CN1129613C - Oleander flower polysaccharide and its derivatives, preparing method and use - Google Patents
Oleander flower polysaccharide and its derivatives, preparing method and use Download PDFInfo
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- CN1129613C CN1129613C CN99125746A CN99125746A CN1129613C CN 1129613 C CN1129613 C CN 1129613C CN 99125746 A CN99125746 A CN 99125746A CN 99125746 A CN99125746 A CN 99125746A CN 1129613 C CN1129613 C CN 1129613C
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- nif
- oleander flower
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Abstract
The present invention relates to oleander flower polysaccharide which has the function of nerve growth factors and a derivative thereof. The oleander flower polysaccharide is extracted and separated from oleander flowers, and through column chromatography and purification, the oleander flower polysaccharide is prepared. Experiments prove that the oleander flower polysaccharide has the obvious function of promoting growth and differentiation of nerve cells, and has the obvious repair functions for impaired nerve cells. The function of the oleander flower polysaccharide is the same as that of nerve growth factors, or is better than that of nerve growth factors. Therefore, the oleander flower polysaccharide can be used for preparing medicine for treating diseases, such as senile dementia, apoplexy, children's hypophrenia, etc.
Description
The present invention relates to polyose and, especially relate to Oleander flower polysaccharide and its production and application as the application of medicine.
Polysaccharide such as heparin has blood coagulation resisting function in blood system be early found, it as medicinal application in clinical.Along with to the going deep into of sugared function understanding, the polysaccharide of progressively finding a fixed structure has significant effect to the immunity system of body after the fifties: as antitumor action, and antiviral particularly AIDS resisting effect, anti-inflammatory action and radiation resistance.These polysaccharide have three kinds as medicine formally use clinically at home and abroad (the also having several of unofficial use).Certainly the polysaccharide of also finding some structure also has effect as emesis to gi system, (Kigohara H.Yamada H.Kitasato Arch Exp.Med.1991,64:167 such as antiulcer agent; Fang Jinian: polyose medicament, Chen Huili chief editor " structure of saccharide complex and function " Shanghai press of medical university, 1997, p325).All are applied to the maximum characteristics of clinical polysaccharide as medicine be that toxic side effect is little.But polysaccharide yet there are no the people so far to neural effect and reported.Biological Characteristics Study according to carbohydrate once foretold at Thomas in 1998, polysaccharide might as the medicinal application of treatment senile dementia in clinical (Thomas.WR, Current Opinion in Biotechnology, 1998,9:74-79).Therefore seeking a kind of polysaccharide that is similar to the nerve growth factor effect that has, is a job highly significant.We from be separated to 30 surplus kind of the polysaccharide, screen a polysaccharide that derives from the flower of Folium seu Cortex Nerii, find that it not only has stronger immunologic enhancement, but also have tangible promotion breeding and Differentiation neurocyte PC12.
For this reason, the object of the invention provides a kind of Oleander flower polysaccharide and derivative thereof with nerve growth factor effect.
Another purpose of the present invention provides the preparation method of Oleander flower polysaccharide, is to be got by extraction separation purifying in the flower of Folium seu Cortex Nerii.
Another object of the present invention provides Oleander flower polysaccharide and is used for preparation treatment senile dementia, apoplexy, the application of the medicine of diseases such as children mental retardation.
Oleander flower polysaccharide of the present invention is the polysaccharide that is obtained by extraction separation in the flower of Folium seu Cortex Nerii.
Can be prepared into sulfation Oleander flower polysaccharide and carboxymethylation Oleander flower polysaccharide by Oleander flower polysaccharide.
The preparation method of Oleander flower polysaccharide:
Oleander flower polysaccharide is to be obtained by extraction separation in the flower of Folium seu Cortex Nerii, and its step is as follows:
1, extract, separate: flower of Folium seu Cortex Nerii is through alcohol reflux, dried flower after the degreasing, through boiling water extraction, filter: (a) get filtrate, concentrating filter liquor, three times of ethanol sedimentations, throw out is water-soluble, adds cetyl trimethyl bromide (CTAB) precipitation, and is centrifugal, supernatant liquor is through dialysis, and alcohol precipitation, drying and other steps obtain a polysaccharide component (NIF
2) crude product.Throw out is through acetate dissolution, alcohol precipitation, and steps such as Deproteinization obtain another polysaccharide component (NIF
1) crude product.(b) residue obtains another polysaccharide component (NIF through steps such as dialysis, alcohol precipitations again through sodium hydroxide solution extraction
3) crude product (seeing Fig. 1 for details).
