Water-soluble lignin-carbohydrate complex in spica prunellae as well as preparation method and application of water-soluble lignin-carbohydrate complex

Abstract

The invention discloses a water-soluble lignin-carbohydrate complex in spica prunellae as well as a preparation method and application of the water-soluble lignin-carbohydrate complex. According to the present invention, the lignin-carbohydrate complex extracted from prunella vulgaris can effectively inhibit herpes simplex viruses, especially HSV-1 / KOS, HSV-2 / G, HSV-106, HSV-BLUE and HSV-153;

Description

Technical Field

[0001] The invention belongs to the field of medicine, and particularly relates to a water-soluble lignin-carbohydrate complex in Prunella vulgaris and a preparation method and application thereof. Background Art

[0002] Herpes simplex virus (HSV) is a neurotropic virus belonging to the family Herpesviridae. There are eight different types of viruses, divided into three types: alpha, beta and gamma. Humans are the main host. HSV belongs to the alpha herpesvirus subgroup, which includes HSV-1 and HSV-2. HSV-1 and HSV-2 mainly cause mucocutaneous diseases because they affect the mucous membranes and skin, especially the oral and genital areas, respectively. Most HSV-1 infections result in oral herpes, while genital herpes is more commonly caused by HSV-2 infection. In 2016 alone, about two-thirds of people under the age of 50 worldwide were infected with herpes simplex virus type 1, an infected number of about 3.7 billion, while herpes simplex virus type 2 infected about 13%.

[0003] So far, only some drugs such as acyclovir, penciclovir, and valacyclovir can relieve symptoms until the virus is reactivated and symptoms recur. In addition, for immunocompromised people, long-term use of these drugs can lead to drug resistance. Therefore, it is urgent to develop new drugs with fewer side effects and different mechanisms to fight HSV.

[0004] Prunella vulgaris L. is a perennial herbaceous plant of the Lamiaceae family, widely distributed in temperate and tropical regions of Eurasia, northwest Africa and North America. Prunella vulgaris has been used in China for thousands of years, mainly to relieve sore throats, relieve fever, and promote wound healing. In traditional Chinese medicine theory, Prunella vulgaris is bitter and cold in nature, enters the liver meridian, and has the effects of clearing the liver, improving eyesight, and reducing swelling and dispersing nodules. Summary of the invention

[0005] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a water-soluble lignin-carbohydrate complex in Prunella vulgaris and a preparation method and use thereof, so as to solve the problem of lack of effective treatment for herpes simplex virus in the prior art.

[0006] To achieve the above-mentioned and other related purposes, the present invention provides a water-soluble lignin-carbohydrate complex from Prunella vulgaris, wherein the content of lignin-carbohydrate in the lignin-carbohydrate complex is 90%, and the remainder is impurities;

[0007] The monomer composition of lignin includes p-hydroxybenzaldehyde (H type), guaiacylpropane (G type), and syringylpropane (S type), among which p-hydroxybenzaldehyde (H type) is 14.2%, guaiacylpropane (G type) is 21.7%, and syringylpropane (S type) is 64.1%;

[0008] The carbohydrate monosaccharide composition includes mannose, ribose, rhamnose, glucose, glucuronic acid, galactose, galacturonic acid, xylose, and arabinose, wherein the mass ratio of each component to carbohydrate is: mannose 10.97%, ribose 0.63%, rhamnose 3.71%, glucose 15.33%, glucuronic acid 0.73%, galactose 20.48%, galacturonic acid 2.39%, xylose 40.74%, and arabinose 5.01%, and the remainder is impurities.

[0009] Among them, 14.2% of p-hydroxyphenylpropane (H type) refers to the mass ratio of p-hydroxyphenylpropane to lignin monomer, and the rest are the same.

[0010] Preferably, the lignin-carbohydrate complex further comprises any one or more of the following technical features:

[0011] a) number average molecular weight is 20225;

[0012] b) weight average molecular weight (Mw) is 37981;

[0013] c) Z-average molecular weight (Mz) is 64217;

[0014] d) The polydispersity index (Mw / Mn) is 1.87.

