solution: precisely weighing 2mg of a sample, precisely weighing 1mL of ultrapure water for dissolving, adding 1mL of 0.02mol/L sodium chloride aqueous solution filtered by a 0.22 mu m water-phase filter membrane to prepare 1mg/mL of standard solution, centrifuging at 10000rpm for 10min after full dissolution, sucking the centrifuged supernatant of the sample, transferring the supernatant into a liquid-phase sample bottle, and sequencing for sample injection.

③ chromatographic conditions: the Sugar KS-802 and the Sugar KS-804 chromatographic columns are connected in series; 0.02mol/L sodium chloride water solution is used as a mobile phase (0.22 mu m water phase filter membrane filtration); the flow rate is 0.8 mL/min; the injection volume is 20 mu L; the analysis time is 25 min; the detector is a differential detector (RID); the temperature of the detection cell is 40 +/-0.1 ℃.

Fourthly, drawing a standard curve: taking Pullulan D1, D2, D3, D4, D5, D6, D7 and D8 standard solutions, filtering the solutions by a 0.45-micron pore filter, adopting the selected chromatographic conditions, collecting data by using GPC software of Agilent company, drawing a standard curve, and performing linear equation fitting by taking the standard molecular weight as an ordinate and the retention time of a corresponding chromatographic peak as an abscissa.

Measuring the molecular weight of the sample to be measured: the molecular weight distribution range of the sample was measured using Agilent GPC software under the above chromatographic conditions.

As a result: HPGPC molecular weight Range determined that PVE30 had a Mw of 42.54kDa, a Mn of 23.71kDa, and a HPGPC chromatogram of FIG. 11.

4. Determination of total sugar, protein and polyphenol content of Prunellae Spica extract (PVE30) with HSV resisting activity

The method comprises the following steps:

firstly, measuring the total sugar content

Preparing a reference substance solution: accurately weighing 12.50mg of glucose standard substance, placing the glucose standard substance in a 25mL measuring flask, adding a proper amount of water to dissolve the glucose standard substance, diluting the glucose standard substance to a scale, and shaking up;

preparing a test solution: accurately weighing 12.5 mg-25 mL volumetric flask (concentration is 0.5mg/mL) of lyophilized powder of Prunellae Spica extract (PVE30) with HSV-resisting activity, adding pure water to constant volume to scale mark, ultrasonically treating to completely dissolve, and shaking.

Drawing a standard curve: precisely measuring reference substance standard solutions 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL, 1.2mL, 1.4mL, 1.6mL, 1.8mL and 2.0mL, respectively placing in test tubes with plugs, respectively adding water to make up to 2.0mL, precisely adding 5% phenol solution 1mL, shaking, rapidly and precisely adding sulfuric acid 5mL, shaking, standing for 30 minutes, cooling to room temperature, taking corresponding reagents as blanks, irradiating with ultraviolet-visible spectrophotometry (general rule 0401), measuring absorbance at 490nm wavelength, taking absorbance as ordinate and concentration as abscissa, and drawing a standard curve.

And (3) total sugar content determination: precisely measuring 1mL of a test solution, placing the test solution in a 10mL test tube with a plug, adding water to supplement the water to 2.0mL, measuring the absorbance according to the method under the preparation item of a standard curve from the point that 1mL of 5% phenol solution is precisely added, reading the weight of total sugar in the test solution from the standard curve, and calculating according to a formula 2 to obtain the glucose sensor.

Measurement of protein content

Preparing a reference substance solution: 10.12mg of bovine serum albumin reference substance is precisely weighed and placed in a 100mL measuring flask, and water is added for dissolving and diluting to a scale mark to prepare a reference substance solution with the concentration of 0.101 mg/mL.

Preparation of a test solution: accurately weighing 12.5 mg-25 mL volumetric flask (concentration is 0.5mg/mL) of lyophilized powder of Prunellae Spica extract (PVE30) with HSV-resisting activity, adding pure water to constant volume to scale mark, ultrasonically treating to completely dissolve, and shaking.

