A kind of kelp polysaccharide and its preparation method and application

Abstract

The invention discloses a kelp polysaccharide and a preparation method and application thereof, belonging to the field of biomedical technology. In vitro experiments have confirmed that kelp polysaccharide LJP‑1 has significant anti-HSV‑1 viral activity, can inhibit virus adsorption, penetration and direct inactivation of viruses, and can be developed as an anti-HSV‑1 drug; animal experiments have confirmed that LJP‑1 has a significant therapeutic effect on herpetic stomatitis in mice caused by HSV‑1 infection, and can be used to prepare drugs for preventing and treating herpetic stomatitis. The polysaccharide provided by the present invention has high purity, does not contain impurities such as protein and nucleic acid, and has small toxic and side effects. It has a wide range of application prospects in the preparation of low-toxic and efficient anti-HSV‑1 drugs and the development of drugs for preventing and treating herpetic stomatitis caused by HSV‑1 infection, and is conducive to the further development and utilization of kelp resources.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to laminarin and a preparation method and application thereof. Background Art

[0002] Herpes simplex virus (HSV) belongs to the family of Herpesviridae and is a neurotoxic double-stranded DNA virus that can cause a variety of diseases in humans. HSV can be divided into two major types: herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2). Among them, HSV-1 is considered to be one of the most common viruses in humans. It is usually transmitted through direct contact with the saliva or other body fluids of the infected host, causing various symptoms in peripheral tissues, including cheilitis, herpetic gingivostomatitis, and herpetic keratitis. In addition, HSV-1 can also cause herpes simplex encephalitis and various neurological diseases. However, there is currently no vaccine available for the treatment of HSV-1 infection. The main drugs used clinically to treat HSV-1 infection are nucleoside analogs, including acyclovir (ACV) and its derivatives. Although these drugs can specifically block the replication of viral DNA, long-term use can cause adverse reactions such as nausea and vomiting. In addition, these drugs are not effective against ACV-resistant HSV-1 viruses. Therefore, searching and discovering safe and effective anti-HSV-1 drugs from natural resources is of great value and far-reaching significance.

[0003] Laminaria japonica is a marine brown algae with both medicinal and edible properties. It is widely distributed along the coast of Liaodong Peninsula and Shandong Peninsula in my country. In China, kelp is often used to treat diseases such as goiter, urinary system diseases, edema and tumors. Among them, polysaccharides are considered to be one of the main bioactive components of kelp, with multiple pharmacological activities such as anti-tumor, anti-inflammatory, immunomodulatory, anticoagulant, antioxidant and antibacterial. Previous studies have shown that kelp polysaccharides have good anti-respiratory syncytial virus activity and anti-enterovirus 71 activity. However, there are no reports on the anti-HSV-1 activity of kelp polysaccharides. Summary of the invention

[0004] The purpose of the present invention is to provide a method for preparing laminarin polysaccharide and its application in anti-HSV-1 drugs and drugs for preventing and treating herpetic stomatitis caused by HSV-1 infection, so as to solve the problems existing in the above-mentioned prior art.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention is a method for preparing laminarin polysaccharide LJP-1, comprising the following steps:

[0007] (1) The kelp medicinal material is crushed, defatted, extracted in a water bath, and the extract is concentrated under reduced pressure to obtain a kelp crude extract;

[0008] (2) After the kelp crude extract is subjected to alcohol precipitation and protein removal, the kelp crude polysaccharide LJP is obtained;

[0009] (3) The crude Laminaria japonica polysaccharide LJP is initially separated by a DEAE-52 anion exchange column to obtain LJP-OM;

[0010] (4) Separating the LJP-0M by Sephacryl S-200 gel chromatography to obtain the laminarin LJP-1.

[0011] The second technical solution of the present invention is the laminarin LJP-1 prepared by the preparation method.

[0012] The third technical solution of the present invention is the use of the laminarin polysaccharide LJP-1 in the preparation of drugs for preventing and treating diseases caused by herpes simplex virus.

