Application of a konjac glucomannan oligosaccharide mixture in the preparation of anti-HSV-1 drugs

Abstract

The invention discloses an application of a konjac glucomannan oligosaccharide mixture in the preparation of an anti-HSV-1 drug. The konjac glucomannan oligosaccharide mixture is obtained by membrane separation and purification of the primary product produced by hydrolyzing white konjac powder produced in Yunnan with recombinant β-mannanase. The konjac glucomannan oligosaccharide mixture mainly contains oligosaccharides such as glucomannobiose, glucomannotriose, glucomannotetraose and glucomannopentaose. Among them, the content of glucomannan oligosaccharides (disaccharides to pentasaccharides) reaches more than 70%. In vitro experiments of the konjac glucomannan oligosaccharide mixture show that it has anti-HSV-1 wild strain and anti-HSV-1ACV drug-resistant strain activity, and has low cytotoxicity and drug-food homology characteristics, providing a new method for preparing drugs for treating and preventing diseases caused by HSV-1ACV drug-resistant strain infection.

Description

Technical Field

[0001] The present application relates to the field of pharmaceutical biotechnology, and in particular to the use of a konjac glucomannan oligosaccharide mixture in the preparation of an anti-HSV-1 drug. Background Art

[0002] Konjac glucomannan oligosaccharides (KGMOS) are low molecular weight polysaccharides, belonging to heteropolysaccharides among homogeneous polysaccharides. They are functional oligosaccharides with an average degree of polymerization of 2 to 10 formed by connecting β-D-glucose and β-D-mannose through β-(1→4) glycosidic bonds. In KGMOS, the molar ratio of glucose to mannose is generally between 1:1.6 and 1.7, and glucose or mannose branches connected by β-(1→3) glycosidic bonds are usually generated at the 3rd C position of mannose in the main chain to form short branched forms. Glucose and / or mannose at the main chain and / or branch positions produce natural acetylation modifications.

[0003] KGMOS is a functional oligosaccharide that has been studied a lot. The functions that have been reported so far can be roughly divided into two categories. One is that it has immunomodulatory function, such as being able to improve the body's immunity and be used as an adjuvant for vaccines (enhancing humoral immunity and cellular immunity) and improving the index of immune organs. The second is that it has the function of regulating the intestinal microbial population and thus improving intestinal function, such as maintaining the stability of the intestinal flora, improving the intestinal structure, and absorbing harmful intestinal bacteria and the toxins they produce. At the same time, there are also literature reports on in vitro and in vivo anti-tumor activity, antioxidant capacity, reduction of NO free radical levels, reduction of weight gain in mice fed a high-fat diet, improvement of animal production performance, and antiviral functions.

[0004] It has been reported that KGMOS sulfate has anti-HSV-2 activity (Yang Min, Meng Yiwen, Preparation of Konjac Oligosaccharide Sulfate and Study on Its Antiviral Activity [J], Natural Product Research and Development, 2002, 1 (14): 17-19). It has also been reported that konjac glucomannan sulfate (HOGS) has an antiviral effect on Coxsackievirus 3 (CVB3), poliovirus 1 (PV1), hepatitis B virus (HBV) and influenza A virus (IAV) (Huang Hao, Study on the Antiviral Properties and Mechanism of Konjac HOGS, Huazhong Agricultural University, 2007).

[0005] Herpes simplex virus type I (HSV-1) is an important human pathogen that is widely spread and carried in all kinds of people. HSV-1 will produce two different infection states in the human body: lytic infection and latent infection. HSV-1 lurking in the motor neurons of the trigeminal ganglion of the human body can change from the latent infection state to the lytic infection state under stress, environmental changes and reduced immunity, producing obvious lesions or clinical symptoms. The clinical symptoms of HSV-1 infection include oral herpes, keratitis and viral encephalitis. Among them, herpes simplex virus keratitis is one of the main causes of blindness in adults. Although the incidence of herpes simplex virus encephalitis is low, the prognosis is poor, resulting in serious sequelae of mental disorders.

