CN1205231C - Water soluble heteropolysaccharide with anti-tumor activity and its preparing method and use - Google Patents
Water soluble heteropolysaccharide with anti-tumor activity and its preparing method and use Download PDFInfo
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- CN1205231C CN1205231C CN 03118416 CN03118416A CN1205231C CN 1205231 C CN1205231 C CN 1205231C CN 03118416 CN03118416 CN 03118416 CN 03118416 A CN03118416 A CN 03118416A CN 1205231 C CN1205231 C CN 1205231C
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Abstract
The present invention discloses water soluble heteropolysaccharides of a tuckahoe mycelium as spawn artificially cultured, a preparation method thereof and purposes thereof. The water soluble heteropolysaccharides have antineoplastic activity. A culture medium is prepared from corn steep liquor as a main ingredient, and the tuckahoe mycelium is cultured through the submerged fermentation of wild tuckahoe spawn; water soluble heteropolysaccharides are respectively extracted from the mycelium with physiological saline and hot water. The obtained heteropolysaccharides are mainly composed of alpha-D-glucose, mannose, galactoside and proteins. Activity experiment results in vivo indicate that the water soluble heteropolysaccharides perform obvious inhibiting action on the growth of an Sarcoma180 tumor implanted in a mouse in vivo and perform no toxic or side effect. Activity experiments results in vitro indicate that the water soluble heteropolysaccharides have a significant inhibiting effect on the proliferation of acute leukemia poison cells (HL-60) of a human body without destroying the proliferation of normal monkey kidney cells. The water soluble heteropolysaccharides can be used as antitumor medicines or as health products for improving the immunological function of organisms.
Description
Technical field
The present invention relates to water-soluble mixed polysaccharide that has anti-tumor activity and its production and use.Belong to the natural polymer field, also belong to biology field.
Background technology
The seventies, because the progress that the research aspects such as chemical functional, phylactic agent and new drug of cytolemma obtain, and the discovery of complex polysaccharides such as multiple glycoprotein, glycolipid, be sure of that polysaccharide has the biological activity of many aspects.Chihara (J.Immunol.Immunopharmacol., IV, 85,1983) research think the activity of polysaccharide compound be not resemble in chemotherapy direct cell killing, but there is a kind of indirect anti-tumor activity, the i.e. activity of being transmitted by immunity system mainly be to the exciting of immune specific non-specific or specific function, so toxicity is minimum.Poria cocos is the traditional Chinese medicine of China, function such as have diuresis, excreting dampness, tonifying spleen, calm the nerves.In recent years, the research of Poria cocos mainly concentrates on its main component Pachymose, and it and it derivative has antimutagenic, antitumor (Yakugaku Zasshi, 106,206,1986) and immunity (Phytother.Res., 9,448,1995) isoreactivity.The polysaccharide that people such as Narui (Carbohydr.Res.87,161,1980) report extracts from poria cocos sclerotium has tangible anti-tumor activity.Yet, because the Poria cocos artificial culture cycle is long, and be difficult to preserve and transportation, make the Pachymose that makes by sclerotium be difficult to promote.
Summary of the invention
Problem to be solved by this invention provides a kind of water-soluble mixed polysaccharide with anti-tumor activity and its production and use, and this mixed polysaccharide is a raw material with the Poria cocos bacterial classification of the artificial culture of easy acquisition, and its method for making is simple, easily with suitability for industrialized production.
For achieving the above object, the technical solution adopted in the present invention is as follows: a kind of water-soluble mixed polysaccharide (ac-PCM2) with anti-tumor activity is made by laxative remedy: Poria cocos bacterial classification (the Poria cocos that adopts artificial culture; DSMZ of Microbe Inst., Chinese Academy of Sciences provides, numbering 5.78) go out Poria mycelium in the liquid nutrient medium mid-deep strata fermentation culture that contains corn steep liquor, Poria mycelium is carried out Soxhlet with ethyl acetate, acetone successively extract removal fat; Be immersed in then in the physiological saline, centrifugal, centrifugal back gained precipitation is soaked extraction with 105～120 ℃ hot water under 0.12～0.20MPa, centrifugal, collects extracting solution; Extracting solution is used H after concentrating
2O
2Decolouring is removed free protein with the Sevag method and is not had the protein absorption peak of 280nm until ultraviolet detection, reverse then flowing water dialysis, and lyophilize at last gets the pure product of water-soluble mixed polysaccharide.