[0015] Furthermore, the two-dimensional heteronuclear single quantum coherence nuclear magnetic resonance image of the lignin-carbohydrate complex showed that in the aromatic region, three basic structural units of lignin were identified, and the signal at the chemical shift δC / δH128.9 / 7.21 was attributed to C 2,6 -H 2,6 , indicating the presence of a p-hydroxyphenyl structure (H unit); guaiacyl propane (G type), δC / δH 110 / 7.0 corresponds to C 2 -H 2 , δC / δH 115.5 / 7.00 and 6.78 and 119.1 / 6.78 are respectively assigned to C 5 -H 5 and C 6 -H 6 The signal of the syringyl unit (S unit) at δC / δH 103.8 / 6.68 corresponds to C 2,6 -H 2,6 ;

[0016] In addition, obvious β-O-4 bond and xyloside signals were identified in the side chain region, with δC / δH 62.6 / 3.37 and 3.82 (C 5 -H 5 )、72.5 / 2.95(C 2 -H 2 )、74.0 / 3.22(C 3 -H 3 ) and 75.9 / 3.49(C 4 -H 4 ) was assigned to methyl β-D-xylopyranoside, δC / δH 75.8 / 4.70 (C 3 -H 3 ) indicated the presence of 3-O-acetyl-modified xyloside; at δC / δH 60.0 / 3.11–3.88 (C γ -H γ ) indicated the presence of a γ-hydroxylated β-O-4 substructure.

[0017] Furthermore, the infrared scanning spectrum of the lignin-carbohydrate complex shows that the absorption peak at 3234.05 cm-1 belongs to OH stretching vibration, the absorption peaks at 1590 cm-1 and 1518 cm-1 belong to the stretching vibration of the aromatic ring C=C skeleton, the absorption peak at 1401 cm-1 belongs to CH bending vibration, the absorption peaks at 1156 cm-1 and 1074 cm-1 belong to COC stretching vibration or CO stretching vibration, the absorption peak at 1011 cm-1 belongs to CO stretching vibration, the absorption peak at 951 cm-1 belongs to sugar ring vibration or CO stretching vibration, and the absorption peaks at 809 cm-1 and 763 cm-1 belong to the out-of-plane bending vibration of the aromatic ring CH.

[0018] Furthermore, the ultraviolet scanning spectrum of the lignin-carbohydrate complex shows that there are absorption peaks at 280 nm and 324 nm.

[0019] Another aspect of the present invention provides the use of the above lignin-carbohydrate complex in the preparation of anti-herpes simplex virus drugs.

[0020] Furthermore, the herpes simplex virus refers to any one or more of HSV-1 / KOS, HSV-2 / G, HSV-106, HSV-BLUE or HSV-153.

[0021] Another aspect of the present invention provides a method for preparing the above-mentioned lignin-carbohydrate complex, the method comprising the following steps:

[0022] a) grinding the alcohol precipitate extract of Prunella vulgaris, adding LiCl / DMSO and stirring in a magnetic stirrer for 24 hours to obtain an extract

[0023] b) adding the extract obtained in step a) to an aqueous solution of 1,4-dioxane and stirring the mixture in a magnetic stirrer for 24 hours to obtain an extract;

[0024] c) centrifuging the extract obtained in step b) and taking the supernatant;

[0025] d) adding the supernatant to a 3500Da dialysis bag and dialyzing it in pure water;

[0026] e) The contents of the dialysis bag are concentrated under reduced pressure, re-dissolved in acetic acid / water solution, dropped into pure water for regeneration, centrifuged, and the supernatant is concentrated by distillation under reduced pressure and freeze-dried to obtain the product.

[0027] Furthermore, the alcohol precipitation extract of Prunella Vulgaris refers to a 30% alcohol precipitation extract.

[0028] Furthermore, the step a) is specifically as follows: 2 g of 30% alcohol precipitate extract of Prunella vulgaris is added into a grinding tube, a 2 mm steel ball is placed in the tube, the tube is ground at 60 Hz using a grinder for 2 hours, 50 ml of 10% LiCl / DMSO is added, and the tube is placed in a magnetic stirrer for stirring and extraction for 24 hours.

[0029] Furthermore, the step b) is specifically as follows: the extract obtained in step a) is mixed into 3 times the volume of 75% (V / V) 1,4-dioxane aqueous solution and placed in a magnetic stirrer for stirring and extraction for 24 hours.