[0006] Acyclovir (ACV) is still the first-line drug for anti-HSV-1 in clinical practice, but years of extensive clinical application have led to serious drug resistance of HSV-1 to ACV. Other anti-HSV-1 nucleotide analogs such as valacyclovir and penciclovir also face similar drug resistance challenges. Therefore, the development of new anti-HSV-1 drugs is one of the main tasks facing current clinical applications. At present, there is no literature reporting that KGMOS prepared by enzymatic method has activity against HSV-1 wild strains and HSV-1 ACV-resistant strains without chemical modification. Summary of the invention

[0007] In view of the deficiencies of the prior art, the present invention provides an application of a konjac glucomannan oligosaccharide mixture (KGMOS) in the preparation of an anti-HSV-1 drug. The konjac glucomannan oligosaccharide mixture is both a medicine and a food, and the mixture has good anti-HSV-1 wild strain and anti-HSV-1 ACV drug-resistant strain activity in in vitro experiments, and can be used to prepare an anti-HSV-1 ACV drug-resistant strain drug to solve the drug resistance problem of treating HSV-1 infection in clinical treatment.

[0008] In order to solve the above technical problems, the technical solution of the present invention is as follows: application of a konjac glucomannan oligosaccharide mixture in the preparation of an anti-HSV-1 drug.

[0009] The present invention also provides application of the konjac glucomannan oligosaccharide mixture in preparing a drug for resisting HSV-1 ACV drug-resistant strains.

[0010] Compared with ACV, KGMOS has lower cytotoxicity. The 50% effective / inhibitory concentration (EC / IC50) of the KGMOS mixture for HSV-1 wild strain in THP-1 is 0.675 mg / mL and 0.4968 mg / mL, respectively, and the selective index (SI) is 24.95 and 33.90, respectively. The IC50 of the KGMOS mixture for HSV-1 ACV resistant strain in THP-1 is 0.6403 mg / mL and 0.5974 mg / mL, respectively; the SI is 26.30 and 28.19, respectively. The IC50 of ACV for HSV-1 ACV resistant strain is 0.3192 mg / mL, and the SI is 5.04. The effect of KGMOS against HSV-1 ACV resistant strain is significantly better than that of ACV.

[0011] The KGMOS mixture can be used not only to prepare drugs for preventing HSV-1 infection, but also to prepare drugs for treating diseases caused by HSV-1 infection, including herpes labialis, eye diseases (keratitis) and viral encephalitis.

[0012] As a further description of the above scheme: the konjac glucomannan oligosaccharide mixture is obtained by using white konjac refined flour as a substrate, and the initial product produced after hydrolysis with recombinant β-mannanase is obtained after membrane separation and purification; the encoding gene of the recombinant β-mannanase has a GeneBank accession number of NCBI official website: MW357596.

[0013] As a further description of the above scheme: the konjac glucomannan oligosaccharide mixture is mainly composed of β-D-glucose, β-D-mannose and oligosaccharides with an average degree of polymerization of 2-5 in which β-D-glucose and β-D-mannose are connected by β-1,4-glycosidic bonds, and its molecular formula can be expressed as [C 6 H 10 O 5 ] n , where n=2~5.

[0014] As a further description of the above scheme: the molecular weight of the konjac glucomannan oligosaccharide mixture is mainly 200-1000Da; the total content of low-polymerization oligosaccharides of di-, tri-, tetra- and pentasaccharides reaches more than 70%.

[0015] As a further description of the above scheme: the konjac glucomannan oligosaccharide mixture can significantly upregulate the levels of cytokines related to natural immunity and inflammation, such as the expression levels of IFNβ, ISG15, ISG56, MxI, IL-1β, IL-6 and TNFα.

[0016] As a further description of the above scheme: the konjac glucomannan oligosaccharide mixture can inhibit the replication, transcription and viral proliferation of HSV-1 ACV drug-resistant strain genes in vitro.