2, purifying: polysaccharide component NIF
2Through diethylaminoethylcellulose (DEAE-Cellulose) column chromatography, water, 0.2M NaCl, 0.4M NaCl, 4.0M NaCl are opened up layer, obtain each polysaccharide component NIF of preliminary purification
2I, NIF
2II, NIF
2III, NIF
2IV, above-mentioned four components through each polysaccharide component of DEAE-Cellulose and/or sephacryl (Sephacryl S-300) and/or sephadex G-200 (Sephadex G-200) column chromatography acquisition final purification, distinguish called after J again
7, J
8, J
6, J
4, J
5And J
1(seeing Fig. 2 for details).NIF
1And NIF
3Also through diethylaminoethyl--sepharose (DEAE-Sepharose) and/or Sephacryl S-300 column chromatography such as NIF
2Similar purge process, obtain other components of pure polysaccharide: J respectively
10, J
3, J
2And J
9(seeing Fig. 3 and Fig. 4 for details).
Each component is through conventional method such as all-hydrolytic, molecular weight determination, and nucleus magnetic resonance, chemistry such as infrared spectra and spectrographic technique prove polysaccharide structures.
10 Oleander flower polysaccharide components that above-mentioned purifying is obtained carry out the purity evaluation: the NaOH solution with 0.001ml/L is moving phase, and the flow velocity that 0.2ml/ divides carries out purity check on KS-805 polysaccharide post, record each polysaccharide component J
1-J
10Be single symmetrical peak, illustrate that all components have all reached single pure product.
The molecular weight determination of Oleander flower polysaccharide: with standard T-dextran series T-500, T-110, T-70, T-40, T-20 is standard substance, and condition is carried out HPLC described in identifying by purity, records retention time, with retention time known standard molecular weight logarithm is mapped, get typical curve and carry out the Oleander flower polysaccharide sample then, the same terms carries out HPLC, records retention time, can try to achieve corresponding molecular weight from typical curve, be respectively J
1(molecular weight＞1 * 10
6), J
2(1.6 * 10
4), J
3(1.6 * 10
5), J
4(＞1 * 10
6), J
5(＞1 * 10
6), J
6(＞1 * 10
6), J
7(5.0 * 10
3), J
8(1.1 * 10
4), J
9(＞1 * 10
6), J
10(＞1 * 10
6).
The preparation of Oleander flower polysaccharide derivative of the present invention:
(1) sulfation Oleander flower polysaccharide preparation: get pure Oleander flower polysaccharide in anhydrous pyridine, with chlorsulfonic acid reaction, NaOH neutralization, dialysis, freeze-drying promptly gets the sulfation Oleander flower polysaccharide.
(2) preparation of carboxymethylation Oleander flower polysaccharide: get pure Oleander flower polysaccharide and be dissolved among the NaOH, with chloroacetate reaction, the acetic acid neutralization, dialysis, freeze-drying promptly get the carboxymethylation Oleander flower polysaccharide.
Oleander flower polysaccharide of the present invention has tangible promotes growth Differentiation through experiment confirm to neurocyte (PC-12 cell), and injured nerve cells is had tangible promotion repairing effect.Oleander flower polysaccharide mainly shows the elongation of irritation cell to the effect of PC-12 cell, has the neural axon growth of active growing tip aixs cylinder sample.As seen from Figure 5, the PC-12 cell places the Oleander flower polysaccharide solution of different concns, (NGF) makes positive control with nerve growth factor, all show the growth that doubles the archeocyte neural axon at least, Oleander flower polysaccharide can induce neuroganglion to stretch when concentration is 10 μ g/ml, proves that thus its effect very is similar to NGF.Experimental result show its effect or with NGF quite or be better than NGF.Therefore Oleander flower polysaccharide can be used for preparation treatment senile dementia, the medicine of diseases such as apoplexy, children mental retardation.Also can resist H
2O
2To nerve cell damage, damaging cells there is tangible promotion repairing effect etc. chemical substance.Oleander flower polysaccharide also can be used for short nerve growth in the experiment, the positive control reagent that differentiation and neural cell injury are repaired.
