CN1279953A - Application of seeweed polyose sulphate - Google Patents
Application of seeweed polyose sulphate Download PDFInfo
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- CN1279953A CN1279953A CN 00120874 CN00120874A CN1279953A CN 1279953 A CN1279953 A CN 1279953A CN 00120874 CN00120874 CN 00120874 CN 00120874 A CN00120874 A CN 00120874A CN 1279953 A CN1279953 A CN 1279953A
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- sulfated polysaccharides
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Abstract
The present invention discloses a new medical application of seaweed polyose sulphate in preparing new medicine for hepatitis B. When the dosage is 750-1500 mg each day in three times, seaweed polyose sulphate has obvious curative effect on hepatitis B.
Description
The present invention relates to a kind of novel medical use of SULFATED POLYSACCHARIDES FROM SEAWEEDS SPS, promptly as the purposes for the treatment of hepatitis B medicament.
Many viral diseases still serious threat human life security, and wherein the hepatitis B (HB) that causes of hepatitis B virus (HBV) is the strong and great infectious disease of hazardness of a kind of infectiousness, does not still have effective medicine at present.
China is the sick big country of sending out of hepatitis B (being called for short hepatitis B later on), and the HBVer reaches 1.2 hundred million, and wherein quite a few patient will be converted into liver cirrhosis and hepatocarcinoma, and it has a strong impact on the healthy of people.The diagnosis and the preventive measure of hepatitis B are mature on the whole, but effectively Drug therapy is unsatisfactory.The most frequently used interferon only reaches 40% to eligible patients's effective percentage in the treatment at present.In view of present employed anti-hbv drug exists therapeutic effect incomplete, phenomenons such as toxic side effect and drug withdrawal bounce-back need be sought medicine effective, low toxicity.
Modern medicinal Sargassum reaches over one hundred kind, has that heat-clearing and toxic substances removing, inducing diuresis to remove edema, the kidney invigorating nourish heart, effects such as activating blood circulation to dissipate blood stasis and the anthelmintic that helps digestion.Contain plurality of active ingredients in the Sargassum, wherein, polysaccharide sulfate is to study comparatively deeply, simultaneously also is to possess the composition of practical value most.Sargassum polysaccharides is a kind of glycoconjugates with special construction, contains the proteoglycan of sulfate group.Drug research based on saccharide has great potential, and carbohydrate molecule plays an important role at aspects such as cell recognition, signal transmission, cell-cell interactions, and sugar moieties has determined the function of whole molecule.The saccharide medicine is at infection, and is anticancer, and all there is exploitation and application in various degree aspects such as antiviral and resisting rheumatoid disease arthritis.Conjugated protein component performance in the polysaccharide suppresses virus breeding and kill virus effect, is that multidigit is put antiviral molecular basis.
To the antivirus action of SULFATED POLYSACCHARIDES FROM SEAWEEDS SPS, existing both at home and abroad (Zheng Yongtang, Ben Kunlong, Jin Shanwei, the HIV (human immunodeficiency virus)-resistant activity of protein extract in 17 kind of plant, Chinese virusology, 1998 reported; 13:312-24; Witvrouw M and De Clercq E.Sulfated polysaccharides extracted fromsea algae as potential antiviral drugs.Gen Pharmacol, 1997; 29:497-511; Witvrouw M, Este JA, Mateu MQ, et al.Activity of a sulfated polysaccharidesextracted from the red seaweed aghardhiella tenera against humanimmunodeficiency virus and enveloped viruses.Antiviral chemistry ﹠chemotherapy, 1994; 5:297-303).But, do not see the research report that its anti-hepatitis B virus effect is arranged.
The present inventor finds that under study for action SULFATED POLYSACCHARIDES FROM SEAWEEDS SPS has the effect of beyond thought significant anti-hepatitis B virus.
The object of the present invention is to provide a kind of new medicine for the treatment of hepatitis B.
SULFATED POLYSACCHARIDES FROM SEAWEEDS SPS of the present invention (Sulfated polysaccharides from seaweeds, SPS) be the protein polysaccharide of a kind of sulfur acid ester group of from Brown algae, extracting, main physicochemical characteristics is: soluble in water, dissolubility 〉=30%, white or yellow polycrystal powder, the distinctive fragrance of tool; SO42-content is 20 ± 5%, and fucose content is 20 ± 5%, protein content 10-18%.
SULFATED POLYSACCHARIDES FROM SEAWEEDS SPS extraction process of the present invention is by the exploitation of the present inventor's independent studies, and it may further comprise the steps:
1. select materials: the light dry seaweed of optional homemade high-quality (also can use Macrocystis pyrifera (L.) Ag.), the main place of production, China north
The marine site, side;
2. soak and cleaning: soak and cleaning the strict time of controlling immersion and cleaning under the room temperature;
3. pulverize;
4. water is carried;
5. low concentration precipitate with ethanol;
6. the high concentration precipitate with ethanol obtains thick product;
7. purification obtains purified product.