[0013] The fourth technical solution of the present invention is a medicine for preventing and treating diseases caused by herpes simplex virus, comprising the laminarin polysaccharide LJP-1.

[0014] Based on the above technical solution, the present invention has the following technical effects:

[0015] (1) The present invention proposes for the first time that a homogeneous polysaccharide LJP-1 in kelp has significant anti-HSV-1 activity. In vitro tests have shown that the kelp polysaccharide LJP-1 exhibits strong anti-adsorption, anti-penetration and direct inactivation effects on HSV-1 virus. 50 The values ​​were 0.6401 μg / mL, 8.082 μg / mL and 7.179 μg / mL respectively.

[0016] (2) The present invention proposes for the first time that a homogeneous polysaccharide LJP-1 in kelp has a significant therapeutic effect on herpetic stomatitis caused by HSV-1 infection. Animal experiments have shown that the kelp polysaccharide LJP-1 has a significant improvement effect on the skin pathological damage, skin inflammatory response, skin virus titer, etc. of herpetic stomatitis mice caused by HSV-1 infection.

[0017] (3) The kelp polysaccharide provided by the present invention has high purity and does not contain impurities such as protein and nucleic acid.

[0018] (4) The polysaccharide provided by the present invention has little toxic and side effects, and has broad application prospects in the preparation of low-toxic and high-efficiency anti-HSV-1 drugs and in the development of drugs for preventing and treating herpetic stomatitis caused by HSV-1 infection, which is conducive to the further development and utilization of kelp resources.

[0019] (5) The method for preparing kelp polysaccharide of the present invention is not only simple to operate and low in cost, but also more efficient and practical, and at the same time better protects the biological activity of the polysaccharide, and is worthy of promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 This is a flow chart for the preparation of kelp polysaccharide LJP-1.

[0022] Figure 2 This is the HPGPC chromatogram of kelp polysaccharide LJP-1.

[0023] Figure 3 This is the infrared spectrum of kelp polysaccharide LJP-1.

[0024] Figure 4 The figure shows the effect of Laminaria japonica acidic polysaccharide LJP-1 on the fluorescence level of EGFP in Vero cells infected with HSV-1 / EGFP. A is the fluorescence intensity graph of EGFP in Vero cells infected with HSV-1 / EGFP; B is the fluorescence quantitative analysis graph of EGFP.

[0025] Figure 5 The anti-adsorption (A), anti-penetration (B) and inactivation (C) effects of kelp polysaccharide LJP-1 on HSV-1 virus.

[0026] Figure 6 The curve diagram of the inhibitory effect of kelp polysaccharide LJP-1 on HSV-1 virus. Among them, A is the EC value of LJP-1's anti-adsorption effect on HSV-1 virus. 50 Figure 2: EC of LJP-1 against HSV-1 virus penetration 50 Figure; C is the EC of LJP-1 inactivating HSV-1 virus 50 picture.

[0027] Figure 7 The effect of kelp polysaccharide LJP-1 on skin lesions in mice with herpetic stomatitis caused by HSV-1 infection. A is a representative photo of mouse skin lesions; B is the pathological lesion score of mouse skin.

[0028] Figure 8 The purpose is to investigate the effects of kelp polysaccharide LJP-1 on pathological tissue damage and inflammatory edema in the skin of mice with herpetic stomatitis caused by HSV-1 infection.

[0029] Fig. 9 The figure shows the effect of kelp polysaccharide LJP-1 on the virus titer of the skin of mice with herpetic stomatitis caused by HSV-1 infection. A is a representative photo of the virus titer in the skin of mice; B is a quantitative analysis of the virus titer in the skin of mice. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0036] The embodiment of the present invention provides a method for preparing laminarin LJP-1, comprising the following steps:

[0037] (1) The kelp medicinal material is crushed, defatted, extracted in a water bath, and the extract is concentrated under reduced pressure to obtain a kelp crude extract;

[0038] (2) After the kelp crude extract is subjected to alcohol precipitation and protein removal, the kelp crude polysaccharide LJP is obtained;

[0039] (3) The crude Laminaria japonica polysaccharide LJP is initially separated by a DEAE-52 anion exchange column to obtain LJP-OM;

[0040] (4) Separating the LJP-0M by Sephacryl S-200 gel chromatography to obtain the laminarin LJP-1.