Oleander flower polysaccharide of the present invention has the effect that is similar to NGF, and NGF content is few in animal body, separation and purification difficulty, and Folium seu Cortex Nerii source is abundant, the separation and purification of Oleander flower polysaccharide is fairly simple comparatively speaking, is convenient to produce.Therefore to the Oleander flower polysaccharide of neural system biologically active, have great academic significance and application prospect.
The present invention is further elaborated by the following drawings and embodiment, but does not limit content of the present invention.
Description of drawings:
Fig. 1 is obtained the crude product NIF of Oleander flower polysaccharide by the flower of Folium seu Cortex Nerii extraction separation
1, NIF
2And NIF
3Schema.
Fig. 2 is by Oleander flower polysaccharide crude product NIF
2Purifying gets polysaccharide component J
1, J
4, J
5, J
6, J
7And J
8Schema.
Fig. 3 is by Oleander flower polysaccharide crude product NIF
1Purifying gets polysaccharide component J
10Schema.
Fig. 4 is by Oleander flower polysaccharide crude product NIF
3Purifying gets polysaccharide component J
2, J
3And J
9Schema.
Fig. 5 is Oleander flower polysaccharide component J
1, J
2, J
3, J
4And J
5Effect synoptic diagram to neurocyte PC-12.
Embodiment 1:
Flower of Folium seu Cortex Nerii 500g places the Sha Shi extractor, with 95% alcohol reflux 8 hours, ethanol liquid discarded, and flower of Folium seu Cortex Nerii places the infrared lamp ethanol that goes down, get dried flower, use 10 liters of boiling water extraction 8 hours then, filter to get filtrate, filtrate decompression is concentrated into 1.5 liters, stir the ethanol that adds 4.5 liter 95% down, must precipitate, precipitate through centrifugal abandoning supernatant.The water-soluble 1000ml of throw out, the cetyl trimethylammonium bromide of Dropwise 5 % concentration (CTAB) 300ml then, centrifugal supernatant liquor and throw out, supernatant liquor is dialysed through water, three times of ethanol sedimentations are centrifugal, vacuum-drying, Crude polysaccharides NIF
24.6g.Residue after the boiling water extraction extracts with the 5%NaOH room temperature and spends the night, and centrifugal, supernatant liquor is transferred PH to 7.0 with hydrochloric acid, and dialysis adds 3 times of ethanol sedimentations, and is centrifugal, and vacuum-drying gets another Crude polysaccharides NIF
33.2g.Throw out with the 5%CTBA precipitation, be dissolved among the 5%HOAC 500ml, add the 1500ml ethanol sedimentation, the centrifugal throw out that gets, throw out is dissolved in the 400ml water, adds 15% trifluoroacetic acid (TCA), the centrifugal precipitation of going, supernatant liquor is through dialysis, and three times of ethanol sedimentations, centrifugal, vacuum-drying get another Crude polysaccharides NIF
12.2g.
Embodiment 2:
NIF
24.6g as shown in Figure 2, behind DEAE-Mierocrystalline cellulose chromatography post, water, 0.2M NaCl, 0.4M NaCl and 4M NaCl open up layer in succession, fraction collection is got the partly positive of sulfuric acid-phynol method colour developing, partly respectively to distill water dialysis 2 days, lyophilize then obtains NIF respectively for each
2I0.83g, NIF
2II 0.65g, NIF
2III 0.94g and NIF
2IV 0.76g.NIF
2I more once through the DEAE-cellulose chromatography, water exhibition layer is got sugar moieties, dialysis, lyophilize gets NIF
2I A0.64g, NIF
2I A again through the DEAE-cellulose chromatography once through water exhibition layer, dialyses as preceding method, and lyophilize gets two components, J
70.19g, J
80.21g.NIF
2II is through Sephacryl S-300 column chromatography, and 0.2M NaCl opens up layer, dialysis then, and lyophilize gets J
60.32g.NIF
2III is through the DEAE-cellulose chromatography, and 0.4M NaCl opens up layer, dialysis, and lyophilize gets NIF then
2IIIB 0.234g, NIF
2IIIC 0.518g.NIF
2IIIB is again through Sephadex G-200 column chromatography, and water exhibition layer is dialysed, and lyophilize gets J
40.18g.NIF
2IIIC is through Sephacryl S-300 column chromatography, and 0.2M NaCl opens up layer, dialysis then, and lyophilize gets J
50.4g.NIF
2IV is through Sephacryl S-300 column chromatography, and 0.2M NaCl opens up layer, dialysis then, and lyophilize gets J
10.35g.