[0030] Furthermore, the step c) is specifically as follows: centrifuging the extract obtained in step b) at 14400rpm for 10min, taking the supernatant, adding the lower precipitate to 50ml of 75% (V / V) 1,4-dioxane aqueous solution and placing it in a magnetic stirrer for stirring and extracting for 24h; centrifuging the obtained extract at 14400rpm for 10min, taking the supernatant, adding the lower precipitate to 50ml of 75% (V / V) 1,4-dioxane aqueous solution and placing it in a magnetic stirrer for stirring and extracting for 24h; centrifuging the obtained extract at 14400rpm for 10min, taking the supernatant, and then combining the obtained supernatants.

[0031] Furthermore, the step d) is specifically to mix the obtained supernatants, add them into a 3500Da dialysis bag, place them in 3L pure water for dialysis, change the water every 8 hours, and dialyze for a total of 72 hours.

[0032] Furthermore, the step e) is specifically as follows: the contents of the dialysis bag are concentrated under reduced pressure, re-dissolved in 20 ml of 90% (V / V) acetic acid / water solution, dripped into 80 ml of pure water for regeneration, centrifuged at 14400 rpm for 10 min, the supernatant is concentrated by reduced pressure distillation, and freeze-dried to obtain LCC-S.

[0033] As described above, the water-soluble lignin-carbohydrate complex in Prunella vulgaris of the present invention and its preparation method and use have the following beneficial effects:

[0034] The water-soluble lignin-carbohydrate complex can effectively inhibit herpes simplex viruses, especially HSV-1 / KOS, HSV-2 / G, HSV-106, HSV-BLUE and HSV-153. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the 2D-HSQC image of LCC-S

[0036] Figure 2 This is the elution spectrum of the high performance gel permeation chromatography of LCC-S

[0037] Figure 3 This is the infrared scan of LCC-S

[0038] Figure 4 This is the UV scanning spectrum of LCC-S

[0039] Figure 5 This is the intuitive appearance of LCC-S

[0040] Figure 6 This is the GC-MS chromatogram of the lignin monomer composition of LCC-S

[0041] Figure 7 This is the HPLC chromatogram of the monosaccharide composition of LCC-S DETAILED DESCRIPTION

[0042] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0043] The high performance gel permeation chromatograph was Waters 1525, the detector was Waters 2414, and the chromatographic column was Agilent PLgel 5um MIXED-C (made in GB)

[0044] The mass spectrometer was Shimadzu GCMS-QP2010 SE quadrupole gas chromatography-mass spectrometry, and the chromatographic column used was Agilrnt DB-5MS (30m*0.250mm).

[0045] The HPLC column used for analysis was a SHISEIDO C18 column (4.6 mm × 250 mm, 5 μm);

[0046] The UV spectrophotometer is Shanghai Meipuda P2 single-beam UV-visible spectrophotometer;

[0047] Infrared spectra were tested using a Perkin-Elmer 577 spectrometer;

[0048] NMR was tested using a BrukerAV-600MHz spectrometer;

[0049] For the liquid phase, acetonitrile was of chromatography grade, water was distilled water, and trifluoroacetic acid was of analytical grade;

[0050] Other reagents were of synthetic grade.

[0051] Example 1 Isolation and purification of LCC-S from Prunella vulgaris

[0052] a) 2 g of 30% alcohol precipitate extract of Prunella vulgaris was added to a grinding tube, a 2 mm steel ball was placed in the tube, and the tube was ground at 60 Hz for 2 hours. 50 ml of 10% LiCl / DMSO was added and the tube was stirred and extracted with a magnetic stirrer for 24 hours.

[0053] b) The extract obtained in step a) was mixed with 3 times the volume of 75% (V / V) 1,4-dioxane aqueous solution and placed in a magnetic stirrer for extraction for 24 hours.

[0054] c) The extract obtained in step b) was centrifuged at 14400 rpm for 10 min, the supernatant was collected, and the lower precipitate was added to 50 ml of 75% (V / V) 1,4-dioxane aqueous solution and stirred in a magnetic stirrer for 24 h.

[0055] d) The extract obtained in step c) was centrifuged at 14400 rpm for 10 min, the supernatant was collected, and the lower precipitate was added to 50 ml of 75% (V / V) 1,4-dioxane aqueous solution and stirred in a magnetic stirrer for 24 h.

[0056] e) The extract obtained in step d) was centrifuged at 14400 rpm for 10 min, and the supernatant was collected.

[0057] f) The supernatants obtained in steps b), c) and d) were mixed, added to a 3500Da dialysis bag, and dialyzed in 3L of pure water, with the water changed every 8 hours for a total of 72 hours.