Drawing a standard curve: taking 0.1mL, 0.2mL, 0.3mL, 0.4mL, 0.5mL, 0.6mL, 0.7mL, 0.8mL, 0.9mL and 1.0mL of bovine serum albumin reference solution with the concentration of 0.101mg/mL into a stopple test tube, adding water to complement to 1.0mL, precisely adding 5mL of Coomassie brilliant blue test solution, shaking uniformly, taking a corresponding reagent as a blank, performing ultraviolet-visible spectrophotometry (2020 edition of pharmacopoeia, IV rules 0401), measuring the absorbance at the wavelength of 595nm, taking the absorbance as the ordinate and the concentration as the abscissa, and drawing a standard curve.

Protein content determination: precisely measuring 1mL of the test solution, placing the test solution in a 10mL test tube with a plug, measuring the absorbance according to the method from the point that 5mL of the Coomassie brilliant blue test solution is precisely added, reading the weight of the protein in the test solution from the standard curve, and calculating according to a formula 3 to obtain the test solution.

③ measurement of polyphenol content

Preparation of a reference solution: accurately weighing gallic acid reference substance 52.00mg, placing into 500mL measuring flask, adding water to dissolve, and diluting to scale mark to obtain reference substance solution with concentration of 0.104 mg/mL.

Preparation of a test solution: accurately weighing 12.5 mg-25 mL volumetric flask of lyophilized powder of Prunellae Spica extract (PVE30) with HSV-resisting activity (concentration is 0.5mg/mL, adding pure water to constant volume to scale mark, ultrasonically treating to completely dissolve, and shaking.

Drawing a standard curve: accurately weighing 0.5mL, 1.0mL, 2.0mL, 3.0mL, 4.0mL and 5.0mL of gallic acid reference substance solution with the concentration of 0.052mg/mL, respectively placing in 25mL brown volumetric flasks, respectively adding 1mL of phosphomolybdic tungstic acid test solution and 10mL of water, diluting to a scale with 29% sodium carbonate solution, shaking uniformly, standing for 30 minutes, measuring absorbance at 760nm wavelength according to an ultraviolet-visible spectrophotometry (2020 edition, China pharmacopoeia, Ministry of Japan 0401), and drawing a standard curve with the absorbance as ordinate and the concentration as abscissa.

And (3) measuring the polyphenol content: precisely measuring 2mL of a selfheal extract (PVE30) test solution with HSV (herpes simplex virus) resistance activity, placing the test solution into a 25mL brown volumetric flask, measuring absorbance according to a method from the point that 1mL of phosphomolybdic tungstic acid test solution is added, reading the weight of polyphenol in the test solution from a standard curve, and calculating according to a formula 4 to obtain the anti-HSV test solution.

As a result: see table 16.

TABLE 16 determination of the content of a Prunella vulgaris extract (PVE30) with anti-HSV activity

5. Monosaccharide composition of Prunellae Spica extract (PVE30) with HSV resisting activity

The method comprises the following steps: the pretreatment method comprises the following steps:

complete acid hydrolysis: taking a proper amount of selfheal extract (PVE30) freeze-dried powder with anti-HSV activity, putting the selfheal extract (PVE30) freeze-dried powder into a hydrolysis tube, adding 1mL of 4mol/L trifluoroacetic acid aqueous solution, standing the mixture in a constant-temperature oven at 120 ℃, hydrolyzing for 2 hours, taking out the mixture, and drying the mixture by nitrogen;

and (3) derivatization reaction: adding 1mL of 0.5mol/L PMP-methanol solution and 0.3mol/L aqueous solution of NaOH into a selfheal extract (PVE30) sample with HSV resistance activity after complete acid hydrolysis and nitrogen blow-drying treatment, standing in a constant-temperature water bath at 70 ℃ for 60 minutes, taking out and cooling, adding 0.3mol/L aqueous solution of LHCl 0.5mL, adding 0.5mL of chloroform, shaking uniformly, standing for 20 minutes, layering the solution, removing the lower layer of chloroform, adding chloroform again, repeatedly extracting for three times as before, taking the upper layer of aqueous solution, diluting to a constant volume of 2mL, appropriately diluting according to the concentration of the sample, filtering with a 0.22 mu m filter membrane, transferring to a sample bottle, and detecting by using a machine.

