TW201515652A - Sargassum polysaccharide for manufacturing a composition for treating or preventing enterovirus 71 infection - Google Patents

Abstract

The present invention relates to use of Sargassum polysaccharide for manufacturing a composition for treating or preventing enterovirus 71 infection. The composition of the invention can be used as a pharmaceutical composition, a food additive or an additive to cleaning and sanitation products.

Description

Use of Sargassum polysaccharide for the treatment or prevention of enterovirus 71 infection

The present invention relates to the use of a Sargassum polysaccharide for the preparation of a composition for the treatment or prevention of an enterovirus 71 infection, wherein the composition is typically useful as an additive to pharmaceutical compositions, food supplements and sanitizers.

EV71 belong Picornaviridae (Picornaviridae), Enterovirus (Enterovirus), it is a kind of intestinal virus known last to be discovered. Enterovirus 71 infection of the brain and brain stem causes sudden central nervous system diseases such as: lethargy, irritability, dyspnea, encephalitis, aseptic meningitis, hand, foot and mouth disease, herpetic angina and heart failure shock, easy It causes severe illness in children and is extremely harmful to children.

In terms of physicochemical properties, enterovirus 71 virus particles are not easily affected by acid and alkali, so their infectivity can be stably retained in an acidic environment. Since Enterovirus 71 does not have an envelope structure formed by lipids and belongs to a naked virus, it is resistant to ethanol, so that alcohol cannot exert an inhibitory effect. In addition, the cleaning agents commonly used in households have no bactericidal effect on enterovirus 71. There are currently no drugs or vaccines that are effective against infections caused by enterovirus 71.

Sargassum is a kind of seaweed in brown algae. It belongs to brown algae, carrageen, and horsetail algae. It is distributed in many sea areas of the world. It can be found in the northern and northeastern coastal areas of Taiwan and in Wuhu, Ludao, Lanyu, etc. The region, especially in the Penghu area, has considerable production. Sargassum polysaccharide is a water-soluble and highly viscous polysaccharide extracted from Sargassum. It has been reported that Sargassum polysaccharide has the pharmacological activities of enhancing immunity, inhibiting tumor cells, anti-oxidation and anti-virus, but the pharmacological mechanism of anti-virus is still unclear, especially for enterovirus.

Therefore, for enterovirus 71, there is still a need to find active ingredients that are effective against infection, especially natural ingredients from daily life, which not only have therapeutic effects, but also have the advantages of being non-toxic and easy to obtain.

The present invention discloses the use of a Sargassum polysaccharide for the preparation of a composition for the treatment or prevention of enterovirus 71 infection.

In some specific examples, the Sargasso polysaccharide of the present invention may be subjected to sulfation treatment, having a sulfate content of 10% (w/w) or more, 20% (w/w) or more, 40% (w/w) or more, Or 60% (w/w) or more. In a specific embodiment, the S. cerevisiae polysaccharide of the present invention has a sulfate content of 60-70% (w/w).

Specifically, the composition of the present invention can increase the inhibition rate against enterovirus 71 infection, for example, reducing the proportion of cells infected with enterovirus, inhibiting the synthesis of enterovirus RNA, and inhibiting enterovirus-induced cytopathic and apoptosis.

Detailed descriptions of various specific examples of the invention are given below. Other features of the present invention will be apparent from the following detailed description and claims.

Without further elaboration, it is believed that those of ordinary skill in the art of Therefore, it is to be understood that the following description is for illustrative purposes only and is not intended to limit the remaining disclosure

Figure 1 shows an HPLC chromatogram of Sargassum polysaccharides, wherein Figure 1 (A) is an HPLC chromatogram of the Sargassum polysaccharide standard, and Figure 1 (B) is an HPLC chromatogram of sulfated Sargassum polysaccharide.

Figure 2 shows the inhibitory effects of different concentrations of Sargassum polysaccharide standard and sulfated Sargassum polysaccharide on enterotoxin type 71.

The invention analyzes the polysaccharide of Sargassum, and proves for the first time that it has a good inhibitory effect on the infection of enterovirus 71 type, especially the Sargassum polysaccharide which increases the sulfate content, and can further improve the effect of inhibiting the virus. Sargassum polysaccharides are non-toxic and readily available, compared to toxic and expensive chemical agents, especially for high-risk infants. Therefore, the present invention has a specific contribution to the treatment and prevention and treatment of enterovirus 71.