[0058] g) The contents of the dialysis bag in step f) were concentrated under reduced pressure, re-dissolved in 20 ml of 90% (V / V) acetic acid / water solution, and dropped into 80 ml of pure water for regeneration. The mixture was centrifuged at 14400 rpm for 10 min, and the supernatant was concentrated by distillation under reduced pressure and freeze-dried to obtain LCC-S.

[0059] in, Figure 1 This is the 2D-HSQC (two-dimensional heteronuclear single quantum coherence nuclear magnetic resonance) image of LCC-S; it can be seen that in the aromatic region, the three basic structural units of lignin are identified, and the signal at the chemical shift δC / δH 128.9 / 7.21 is attributed to C 2,6 -H 2, 6, indicating the presence of a p-hydroxyphenyl structure (H unit). Guaiacyl unit (G unit), δC / δH 110 / 7.0 corresponds to C 2 -H 2 , δC / δH 115.5 / 7.00 and 6.78 and 119.1 / 6.78 are respectively assigned to C 5 -H 5 and C 6 -H 6 , which is consistent with the typical characteristics of the guaiacyl unit. The signal of the syringyl unit (S unit) at δC / δH 103.8 / 6.68 corresponds to C 2,6 -H 2,6 In addition, it can be observed that the lower structure signals of p-coumarin ester (pCA) and ferulic acid ester (FA) are more prominent, with δC / δH 145.1 / 7.49 (C α -H α )、130.5 / 7.49(C 2,6 -H 2,6 )、116.8 / 6.46(C β -H β ) and 115.3 / 6.72(C 3,5 -H 3,5 ) are consistent with the structure of esterified p-coumaric acid. δC / δH 122.5 / 7.12 (C 6 -H 6 )、111.5 / 7.29(C 2 -H 2 ) and 116.8 / 6.46(C β -H β ) indicates that the ferulic acid ester is connected by an ester bond. In the side chain region, obvious β-O-4 bonds and xyloside signals were identified, with δC / δH 62.6 / 3.37 and 3.82 (C 5 -H 5 )、72.5 / 2.95(C 2 -H 2)、74.0 / 3.22(C 3 -H 3 ) and 75.9 / 3.49(C 4 -H 4 ) is attributed to methyl β-D-xylopyranoside. δC / δH 75.8 / 4.70 (C 3 -H 3 ) indicates the presence of 3-O-acetyl-modified xyloside. γ -H γ ) indicated the presence of a γ-hydroxylated β-O-4 substructure, which is a typical connection mode of lignin-carbohydrate complexes.

[0060] Figure 3 This is the infrared scanning spectrum of LCC-S, which shows:

[0061] The absorption peak at 3234.05 cm-1 is attributed to OH stretching vibration, indicating the presence of hydroxyl groups in the sample, which may be phenolic hydroxyl groups in lignin or alcoholic hydroxyl groups in carbohydrates, and may be related to intermolecular hydrogen bonds. The absorption peaks at 1590 cm-1 and 1518 cm-1 are attributed to the stretching vibration of the aromatic ring C=C skeleton, which is the characteristic absorption of the benzene ring in the guaiacyl and syringyl groups in lignin. The absorption peak at 1401 cm-1 is attributed to CH bending vibration, which may come from the methoxy group (-OCH 3 ) or CH in carbohydrates 2 / CH 3 Deformation vibration. The absorption peaks at 1156 cm-1 and 1074 cm-1 are attributed to COC stretching vibration or CO stretching vibration, which may be due to the ether bonds in carbohydrates or the aliphatic / aromatic ether bonds in lignin.

[0062] The absorption peak at 1011cm-1 is attributed to CO stretching vibration, which may come from the COC of the sugar ring in carbohydrates or the CO vibration of the lignin side chain. The absorption peak at 951cm-1 is attributed to the sugar ring vibration or CO stretching vibration, which may be the vibration characteristic of the xylose or arabinose pyranose ring. The absorption peaks at 809cm-1 and 763cm-1 are attributed to the out-of-plane bending vibration of the aromatic ring CH.

[0063] Figure 4 This is the UV scanning spectrum of LCC-S, which shows:

[0064] The absorption at 280nm is more obvious, which is the characteristic absorption of non-conjugated phenolic groups, and the absorption at 324nm is caused by conjugated phenolic groups p-coumaric acid and ferulic acid.