Above-mentioned water-soluble mixed polysaccharide mainly is made up of alpha-D-glucose, seminose, semi-lactosi and protein.Wherein contain protein 18.1%, polysaccharide 81.9% (alpha-D-glucose accounts for 51.4% in the polysaccharide, semi-lactosi accounts for 29.7%, seminose accounts for 19.1%, Fucose account for 0.8%), weight-average molecular weight is 17.0 * 10
4Below all be weight percentage.
The present invention also provides the preparation method of above-mentioned water-soluble mixed polysaccharide, adopts Poria cocos bacterial classification (the Poria cocos of artificial culture; DSMZ of Microbe Inst., Chinese Academy of Sciences provides, numbering 5.78) go out Poria mycelium in the liquid nutrient medium mid-deep strata fermentation culture that contains corn steep liquor, Poria mycelium is carried out Soxhlet with ethyl acetate, acetone successively extract removal fat; Be immersed in then in the physiological saline, centrifugal, centrifugal back gained precipitation is soaked extraction with 105～120 ℃ hot water under 0.12～0.20MPa, centrifugal, collects extracting solution.After extracting solution concentrates, with 20%～30% (weight percent) H
2O
2Decolouring is removed free protein with the Sevag method and is not had the protein absorption peak of 280nm until ultraviolet detection, dialyses then, lyophilize gets the pure product of required water-soluble mixed polysaccharide (ac-PCM2).
The 1L distilled water that basic composition is of above-mentioned substratum contains corn steep liquor 5～15mL, D-glucose 20～30g, yeast extract paste 3～3.5g, KH
2PO
40.8～1.2g, CaCl
22H
2O 0.04～0.08g, MnCl
24H
2O 3～7mg, MgSO
47H
2O 0.3～0.7g, ZnCl
22～6mg, Fe
2(SO
4)
33～7mg, vitamins B
10.08～0.12mg, distilled water 1L.
Above-mentioned Poria mycelium Soxhlet is extracted and is removed the fat time at 4～8 hours, and its extraction time in physiological saline was at 8～12 hours; Dialysis time was at 5～10 days.
The purposes of above-mentioned water-soluble mixed polysaccharide in the healthcare products of preparation antitumor drug or enhance immunity function.
Take into account light scattering apparatus with infrared spectra, nucleus magnetic resonance, gas-chromatography, protein analyzer, viscosity and determined above-mentioned water-soluble mixed polysaccharide chemical constitution and secondary structure.
This water-soluble mixed polysaccharide, and has no side effect apparently higher than other mixed polysaccharide to its anti-tumor activity as a result of implanting intravital Sarcoma 180 tumor control rates of mouse, can be used as healthcare products, also has application prospect as antitumor natural drug.
Compared with the prior art, innovation of the present invention is as follows: the water-soluble mixed polysaccharide of the Poria mycelium that is provided is the clearly brand-new material of a kind of structure, and it has tangible anti-tumor activity, and has no side effect.This Poria mycelium mixed polysaccharide is expected to become the antitumor drug that DEVELOPMENT PROSPECT is arranged or the healthcare products of enhance immunity function.The preparation method of this polysaccharide is that the cheap corn steep liquor of employing is a main raw material in the substratum, utilizes the biotechnology fermentation technique to cultivate Poria mycelium, and therefrom isolates the polysaccharide with anti-tumor activity.It has more notable antitumor activity than the mycelium polysaccharides that the substratum that with wheat bran is main raw material obtains.Its chemical constitution of mycelium polysaccharides, protein content and molecular weight that different substratum obtain are all different, therefore have influence on bioactive difference.Method for making of the present invention is simple, easily with suitability for industrialized production.