[0017] The present invention is characterized in that: the present invention first discovered that the KGMOS mixture prepared by genetically engineered β-mannanase has the activity against HSV-1 wild strain and HSV-1 ACV resistant strain without chemical derivatization, and this feature makes it have a very obvious advantage in the process of preparing drugs against HSV-1 ACV resistant strain. Part of the reason why this KGMOS mixture without chemical derivatization can resist HSV-1 ACV resistant strain may be that the functional KGMOS content in the mixture is relatively high, which activates the antiviral natural immune response of the cell and thus obtains the function of inhibiting the replication and proliferation of the virus in the cell.

[0018] The chemically derived KGMOS mixture exerts its antiviral effect mainly by inhibiting the binding of the virus capsid protein to the cell surface receptor, that is, inhibiting the adsorption of the virus to the cell and thus inhibiting the virus from entering the cell, thereby inhibiting the virus from infecting the cell. Its ability to inhibit the virus from replicating in the cell is relatively weak.

[0019] The KGMOS mixture provided by the present invention can inhibit the replication of HSV-1 ACV resistant strains in cells by activating the natural immunity of cells in in vitro experiments, rather than an antiviral mechanism that mainly inhibits the adsorption process of viruses and cells. Pre-treating cells with KGMOS in advance to activate the natural immune response of cells can also exert the ability to resist HSV-1. This feature allows KGMOS to be used to prepare drugs for preventing HSV-1 infection and to prepare drugs for treating diseases caused by HSV-1 infection.

[0020] In the present application, human THP-1 cells differentiated by PMA were used, which have the characteristics of immune cells. Using this cell model to test the anti-HSV-1 activity of KGMOS has obvious advantages. Compared with African green monkey kidney cells (Vero) commonly used in previous reports to test anti-HSV-1 activity, THP-1 cells have a complete natural immune signaling pathway and can show a response to stimulation by immune active substances. At the same time, THP-1 is a human-derived cell, which is closer to the actual application of the human body.

[0021] Compared with the prior art, the present invention has the following beneficial effects: ① The KGMOS mixture provided by the present invention has anti-HSV-1 activity without chemical derivatization, and thus can be used to prepare drugs for treating and / or preventing diseases caused by HSV-1 infection; ② The KGMOS mixture provided by the present invention achieves the anti-HSV-1 function by regulating the host's natural immunity, and thus does not cause drug resistance problems; ③ The KGMO mixture provided by the present invention has anti-HSV-1 ACV resistant strain activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the mass spectrum of KGMOS;

[0023] Figure 2 It is the TCL diagram of KGMOS;

[0024] Figure 3 For the cytotoxicity experiments of KGMOS and ACV;

[0025] Figure 4 is the IC50 of KGMOS in the prevention mode;

[0026] Figure 5 is the IC50 of KGMOS in therapeutic mode;

[0027] Figure 6 is the IC50 of ACV;

[0028] Figure 7 For determination of virus titer;

[0029] Figure 8 Quantification of gene expression. DETAILED DESCRIPTION

[0030] The present invention uses a beta-mannan enzyme prepared by a genetic engineering method to hydrolyze konjac flour to obtain a primary product, and then obtains a KGMOS mixture after solid-liquid centrifugation, inorganic ceramic membrane fractionation and spray drying. The KGMOS mixture is analyzed by mass spectrometry and thin layer chromatography (TCL) and shows that it mainly contains a mixture of glucomannobiose to pentasaccharides. The KGMOS mixture is tested for its anti-HSV-1 activity in THP-1 cells, and the results show that it has anti-HSV-1 ACV drug-resistant strain activity. The following examples will be further described in detail.

[0031] Example 1 Preparation and composition analysis of KGMOS mixture

[0032] Materials and methods

[0033] Konjac flour was first-class product with a purity of 85%; TCL silica gel plates and developing agents were conventional laboratory supplies; enzymatic hydrolysis tanks and inorganic ceramic membrane filtration equipment were from the Enzyme Engineering Laboratory of Yunnan Normal University; and matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS, Bruker, Germany) was from the Kunming Institute of Botany.