This preparation method mild condition is not destroyed the contained various natural nutrition compositions of Sargassum frond, and is simple to operate, improved the Sargassum comprehensive utilization degree, productive rate higher (2%), and cost is lower, is suitable for suitability for industrialized production.
Pharmaceutical composition of the present invention can be made into tablet, pill, injection and capsule (soft, hard), and every dose contains SULFATED POLYSACCHARIDES FROM SEAWEEDS SPS 250mg, and it is oral to follow the doctor's advice.Every day, consumption 750mg～1500mg can divide to give for three times, each 250mg～500mg.
With reference to State Standard of the People's Republic of China GB15193.3-94, GB15193.5-94, GB15193.8-94, GB15193.7-94, GB15193.4-94, GB15193.14-94 and GB15193.13-94, carry out rat acute toxicity test, acute toxicity test in mice, mouse bone marrow cells micronucleus test (male), mouse bone marrow cells micronucleus test (female), mouse sperm deformity test, mouse testis chromosomal aberration test, Salmonella reversion test, the tertogenicity test of rat system and 30 days feeding trials of rat, the result shows large and small Mus LD
50＞21.50g/kg; Capsule experimental group and group of solvents compare, and the mouse Bone marrow cells micronucleus rate does not have significant difference; Capsule experimental group and group of solvents compare, mouse sperm deformity rate there was no significant difference; Capsule experimental group and group of solvents compare, mice primary spermatocyte chromosome aberrations rate, no significant difference; Salmonella reversion test, experimental group return and become clump count and be less than or equal to nature and return and become 2 times of clump counts; In the rat tertogenicity test, experimental group and group of solvents compare, and index differences such as pregnant Mus pregnancy period weight gain and reproduction embryo inspection, tire Mus growth promoter and skeleton deformity inspection are not remarkable; Rat was fed in 30 days, compared clinical examination with the normal feedstuff matched group: internal organs weighing, blood biochemical are learned inspection, hematological examination, histopathologic examination, difference that there are no significant.It is nontoxic that this result shows that SULFATED POLYSACCHARIDES FROM SEAWEEDS SPS belongs to, hereditary-less toxicity and no teratogenesis material.
The oral SULFATED POLYSACCHARIDES FROM SEAWEEDS SPS capsule of rat (4000mg/kg), one day 2 times, serve on 10 days, under waking state, observe its spiritual nervous system, cardiovascular system respiratory system of unifying, all no abnormal performance of result.
Try clothes through this laboratory scientific and technical personnel and trial volunteer worker 30 people, SULFATED POLYSACCHARIDES FROM SEAWEEDS SPS does not all cause bad variation to digestive system, nervous system, respiratory system and blood circulation, and majority are reflected full of vitality, and appetite increases.5 routine physical weakness, the experimenter who easily catches a cold reflect that times of common cold obviously reduces
Describe the present invention in detail with embodiment below, but these embodiment must not be interpreted as the limitation of the present invention that goes up in all senses.
Embodiment 1: the preparation of SULFATED POLYSACCHARIDES FROM SEAWEEDS SPS
The 100kg Sargassum through soaking, clean, pulverizing, adds water 800kg, and 90-100 ℃ extracts 3.5h 3 times.Get the supernatant concentrating under reduced pressure to 25L, precipitate under 30% ethanol condition, get supernatant again, reduction vaporization adds in the ethanol of 85L 95% to 10L, filters, and gets thick product.60 ℃ of dryings, purification, 100 orders are pulverized.
Embodiment 2: the effect of SULFATED POLYSACCHARIDES FROM SEAWEEDS SPS resisting HBV virus
SPS has anti-preferably HBV effect in vivo, outward.
One, the external anti-HBV effect research of SPS
1.SPS in the 2.2.15 cell culture to the inhibitory action of HBeAg and HBsAg
(1) cell culture: cover with in the culture bottle of 2.2.15 cell and add 0.25% pancreatin (preparation of Hanks liquid), 37 ℃ digested 3 minutes, add the MEM culture fluid and (contain hyclone 10%, 3% glutamine 1%, G418 380g/ml, kanamycin 50U/ml) piping and druming, go down to posterity at 1: 3, covered with in 10 days, and added the cell counting count board counting, be mixed with every milliliter of 100,000 cell inoculation Tissue Culture Plates, the every hole 0.2ml of 96 orifice plates, the every hole 1ml of 24 orifice plates, 37 ℃ of 5%CO2 cultivated 24 hours, and cell experimentizes after growing up to monolayer.