[0041] In some specific embodiments, in step (1), the degreasing method is: degreasing by Soxhlet reflux with petroleum ether 2-3 times, and collecting the residue;

[0042] The water bath extraction method is as follows: the medicinal residue is mixed with water in a mass ratio of 1:30 and then subjected to water bath extraction at a temperature of 80-85° C. for 2-3 hours, and the extraction is repeated 2-3 times before combining the extracts.

[0043] In some specific embodiments, in step (2), the alcohol precipitation method is: adding anhydrous ethanol to the kelp crude extract, refrigerating and standing overnight, and collecting the precipitate by centrifugation;

[0044] The method for removing protein is: removing protein by using Savag method.

[0045] The embodiment of the present invention also provides laminarin LJP-1 prepared by the preparation method.

[0046] In some specific embodiments, the laminarin LJP-1 is an acidic polysaccharide composed of seven monosaccharides with a molecular weight of 272,231 Da;

[0047] The total sugar content of the kelp polysaccharide LJP-1 is 91.15%, the uronic acid content is 7.68%, and the sulfate content is 24.2%;

[0048] The molar ratio of monosaccharides in the kelp polysaccharide LJP-1 is galactose:fucose:glucuronic acid:mannose:rhamnose:xylose:glucose=0.314:0.281:0.152:0.092:0.047:0.043:0.036.

[0049] In some specific embodiments, the primary structure of LJP-1 is mainly composed of terminally linked galactose, 1,4-linked galactose, 1,6-linked galactose, 1,3,6-linked galactose, 1,4,6-linked galactose, 1,3-linked fucose, 1,4-linked glucose, 1,3,4-linked glucose, terminally linked mannose, 1,3-linked rhamnose, terminally linked xylose and 1,4-linked xylose.

[0050] The embodiment of the present invention also provides the use of laminarin polysaccharide LJP-1 in preparing medicine for preventing and treating diseases caused by herpes simplex virus.

[0051] In some specific embodiments, the herpes simplex virus causes a disease comprising herpetic stomatitis.

[0052] The embodiment of the present invention also provides a medicine for preventing and treating diseases caused by herpes simplex virus, comprising the laminarin polysaccharide LJP-1.

[0053] In some specific embodiments, a pharmaceutically acceptable excipient is also included.

[0054] Example 1

[0055] Preparation of Laminaria japonica polysaccharide LJP-1

[0056] 10kg of kelp medicinal material was crushed, passed through a 50-mesh sieve, and defatted by petroleum ether Soxhlet reflux (the temperature of petroleum ether Soxhlet extraction and defatting was 60-80°C, and the amount of petroleum ether added was 6 times the weight of the kelp medicinal material) for 3 times, then filtered, and the residue was added with deionized water in a ratio of 1:30, and extracted 3 times at 80°C in a water bath, each time for 2 hours, and the extracts were combined and concentrated under reduced pressure to obtain a crude kelp extract. 4 times the volume of anhydrous ethanol was added to the crude kelp extract, and it was allowed to stand overnight in a refrigerator at 4°C. The supernatant was removed by centrifugation, the precipitate was collected, and it was re-dissolved with deionized water. The Savag method was further used to remove protein, centrifuged, and the precipitate was discarded, and this was repeated 5-6 times until there was no more precipitate after centrifugation, and then concentrated under reduced pressure and freeze-dried to obtain crude kelp polysaccharide LJP (780g, yield: 7.8%).