[0065] Figure 5It is an intuitive appearance of LCC-S.

[0066] Example 2 HPGPC determination of the molecular weight of lignin-carbohydrate complex LCC-S

[0067] The molecular weight distribution of the water-soluble lignin-carbohydrate complex LCC-S was analyzed using a Waters 1525 room temperature gel chromatograph equipped with a differential refractive index detector (Waters 2414). The analysis conditions were as follows:

[0068] Mobile phase: DMSO; Flow rate: 1mL / min; Injection volume: 100μL; Chromatographic column: Agilent PLgel5umMIXED-C (made in GB); Column temperature: 35℃; Standard sample: polymethyl methacrylate. The analysis results were calculated using the absolute molecular weight method. The results are as follows ( Figure 2 ):

[0069] Number average molecular weight (M n )=20225

[0070] Weight average molecular weight (M w )=37981

[0071] Z-average molecular weight (M z )=64217

[0072] Polydispersity index (M w / M n )=1.87.

[0073] Example 3 Analysis of the ratio of lignin monomers in LCC-S using nitrobenzene oxidation method

[0074] Weigh LCC-S (30 mg) in a centrifuge tube, add nitrobenzene (0.25 mL), 4 mol / L sodium hydroxide solution (2 mL) and seal and hydrolyze at 95 ° C for 24 hours. Take out and cool, add 6 mol / L hydrochloric acid (1.6 mL), and shake well. Centrifuge the centrifuge tube at 13000 r / min for 5 minutes. Take 500 μL of the supernatant in a microtube, add ethyl acetate (2 mL) and extract twice, and take the organic phase to dry with nitrogen.

[0075] Weigh 0.05 g (accurate to 0.1 mg) of p-hydroxybenzaldehyde, vanillin, and syringaldehyde standards respectively, dissolve them in methanol and make up to 50 mL, and seal them at 4°C. Pipette 62.5 μL, 125 μL, 250 μL, 500 μL, and 2000 μL of the standard stock solution (1.0 mg / mL) respectively, and make up to 10 mL with methanol. The concentrations of the standard series working solutions are 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, and 200 μg / mL, respectively.

[0076] 1 mg of sample was re-dissolved in methanol, filtered with a 0.45 μm microporous membrane, and analyzed by GC-MS. The chromatographic conditions were as follows: carrier gas: helium; chromatographic column: Agilent DB-5MS (30 m*0.250 mm); column temperature: 70 °C; injection temperature: 250 °C; split ratio: 1:10; heating rate: 20 °C / min; heating program: 70 °C (4 min), 70 °C-240 °C (7.5 min) The results are as follows (Table 1 and Figure 6 )

[0077] The mass spectrometry conditions were as follows:

[0078] Ion source temperature: 200°C

[0079] Interface temperature: 250℃

[0080] Solvent delay: 4min

[0081] Table 1 Lignin monomer composition ratio of LCC-S

[0082]

[0083] Example 4 Analysis of Monosaccharide Composition in LCC-S Using PMP Pre-column Derivatization

[0084] Weigh LCC-S (20 mg) in a 20 ml reaction tube, add 2 mol / L trifluoroacetic acid solution (5 ml), seal and hydrolyze at 120°C for 4 h. Take out and cool, pipette into 1 ml reaction tube and concentrate under reduced pressure, add methanol and evaporate until there is no sour taste, add 1 ml pure water to re-dissolve, pipette 200 μL into a 2 ml centrifuge tube, add 100 μL of 1-phenyl-3-methyl-5-pyrazolone (PMP) methanol solution, 0.3 mol / l NaOH (100 μl), derivatize at 70°C for 60 min, cool, add 0.3 mol / l HCl (100 μl) to neutralize, extract with chloroform 3 times, and filter the water layer with a 0.22 um filter membrane.

[0085] Accurately weigh appropriate amounts of D-Man, D-Rib, Rha, D-GlcUA, D-GalUA, D-Glc, Gal, D-Xyl, and Ara, and dissolve them in water to prepare five different concentrations of 20%, 50%, 100%, 150%, and 200% with reference to the concentrations of each component in the test sample. Take 200 μl and add 0.5 mol / l PMP (100 μ) and 0.3 mol / l NaOH (100 μl). Derivatize at 70°C for 60 min, cool, and then add 0.3 mol / l HCl (100 μl) to neutralize. Extract with chloroform three times and filter the aqueous layer through a 0.22 um filter membrane.