Embodiment 3
NIF
12.2g be dissolved in the 200ml water, in stirring, progressively add 100% ethanol, make the ultimate density of solution be 45%, centrifugal, remove supernatant liquor, must precipitate.Be dissolved in water, through the DEAE-Sepharose column chromatography, 0.4M NaCl opens up layer, then to distill water dialysis 2 days, sees through liquid, and lyophilize gets NIF
1E
450.53g.And then through Sephacryl S-300 column chromatography, 0.2M NaCl opens up layer, dialysis, and lyophilize gets J
100.2g.
Embodiment 4
NIF
33.2g be dissolved in the less water, DEAE-Sepharose column chromatography then, water and 0.4M NaCl exhibition layer in succession, respectively NIF
3I, NIF
3Each 1.2g of III and 0.61g, NIF
3I is again through the DEAE-Sepharose column chromatography, and water exhibition layer gets NIF
3IA, DEAE-Sepharose column chromatography again, water exhibition layer J
30.16g, J
20.42g.
NIF
3III is through Sephacryl S-300 column chromatography, and 0.2M NaCl wash-out is dialysed then, and lyophilize gets J
90.29g.
Claims (3)
1, a kind of preparation method who prepares Oleander flower polysaccharide is characterized in that, is that its step is as follows by extraction separation Oleander flower polysaccharide in the flower of Folium seu Cortex Nerii:
A. extraction separation through alcohol degreasing, obtains filtrate and residue by flower of Folium seu Cortex Nerii behind the water extraction:
Residue obtainedly extract to handle with NaOH, its supernatant liquor is again through dialysis, alcohol precipitation, centrifugal, a component NIF that vacuum drying treatment obtains Oleander flower polysaccharide
3Crude product;
The gained concentrating filter liquor, with three times of ethanol sedimentations, abandoning supernatant that the gained throw out is water-soluble, add cetyl trimethyl bromide precipitation, centrifugal, the gained supernatant liquor obtains another polysaccharide fraction NIF after dialysis, alcohol precipitation, centrifugal, vacuum drying treatment
2Crude product; The gained throw out is through acetate dissolution, alcohol precipitation, and Deproteinization obtains another polysaccharide fraction NIF
1Crude product;
B. purifying is selected the crude product of three Oleander flower polysaccharides of the extraction separation among the step a for use following chromatography column:
Through diethylaminoethyl--Mierocrystalline cellulose and/or diethylaminoethyl--sepharose,, carry out the pure products that 2-3 chromatography purification obtains Oleander flower polysaccharide earlier again through sephacryl S-300 and/or sephadex G-200.
2, a kind of Oleander flower polysaccharide by the described method preparation of claim 1.
3, the application of Oleander flower polysaccharide as claimed in claim 2 is characterized in that, described Oleander flower polysaccharide can be used for preparing the medicine for the treatment of senile dementia, apoplexy or children mental retardation disease.