All technical and scientific terms used herein have the meaning as commonly understood by one of ordinary skill in the art to which the invention pertains, unless otherwise indicated.

The term "a" as used herein, unless otherwise specified, refers to the quantity of at least one (one or more).

In one aspect, the invention features the use of a Sargassum polysaccharide for the preparation of a composition for treating or preventing an enterovirus 71 infection.

Sargassum polysaccharide is a polysaccharide extracted from Sargassum. Chemically, it is a polysaccharide based on "fucose", which is a water-soluble solution composed of fucose and sulphate. a heteropolypolysaccharide component in which fucose is bonded to each other as a primary bond by α-(1→2), α-(1→3) or α-(1→4), and sulfate is bonded to C-2 or C- On the restore side of 4. Specifically, it has a unit structure of the following formula (I):.

The Sargassum polysaccharide can be obtained commercially, or can be extracted from Sargassum by a conventional method, for example, by soaking the algae with hot water to soften it (for example, immersing in hot water of about 100 ° C for about 1-3 hours). After treatment with an enzyme or acidification step, centrifugation, filtration, and collection of the supernatant. After the extraction, the supernatant is further freeze-dried for storage. In some embodiments, the Sargasso polysaccharide used in the present invention has a molecular weight ranging from 50 kDa to 1,000 kDa, from 250 kDa to 950 kDa, or from 450 kDa to 850 kDa. In a particular embodiment, the S. cerevisiae polysaccharide used in the present invention has a molecular weight of about 800 kDa.

Preferably, the step of increasing the sulfate content can be carried out to increase the sulfate content of the Sargassum polysaccharide. In some embodiments, the present invention may employ a chemical addition of a sulfate, for example, by adding a Chlorsulfonic acid-N (N-dimethylformamide, HClSO3/DMF) reagent. The base (such as NaOH) is neutralized, then centrifuged, filtered, and the supernatant is collected. In some specific examples, the Sargasso polysaccharide of the present invention has a sulfate content of 10% (w/w) or more, 20% (w/w) or more, 40% (w/w) or more, or 60% (w/ w) above. In a specific embodiment, the S. cerevisiae polysaccharide used in the present invention has a sulfate content of 60-70% (w/w).

The compositions of the present invention can be formulated with any one or more carriers in accordance with conventional conventional methods. For example, the Sargasso polysaccharide is mixed with a suitable carrier and then formulated into the desired dosage form.

As used herein, the term "carrier" means an inactive ingredient that is used to carry a polysaccharide of Sargasso to form a desired formulation, including any standard pharmaceutically acceptable carrier, which is compatible with the active ingredients of the formulation, and It is not harmful to the individual to be administered, for example, water, physiological saline, glycerin, ethanol, glucose water, dextran, lactose, sucrose, mannitol, analogs thereof or a combination thereof.

The compositions of the present invention may also include binders, lubricants, antioxidants, fillers, and preservatives. Useful binders include gum arabic, sodium alginate, ethyl cellulose, agar, gelatin, starch, hydroxy cellulose, hydroxypropyl cellulose, analogs thereof, or combinations thereof. Useful lubricants include stearic acid, calcium stearate, magnesium stearate, talc, hydrogenated oils, waxes, analogs thereof, or combinations of the foregoing. Useful antioxidants include tocopherols, dibutylhydroxytoluene, butylhydroxymethoxybenzene, ascorbic acid, analogs thereof, or combinations thereof. Useful fillers include microcrystalline cellulose, lactose, mannitol, analogs thereof, or combinations of the foregoing. Useful preservatives include benzoic acid and its salts, sorbic acid and its salts, parabens, sodium acetate dehydrated, hydrogen peroxide, sodium hypochlorite, analogs thereof or combinations thereof.

In one embodiment, the compositions of the present invention are used as pharmaceutical compositions which can be formulated into a variety of dosage forms depending on the route of administration. For example, the pharmaceutical compositions of the present invention can be formulated into capsules or lozenges for oral administration. The capsules may contain any standard pharmaceutical acceptable substance such as gelatin or cellulose. Tablets can be combined with solid carriers and lubricated according to conventional methods The agents are co-pressed and formulated. The pharmaceutical compositions of the present invention may also be formulated in injectable formulations, including aqueous solutions, isotonic saline or dextrose or other well known pharmaceutical acceptable carriers. The pharmaceutical compositions of the present invention may also be formulated as creams, oils, gels, lotions or sprays for topical application.