[0086] Accurately pipette 10 μl of the standard curve solution and the test solution respectively, inject into the liquid chromatograph, measure, and record the chromatogram.

[0087] Chromatographic conditions: Agilent 5TC-C18 (250*4.6mm, 5um) chromatographic column was used for injection detection on HPLC-PDA, the mobile phase was phosphate buffer (pH=6.8) as the aqueous phase, acetonitrile as the organic phase, the aqueous phase: organic phase was 82:18 (V:V) isocratic elution, the flow rate was 0.8ml / min, the column temperature was 35℃, the injection volume was (10μl), and the detection wavelength was 250nm. The results are shown in Table 2 and Figure 7 .

[0088] Table 2 LCC-S monosaccharide composition

[0089]

[0090] Example 5 Analysis of total sugar content in LCC-S using phenol-concentrated sulfuric acid method

[0091] Accurately weigh LCC-S (30 mg), add water to make up to 100 ml, and the test solution is obtained. Take an appropriate amount of anhydrous glucose reference substance, accurately weigh it, add water to make 1 ml of a solution containing 0.2 mg / ml, and the standard solution is obtained. Accurately measure 0.2, 0.4, 0.6, 0.8, and 1.0 ml of the reference solution, respectively, and place them in 10 ml stoppered test tubes, add water to make up to 2.0 ml, accurately add 5% phenol solution (1 ml) (prepare before use), shake well, then accurately add sulfuric acid (5 ml), shake well, cool at room temperature, use the corresponding reagent as blank, measure the absorbance at a wavelength of 490 nm, and draw a standard curve with absorbance as the ordinate and concentration as the abscissa. Accurately measure the test solution (1.0 ml) and place it in a 10 ml stoppered test tube. Add water to each tube to make up to 2.0 ml. Accurately add 5% phenol solution (1 ml), shake well, then accurately add sulfuric acid (5 ml), shake well, cool at room temperature, and use the corresponding reagent as a blank to measure the absorbance at a wavelength of 490 nm. The total sugar content of LCC-S was measured to be 22.5%. Example 6 CCK-8 colorimetric method to detect the in vitro inhibitory effect of LCC-S on standard virus strains HSV-1 / KOS and HSV-2 / G.

[0092] 1. Experimental Materials

[0093] African green monkey kidney cells Vero, herpes simplex virus type 1 KOS strain (HSV-1 / KOS), and herpes simplex virus type 2 G strain (HSV-2 / G) were purchased from ATCC cell bank.

[0094] 2. Experimental Methods

[0095] Vero cells were cultured at 2×10 4Each well was plated in a 96-well plate; 37°C / 5% CO 2 After 24 h of incubation, 100 TCID 50 100 μL / well of HSV virus dilution, 200 μL / well of maintenance culture medium added to blank control wells and cell control wells; incubate at 37℃ for 1 hour, discard the culture medium, wash twice with PBS, add 100, 50, 25, 12.5, 6.25, 3.125, 0 μg / mL of LCC-S 200 μL / well to the experimental wells, 200 μL / well of maintenance culture medium added to blank control wells and cell control wells, at least 3 replicates were set. 37℃ / 5% CO 2 After 72 h of culture, 100 μL / well of maintenance medium and 10 μL / well of CCK-8 colorimetric solution were added; 37°C / 5% CO 2 After 3 h of incubation, the absorbance at 450 / 630 nm (A450 / 630 nm) was measured using an enzyme reader, and the virus inhibition rate was calculated according to the formula. The experiment was repeated at least 3 times; EC was calculated using GraphPadPrism. 50 .

[0096] Inhibition rate % = (sample group - virus control group) / (cell control group - virus control group) × 100%

[0097] 3. Experimental results

[0098] LCC-S has good antiviral activity against HSV-1 / KOS and HSV-2 / G. The results are shown in Table 3.

[0099] Table 3 In vitro inhibitory effect of LCC-S on standard virus strains HSV-1 / KOS and HSV-2 / G

[0100]

[0101] Example 7 CCK-8 colorimetric assay was used to detect the in vitro inhibitory effect of LCC-S on drug-resistant virus strains HSV-106, HSV-BLUE and HSV-153.

[0102] 1. Experimental Materials

[0103] African green monkey kidney cells Vero, drug-resistant strains HSV-106, HSV-BLUE and HSV-153 were purchased from ATCC cell bank.