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|---|---|---|---|
| CN99125746A CN1129613C (en) | 1999-12-24 | 1999-12-24 | Oleander flower polysaccharide and its derivatives, preparing method and use |
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|---|---|---|---|
| CN99125746A CN1129613C (en) | 1999-12-24 | 1999-12-24 | Oleander flower polysaccharide and its derivatives, preparing method and use |
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|---|---|
| CN1301774A CN1301774A (en) | 2001-07-04 |
| CN1129613C true CN1129613C (en) | 2003-12-03 |
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|---|---|---|---|---|
| CN100387703C (en) * | 2003-06-11 | 2008-05-14 | 上海高科联合生物技术研发有限公司 | Fatly phospho embrella Pa-I.H bacterial strain and method of culturing fatty phospho embrella and medicine made from its active component |
| CN101613414B (en) * | 2008-06-26 | 2011-09-07 | 中国科学院上海药物研究所 | Oleander oligosaccharide, preparation method thereof and use thereof |
| US10307450B2 (en) | 2010-01-11 | 2019-06-04 | Phoenix Biotechnology, Inc. | Method of treating neurological conditions with extract of Nerium species or Thevetia species |
| US10383886B2 (en) | 2010-01-11 | 2019-08-20 | Phoenix Biotechnology, Inc. | Method of treating neurological conditions with oleandrin |
| US9011937B2 (en) | 2010-11-22 | 2015-04-21 | Phoenix Biotechnology, Inc. | Method of treating neurological conditions with extract of Nerium species or Thevetia species |
| KR20170049623A (en) * | 2010-11-22 | 2017-05-10 | 피닉스 바이오테크놀러지 인코포레이티드. | Method of treating neurological conditions with extract of nerium species or thevetia species |
| US10702567B2 (en) | 2016-09-14 | 2020-07-07 | Phoenix Biotechnology, Inc. | Method and compositions for treating viral infection |
| US10596186B2 (en) | 2016-09-14 | 2020-03-24 | Phoenix Biotechnology, Inc. | Method and compositions for treating viral infections |
| US10729735B1 (en) | 2016-09-14 | 2020-08-04 | Phoenix Biotechnology, Inc. | Method and compostitions for treating coronavirus infection |
| US11331291B2 (en) | 2017-09-14 | 2022-05-17 | Phoenix Biotechnology, Inc. | Method of and improved composition for treating triterpene-responsive conditions, diseases or disorders |
| JP7370966B2 (en) | 2017-09-14 | 2023-10-30 | フェニックス・バイオテクノロジー・インコーポレイテッド | Methods and improved neuroprotective compositions for treating neurological conditions |
| US11806359B2 (en) | 2020-03-31 | 2023-11-07 | Phoenix Biotechnology, Inc. | Method and compositions for treating Coronavirus infection |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4533548A (en) * | 1981-12-02 | 1985-08-06 | Kitasato Institute | Acidic polysaccharide CH-1 isolated from Chlorella pyrenoidosa and the use thereof |
| EP0246069A2 (en) * | 1986-05-13 | 1987-11-19 | Hüseyin Ziya Dr. Özel | Extracts of nerium species, methods of preparation, and use therefore |
| EP0398313A2 (en) * | 1989-05-16 | 1990-11-22 | Hüseyin Ziya Dr. Özel | Polysaccharides with an immunostimulating and antiproliferative activity, process for their recovery and medicines containing these substances |
| DE3915929A1 (en) * | 1989-05-16 | 1990-11-22 | Hueseyin Ziya Prof Dr Oezel | Polysaccharide from Nerium oleander - having immune-stimulating effect and stimulating tumour necrosis factor synthesis |
-
1999
- 1999-12-24 CN CN99125746A patent/CN1129613C/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4533548A (en) * | 1981-12-02 | 1985-08-06 | Kitasato Institute | Acidic polysaccharide CH-1 isolated from Chlorella pyrenoidosa and the use thereof |
| EP0246069A2 (en) * | 1986-05-13 | 1987-11-19 | Hüseyin Ziya Dr. Özel | Extracts of nerium species, methods of preparation, and use therefore |
| EP0398313A2 (en) * | 1989-05-16 | 1990-11-22 | Hüseyin Ziya Dr. Özel | Polysaccharides with an immunostimulating and antiproliferative activity, process for their recovery and medicines containing these substances |
| DE3915929A1 (en) * | 1989-05-16 | 1990-11-22 | Hueseyin Ziya Prof Dr Oezel | Polysaccharide from Nerium oleander - having immune-stimulating effect and stimulating tumour necrosis factor synthesis |
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| CN1301774A (en) | 2001-07-04 |
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