In another embodiment, the composition of the invention is used as a food additive. The polysaccharide of Sargasso is taken from natural foodstuffs and is not toxic. It can be directly added to foods or nutrients, and it can easily achieve the effect of preventive health care. For example, it can be added to milk powder or other dairy products to provide infants and young children.

In yet another embodiment, the composition of the present invention is an additive to a sanitary article, for example, can be added to a face, body or hand cleaning product, or an environmental sanitizer, or an inlet cover, a cleaning paper towel, a toilet paper Or cotton swabs.

Example 1: Preparation of Sargassum Polysaccharide

The sargasso is washed with water and dried, and then ground. Then prepare a 5% (w/w) aqueous solution of Sargasso to carry out hot water extraction: firstly soak the aqueous solution of Sargasso in a hot water bath (about 100 ° C, soak for about 1-3 hours) to soften the algae, and then use the enzyme Or acid hydrolysis treatment, after the completion of the reaction, high-speed centrifugation, followed by filtration and collection of the supernatant. The supernatant was transferred to -80 ° C, and then freeze-dried by a vacuum freeze dryer (LABCONCO) to obtain a Sargassum polysaccharide.

Example 2: Sulfation reaction

The above-mentioned extracted Sargassum polysaccharide is treated by sulfate chemical method to increase the sulfate content: adding Chlorsulfonic acid-N (N-dimethylformamide, HClSO3/DMF) reagent to the polysaccharide of Sargassum The reaction was carried out, and the reaction was made neutral with NaOH, followed by high-speed centrifugation, followed by filtration and collection of the supernatant. The supernatant was transferred to -80 ° C, and then freeze-dried in a vacuum freeze dryer to obtain a sulfated Sargassum polysaccharide.

Example 3: Analysis of sulfate content

A commercially available Sargasso polysaccharide standard or a sample of sulfated Sargasso polysaccharide to be tested is dissolved in 0.5 ml of HCl at a concentration of 1 N, reacted at 100 ° C for 1 hour, and then evaporated to dryness in an oven. . After adding 0.5ml of deionized water (ddH ₂ O) to dissolve, add 2ml of absolute alcohol, 1ml of cesium chloride buffer (BaCl ₂ buffer, which contains 2M acetic acid 10ml, 0.05M cesium chloride 2ml, 0.02 M sodium bicarbonate 8 ml, and absolute alcohol to 100 ml) and 1.5 ml sodium palmitate solution (Na-rhodizonate solution containing 0.25 mg / ml of Na ₂ C ₆ O ₆ , 100 mg ascorbic acid, and absolute alcohol Mix to 100 ml). The light-proof reaction was carried out for 20 minutes, and finally A520 was measured and converted into a sulfate content. The above method was used to detect the S. cerevisiae polysaccharide standard and the sulfated S. salina polysaccharide sample to be tested, and the sulfate content thereof is shown in Table 1.

Example 4: Molecular Weight Analysis of Polysaccharides

Pullulan was used as a standard (the molecular weight of the standard was 788 kDa, 404 kDa, 212 kDa, 112 kDa, 47.3 kDa, 22.8 kDa, 11.8 kDa, and 5.9 kDa), and 20 μL was taken for HPLC molecular weight analysis. The GPC separation column (OHpak SB-804 HQ) was operated at a flow rate of 0.8 ml/min, the extract was deionized water, and the molecular weight distribution analysis of the polysaccharide was carried out by an Evaporative Light Scattering Detector (ELSD). The analyzed spectrum calculated the retention time of the EC 2000 GPC software to the molecular weight (Mw) of the polysaccharide as the basis for the molecular weight analysis of the polysaccharides of Sargassum.

Figure 1 shows the HPLC results of S. cerevisiae polysaccharides, wherein Figure 1 (A) is a horse HPLC chromatogram of the standard of polysaccharides from S. cerevisiae, Figure 1 (B) is the HPLC chromatogram of sulfated polysaccharides of Sargassum. After comparing the standard curve, the molecular weight of S. cerevisiae is about 800kDa, and Figure 1(B) The residence time shifts to the right, indicating that after the sulfation reaction, the molecular weight increases due to the addition of sulfate.