The instrument method comprises the following steps:

and (3) chromatographic column: a SHISEIDO C18 column (4.6 mm. times.250 mm, 5 μm);

mobile phase: a (0.1mol/L phosphoric acid aqueous solution, pH 6.8): b (acetonitrile) ═ 82: 18

Flow rate: 1.0 mL/min; column temperature: 25 ℃;

sample injection amount: 10 mu L of the solution;

wavelength: 245 nm.

As a result: a Prunellae Spica extract (PVE30) with anti-HSV activity is prepared by acid hydrolyzing and derivatizing, and determining monosaccharide composition and mole percentage by HPLC, wherein HPLC chromatogram of monosaccharide is shown in FIG. 12, and calculated according to monosaccharide standard, each monosaccharide content in PVE30 is from large to small respectively galacturonic acid (GalUA)208.10mg/g, galactose (Gal)32.79mg/g, glucose (Glc)23.56mg/g, arabinose (Ara)15.80mg/g, rhamnose (Rha)15.46mg/g, xylose (Xyl)15.32mg/g, mannose (Man)14.32mg/g, ribose (Rib)8.46mg/g, glucuronic acid (GlcUA)3.70mg/g, fucose (Fuc)2.87 mg/g; the molar percentage of each monosaccharide was calculated (see table 17).

TABLE 17 mole percent of monosaccharide composition of Prunella vulgaris extract (PVE30) having anti-HSV activity (%)

Amino acid composition of Prunellae Spica extract (PVE30) with HSV resisting activity

The method comprises the following steps: a Prunellae Spica extract (PVE30) with HSV resisting activity is subjected to pretreatment such as acid hydrolysis, and amino acid type and content of protein therein are analyzed by automatic amino acid tester.

The pretreatment method comprises the following steps:

weighing a proper amount of selfheal extract (PVE30) freeze-dried powder with anti-HSV activity, adding 10-15mL of 6mol/L hydrochloric acid aqueous solution into a hydrolysis tube, putting the hydrolysis tube into a dry ice refrigerant, standing for 3-5min, introducing nitrogen for protection, screwing a bottle cap, putting the hydrolysis tube into a constant-temperature electric heating blast box at 110 +/-1 ℃, hydrolyzing for 22 hours, taking out, standing and cooling to room temperature. Open the pipe of hydrolysising, wherein the hydrolysate filters, shifts to in the 50mL volumetric flask, then with the washing pipe of rinsing of a small amount of many times of purified water, the washing liquid shifts to above-mentioned 50mL volumetric flask in the same, adds the constant volume of purified water to the scale mark, shakes and makes the misce bene. Accurately sucking 1.0mL of filtrate in a 50mL volumetric flask, transferring to a 15mL test tube, drying at 40 ℃ under reduced pressure, adding 1.0mL of sodium citrate buffer solution with pH 2.2 for redissolving, fully shaking and mixing uniformly, filtering through a 0.22-micron filter membrane, and measuring on a machine.

The instrument method comprises the following steps:

a chromatographic column: a sulfonic acid type cationic resin;

wavelength: 570nm and 440 nm;

sample introduction amount: 500 mu L of the solution;

reaction temperature: 135 + -5 deg.C

As a result: amino acid composition detection of Prunellae Spica extract (PVE30) with anti-HSV activity shows that Prunellae Spica ethanol precipitation sample PVE30 contains protein, and main amino acids constituting the protein are glutamic acid, aspartic acid, arginine, glycine and serine (see Table 18, figure 13).