Embodiment
Below in conjunction with concrete example technical scheme of the present invention is described further:
Embodiment
The Poria cocos bacterial classification provides for DSMZ of Microbe Inst., Chinese Academy of Sciences, numbering 5.78 (public can buy at any time).At first in slant medium, cultivate bacterial classification.Cultivate the test tube of usefulness and tampon earlier at 121 ℃ of sterilizations 30 minutes (sky disappears), reinstall substratum, tiltedly put cooling and make it be frozen into the inclined-plane, inoculate at sterilisable chamber then 121 ℃ of sterilizations 30 minutes.Slant culture carries out 7 days by a definite date under 28 ℃ of constant temperature.The liquid culture based formulas is as follows: contain corn steep liquor (10mL) in the 1L distilled water, D-glucose (25g), yeast extract paste (3.2g), KH
2PO
4(1.0g), CaCl
22H
2O (0.06g), MnCl
24H
2O (5mg), MgSO
47H
2O (0.5g), ZnCl2 (4mg), Fe
2(SO
4)
3(5mg), vitamins B
1(0.1mg).That cultivates usefulness shakes bottle (250mL) and tampon in advance 121 ℃ of sterilizations 30 minutes, and again 121 ℃ of sterilizations 30 minutes, cooling is then inoculated at sterilisable chamber behind the liquid nutrient medium of packing into.Leave standstill and will shake bottle after one day and place on the shaking table, concussion was cultivated 12 days under 25 ± 1 ℃ of conditions.The gained mycelium is light yellow, and a kind of light fragrance is arranged.
From substratum after the filtering separation, the powder that obtains through lyophilize carries out Soxhlet with ethyl acetate, acetone successively and extracted each 8 hours with Poria mycelium.Extract three times with the 9g/L sodium chloride aqueous solution then: be immersed in the 9g/L sodium chloride aqueous solution and spend the night, centrifugal, collect supernatant liquor (ac-PCM1).Residue extracts three times down for 120 ℃ in high pressure with hot water, and is centrifugal each half an hour, collects supernatant liquor (ac-PCM2).Extracting solution is all used 30% (weight percent) H after concentrating
2O
2Decolouring is removed free protein with the Sevag method, carries out 9 times repeatedly and does not have the absorption peak at 280nm place until ultraviolet detection, and respectively with clear water and redistilled water dialysis 5 days and 3 days, concentrated, lyophilize at last gets the pure product of polysaccharide then.The Poria mycelium nutrient solution filters, and uses 30%H after concentrating
2O
2Decolouring is removed free protein with the Sevag method, carries out 9 times repeatedly and does not have the absorption peak at 280nm place until ultraviolet detection, and respectively with clear water and redistilled water dialysis 5 days and 3 days, concentrated, lyophilize at last gets exocellular polysaccharide (ac-PCM0) then.The gained polysaccharide is light yellow powdery solid.
Control group liquid culture based formulas is: contain the extracting solution of wheat bran (200g) in the 1L distilled water, D-glucose (25g), yeast extract paste (3.2g), KH
2PO
4(1.0g), CaCl
22H
2O (0.06g), MnCl
24H
2O (5mg), MgSO
47H
2O (0.5g), ZnCl
2(4mg), Fe
2(SO
4)
3(5mg), vitamins B
1(0.1mg).The water-soluble mixed polysaccharide extracting method of mycelium cultural method and Poria mycelium is with on ac-PCM0 and the ac-PCM1.The gained polysaccharide is light yellow solid powder difference called after: ab-PCM0 and ab-PCM1.
The composition of these polysaccharide, protein content, molecular weight and productive rate are listed in subordinate list 1, and the polysaccharide sample carries out intraperitoneal injection test in the body through biology department of Hong Kong Chinese University to implanting Sarcoma 180 mice with tumor, draws anti-tumor activity and the results are shown in subordinate list 2.Show thus by the mycelium polysaccharides ac-PCM2 that contains the corn steep liquor culture medium culturing and have tangible anti-tumor activity.The antiproliferative experimental result that water-soluble mixed polysaccharide of the present invention is carried out human body acute leukemia poison cell (HL-60) shows that it has significant inhibition effect to propagation simultaneously, and can not destroy the propagation of normal monkey-kidney cells.This polysaccharide can be used for preparing the healthcare products of antitumor drug and enhance immunity function.
