CN1478902A - Ganoderma mycellium antitumour water soluble neteropolysaccharide and its preparation method and use - Google Patents
Ganoderma mycellium antitumour water soluble neteropolysaccharide and its preparation method and use Download PDFInfo
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- CN1478902A CN1478902A CNA031282903A CN03128290A CN1478902A CN 1478902 A CN1478902 A CN 1478902A CN A031282903 A CNA031282903 A CN A031282903A CN 03128290 A CN03128290 A CN 03128290A CN 1478902 A CN1478902 A CN 1478902A
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Abstract
A process for preparing the water-soluble heteropolyose with antineoplastic activity from ganoderma mycelia includes such steps as preparing culture medium, deep fermenting to obtain mycelia, and using the buffering phosphate solution to extract two water-soluble heteropolyose-protein compounds and one glucosan. Its advantages are high suppression to repdocution of tumor cell, and no toxic by-effect.
Description
Technical field the present invention relates to water-soluble mixed polysaccharide of Ganoderma mycelium that has anti-tumor activity and its production and use.It belongs to chemical field, also belongs to biology field.
The background technology malignant tumour has become the highest disease of human mortality ratio, and countries in the world are all studied tumor treatment in the cost huge fund.At the low phenomenon of cancer patients's immunizing power, people actively seek a kind of not only had antitumous effect but also medicine that can the enhance immunity function.Studies show that since the eighties, polysaccharide all have significant curative effect to the disease of multiple harm humans health as immunologic derangement, cancer, diabetes, hypertension, hepatitis, pneumonia etc.Polysaccharide compound is a kind of immunomodulator, it can strengthen scavenger cell and leukocytic phagocytic function, induce and produce tumour necrosis factor (TNF) and leukin, thereby improve the immunity function of human body, and normal cell there is not toxic side effect (Intern.J.Orient.Med., 17,57,1992).People (Nipon Nogeikagaku Kaishi, 42,2641,1985) such as people such as Sone (Agric.Biol.Chem., 49,2461,1985) and Mizuno have reported that respectively the polysaccharide that extracts has tangible anti-tumor activity from Ganoderma sporophore and mycelium.In recent years the report about glossy ganoderma medicine and healthcare products is a lot, but the I and II structure of lucidum mycelium polysaccharide also imperfectly understands, in addition because wild Ganoderma sporophore resource-constrained, and the artificial culture growth cycle is long, the wall breaking technology of Ganoderma spore remains to be broken through simultaneously, makes Ganoderma sporophore and applying of Ganoderma spore active polysaccharide be restricted.And adopt biotechnology to cultivate Ganoderma mycelium, and therefrom isolate polysaccharide and can overcome these shortcomings.
Summary of the invention problem to be solved by this invention provides antitumor water-soluble mixed polysaccharide of a kind of Ganoderma mycelium and its production and use, the water-soluble mixed polysaccharide of gained has obvious anti-tumor activity, and its preparation method is simple, with short production cycle, lower cost.
For achieving the above object, the technical solution adopted in the present invention is as follows:
The antitumor water-soluble mixed polysaccharide of Ganoderma mycelium, made by laxative remedy: the Ganderma lucidum strain (Ganoderma tsugae) that adopts using fungus institute of Hua Zhong Agriculture University to provide goes out Ganoderma mycelium in the liquid nutrient medium mid-deep strata fermentation culture that contains glucose, Ganoderma mycelium is carried out Soxhlet with ethyl acetate, acetone successively extract removal fat; Soaking at room temperature is in phosphate buffered saline buffer then, and is 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 water-soluble mixed polysaccharide-protein (GM1).Above-mentioned centrifugal back gained mycelium residue soaks extraction with 20～50 ℃ phosphate buffered saline buffer, and is 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 water-soluble mixed polysaccharide-protein (GM2).Above-mentioned centrifugal back gained mycelium residue soaks extraction with 50～100 ℃ phosphate buffered saline buffer, and is 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 water-soluble glucan (GM3).
Above-mentioned two kinds of water-soluble mixed polysaccharide-protein complexes mainly are made up of the protein of alpha-D-glucose, seminose, semi-lactosi and different content, and conjugated protein content is respectively 13.5% and 20.1%, and its weight-average molecular weight is respectively 62.8 * 10
4With 81.8 * 10
4, and the weight-average molecular weight 604.7 * 10 of water-soluble glucan
4
Above-mentioned substratum basic composition is D-glucose (10～20%), peptone (0.6～1.2%), yeast extract paste (0.5～1.5%), KH
2PO
4(0.03～0.07%), K
2HPO
4(0.03～0.07%), MgSO
4(0.03～0.07%); Above per-cent all is weight percentage.
