CN104922146B - Sargassan is in preparation for treating or preventing the purposes in the composition that enteric virus71 type infects - Google Patents
Sargassan is in preparation for treating or preventing the purposes in the composition that enteric virus71 type infects Download PDFInfo
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- CN104922146B CN104922146B CN201410108714.4A CN201410108714A CN104922146B CN 104922146 B CN104922146 B CN 104922146B CN 201410108714 A CN201410108714 A CN 201410108714A CN 104922146 B CN104922146 B CN 104922146B
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Abstract
The invention discloses sargassan in preparation for treating or preventing the purposes in the composition that enteric virus71 type infects.Composition of the invention can be used as the additive of medical composition, food additives or sanitary product.
Description
Technical field
The present invention relates to a kind of sargassan in preparation for treating or preventing in the composition that enteric virus71 type infects
Purposes, wherein the composition typically can be used as the additive of medical composition, food additives and sanitary product.
Background technique
Enteric virus71 type belongs to Picornaviridae (Picornaviridae), enterovirus category (Enterovirus),
It is the one kind being finally found in the enterovirus being currently known.Enteric virus71 type infection brains and brain stem cause burst sexual centre mind
The illness of warp, such as: lethargic sleep, irritability, expiratory dyspnea, encephalitis, aseptic meningitis, brothers mouthful disease, herpangina and the heart
Dirty failure shock, be easy to cause children's severe, very harmful to children.
In physicochemical characteristics, enteric virus71 type virion is not influenced vulnerable to soda acid, therefore its appeal is in acyclic acidic
Under border, it can be remained by stable.Outer embrane (envelope) structure is formed by by lipid since enteric virus71 type does not have,
Belong to kind of a naked virus, therefore there is tolerance to ethyl alcohol, so that alcohol can not play inhibiting effect.In addition, general family is common
Detergent is to enteric virus71 type also without bactericidal effect.Enteric virus71 type institute can be effectively also fought without drug or vaccine at present
Caused infection.
Sargassum (Sargassum) is a kind of seaweed in brown alga, belongs to Phaeophyta, pelvetia silquosa mesh, Sargassaceae, distribution
In many sea areas in the world, be found in the regions such as northern, northeast littoral sea and Penghu, Lutao, Lanyu in Taiwan, especially in
Penghu sea area has considerable yield.Sargassan (Sargassum polysaccharide) is extracted from sargassum
A kind of soluble easily in water, higher polysaccharose substance of viscosity.There is report display sargassan that there is strengthen immunity, inhibit swollen
Oncocyte, anti-oxidant and antiviral etc. pharmacological activity, but antiviral pharmacological mechanism is still not clear, especially for enterovirus
Rare further investigated.
Therefore, for enteric virus71 type, still a need to the active constituent that can effectively antagonize its infection is found out, especially from
The natural food materials of daily life also have nontoxic and handy advantage not only up to therapeutic efficiency.
Summary of the invention
The present invention provides a kind of sargassan in preparation for treating or preventing the composition of enteric virus71 type infection
In purposes.
In some embodiments, sargassan of the invention can be handled through sulphation, so that treated horse hair
In polysaccharides, sulfate radical content reach 10%(w/w) more than, 20%(w/w) more than, 40%(w/w) more than or 60%(w/w) more than.?
In one certain specific embodiments, in sargassan of the invention, sulfate radical content reaches 60-70%(w/w).
Specifically, composition of the invention can increase the inhibiting rate for the infection of enteric virus71 type, for example, reducing cell
The ratio of enteric virus71 type is infected, inhibits the RNA synthesis of enteric virus71 type and inhibits cytopathy caused by enteric virus71 type
And Apoptosis.
The specification specified of each specific embodiment of the invention is as after.Other features of the invention will be via following each
Detailed description and claim in specific embodiment and clearly appear from.
It need not be further elucidated above, it is believed that the technical staff in the technical field of the invention based on the foregoing description can benefit
With the present invention to most wide degree.It is used accordingly, it is to be understood that the following description is merely possible to illustrate, rather than with any
Mode limits remaining disclosure
Detailed description of the invention
Fig. 1 shows the HPLC map of sargassan, and wherein Fig. 1 (A) is the HPLC map of sargassan standard items, and
Fig. 1 (B) is the HPLC map of sulphation sargassan.
Fig. 2 shows the suppression of the sargassan standard items and sulphation sargassan of various concentration for 71 type of intestines viral disease
Effect processed.