(2) medicine pair cell toxicity test: SPS is mixed with 20mg/ml solution with culture fluid, is diluted to 1.25mg/ml with 2 times and adds 96 porocyte culture plates, and same concentration liquid was changed in per 4 days in every concentration 4 holes, established no drug cell matched group.With the observation of cell pathological changes is index, 8 days microscopically observation of cell pathological changes, and destroying fully is 4; 75% is 3; 50% is 2; 25% is 1; Anosisly become 0.Calculate every concentration liquid average cell lesion degree and suppression ratio (%).Press Reed Muench method and calculate the poisonous concentration of half (TC50), maximal non-toxic concentration (TC0).
(3) medicine is to HBeAg, HBsAg inhibition test: 2.2.15 cell 104/ml inoculates 24 porocyte culture plates, every hole 1ml, 37 ℃ of 5%CO2 cultivated 24 hours, add the following 2 times of dilution test medicinal liquids of non-toxic concn, 5 dilution factors are respectively 10,5,2.5,1.25 and 0.625mg/ml, every concentration 3 holes, 37 ℃ of 5%CO2 cultivate, changed the original content medicinal liquid in per 4 days and cultivate, results culture fluid in the time of the 8th day ,-20 ℃ of stored frozen.The solid phase radioimmunoassay box is measured HBsAg and HBeAg.Effect of drugs calculates: calculate cell contrast and every concentration c pm average and standard deviation, P/N value and inhibition percentage rate (%), medium effective concentration (IC50) and selection index (SI).
2.SPS the inhibitory action ultracentrifugation to hepatitis b virus dna polymerase (HBV DNAp) prepares the HBV granule, add employing virus cracking liquid (0.02M Tris-HCl pH7.6,0.05M NaCl, 0.001M EDTA, 0.1%-mercaptoethanol [ME], 0.01%Triton X-100).Ice bath adds 60ul and mixes liquid (0.2M Tris-HCl pH7.6,0.2M NH4Cl, 0.06M MgCl2,2%NP-40,0.6%-ME, 0.1mM dNTP), (concentration is respectively 201 SPS: 20,4,0.8,0.16,0.032mg/ml), 30ul lytic virus, 1.5Ci 3H-dTTP, 4 ℃ of abundant mixings, move to 45 ℃ of reactions 2.5 hours, after reaction finishes, add 10% ice-cold trichloroacetic acid precipitation DNA of 3ml, sedimentary DNA closes on the plain film of glass fibre, wash film with 5% trichloroacetic acid and 70% ethanol then,, add the 8ml scintillation solution and (contain 0.5%PPO the film oven dry, the xylene solution of 0.015%POPOP) liquid scintillation counter is measured cpm, calculates suppression ratio.
3.SPS to HBsAg and the bonded inhibition experiment of anti--HBs: with concentration be respectively 20,4,0.8,0.16, the SPS of 0.032mg/ml mixes as testing sample with isopyknic Purification of HBsAg, negative if (no Purification of HBsAg) and positive (no SPS) contrast, with HBsAg content in the solid phase radioimmunological kit working sample, calculate medicine to HBsAg and anti--bonded suppression ratio of HBs.
4. experimental result (1) SPS has inhibitory action to HBV in 2215 cell culture
(2) SPS has inhibitory action ((IC to the HBV archaeal dna polymerase
50=225 μ g/ml) (3) SPS inhibition HBsAg combines (IC with anti-HBs
50=200 μ g/ml)
| Cytotoxicity (mg/ml) | HbeAg | HBsAg |
| IC 50??????????TC 016．32±0．65?10±0 | IC 50(mg／ml)??SI 4．88±0．42??3．37±0．321 | IC 50(mg／ml)??SI 4．26±1．04??4．0±1．01 |
Two, anti-HBV effect research in the SPS body
1. duck hepatitis B virus infection: 1 age in days Beijing duck, clear through the positive Sanguis Anas domestica of lower limb shin intravenous injection Shanghai sheldrake DHBV-DNA, every 0.2ml got blood in back 7 days in infection, separation of serum ,-70 ℃ of preservations are to be checked.
2. Drug therapy test: DHBV infect duckling after 7 days random packet carry out the Drug therapy test, every group of 6 ducklings, 3 dosage groups of administration component, the sulphuric acid Sargassum polysaccharides is respectively 25,50 and 100mg/kg group, lumbar injection 1 day 2 times, 10 days, establish virus control group (DHBV), with the physiologic saline for substitute medicine.Positive drug is made positive control with acyclovir 100mg/kg group.The 7th day is (T0) before the medication after infection, and medication the 5th day (T5) after medication the 10th day (T10) and the drug withdrawal the 3rd day (P3), is got blood from duck lower limb shin vein, separation of serum, and-70 ℃ of preservations are to be checked.