Above-mentioned culture condition is that 25～30 ℃ of shaking tables were cultivated 10～15 days, and shaking speed is 150～220rpm.The gained mycelium is light yellow, and a kind of light fragrance is arranged.Filter out mycelium and cryodesiccated Ganoderma mycelium powder.Productive rate is the dried mycelium of every liter of nutrient solution 5～8 grams.
Above-mentioned Ganoderma mycelium Soxhlet is extracted and is removed the fat time at 4～8 hours, extraction time under its room temperature in phosphate buffered saline buffer was at 8～12 hours, extraction time under 20～50 ℃ is 2～4 hours, and the extraction time under 50～100 ℃ is 0.5～1.5 hour; Dialysis time was at 5～10 days.
Above-mentioned water-soluble polysaccharide can be used as the healthcare products of antitumor drug or enhance immunity function.
Above-mentioned water-soluble polysaccharide is taken into account light scattering apparatus with infrared, nucleus magnetic resonance, gas-chromatography, protein analyzer, viscosity and has been determined its chemical constitution and secondary structure.
The water-soluble mixed polysaccharide of Ganoderma mycelium of the present invention is the clearly new material of structure, and they have obvious anti-tumor activity.Owing to understood fully its chemical constitution and secondary structure, without any side effects in addition, this water-soluble Ganoderma mycelium mixed polysaccharide can become ideal and the antitumor drug of DEVELOPMENT PROSPECT or the healthcare products of enhance immunity function are arranged.Utilize the biotechnology fermentation technique to cultivate Ganoderma mycelium, and therefrom isolate polysaccharide with anti-tumor activity.Therefore have broad application prospects and the potential economic benefit.Water-soluble mixed polysaccharide preparation method of the present invention is simple, with low cost, with short production cycle, be easy to suitability for industrialized production.
Embodiment is described further technical scheme of the present invention below in conjunction with concrete example:
Embodiment
Ganderma lucidum strain (the Ganoderma tsugae that Ganderma lucidum strain provides for using fungus institute of Hua Zhong Agriculture University; The 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 121 ℃ of sterilizations 30 minutes.Inoculate at sterilisable chamber then.Slant culture carries out 7 days by a definite date under 28 ℃ of constant temperature.The liquid culture based formulas is as follows: 15% D-glucose, 1% peptone, 1% yeast extract paste, 0.05% KH
2PO
4, 0.05% K
2HPO
4, 0.05% MgSO
4, all be weight percentage.That cultivates usefulness shakes bottle (150mL) and tampon in advance 121 ℃ of sterilizations 30 minutes, behind the liquid nutrient medium of packing into again 121 ℃ of sterilizations 30 minutes.The cooling back is inoculated at sterilisable chamber.Leave standstill and will shake bottle after one day and place on the shaking table, concussion was cultivated 12 days under 28 ± 1 ℃ of conditions.The gained mycelium is light yellow, and a kind of light fragrance is arranged.Filter out mycelium and cryodesiccated Ganoderma mycelium powder.Productive rate is the dried mycelium of every liter of nutrient solution 5.6 grams.
Ganoderma mycelium powder (100 gram) is carried out Soxhlet with ethyl acetate, acetone successively extracted each 8 hours.Room temperature 0.2M phosphate buffered saline buffer (pH=7.0) extraction with aqueous solution three times then, each 12 hours, centrifugal, merge and collect supernatant liquor (GM1).The residue obtained 0.2M phosphate buffered saline buffer of using is in 20～50 ℃ of extractions three times, and each 4 hours, centrifugal, supernatant liquor (GM2) was collected in merging.The residue obtained 0.2M phosphate buffered saline buffer of using is in 50～100 ℃ of extractions three times, and each 1 hour, centrifugal, supernatant liquor (GM3) was collected in merging.Extracting solution is all used 30% H after concentrating
2O
2Decolouring is to light yellow, remove free protein with the Sevag method, carry out not having until ultraviolet detection more than 9 times the protein absorption peak of 280nm repeatedly, dialysed 5 days and 3 days with clear water and redistilled water respectively then, concentrate, last lyophilize gets the pure product GM1 of white powdery polysaccharide, GM2 and GM3 is respectively 1.3 grams, 0.5 gram and 0.7 gram.