[0057] Take kelp crude polysaccharide (2.0g), add 300mL deionized water to dissolve, centrifuge, and use DEAE-52 anion exchange column for preliminary separation of the supernatant, and use deionized water and different concentrations of NaCl solution (0.2, 0.4, 0.6, 0.8, 1.0, 2.0mol / L) as eluent for gradient elution, the flow rate is 12mL / 15min to collect each flow fraction, and detect the absorbance value at 490nm (sulfuric acid-phenol method color development). According to the polysaccharide color development reaction, each elution component is combined, concentrated, dialyzed, and freeze-dried to obtain a secondary component LJP-OM (224mg, yield: 11.2%).

[0058] Dissolve LJP-0M (160 mg) in deionized water, centrifuge, and separate the supernatant using Sephacryl S-200 gel chromatography. Elute with deionized water at a flow rate of 4 mL / 15 min, and collect each fraction. Detect the absorbance at 490 nm (sulfuric acid-phenol method for color development). According to the color development reaction of the polysaccharide, combine the eluted components, concentrate, dialyze (the molecular weight cutoff during dialysis is 1000 Da), and freeze-dry to obtain the kelp uniform polysaccharide LJP-1 (120 mg, yield: 75%). The results are as follows Figure 1 shown.

[0059] Example 2

[0060] Structural Characterization of Laminaria japonica polysaccharide LJP-1

[0061] (1) Molecular weight determination

[0062] The molecular weight of kelp polysaccharide LJP-1 was determined by high performance gel permeation chromatography (HPGPC). Chromatographic conditions: separation was performed using a BRT105-103-101 series gel column, a flow rate of 0.8 mL / min, an injection volume of 25 μL, a 0.05 mol / L NaCl solution as the mobile phase, a column temperature of 40°C, and a differential refractive index detector (RID-20A).

[0063] Experimental method: Accurately weigh 5 mg each of kelp polysaccharide LJP-1 and Dextrans series standards (1152Da, 5000Da, 11,600Da, 23,800Da, 48,600Da, 80,900Da, 148,000Da and 273,000Da), dissolve them in 1 mL of mobile phase to make a 5 mg / mL solution, and centrifuge at 12,000 r / min for 10 min. Extract the supernatant, filter it with a 0.22 μm microporous filter membrane, and then detect it, and record the retention time of the polysaccharide molecules. Draw a standard curve with the retention time as the horizontal axis and the logarithm (Log) of the molecular weight of the standard polysaccharide as the vertical axis to obtain the corresponding linear regression equation. Subsequently, the retention time of the polysaccharide sample to be tested was substituted into the regression equation, and the molecular weight of the polysaccharide LJP-1 was obtained to be 272,231 Da. The results are as follows Figure 2 shown.

[0064] (2) Determination of total sugar, uronic acid, sulfate and protein content

[0065] The total sugar content of LJP-1 was determined to be 91.15% by the phenol-sulfuric acid method; the uronic acid content of LJP-1 was determined to be 7.68% by the m-hydroxybiphenyl colorimetric method; the sulfate content of LJP-1 was determined to be 24.2% by the barium chloride-gelatin turbidimetric method; and LJP-1 was basically protein-free by the bicinchoninic acid method.

[0066] (3) Monosaccharide composition analysis

[0067] Ion chromatography (IC) was used to determine the monosaccharide composition of kelp polysaccharide LJP-1. About 5 mg of LJP-1 and 16 monosaccharide standards (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, galactosamine hydrochloride, glucosamine hydrochloride, N-acetyl-D-glucosamine, guluronic acid, mannuronic acid) were accurately weighed, 2 mL TFA (3 mol / L) was added to hydrolyze at 120 ° C for 3 h to obtain the hydrolyzate, deionized water was added to dissolve, and the supernatant was centrifuged for IC analysis.

[0068] LJP-1 is a polysaccharide composed of seven monosaccharides, and the molar ratio of monosaccharides is galactose:fucose:glucuronic acid:mannose:rhamnose:xylose:glucose=0.314:0.281:0.152:0.092:0.047:0.043:0.036.