TABLE 18 amino acid composition of PVE30 (mg/g)

Example 9

Comparison of the composition of water-soluble polysaccharides prepared in "ethanol fractionation and purification of selfheal water-soluble polysaccharides and research on their physicochemical properties" (33, 4.1.1.2016 (No. 20,2012. kalasxia, ney heiping, li chang, xigming)) with the monosaccharide of PVE 30:

according to the descriptions in the research on the ethanol fractional purification and physical and chemical properties of the water-soluble polysaccharide of selfheal: the six components all contain rhamnose, arabinose, xylose, mannose, glucose and galactose, wherein the xylose and arabinose have the highest content, the mannose has the lowest content, ribose and fucose are not contained, but the specific molar composition proportion of monosaccharide in each component is different. Wherein the monosaccharide composition weight ratio of the 30% alcohol precipitation part PVLP-2 is as follows: rhamnose: arabinose: xylose: mannose: glucose: galactose ═ 1.20: 2.57: 3.63: 1.00: 0.94: 1.83.

the PVE30 provided by the invention contains galacturonic acid, galactose, glucose, arabinose, rhamnose, xylose, mannose, ribose, glucuronic acid and fucose, wherein the content of galacturonic acid and galactose is the highest, and the content of fucose is the lowest. After acid hydrolysis and derivatization, PVE30 measures monosaccharide composition and mole percentage by HPLC, wherein HPLC chromatogram of monosaccharide is shown in figure 16, and calculated according to monosaccharide standard products, the monosaccharide content in PVE30 is from large to small, and respectively comprises galacturonic acid (GalUA)208.10mg/g, galactose (Gal)32.79mg/g, glucose (Glc)23.56mg/g, arabinose (Ara)15.80mg/g, rhamnose (Rha)15.46mg/g, xylose (Xyl)15.32mg/g, mannose (Man)14.32mg/g, ribose (Rib)8.46mg/g, glucuronic acid (GlcUA)3.70mg/g and fucose (Fuc)2.87 mg/g; the molar percentages of the monosaccharides are shown in Table 17.

Claims (7)

1. A selfheal extract with HSV (herpes simplex virus) resisting activity is characterized by being prepared by the following method: taking selfheal fruit clusters or powder thereof, adding 10-22 volume times of water, heating to 80-100 ℃, reflux extracting for 2-4 times, each time for 0.5-2h, filtering, combining filtrates, concentrating under reduced pressure, then adding 0.2-1 volume times of ethanol into the concentrated solution to make the ethanol concentration be 30% v/v, standing for 6-24 h at 4 ℃, centrifuging, washing the obtained precipitate with 30% v/v ethanol for at least two times, collecting the precipitate, and drying.

2. A prunella vulgaris extract having anti-HSV activity according to claim 1 wherein: the selfheal extract with the anti-HSV activity contains 20-40% of total sugar, 5-25% of protein and 15-30% of polyphenol by mass.

3. A prunella vulgaris extract having anti-HSV activity according to claim 1 wherein: the molecular weight is more than 2.0 kDa.

4. A preparation method of a selfheal extract with HSV (herpes simplex virus) resisting activity is characterized by comprising the following steps: taking selfheal fruit clusters or powder thereof, adding 10-22 volume times of water, heating to 80-100 ℃, reflux extracting for 2-4 times, each time for 0.5-2h, filtering, combining filtrates, concentrating under reduced pressure, then adding 0.2-1 volume times of ethanol into the concentrated solution to make the ethanol concentration be 30% v/v, standing for 6-24 h at 4 ℃, centrifuging, washing the obtained precipitate with 30% v/v ethanol for at least two times, collecting the precipitate, and drying.

5. A pharmaceutical composition having anti-HSV activity, wherein the active ingredient of the pharmaceutical composition is the prunella vulgaris extract according to any one of claims 1 to 3.

6. The pharmaceutical composition of claim 5, further comprising a pharmaceutically acceptable excipient.

7. Use of an extract of Prunella vulgaris according to claim 1 having anti-HSV activity in the manufacture of a medicament for the prevention or treatment of herpes simplex virus infection.

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