3. detection method: get above-mentioned Sanguis Anas domestica to be checked and check film, measure Sanguis Anas domestica clear in the DHBV-DNA level.Press nick translation test kit description method,, and make the clear dot blot hybridization of Sanguis Anas domestica with 32P labelling DHBV-DNA probe, autoradiography diaphragm speckle, measure OD value (optical filter is 490nm) in microplate reader, calculating serum DHBV-DNA density, with hybridization spot OD value as specimen DHBV-DNA level value.Calculate the inhibition % of (T0) before the 3rd day (P3) serum DHBV-DNA of different time (T5, T10) and drug withdrawal after every group of duck medication and the administration, and mapping, respectively organize the clear DHBV-DNA suppression ratio of Sanguis Anas domestica dynamically.
4. experimental result
DHB (DHBV) infected duck hepatitis animal lumbar injection SPS (50,100 μ g/kg), Bid * 10, with the normal saline group is virus control, do the positive drug contrast with acyclovir (ACV), SPS group (100 μ g/kg) compares with the virus control group, can significantly reduce DHBV infected duck serum DHBV-DNA level.Especially do not have a rebound after it should be noted that drug withdrawal.
Claims (8)
1. the application of SULFATED POLYSACCHARIDES FROM SEAWEEDS SPS in preparation treatment hepatitis B medicament.
2. purposes according to claim 1 is characterized in that said SULFATED POLYSACCHARIDES FROM SEAWEEDS SPS is the Brown algae sulfated polysaccharide.
3. purposes according to claim 2 is characterized in that said SULFATED POLYSACCHARIDES FROM SEAWEEDS SPS water solubility more than or equal to 30%, is white or yellow polycrystalline, fragranced is arranged, SO42-content is 20 ± 5%, and fucose content is 20 ± 5%, and protein content is 10-18%.
4. purposes according to claim 1 and 2 is characterized in that said SULFATED POLYSACCHARIDES FROM SEAWEEDS SPS gives with tablet form.
5. purposes according to claim 1 and 2 is characterized in that said SULFATED POLYSACCHARIDES FROM SEAWEEDS SPS gives with capsule form.
6. purposes according to claim 1 and 2 is characterized in that said SULFATED POLYSACCHARIDES FROM SEAWEEDS SPS gives with pill.
7. purposes according to claim 1 and 2 is characterized in that said SULFATED POLYSACCHARIDES FROM SEAWEEDS SPS gives with the injection form.
8. purposes according to claim 1 and 2 is characterized in that SULFATED POLYSACCHARIDES FROM SEAWEEDS SPS consumption every day is 750mg～1500mg.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 00120874 CN1118281C (en) | 2000-08-04 | 2000-08-04 | Application of seeweed polyose sulphate |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 00120874 CN1118281C (en) | 2000-08-04 | 2000-08-04 | Application of seeweed polyose sulphate |
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| Publication Number | Publication Date |
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| CN1279953A true CN1279953A (en) | 2001-01-17 |
| CN1118281C CN1118281C (en) | 2003-08-20 |
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| CN 00120874 Expired - Fee Related CN1118281C (en) | 2000-08-04 | 2000-08-04 | Application of seeweed polyose sulphate |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105273093A (en) * | 2014-07-10 | 2016-01-27 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Preparation method for sulfated polysaccharides from seaweed |
| CN105343121A (en) * | 2015-09-28 | 2016-02-24 | 青岛海洋生物医药研究院股份有限公司 | Application of polyguluronate sulfate to anti-hepatitis B virus medicine preparation |
| CN107400172A (en) * | 2017-07-20 | 2017-11-28 | 房俊英 | A kind of bioactive substance extracting method |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101361799B (en) * | 2008-09-01 | 2011-07-20 | 北京世纪博康医药科技有限公司 | Composition containing brown alga polysaccharide sulfuric ester and schisandra chinensis and use thereof |
-
2000
- 2000-08-04 CN CN 00120874 patent/CN1118281C/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105273093A (en) * | 2014-07-10 | 2016-01-27 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Preparation method for sulfated polysaccharides from seaweed |
| CN105273093B (en) * | 2014-07-10 | 2018-01-23 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | A kind of preparation method of seaweed polysaccharide sulfate |
| CN105343121A (en) * | 2015-09-28 | 2016-02-24 | 青岛海洋生物医药研究院股份有限公司 | Application of polyguluronate sulfate to anti-hepatitis B virus medicine preparation |
| CN107400172A (en) * | 2017-07-20 | 2017-11-28 | 房俊英 | A kind of bioactive substance extracting method |
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| Publication number | Publication date |
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| CN1118281C (en) | 2003-08-20 |
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