[0069] (4) Methylation analysis

[0070] Gas chromatography-mass spectrometry (GC-MS) was used to analyze the methylation of kelp polysaccharide LJP-1. About 3 mg of LJP-1 was accurately weighed, 1 mL of DMSO solution was added to dissolve it, and then CMC-NaBH 4 After reduction, dialysis, and freeze-drying, the methylation product was methylated by the modified Hakomori method. The methylated product was hydrolyzed with 2M TFA for 90 min and then treated with NaBH 4 After reduction for 8 h, the product was acetylated with acetic anhydride-pyridine at 100 °C for 1 h to obtain a partially methylated alditol acetate derivative, which was then analyzed by GC-MS.

[0071] The main connection modes of the sugar residues of LJP-1 are: terminally linked galactose, 1,4-linked galactose, 1,6-linked galactose, 1,3,6-linked galactose, 1,4,6-linked galactose, 1,3-linked fucose, 1,4-linked glucose, 1,3,4-linked glucose, terminally linked mannose, 1,3-linked rhamnose, terminally linked xylose and 1,4-linked xylose.

[0072] (5) Infrared spectroscopy analysis

[0073] The dried KBr powder was mixed with the dried kelp polysaccharide LJP-1, ground and pressed into granules with a thickness of 1 mm. Subsequently, the FT-IR spectrometer was used to analyze the granules at 400-4000 cm -1 Scan and analyze samples within the range.

[0074] The results of the test are as follows Figure 3 As shown, 3464cm-1 The absorption peak at 2936cm is caused by the stretching vibration of –OH; -1 and 1411cm -1 The absorption peak at 1626cm is caused by C–H bending vibration and stretching vibration; -1 The absorption peak at 1250cm is caused by C=O stretching vibration; -1 and 830cm -1 The absorption peak at 1138 cm is caused by the S=O and C–O–S stretching vibrations in the sulfate group, indicating that LJP-1 contains sulfate groups. -1 and 1050cm -1 The absorption peaks at are caused by C–O–C and C–O–H stretching vibrations.

[0075] Example 3

[0076] Determination of Anti-HSV-1 Activity of Laminaria japonica Polysaccharide LJP-1

[0077] (1) Before exposing Vero cells to HSV-1 / recombinant enhanced green fluorescent protein (EGFP), they were pretreated with different concentrations of LJP-1 (10, 25, 50 μg / mL) or the positive drug acyclovir (50 μmol / L) for 12 h. After infection, the cells were rinsed with PBS and co-incubated with LJP-1. After 48 h, the presence of EGFP was observed under a fluorescence microscope. In addition, Vero cells infected with HSV-1 / EGFP were used as the model group. The results are shown in Figure 2. Figure 4 As shown in the figure, compared with the virus model group, the LJP-1 treatment group can reduce the fluorescence level of EGFP in a dose-dependent manner. At a concentration of 50 μg / mL, the antiviral effect of LJP-1 is equivalent to that of the positive drug acyclovir.

[0078] (2) In the anti-adsorption test, Vero cells were exposed to a series of dilutions of LJP-1 (10, 25, 50 μg / mL) or the positive drug punicalagin (50 μmol / L) and cooled at 4°C for 1 h. Subsequently, Vero cells were infected with HSV-1 / EGFP at 4°C for 2 h. Finally, the cells were rinsed with PBS to remove unattached viruses. The results are shown in Figure 2. Figure 5 As shown, LJP-1 exhibited a strong anti-adsorption effect on HSV-1 / EGFP infection in a dose-dependent manner.

[0079] (3) In the anti-penetration test, pre-cooled Vero cells were exposed to a series of dilutions of LJP-1 (10, 25, 50 μg / mL) or the positive drug punicalagin (50 μmol / L) and infected with HSV-1 / EGFP for 2 h at 4°C. After the attachment period, the cells were washed with PBS and treated with pH 3.0 citrate buffer for 1 min to inactivate any virus that did not penetrate the cells. The results are shown in Figure 2. Figure 5 As shown, LJP-1 exhibited a strong anti-penetration effect on HSV-1 / EGFP infection in a dose-dependent manner.