[0034] Preparation of recombinant β-mannanase

[0035] The recombinant β-mannanase was prepared according to the method disclosed in the existing document "Zhe Yuanyuan, Miao Huabiao, Wu Qian, Mu Yuelin, Huang Zunxi, et al. Study on heterologous expression of thermostable β-mannanase and preparation of oligosaccharides".

[0036] The characteristics of the prepared recombinant β-mannanase are as follows: the recombinant mannanase DL4man gene is 1104bp, as shown in SEQ ID NO.1, the accession number is: MW357596, the protein molecular weight is 41.5kDa, and it belongs to the glycoside hydrolase 26 family; the optimal reaction pH value is 5.5, the optimal reaction temperature is 55°C, and it is a high-temperature resistant mannanase.

[0037] Enzymatic hydrolysis of konjac flour

[0038] Add 100kg of pure water to the enzymolysis tank and heat it to 55-60℃. Then add recombinant β-mannanase to make the enzyme activity in the aqueous solution 50IU / mL. Weigh 12.5kg of first-grade konjac flour and slowly add it to the enzymolysis tank (completed within 10min), mixing while hydrolyzing. Then continue to add 12.5kg of first-grade konjac flour (completed within 10min) and stir and mix continuously for about 1h, and end the enzymolysis process when the viscosity of the solution is reduced to 50 centipascals (cP). Then heat up the hydrolyzate in the enzymolysis tank to 80℃, and keep it at this temperature for 10min to terminate the enzymolysis reaction, and then pass cooling water to cool the enzymolysis solution to 45℃.

[0039] Ceramic membrane filtration of enzymatic hydrolysates

[0040] The crude fiber in the reaction system is filtered and retained by the primary screen at the bottom of the enzymolysis tank to obtain the primary filtrate. The centrifugal filtrate is obtained by separating and removing the starch, small molecular weight fiber, suspended matter, etc. in the primary filtrate using a disc centrifuge with a rotation speed of 8000r / min. The centrifugal filtrate is then microfiltered through an inorganic ceramic membrane assembly with a pore size of 0.2μm to remove waste residue, protein and macromolecular polysaccharides to obtain a ceramic membrane filtrate. The ceramic membrane filtrate is passed through an organic ultrafiltration membrane (molecules with a molecular weight cutoff greater than 5000Da) assembly to obtain an ultrafiltration permeate, and the ultrafiltration permeate is then filtered through an organic nanofiltration membrane (molecules with a molecular weight cutoff greater than 100Da) to obtain a nanofiltration concentrate, which is spray-dried to obtain a powdered konjac glucomannan oligosaccharide.

[0041] TCL Analysis of Konjac Glucomannan Oligosaccharide

[0042] The silica gel plate (G60) was placed in an oven at 100°C for 0.5h for activation before loading. The powdered konjac glucomannan oligosaccharide obtained above was dissolved in water and loaded onto the silica gel plate using a capillary. The plate was then placed in a chromatography cylinder containing a developing agent (n-butanol: formic acid: water = 4:6:1 (V / V)). When the front edge of the developing agent reached about 2cm from the edge of the plate, the chromatography was terminated and sprayed with concentrated sulfuric acid for color development.

[0043] Analysis of Konjac Glucomannan Oligosaccharide by MALDI-TOF-MS

[0044] The concentrated sample of konjac glucomannan oligosaccharide was diluted 5 times to make the oligosaccharide concentration in the solution 40g / L. The sample and 2,5-dihydroxybenzoic acid (DHB) matrix solution were diluted 1:1 and mixed on a MALDI plate and dried at room temperature. The composition of oligosaccharides was analyzed using a matrix-assisted laser desorption ionization time-of-flight mass spectrometer MALDI-TOF / TOF. The instrument used MALDI ionization, and each mass spectrum obtained a total of 10,000 to 20,000 laser emissions. The mass spectrometer was collected using an ultra-light MALDI-TOF / TOF Bruker mass spectrometer in positive ion mode.