Subordinate list 1. Poria mycelium polysaccharide monose compositions, protein content, productive rate and molecular weight
Contents of monosaccharides in the polysaccharide (%)
Protein yields M
w* 10
-4
Sample
(％) (％) (gmol
-1)
Fucose pectinose wood sugar seminose semi-lactosi glucose
ac-PCM0 1.4 1.0 - 43.0 27.4 27.2 25.5 10.1
ac-PCM1 4.5 - + 15.8 23.9 53.4 42.9 1.7 12.5
ac-PCM2 0.8 - - 19.1 29.7 51.4 18.1 1.7 17.0
ab-PCM0 - 8.3 10.0 19.4 11.5 50.6 11.6 7.7
ab-PCM1 - 3.8 2.5 10.0 26.3 49.9 - 1.5 5.2
-: do not detect; +: trace
The anti-tumor activity of subordinate list 2. Poria mycelium polysaccharide
Dosage
Sample inhibiting rate (%) suppresses fully
(mg/kg×days)
ac-PCM0 20×10 - 0/7
Real
ac-PCM1 20×10 12.7 1/7
Example
ac-PCM2 20×10 42.3
* 0/7
Ratio
ab-PCM0 20×10 8.33 0/8
{。##.##1},
ab-PCM1 20×10 4.17 0/8
Group
-intravital Sarcoma 180 growth of tumor of mouse there is not the obvious suppression effect
*p＜0.01
Claims (6)
1. water-soluble mixed polysaccharide with anti-tumor activity, made by laxative remedy: the Poria cocos bacterial classification (Poria cocos) of DSMZ of employing Microbe Inst., Chinese Academy of Sciences artificial culture that provide, numbering 5.78 goes out Poria mycelium in the liquid nutrient medium mid-deep strata fermentation culture that contains corn steep liquor, Poria mycelium is carried out Soxhlet with ethyl acetate, acetone successively extract removal fat; Be immersed in then in the physiological saline, centrifugal, centrifugal back gained precipitation is soaked extraction with 105～120 ℃ hot water under 0.12～0.20MPa, centrifugal, collects extracting solution; Extracting solution is used H after concentrating
2O
2Decolouring is removed free protein with the Sevag method and is not had the protein absorption peak of 280nm until ultraviolet detection, dialyses then, lyophilize gets the pure product of water-soluble mixed polysaccharide.
2. the preparation method of the described water-soluble mixed polysaccharide of claim 1, it is characterized in that: the Poria cocos bacterial classification (Poria cocos) of DSMZ of employing Microbe Inst., Chinese Academy of Sciences artificial culture that provide, numbering 5.78 goes out Poria mycelium in the liquid nutrient medium mid-deep strata fermentation culture that contains corn steep liquor, Poria mycelium is carried out Soxhlet with ethyl acetate, acetone successively extract removal fat; Be immersed in then in the physiological saline, centrifugal, centrifugal back gained precipitation is soaked extraction with 105～120 ℃ hot water under 0.12～0.20MPa, centrifugal, collects extracting solution; After extracting solution concentrates, with 20wt%～30wt%H
2O
2Decolouring is removed free protein with the Sevag method and is not had the protein absorption peak of 280nm until ultraviolet detection, dialyses then, lyophilize gets the pure product of polysaccharide.
3. method according to claim 2 is characterized in that: the 1L distilled water that basic composition is of described substratum contains corn steep liquor 5～15mL, D-glucose 20～30g, yeast extract paste 3～3.5g, KH2PO4 0.8～1.2g, CaCl22H2O 0.04～0.08g, MnCl24H2O 3～7mg, MgSO47H2O 0.3～0.7g, ZnCl2 2～6mg, Fe2 (SO4) 3 3～7mg, VITMAIN B1 0.08～0.12mg.
4. according to claim 2 or 3 described methods, it is characterized in that: above-mentioned Poria mycelium Soxhlet is extracted and is removed the fat time at 4～8 hours, and its extraction time in physiological saline was at 8～12 hours; Dialysis time was at 5～10 days.
5. the described water-soluble mixed polysaccharide of claim 1 is in the purposes of preparation in the antitumor drug.
6. the purposes of the described water-soluble mixed polysaccharide of claim 1 in the healthcare products of preparation enhance immunity function.
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1308350C (en) * | 2004-07-28 | 2007-04-04 | 武汉大学 | Process for preparing tuckahoe mycelium extracellular polysacchride with anti-tumor activity |
| CN105237649B (en) * | 2014-07-11 | 2018-01-16 | 中国人民解放军军事医学科学院毒物药物研究所 | A kind of pachymaran, preparation method and the usage |
| CN105585638B (en) * | 2014-10-22 | 2017-12-15 | 北京中安佐际生物科技有限公司 | Pachymaran active component and composition, preparation method and the usage |
| CN105061627A (en) * | 2015-08-31 | 2015-11-18 | 桂林茗兴生物科技有限公司 | Pachymaran extraction method |
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