[0080] (4) In the virus inactivation test, HSV-1 / EGFP was incubated with LJP-1 at 37°C for 2 h, and then the mixture was added to Vero cells and diluted 100-fold. At 72 h after infection, the inhibition rate was evaluated using the plaque forming unit (PFU) assay. The results are shown in Figure 5 As shown, LJP-1 exhibited a strong inactivation effect on HSV-1 / EGFP infection in a dose-dependent manner.

[0081] (5) In the cytotoxicity test, Vero cells were seeded in 96-well plates. Subsequently, different concentrations of LJP-1 (10, 25, 50 μg / mL) were added to the cells. After incubation for 72 h, cell counting kit-8 (CCK-8) was used to evaluate cell viability. The results are shown in Table 1. Figure 6 As shown in Figure 2, LJP-1 has significant anti-adsorption, anti-penetration and inactivation effects on HSV-1 virus. 50 The values ​​were 0.6401 μg / mL, 8.082 μg / mL and 7.179 μg / mL respectively.

[0082] Example 4

[0083] Therapeutic effect of Laminaria japonica polysaccharide LJP-1 on herpetic stomatitis induced by HSV-1 infection in mice

[0084] Forty BALB / c male mice (body weight: 15-19 g) were randomly divided into five groups according to body weight, namely, group A, group B, group C, group D, and group E, with 8 mice in each group.

[0085] Among them, group A was the normal control group, and the mice in this group did not receive any treatment.

[0086] Group B was the HSV-1 virus model group. The method for constructing the virus model was as follows: BALB / c male mice were anesthetized by intraperitoneal injection of sodium pentobarbital, the chin was shaved, hair was removed, and a small piece of skin (2-4 mm) was cut with a scalpel. 2 ) and drip 20 μL of 10 6PBS drops of PFU HSV-1. After HSV-1 infection, the weight of mice in the model group decreased significantly, and their skin tissues showed obvious lesions, indicating that the model was successfully established.

[0087] Group C is the low-dose group of kelp polysaccharide LJP-1 (10 μg / mL): low-dose LJP-1 was applied to the skin affected areas of mice infected with HSV-1 virus for 7 consecutive days.

[0088] Group D was the high-dose group of kelp polysaccharide LJP-1 (50 μg / mL): high-dose LJP-1 was applied to the skin affected areas of mice infected with HSV-1 virus for 7 consecutive days.

[0089] Group E was the positive control acyclovir group (50 μmol / L): acyclovir was applied to the skin affected areas of mice infected with HSV-1 virus for 7 consecutive days.

[0090] After 7 days, the mice in each group were weighed, and skin tissue samples infected with HSV-1 virus were extracted for pathological analysis, virus titer determination, etc.

[0091] (1) Effect of kelp polysaccharide LJP-1 on skin lesions in mice with herpetic stomatitis caused by HSV-1 infection.

[0092] Skin tissue symptom damage score is one of the important indicators for evaluating the severity of skin tissue lesions. The larger the value, the more serious the skin tissue lesions. Figure 7 As shown, compared with the normal control group, the skin tissue symptom damage score of mice in the virus model group was higher. Compared with the virus model group, both the high and low dose treatment groups of kelp polysaccharide LJP-1 could significantly reduce the skin tissue symptom score of mice with herpetic stomatitis.

[0093] (2) Effects of kelp polysaccharide LJP-1 on pathological tissue damage and inflammatory edema in the skin of mice with herpetic stomatitis caused by HSV-1 infection.

[0094] The results of histopathological analysis showed that compared with the normal control group, the skin tissue of mice with herpetic stomatitis caused by HSV-1 infection had obvious pathological damage and obvious inflammatory edema. Compared with the virus model group, each drug-treated group could significantly improve the pathological damage of the skin tissue of mice with herpetic stomatitis and significantly alleviate the inflammatory edema ( Figure 8 ).