[0045] result

[0046] The product obtained by enzymatic hydrolysis of konjac flour was subjected to TCL analysis and mass spectrometry analysis. The TCL analysis results showed that the main components were a mixture of oligosaccharides with different polymerization degrees, such as monosaccharides, disaccharides, trisaccharides, tetrasaccharides and pentasaccharides. Among them, there may be various modifications at different positions of certain sugar groups, which may cause the chromatography to move to different positions of the chromatography plate (such as Figure 2 The mass spectrum results are shown in Figure 1 The relative content of each component was calculated based on the peak area at the position of each oligosaccharide as shown in Table 1.

[0047] Table 1 Relative content of various oligosaccharides

[0048] Molecular weight (MW, Da) Oligosaccharides Relative content (%) 212 Monosaccharide (M1) 0.097(22.4％) 381 Disaccharide (M2) 0.193(44.6％) 543 Trisaccharide (M3) 0.098(22.6％) 705 Tetrasaccharide (M4) 0.031(7.2％) 867 Pentasaccharide (M5) 0.010(2.3％) 1030 Hexasaccharide (M6) 0.003(0.7％) 1192 Seven sugars (M7) 0.001(0.2％)

[0049] The product components of the recombinant β-mannanase used in this application to hydrolyze konjac flour are mainly monosaccharides, mannobiose and mannotriose. The main products of konjac flour hydrolyzed by β-mannanase from Penicillium chrysogenum include manno-oligosaccharides with a degree of polymerization greater than 4, as well as a small amount of mannose (Zhen Hongmin, Hua Xiaohan, Ma Junwen, et al. Efficient expression, properties and applications of β-mannanase from Penicillium chrysogenum [J]. Food Science, 2021, 42 (8): 98-105.). Therefore, when mannanases from different sources hydrolyze the same substrate, the product components are also different, the binding of enzymes with different structures to substrates is different, and the cleavage of glycosidic bonds is also different.

[0050] Example 2 KGMOS inhibits HSV-1 replication in TPH-1 cells

[0051] Materials and methods

[0052] Acyclovir (ACV) dry powder, product of MCE; phorbol myristate ester (PMA), product of MCE, dissolved in DMOS and then filtered and sterilized for later use; KGMOS was prepared using the above method and dissolved in RPMI-1640 medium; CCK8, product of Quanshijin Company; RNA extraction and RT-qPCR reagents, products of Bao Biotechnology Company; THP-1 and Vero cells were preserved by this laboratory; cell culture medium, fetal bovine serum, double antibody, etc. were all routine laboratory supplies; HSV-1 wild strain was F type; HSV-1-EGFP was a TK gene mutant strain, in which the CDS of the EGFP gene was inserted into the coding region of the TK gene, and it could express green fluorescent protein in infected cells.

[0053] PMA (phorbol 12-myristate 13-acetate, abbreviated as phorbol ester, also known as TPA) induced differentiation of THP-1 cells

[0054] After adding PMA at a final concentration of 160 nM to the THP-1 cell culture medium for 12 h, it was observed that the THP-1 cells changed from a non-adherent state to an adherent state, and the culture medium was replaced with a PMA-free medium for subsequent experiments.

[0055] Toxicity experiment of KGMOS on THP-1 cells

[0056] THP-1 cells differentiated by PMA induction were cultured in 96-well plates, and 200 μL of complete RPMI-1640 culture medium containing different concentrations of KGMOS (128, 64, 32, 16, 8, 4, 2, 1 mg / mL) or ACV (16, 8, 4, 2, 1, 0.5, 0.25, 0.125 mg / mL) was added to each well. After culturing in a 37°C 5% carbon dioxide cell culture incubator for 24 hours, 10 μL of CCK-8 reagent was added to each well, and then the cells were cultured in a cell culture incubator for another 4 hours, and the absorbance at a wavelength of 450 nm was measured on an ELISA reader. The CC50 value was calculated using GraphPad Prism 6.0 software. Each group contained 4 biological replicates.