Example 5: Cell and virus culture

The enterovirus 71 virus strain was obtained from a throat specimen of a two-year-old baby girl who died of EV71 in July 2001. The strain was MEL701 and was provided by the laboratory of Dr. Chen Quanmu from Zhongxing University.

On the other hand, African green monkey kidney cells (VERO cells, ATCC CCL-18) were cultured in RPMI-1640 medium (containing 2% FBS, pH 7.0), when the cells grew about eight minutes. Subculture is carried out at full time. First remove the cell culture medium, wash away the residual serum and cell metabolites with phosphate buffer saline (PBS), and add trypsin and ethylene diamine tetraacetic acid (EDTA) to 3 to 5 Minutes, the reaction was stopped with fetal bovine serum (FBS) and the cells were washed with cell culture medium, collected in a centrifuge tube, centrifuged, and the supernatant was removed to collect the cell pellet, and then reconstituted with the culture solution, and then used as a cell. count.

The MEL701 strain was inoculated into VERO cells (containing 2% FBS RPMI-1640 medium), and the cell type was observed by an inverted microscope. When more than 80% of the cytopathic effect (CPE) occurred, the virus was collected. The culture medium was stored in a -80 ° C refrigerator for subsequent experiments.

Example 6: Viral plaque inhibition test

The VERO cells were seeded in a 6-well culture dish (1×10 ⁵ cells/well), placed in a 37° C. incubator containing 5% CO ₂ , and the cells were cultured until six or seven minutes, and the culture solution was removed. Rinse the cells once with PBS. The Sargassum polysaccharide standard and the sulfated Sargassum polysaccharide (sample with a sulfate content of 67.6%) were separately diluted to different concentrations in RPMI-1640 medium (without fetal bovine serum), and each was cultured at 37 ° C with the cells. After 1 hour of action, virus solution (50 PFU/well) was added and infected at 37 ° C for 1 hour. Finally, RPMI-1640 medium containing 2% agarose gel was added, and it was solidified. After being cultured for 4 to 5 days in a 37 ° C incubator containing 5% CO ₂ , it was fixed with 10% of formalin. In addition to the gel, it was stained with 1% crystal violet, and the number of plaques was counted after washing with tap water.

The plaque inhibition rate (%) is calculated as: (VC-VCS)/VC×100%, wherein the VC is the number of virus spots in the control virus-infected group in which the S. salina polysaccharide standard or the sulfated Sargasso polysaccharide is not added; VCS is the number of virus spots in the experimental virus-infected group in which the Sargassum polysaccharide standard or the sulfated Sargassum polysaccharide was added. Experimental data were obtained from three independent experiments and are shown as mean ± standard error (mean ± SEM).

Figure 2 shows the results of the plaque inhibition test. The results showed that the polysaccharide of Sargasso showed good anti-infection effect of EV71, and the inhibition rate was about 30-45%. Especially after the increase of sulfate content in Sargasso polysaccharide, the antiviral effect increased by about 28-47%. The inhibition rate is increased to about 60-80% or more, and the antiviral effect is quite excellent.

Claims (10)

Use of a Sargasso polysaccharide for the preparation of a composition for treating or preventing an enterovirus 71 infection. The use of the first aspect of the patent application, wherein the Sargassum polysaccharide has a sulfate content of 10% (w/w) or more. The use of the second aspect of the patent application, wherein the Sargassum polysaccharide has a sulfate content of 20% (w/w) or more. The use of the second aspect of the patent application, wherein the Sargassum polysaccharide has a sulfate content of 40% (w/w) or more. The use of the second aspect of the patent application, wherein the Sargassum polysaccharide has a sulfate content of 60% (w/w) or more. The use of the second aspect of the patent application, wherein the Sargassum polysaccharide has a sulfate content of 60-70% (w/w). The use of the first aspect of the patent application, wherein the Sargassum polysaccharide is extracted from Sargassum and treated by a step of increasing the sulfate content. The use of the seventh aspect of the patent application, wherein the step of increasing the sulfate content is achieved by chemically adding sulfate. The use of any one of claims 1 to 8 wherein the composition increases the rate of inhibition of enterovirus 71 infection. The use of claim 9, wherein the composition is an additive to a pharmaceutical composition, a food additive or a sanitary product.