[0095] (3) Effect of kelp polysaccharide LJP-1 on viral titer in the skin of mice with herpetic stomatitis caused by HSV-1 infection.

[0096] The PFU method was used to determine the HSV-1 virus titer in the skin of mice with herpetic stomatitis. The results showed that compared with the normal control group, the virus titer in the skin tissue of mice with herpetic stomatitis was significantly increased. Compared with the virus model group, both low-dose LJP-1 (10μg / mL) and high-dose LJP-1 (50μg / mL) significantly reduced the virus titer of HSV-1 in a dose-dependent manner ( Fig. 9 ).

[0097] In summary, the present invention provides a kelp polysaccharide LJP-1, including: galactose, fucose, glucuronic acid, mannose, rhamnose, xylose and glucose, with a molecular weight of 272,231Da, a total sugar content of 91.15%, a uronic acid content of 7.68%, a sulfate content of 24.2%, and substantially no protein. In vitro experiments have confirmed that LJP-1 has significant anti-HSV-1 viral activity, can inhibit virus adsorption, penetration and directly inactivate viruses, and can be developed as an anti-HSV-1 drug; animal experiments have confirmed that LJP-1 has a significant therapeutic effect on herpetic stomatitis in mice caused by HSV-1 infection, and can be used to prepare drugs for preventing and treating herpetic stomatitis. The polysaccharide provided by the present invention has high purity, does not contain impurities such as protein and nucleic acid, and has small toxic and side effects. It has broad application prospects in the preparation of low-toxic and efficient anti-HSV-1 drugs and the development of drugs for preventing and treating herpetic stomatitis caused by HSV-1 infection, and is conducive to the further development and utilization of kelp resources.

[0098] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A kelp polysaccharide LJP-1, characterized in that: The kelp polysaccharide LJP-1 is an acidic polysaccharide composed of seven monosaccharides with a molecular weight of 272,231Da; The total sugar content of the kelp polysaccharide LJP-1 is 91.15%, the uronic acid content is 7.68%, and the sulfate content is 24.2%; The molar ratio of monosaccharides in the kelp polysaccharide LJP-1 is galactose:fucose:glucuronic acid:mannose:rhamnose:xylose:glucose=0.314:0.281:0.152:0.092:0.047:0.043:0.

036.

2. The laminarin LJP-1 according to claim 1, characterized in that The preparation method comprises the following steps: (1) The kelp medicinal material is crushed, defatted, and then extracted in a water bath, and the extract is concentrated under reduced pressure to obtain a kelp crude extract; (2) After the kelp crude extract is subjected to alcohol precipitation and protein removal, the kelp crude polysaccharide LJP is obtained; (3) The crude Laminaria japonica polysaccharide LJP is initially separated by a DEAE-52 anion exchange column to obtain LJP-OM; (4) Separating the LJP-0M by Sephacryl S-200 gel chromatography to obtain the laminarin LJP-1.

3. The laminarin LJP-1 according to claim 2, characterized in that In step (1), the degreasing method is: degreasing by Soxhlet reflux with petroleum ether 2-3 times, and collecting the residue; The water bath extraction method is as follows: the medicinal residue is mixed with water in a mass ratio of 1:30 and then subjected to water bath extraction at a temperature of 80-85° C. for 2-3 hours, and the extraction is repeated 2-3 times before combining the extracts.

4. The laminarin LJP-1 according to claim 2, characterized in that In step (2), the alcohol precipitation method is: adding anhydrous ethanol to the kelp crude extract, refrigerating and standing overnight, and collecting the precipitate by centrifugation; The method for removing protein is: removing protein by using Savag method.

5. Use of the laminarin LJP-1 as claimed in any one of claims 1 to 3 in the preparation of drugs for preventing and treating diseases caused by herpes simplex virus.

6. A drug for preventing and treating diseases caused by herpes simplex virus, characterized in that: The invention comprises the laminarin LJP-1 as described in any one of claims 1 to 3.

7. The drug according to claim 6, characterized in that Pharmaceutically acceptable excipients are also included.

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