Specific embodiment
The present invention analyzes sargassan, confirm for the first time its for enteric virus71 type infected with good inhibitory effect,
The sargassan for especially increasing sulfate radical content can further improve its effect for inhibiting enteric virus71 type.Sargassum is more
Sugared nontoxic and easy acquirement, it is high compared to toxic and expensive chemicals acceptance, especially for the baby children of high-risk
For youngster.Therefore, the present invention is made that tremendous contribution for the treatment and prevention and treatment of disease caused by enteric virus71 type.
Unless otherwise specified, all technical and scientific terms used here have such as fields of the present invention
The meaning that technical staff is generally understood.
" one " word used herein, it is such as not specified, refer to the quantity of at least one (one or more).
On the one hand, the present invention is to disclose a kind of sargassan in preparation for treating or preventing the infection of enteric virus71 type
Composition in purposes.
Sargassan is polysaccharide obtained by being extracted as sargassum, and chemically, it is a kind of is with " fucose "
Main polysaccharide body, the heteromeric polysaccharide component of water solubility being made of fucose (fucose) and sulfate (sulphate), wherein
Fucose is bonded into major key mutually with α-(1 → 2), α-(1 → 3) or α-(1 → 4), and sulfate is incorporated in going back for C-2 or C-4
On former end.Specifically, its following formula (I) of chemical structure for having:
Formula (I)
Sargassan can also be extracted from sargassum by conventional method and be obtained, for example, with hot water by commercially obtaining
Impregnating frond makes its softening (for example, being soaked in about 100 DEG C or so of hot water about 1-3 hour), through ferment or acidification step processing,
It is then centrifuged for, filters, collect supernatant.The supernatant obtained after extraction can be further freeze-dried, and be saved with benefit.Have in part
In body embodiment, the sargassan that the present invention uses, molecular weight ranges are 50kDa to 1, and 000kDa, 250kDa are extremely
950kDa or 450kDa to 850kDa.In a certain specific embodiments, the sargassan tool molecular weight that the present invention uses is about
800kDa。
Preferably, the step of increase sulfate radical content can be carried out, to increase the sulfate radical content of sargassan.In part
In specific embodiment, chemical method addition sulfate radical is can be used in the present invention, for example, chlorosulfonic acid-dimethylformamide is added
(Chlorsulfonic acid-N, N-dimethylformamide, HClSO3/ DMF) reagent reacted, with alkali after reaction
(such as NaOH) is neutralized, and is then centrifuged for, is filtered, and supernatant is collected.In some embodiments, in sargassan of the invention
Sulfate radical content reach 10%(w/w) more than, 20%(w/w) more than, 40%(w/w) more than or 60%(w/w) more than.In a specific tool
In body embodiment, sulfate radical content reaches 60-70%(w/w in the sargassan that the present invention uses).
Composition of the invention can be deployed according to conventional method traditionally with any one or more of carrier.For example,
Sargassan is mixed with suitable carrier, then is made into required dosage form.
" carrier " word used herein means to form the non-live of be intended to formulation to carry sargassan
Acceptable carrier on property ingredient, including any standard pharmaceutical, can be compatible with the active constituent of composite, and to being applied
Individual is harmless, for example, water, physiological saline, glycerol, ethyl alcohol, glucose water, glucan, lactose, sucrose, mannitol, its analog
Or above combination.
Composition of the invention still includes adhesive, lubricant, antioxidant, filler and preserving agent.It is available glutinous
Knot agent include Arabic gum, sodium alginate, ethyl cellulose, agar, gelatin, starch, hydroxylated cellulose, hydroxypropyl cellulose,
Its analog or more than combination.Available lubricant include stearic acid, calcium stearate, magnesium stearate, talcum, hydrogenated oil and fat, it is cured,
Its analog or more than combination.Available antioxidant include Tocopherol, dibutyl hydroxy toluene, butylhydroxy methoxy benzene,
Ascorbic acid, its analog or more than combination.Available filler includes microcrystalline cellulose, lactose, mannitol, its analog
Or above combination.Available preserving agent include benzoic acid and its esters, sorbic acid and its esters, parabens,
Go water acetic acid sodium, hydrogen peroxide, sodium hypochlorite, its analog or more than combination.