Monose composition, conjugated protein content, molecular weight and the productive rate of these polysaccharide are listed in subordinate list 1.Through Tongji Medical College, Huazhong Science and Technology Univ. the biological activity that the mouse of implanting Sarcoma 180 tumours carries out abdominal injection experiment in the body be the results are shown in subordinate list 2.Obviously, the Ganoderma mycelium water-soluble polysaccharide all has tangible antitumous effect.The mouse body weight all has increase during the administration simultaneously, shows that thus this polysaccharide sample has no side effect, and particularly molecular weight is no more than 1 * 10
6Two kinds of water-soluble Ganoderma mycelium mixed polysaccharide-protein complexes have higher anti-tumor activity.This polysaccharide can be used for preparing the healthcare products of antitumor drug and enhance immunity function, has great promotion and application prospect.
The water-soluble mixed polysaccharide monose of subordinate list 1. Ganoderma myceliums composition, protein content, productive rate and the molecular weight sample white M that lays eggs
w* 10
-4Monose is formed (%)
Rate matter (g mouse rock wood sweet dew gala grape glucose
(%) (%) mol
-1) sugared ammonia GM1 1.3 13.54 62.8-6.7 0.4 26.1 48.3 10.1 8.5GM2 0.5 20.11 81.8-6.2 nd of Lee sugar algae 4.8 34.3 46.8 7.9GM3 0.7-604.7-0.6 0.5 1.9 5.2 86.5 5.2
-: do not detect;
Anti-S180 tumor promotion sample dose inhibiting rate weight increase rate (%) in the water-soluble mixed polysaccharide mouse of the subordinate list 2. Ganoderma myceliums body
(mg/kg×da (％)
ys)GM1 16×10 54.06 27.5
32×10 57.30 21.8GM2 16×10 73.03 4.7
32×10 68.54 4.9GM3 5×10 43.07 33.3
16×10 55.06 23.3
32×10 51.46 11.5
Claims (10)
1. antitumor water-soluble mixed polysaccharide of Ganoderma mycelium, make by laxative remedy: adopt Ganderma lucidum strain (Ganoderma tsugae) to go out Ganoderma mycelium, Ganoderma mycelium is carried out Soxhlet with ethyl acetate, acetone successively extract removal fat in the liquid nutrient medium mid-deep strata fermentation culture that contains glucose; Soaking at room temperature is in phosphate buffered saline buffer then, and is centrifugal, collects extracting solution; Extracting solution is used H after concentrating
2O
2Decolouring is removed free protein with the Sevag method, and dialysis, lyophilize get the pure product of polysaccharide then.
2. antitumor water-soluble mixed polysaccharide of Ganoderma mycelium, make by laxative remedy: adopt Ganderma lucidum strain (Ganoderma tsugae) to go out Ganoderma mycelium, Ganoderma mycelium is carried out Soxhlet with ethyl acetate, acetone successively extract removal fat in the liquid nutrient medium mid-deep strata fermentation culture that contains corn steep liquor; The mycelium residue of gained extracts with 20～50 ℃ phosphate buffered saline buffer immersion after the phosphate buffered saline buffer room temperature is extracted, and is centrifugal, collects extracting solution; Extracting solution is used H after concentrating
2O
2Decolouring is removed free protein with the Sevag method, and dialysis, lyophilize get the pure product of polysaccharide then.
3. antitumor water-soluble mixed polysaccharide of Ganoderma mycelium, make by laxative remedy: adopt Ganderma lucidum strain (Ganoderma tsugae) to go out Ganoderma mycelium, Ganoderma lucidum mycelium body and function ethyl acetate, acetone are carried out Soxhlet extract removal fat in the liquid nutrient medium mid-deep strata fermentation culture that contains corn steep liquor; Mycelium residue after phosphate buffered saline buffer room temperature and 20～50 ℃ extract successively soaks with 50～100 ℃ phosphate buffered saline buffer and extracts, and is centrifugal, the collection extracting solution; Extracting solution is used H after concentrating
2O
2Decolouring is removed free protein with the Sevag method, and dialysis, lyophilize get the pure product of polysaccharide then.
4. the preparation method of the described water-soluble mixed polysaccharide of claim 1, it is characterized in that: adopt Ganderma lucidum strain (Ganoderma tsugae) to go out Ganoderma mycelium, Ganoderma mycelium is carried out Soxhlet with ethyl acetate, acetone successively extract removal fat in the liquid nutrient medium mid-deep strata fermentation culture that contains glucose; Soaking at room temperature is in phosphate buffered saline buffer then, and is centrifugal, collects extracting solution; After extracting solution concentrates, with the H of 20%～30% (weight percent)
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.