[0104] 2. Experimental Methods

[0105] Vero cells were cultured at 2×10 4 Each well was plated in a 96-well plate; 37°C / 5% CO 2 After 24 h of incubation, 100 TCID 50100 μL / well of HSV virus dilution, 200 μL / well of maintenance culture medium added to blank control wells and cell control wells; incubate at 37℃ for 1 hour, discard the culture medium, wash twice with PBS, add 100, 50, 25, 12.5, 6.25, 3.125, 0 μg / mL of LCC-S 200 μL / well to the experimental wells, 200 μL / well of maintenance culture medium added to blank control wells and cell control wells, at least 3 replicates were set. 37℃ / 5% CO 2 After 72 h of culture, 100 μL / well of maintenance medium and 10 μL / well of CCK-8 colorimetric solution were added; 37°C / 5% CO 2 After 3 h of incubation, the absorbance at 450 / 630 nm (A450 / 630 nm) was measured using an enzyme reader, and the virus inhibition rate was calculated according to the formula. The experiment was repeated at least 3 times; EC was calculated using GraphPadPrism. 50 .

[0106] Inhibition rate % = (sample group - virus control group) / (cell control group - virus control group) × 100%

[0107] 3. Experimental results

[0108] LCC-S has good antiviral activity against drug-resistant strains HSV-106, HSV-BLUE and HSV-153. The results are shown in Table 4.

[0109] Table 4 In vitro inhibitory effect of LCC-S on standard virus strain HSV-2 / G

[0110]

[0111] The above examples are for the purpose of illustrating the embodiments disclosed by the present invention and are not to be construed as limitations of the present invention. In addition, the various modifications listed herein and the variations of methods and compositions in the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications obvious to those skilled in the art as described above to obtain the invention should be included within the scope of the present invention.

Claims

1. A water-soluble lignin-carbohydrate complex in Prunella vulgaris, characterized in that: The content of lignin-carbohydrate in the lignin-carbohydrate complex is 90%, and the remainder is impurities; Among them, lignin includes p-hydroxybenzaldehyde (H type), guaiacyl propane (G type), and syringyl propane (S type), among which p-hydroxybenzaldehyde (H type) is 14.2%, guaiacyl propane (G type) is 21.7%, and syringyl propane (S type) is 64.1%; Carbohydrates include mannose, ribose, rhamnose, glucose, glucuronic acid, galactose, galacturonic acid, xylose, and arabinose, wherein the mass ratio of each component to carbohydrate is: mannose 10.97%, ribose 0.63%, rhamnose 3.71%, glucose 15.33%, glucuronic acid 0.73%, galactose 20.48%, galacturonic acid 2.39%, xylose 40.74%, and arabinose 5.01%, and the remainder is impurities.

2. The water-soluble lignin-carbohydrate complex in Prunella vulgaris according to claim 1, characterized in that: The lignin-carbohydrate complex also includes any one or more of the following technical features: a) number average molecular weight is 20225; b) weight average molecular weight (Mw) is 37981; c) Z-average molecular weight (Mz) is 64217; d) The polydispersity index (Mw / Mn) is 1.

87.

3. The water-soluble lignin-carbohydrate complex in Prunella vulgaris according to claim 1, characterized in that: The two-dimensional heteronuclear single quantum coherence nuclear magnetic resonance image of the lignin-carbohydrate complex shows that in the aromatic region, three basic structural units of lignin are identified: the signal at the chemical shift δC / δH128.9 / 7.21 is assigned to C 2,6 -H 2, 6, indicating the presence of a p-hydroxyphenyl structure (H unit); guaiacyl propane (G type), δC / δH 110 / 7.0 corresponds to C2-H2, δC / δH 115.5 / 7.00 and 6.78 and 119.1 / 6.78 are assigned to C5-H5 and C6-H6, respectively; the signal of the syringyl unit (S unit) at δC / δH 103.8 / 6.68 corresponds to C 2,6 -H 2,6 ; In addition, obvious β-O-4 bond and xyloside signals were identified in the side chain region. δC / δH 62.6 / 3.37 and 3.82 (C5-H5), 72.5 / 2.95 (C2-H2), 74.0 / 3.22 (C3-H3) and 75.9 / 3.49 (C4-H4) were assigned to methyl β-D-pyranoxyloside, δC / δH 75.8 / 4.70 (C3-H3) indicated the presence of 3-O-acetyl-modified xyloside; δC / δH 60.0 / 3.11–3.88 (C γ -H γ ) indicated the presence of a γ-hydroxylated β-O-4 substructure.