In one embodiment, composition of the invention can be deployed into according to administration approach as medical composition
Various dosage forms.For example, medical composition of the invention can be formulated into as capsule or pastille, to carry out oral administration medicine supplying.Capsule
The acceptable substance of medicine containing any standard, for example, gelatin or cellulose.Pastille will can combine according to conventional methods
Object is suppressed with solid-state carrier and lubricant jointly and is deployed.Medical composition of the invention also can be formulated into injection type, be made
Carrier includes aqueous solution, isotonic salt water or Glucose Liquid or the acceptable carrier of medicine known to other.Of the invention
Medical composition is still deployed into emulsifiable paste, finish, gel, lotion or spray, for local application.
In another embodiment, composition of the invention is as food additives.Sargassan is derived from naturally
Food materials, it is not toxic, the effect of being added directly in food or nutriment, simply reach prevention and health care, for example, being added to milk
Infants are provided in powder or other dairy products.
In still another embodiment, composition of the invention is the additive as sanitary product, for example, can add
Add to face, body or hand cleaning articles or environment disinfected liquid or simultaneously inlet shroud, cleaning wipes, toilet paper or cotton rod.
Embodiment 1: the preparation of sargassan
Sargassum is cleaned and dried with flowing water, then is ground.Then prepare 5%(w/w) sargassum aqueous solution carry out
Water hot extraction's method: keep frond soft in hot bath (about 100 DEG C or so, impregnate about 1-3 hours) sargassum aqueous solution soaking first
Change, then with ferment or sour hydrolysis process, carries out high speed centrifugation after the reaction was completed, filter later and collect supernatant.By the supernatant
Liquid moves to -80 DEG C, then is freeze-dried with vacuum freezing drying machine (LABCONCO), and sargassan is obtained.
Embodiment 2: sulfating reaction
The sargassan that above-mentioned extraction is obtained increases sulfate radical content with sulfate radical chemical Treatment: in sargassan
Middle addition chlorosulfonic acid-dimethylformamide (Chlorosulfonic acid-N, N-dimethylformamide, HClSO3/
DMF) reagent is reacted, and is tuned into neutrality after reaction with NaOH, is then carried out high speed centrifugation, is filtered later and collect supernatant.
The supernatant is moved to -80 DEG C, then is freeze-dried with vacuum freezing drying machine to get sulphation sargassan.
Embodiment 3: sulfate radical content analysis
It is using commercial obtainable sargassan standard items or sulphation sargassan sample to be measured, its is molten
In the 0.5ml HCl of concentration 1N, after 100 DEG C are reacted 1 hour, it is evaporated with baking oven to no moisture.The deionization of 0.5ml is added
Water (ddH2O) after back dissolving, the barium chloride buffer (BaCl of 2ml absolute alcohol, 1ml are added2Buffer, the acetic acid containing 2M
The sodium bicarbonate 8ml of barium chloride 2ml, 0.02M of 10ml, 0.05M, and it is quantitative to 100ml) and 1.5ml rose with absolute alcohol
Brown acid sodium solution (Na-rhodizonate solution, the Na containing 0.25mg/ml2C6O6, 100mg ascorbic acid, and with
Absolute alcohol is quantitative to 100ml) mixing.Be protected from light 20 minutes, finally measures A520 and be converted into sulfate radical content.Benefit
Sargassan standard items and sulphation sargassan sample to be measured, sulfate radical content such as table 1 are detected in aforementioned manners
It is shown.
Table 1
Embodiment 4: polysaccharide molecular weight analysis
With Propiram (Pullulan) be standard items (molecular weight of standard items is arrogant to small be followed successively by 788kDa, 404kDa,
212kDa, 112kDa, 47.3kDa, 22.8kDa, 11.8kDa and 5.9kDa), take 20 μ L to carry out the analysis of HPLC molecular weight.GPC points
It is flow velocity 0.8ml/min from tubing string (OHpak SB-804HQ) operating condition, purging with liquid is deionized water, and with evaporative light-scattering
Detector (Evaporative Light Scattering Detector, ELSD) carries out the analysis of polysaccharide body molecular weight distribution.Point
Map after analysis calculates the residence time (retention time) to the standard of polysaccharide molecular weight (Mw) with EC2000GPC software
The foundation that curve is analyzed as sargassan molecular weight.
Fig. 1 shows the HPLC profiling results of sargassan, and wherein Fig. 1 (A) is the HPLC figure of sargassan standard items
Spectrum, Fig. 1 (B) is the HPLC map of sulphation sargassan, can obtain sargassan molecular weight about after comparing standard curve
Fall in about 800kDa, but wherein the residence time of Fig. 1 (B) move right, indicate after sulfating reaction, because of the addition of sulfate radical,
Molecular weight is caused to increase.