5. method according to claim 4 is characterized in that: described substratum basic composition is 10～20% D-glucose, 0.6～1.2% peptone, 0.5～1.5% yeast extract paste, 0.03～0.07% KH
2PO
4, 0.03～0.07% K
2HPO
4, 0.03～0.07% MgSO
4, above per-cent is weight percentage.
6. the preparation method of the described water-soluble mixed polysaccharide of claim 2, it is characterized in that: adopt Ganderma lucidum strain (Ganoderma tsugae) to go out Ganoderma mycelium, Ganoderma mycelium is carried out Soxhlet with ethyl acetate, acetone successively extract removal fat in the liquid nutrient medium mid-deep strata fermentation culture that contains glucose; Mycelium residue after the phosphate buffered saline buffer room temperature is extracted soaks extraction with 20～50 ℃ phosphate buffered saline buffer, and is centrifugal, collects extracting solution; After extracting solution concentrates, with the H of 20%～30% (weight percent)
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.
7. method according to claim 6 is characterized in that: described substratum basic composition is 10～20% D-glucose, 0.6～1.2% peptone, 0.5～1.5% yeast extract paste, 0.03～0.07% KH
2PO
4, 0.03～0.07% K
2HPO
4, 0.03～0.07% MgSO
4, above per-cent is weight percentage.
8. the preparation method of the described water-soluble mixed polysaccharide of claim 3, it is characterized in that: adopt Ganderma lucidum strain (Ganoderma tsugae) to go out Ganoderma mycelium, Ganoderma mycelium is carried out Soxhlet with ethyl acetate, acetone successively extract removal fat in the liquid nutrient medium mid-deep strata fermentation culture that contains glucose; Extract centrifugal back gained mycelium residue through phosphate buffered saline buffer room temperature and 20～50 ℃ and soak extraction with 50～100 ℃ phosphate buffered saline buffer, centrifugal, the collection extracting solution; After extracting solution concentrates, with the H of 20%～30% (weight percent)
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.
9. method according to claim 8, described substratum basic composition is 10～20% D-glucose, 0.6～1.2% peptone, 0.5～1.5% yeast extract paste, 0.03～0.07% KH
2PO
4, 0.03～0.07% K
2HPO
4, 0.03～0.07% MgSO
4, above per-cent is weight percentage.
10. claim 1,2 or 3 described water-soluble mixed polysaccharide are used as the healthcare products of antitumor drug or enhance immunity function.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100424098C (en) * | 2006-10-25 | 2008-10-08 | 南京中科集团股份有限公司 | Process for refining glossy ganoderma spore polysaccharide |
| CN101857645A (en) * | 2010-05-20 | 2010-10-13 | 无限极（中国）有限公司 | Water-soluble neutral Ganoderma lucidum polysaccharide GLP1 and preparation method thereof |
| CN103880970A (en) * | 2012-12-20 | 2014-06-25 | 上海医药工业研究院 | Method utilizing ion-exchange resin to decolor crude polysaccharide in ganoderma sinensis submerged fermentation mycelium |
| CN104357510A (en) * | 2014-10-21 | 2015-02-18 | 中南林业科技大学 | Method for increasing yield of beta-glucan in ganoderma lucidum fermentation product |
-
2003
- 2003-07-08 CN CN 03128290 patent/CN1228449C/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100424098C (en) * | 2006-10-25 | 2008-10-08 | 南京中科集团股份有限公司 | Process for refining glossy ganoderma spore polysaccharide |
| CN101857645A (en) * | 2010-05-20 | 2010-10-13 | 无限极（中国）有限公司 | Water-soluble neutral Ganoderma lucidum polysaccharide GLP1 and preparation method thereof |
| CN101857645B (en) * | 2010-05-20 | 2011-09-21 | 无限极（中国）有限公司 | Water-soluble neutral Ganoderma lucidum polysaccharide GLP1 and preparation method thereof |
| CN103880970A (en) * | 2012-12-20 | 2014-06-25 | 上海医药工业研究院 | Method utilizing ion-exchange resin to decolor crude polysaccharide in ganoderma sinensis submerged fermentation mycelium |
| CN104357510A (en) * | 2014-10-21 | 2015-02-18 | 中南林业科技大学 | Method for increasing yield of beta-glucan in ganoderma lucidum fermentation product |
| CN104357510B (en) * | 2014-10-21 | 2017-07-28 | 中南林业科技大学 | A kind of method for improving beta glucan yield in Ganoderma Lucidum fermentate |
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