[0057] Inhibitory effect of THP-1 pretreated with KGMOS on HSV-1 (preventive mode)

[0058] THP-1 cells differentiated by PMA were cultured in 96-well plates, and 200 μL of KGMOS complete nutrition RPMI-1640 culture medium with concentrations of 8, 4, 2, 1, and 0.5 mg / mL was added to each well. After culturing in a cell culture incubator at 37°C and 5% carbon dioxide for 24 hours, the original KGMOS culture medium was replaced with ordinary culture medium. In the ACV treatment group, 200 μL of ACV with concentrations of 1.0, 0.5, 0.25, 0.125, and 0.0625 mg / mL was added to each well. Subsequently, HSV-1 wild-type strain and HSV-1 ACV-resistant strain were infected with MOI=0.1. After 48 hours, 10 μL of CCK-8 reagent was added to each well, and then the cells were cultured in a cell culture incubator for another 4 hours, and the absorbance at a wavelength of 450 nm was measured with an enzyme reader. The IC50 value was calculated using GraphPad Prism 6.0 software. Each group contained 4 biological replicates.

[0059] Inhibitory effect of THP-1 cells cultured with KGMOS on HSV-1 (therapeutic model)

[0060] The treatment method was the same as that in the prevention mode. The difference was that the THP-1 cells in the KGMOS group were not pretreated with KGMOS, but were cultured in medium containing different concentrations of KGMOS after HSV-1 infection. The ACV group was treated the same as the ACV group in the prevention mode.

[0061] KGMOS inhibits the proliferation of HSV-1

[0062] THP-1 cells differentiated by PMA were cultured in 24-well plates. In the presence of KGMOS at a final concentration of 1 mg / mL, cells were infected with HSV-1 wild strain at MOI = 0.1 for 24 hours, and the cultured cell supernatant was harvested. The proliferation of HSV-1 was determined in Vero cells using the 50% tissue culture infective dose (TCID50) and plaque forming unit (PFU) methods. At the same time, the treatment without KGMOS was used as the control group.

[0063] KGMOS regulate the expression of HSV-1 and host genes

[0064] THP-1 cells differentiated by PMA were cultured in 6-well plates. After infection with HSV-1 wild strain at MOI = 0.1 for 24 hours in the presence of KGMOS at a final concentration of 1 mg / mL, the cultured cells were harvested and total RNA of the cells was extracted using RNAiso Plus, which was then reverse transcribed into cDNA and Real-time PCR was performed using SYBR Green II as a dye to quantify the expression levels of IFNβ, ISG15, ISG56, MxI, IL-1β, IL-6, TNFα and viral ICP0, TK, and gC genes in the cells, with the cell gene GAPDH as an internal reference. The quantitative primer sequences of each gene used in this case are listed in Table 2 below.

[0065] Table 2 Primer pairs used to quantify gene expression

[0066]

[0067]

[0068] result

[0069] After being induced by PMA, THP-1 cells changed from the original suspension state to the adherent growth state, and the cells produced antennae, indicating that they had differentiated into the phenotype of macrophages.

[0070] The CC50 value of KGMOS on THP-1 cells was 16.84 mg / mL, which was much higher than the CC50 value of ACV on THP-1 cells, 1.609 mg / mL. This indicates that KGMOS has low toxicity to THP-1 cells ( Figure 3 ).

[0071] In the preventive mode, i.e., when THP-1 cells were pretreated with KGMOS, the IC50 values ​​of KGMOS against HSV-1 wild-type strain and HSV-1 ACV-resistant strain were 0.675 mg / mL and 0.6403 mg / mL, respectively. Figure 4 ), SI were 24.95 and 26.30 respectively. The IC50 of ACV against HSV-1 wild-type strain and HSV-1 ACV-resistant strain were 0.05768 mg / mL and 0.3192 mg / mL respectively ( Figure 6 ), SI were 27.90 and 5.04 respectively. The experimental results showed that KGMOS had a high degree of inhibition on both HSV-1 wild strain and HSV-1 ACV resistant strain, while ACV only had a high inhibition rate on HSV-1 wild strain, and had a weaker inhibition ability on HSV-1 ACV resistant strain.