Embodiment 5: cell and Virus culture
The Strain of enteric virus71 type is taken from one between in July, 2001 because of two years old of the severe death of enteric virus71 type
The larynx specimen of girl baby, the Strain are MEL701, are provided by Chung Hsing University teacher Chen Quanmu laboratory.
On the other hand, by African green monkey kidney cell (African green monkey kidney cells；VERO cell,
ATCC CCL-18), it is incubated in RPMI-1640 culture solution (containing 2%FBS, pH7.0), when cell grows about eight points of full Shi Jinhang
Squamous subculture.Cell culture fluid is first removed, is washed away with phosphate buffer (phosphate buffer saline, PBS) remaining
Serum and cell metabolite add trypsase and ethylenediamine tetra-acetic acid (ethylene diamine tetraacetic
Acid, EDTA) effect 3 to 5 minutes, with fetal calf serum (fetal bovine serum, FBS) stopped reaction and use cell culture
Liquid sweeps away cell, is collected in centrifuge tube, centrifugation, remove supernatant collection cell precipitation, then with culture solution back dissolving after, carry out
Make cell count.
MEL701 Strain is inoculated in VERO cell (culture solution of RPMI-1640 containing 2%FBS), with inverted micro-
Sem observation cell kenel is collected when there is 80% or more cytopathic phenomena (cytopathic effect, CPE) containing virus
Culture solution, be stored in -80 DEG C of refrigerators, be used for subsequent experimental.
Embodiment 6: viral plaque inhibits test
By VERO cell kind (1 × 10 in 6 hole culture dishes5Cells/well), it is placed in containing 5%CO237 DEG C of incubators in, to
Cell culture completely, takes out culture solution to six, seven points, and it is primary to rinse cell with PBS.By sargassan standard items and sulphation horse
Tail polysaccharides (taking sulfate radical content is 67.6% sample) are diluted to respectively with RPMI-1640 culture solution (without fetal calf serum)
Various concentration respectively after culture effect 1 hour, adds virus liquid (hole 50PFU/), feels at 37 DEG C with cell at 37 DEG C
Dye 1 hour.It is eventually adding the RPMI-1640 culture solution containing 2% agarose gel (agarose), to its solidification, is placed in containing 5%CO2's
It after being cultivated four to five days in 37 DEG C of incubators, is fixed with 10% formalin, excavates gel, with 1% violet staining, with certainly
Water counts viral plaque number after rinsing.
The calculation of viral plaque inhibiting rate (%) are as follows: (VC-VCS)/VC × 100%, wherein VC is that sargassum is not added is more
The viral plaque number of saccharide or the control infection group and viral infection group of sulphation sargassan；And VCS is that sargassan is added
The viral plaque number of the experimental virus infected group of standard items or sulphation sargassan.Experimental data is obtained by independent experiment three times
, with mean+/-standard error (mean ± SEM) display.
Fig. 2 shows that viral plaque inhibits test result.The results show that sargassan shows good confrontation enteric virus71 type
The effect of infection, inhibiting rate is of about 30-45%, and after especially sargassan increases sulfate radical content, antiviral effect is promoted again again
About 28-47%, i.e. inhibiting rate are promoted to about 60-80% or more, and antiviral effect is quite excellent.
Claims (10)
1. sargassan is in preparation for treating or preventing the purposes in the composition that enteric virus71 type infects.
2. purposes according to claim 1, wherein in the sargassan, sulfate radical content up to 10% weight percent with
On.
3. purposes according to claim 2, wherein in the sargassan, sulfate radical content up to 20% weight percent with
On.
4. purposes according to claim 2, wherein in the sargassan, sulfate radical content up to 40% weight percent with
On.
5. purposes according to claim 2, wherein in the sargassan, sulfate radical content up to 60% weight percent with
On.
6. purposes according to claim 2, wherein in the sargassan, sulfate radical content reaches 60-70% weight percent
Than.
7. purposes according to claim 1, wherein the sargassan is to extract from sargassum and contain through increasing sulfate radical
The step of amount, is handled.
8. purposes according to claim 7, wherein the step of increase sulfate radical content is to add sulfate radical with chemical method
And reach.
9. purposes according to any one of claim 1 to 8, wherein the composition is to increase to infect enteric virus71 type
Inhibiting rate.
10. purposes according to claim 9, wherein the composition is adding as medical composition or sanitary product
Add object.
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| CN201410108714.4A CN104922146B (en) | 2014-03-21 | 2014-03-21 | Sargassan is in preparation for treating or preventing the purposes in the composition that enteric virus71 type infects |
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