4. The water-soluble lignin-carbohydrate complex in Prunella vulgaris according to claim 1, characterized in that: The infrared scanning spectrum of the lignin-carbohydrate complex shows that the absorption peak at 3234.05 cm-1 belongs to OH stretching vibration, the absorption peaks at 1590 cm-1 and 1518 cm-1 belong to the stretching vibration of the aromatic ring C=C skeleton, the absorption peak at 1401 cm-1 belongs to CH bending vibration, the absorption peaks at 1156 cm-1 and 1074 cm-1 belong to COC stretching vibration or CO stretching vibration, the absorption peak at 1011 cm-1 belongs to CO stretching vibration, the absorption peak at 951 cm-1 belongs to sugar ring vibration or CO stretching vibration, and the absorption peaks at 809 cm-1 and 763 cm-1 belong to the out-of-plane bending vibration of the aromatic ring CH.

5. Use of the lignin-carbohydrate complex according to any one of claims 1 to 4 in the preparation of an anti-herpes simplex virus drug.

6. The use according to claim 5, characterized in that: The herpes simplex virus refers to any one or more of HSV-1 / KOS, HSV-2 / G, HSV-106, HSV-BLUE or HSV-153.

7. The method for preparing the lignin-carbohydrate complex according to any one of claims 1 to 4, comprising the following steps: a) grinding the alcohol precipitate extract of Prunella vulgaris, adding LiCl / DMSO and stirring in a magnetic stirrer for 24 hours to obtain an extract; b) adding the extract obtained in step a) to an aqueous solution of 1,4-dioxane and stirring the mixture in a magnetic stirrer for 24 hours to obtain an extract; c) centrifuging the extract obtained in step b) and taking the supernatant; d) adding the supernatant to a 3500Da dialysis bag and dialyzing it in pure water; e) The contents of the dialysis bag are concentrated under reduced pressure, re-dissolved in acetic acid / water solution, dropped into pure water for regeneration, centrifuged, and the supernatant is concentrated by distillation under reduced pressure and freeze-dried to obtain the product.

8. The method according to claim 7, characterized in that: The alcohol precipitation extract of Prunella Vulgaris refers to a 30% alcohol precipitation extract.

9. The method according to claim 7, further comprising any one or more of the following technical features: a) The step a) is specifically as follows: 2 g of 30% alcohol precipitate extract of Prunella vulgaris is added into a grinding tube, a 2 mm steel ball is placed in the tube, the tube is ground using a grinder at 60 Hz for 2 hours, 50 ml of 10% LiCl / DMSO is added, and the tube is placed in a magnetic stirrer for stirring and extraction for 24 hours; b) The step b) is specifically as follows: adding the extract obtained in step a) to 3 times the volume of 75% (V / V) 1,4-dioxane aqueous solution and placing it in a magnetic stirrer for stirring and extracting for 24 hours; c) The step c) is specifically as follows: centrifuging the extract obtained in step b) at 14400rpm for 10min, taking the supernatant, adding the lower precipitate to 50ml of 75% (V / V) 1,4-dioxane aqueous solution, placing it in a magnetic stirrer for stirring and extracting for 24h; centrifuging the obtained extract at 14400rpm for 10min, taking the supernatant, adding the lower precipitate to 50ml of 75% (V / V) 1,4-dioxane aqueous solution, placing it in a magnetic stirrer for stirring and extracting for 24h; centrifuging the obtained extract at 14400rpm for 10min, taking the supernatant, and then combining the obtained supernatants.

10. The method according to claim 7, further comprising any one or more of the following technical features: a) Step d) is specifically to mix the obtained supernatants, add them into a 3500Da dialysis bag, place them in 3L pure water for dialysis, change the water every 8h, and dialyze for a total of 72h; b) The step e) is specifically as follows: the contents of the dialysis bag are concentrated under reduced pressure, re-dissolved in 20 ml of 90% (V / V) acetic acid / water solution, dripped into 80 ml of pure water for regeneration, centrifuged at 14400 rpm for 10 min, the supernatant is concentrated by reduced pressure distillation, and freeze-dried to obtain LCC-S.

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