[0072] In the treatment mode, the IC50 of KGMOS against HSV-1 wild-type strain and HSV-1 ACV-resistant strain were 0.4968 mg / mL and 0.5974 mg / mL, respectively. Figure 5 ), SI were 33.90 and 28.19 respectively.

[0073] The experimental results of this section are summarized in Table 3.

[0074] Table 3 Anti-HSV-1 effect of KGMOS

[0075]

[0076] Compared with the control group, the titer of HSV-1 in the cell supernatant was significantly decreased after KGMOS treatment ( Figure 7 A), plaque forming units also decreased significantly ( Figure 7 B). This indicates that cells treated with KGMOS can inhibit the proliferation of HSV-1.

[0077] Compared with the control group, after KGMOS treatment, the expression levels of genes related to innate immunity and inflammation in cells were upregulated, and the expression levels of viral ICP0 (immediate early gene), gC (early gene) and TK (late gene) were downregulated to varying degrees ( Figure 8 ). This experimental result shows that KGMOS inhibits the proliferation of HSV-1 by upregulating the cell's natural immunity and inflammation-related signaling pathways.

[0078] In summary, the IC50 of KGMOS mixture against HSV-1 ACV resistant strains in THP-1 in the prevention and treatment modes were 0.6403 mg / mL and 0.5974 mg / mL, respectively; SI were 26.30 and 28.19, respectively. The IC50 of ACV against HSV-1 ACV resistant strains was 0.3192 mg / mL, and SI was 5.04. The effect of KGMOS against HSV-1 ACV resistant strains was significantly better than that of ACV.

[0079] After THP-1 cells were treated with the KGMOS mixture, the expression levels of cytokines related to innate immunity and inflammation, such as IFNβ, ISG15, ISG56, MxI, IL-1β, IL-6 and TNFα, were significantly upregulated.

[0080] The KGMOS mixture has not only anti-HSV-1 wild strain activity, but also anti-HSV-1 ACV resistant strain activity. The KGMOS mixture can be used not only to prepare drugs for preventing HSV-1 infection, but also to prepare drugs for treating diseases caused by HSV-1 infection.

[0081] The above-mentioned implementation cases are preferred implementations of the present invention, but the implementations of the present invention are not limited to the above-mentioned implementation cases. Any other changes made without departing from the spirit and principle of the present invention shall be equivalent replacement modes and shall be included in the protection scope of the present invention.

Claims

1. Use of a konjac glucomannan oligosaccharide mixture in the preparation of an anti-HSV-1 drug, characterized in that: The konjac glucomannan oligosaccharide mixture mainly consists of beta-D-glucose, beta-D-mannose and oligosaccharides in which beta-D-glucose and beta-D-mannose are connected by beta-1,4-glycosidic bonds, and has a molecular weight of 200-1000 Da.

2. The use of the konjac glucomannan oligosaccharide mixture according to claim 1 in the preparation of a drug against HSV-1, characterized in that: The konjac glucomannan oligosaccharide mixture is used for preparing a medicine for resisting HSV-1 ACV drug-resistant strains.

3. The use according to claim 1 or 2, characterized in that: The konjac glucomannan oligosaccharide mixture is obtained by using white konjac refined flour as substrate, and the primary product produced by hydrolysis with recombinant β-mannanase is obtained by membrane separation and purification; the encoding gene of the recombinant β-mannanase has a GeneBank accession number of MW357596 on the official website of NCBI.

4. The use according to claim 3, characterized in that: The konjac glucomannan oligosaccharide mixture is mainly composed of β-D-glucose, β-D-mannose, and oligosaccharides with an average polymerization degree of 2-5 in which β-D-glucose and β-D-mannose are connected by β-1,4-glycosidic bonds, and its molecular formula is expressed as [C6H 10 O5] n , where n=2~5.

5. The use according to claim 3, characterized in that: The total content of di-, tri-, tetra- and penta-saccharide oligosaccharides with low polymerization degree in the konjac glucomannan oligosaccharide mixture is more than 70%.

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