CN101919875B - Preparation method of enteromorpha mushroom polysaccharide compound - Google Patents
Preparation method of enteromorpha mushroom polysaccharide compound Download PDFInfo
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Abstract
The invention provides a preparation method of an enteromorpha mushroom polysaccharide compound and application as a health-care medicament thereof. The enteromorpha mushroom polysaccharide compound of the invention consists of enteromorpha polysaccharide and mushroom polysaccharide. By adopting a modern scientific method, the enteromorpha polysaccharide and the mushroom polysaccharide are separated, purified and mixed in a certain ratio to form a polysaccharide compound. The prepared polysaccharide compound has the characteristics of safety, no toxicity, easily obtained raw materials, reliable preparation process and suitability for large-scale industrial production.
Description
Technical field
The present invention relates to the bioactive ingredients in Sargassum and the edible fungi; More specifically; Relate to complex, sea grass polysaccharide and lentinan method for preparing that sea grass polysaccharide and lentinan are formed, and be used to improve the health care medicine that contains the Entermorpha mushroom polysaccharide compound that human immunity perhaps suppresses the tumor purposes.
Background technology
Polysaccharide is as from the natural polymer in higher plant, animal and the microbial cell, is the molecule complex structure that formed by the condensation of a plurality of monosaccharide molecule, dehydration and huge glucide, is one of four big base substances that constitute life.Polysaccharide is because its important physical function and application are widely constantly excavated, and polysaccharide starts from nineteen forty-three as medicine, and people have successfully extracted polysaccharide from many biomaterials at present, and these polysaccharide have extremely important and special physiologically active.
Entermorpha (Enteromorpha) is edible and medicinal algae from ancient times, and Enteromorpha Chlorophyta Ulvales Ulvaceae plant is long in the Intertidal zone, and aboundresources in the wild algae of China especially distributes extensively at southeastern coast one band.Lentinus Edodes (lentinus edodes) delicious flavour is the medicine-food two-purpose fungus.
Up to the present; Plant STUDY ON POLYSACHAROSE and use existing many reports single; But all be with distinctive its single effect separately of drug effect performance separately; And the polysaccharide that multiple biomaterial is extracted especially carries out compound research and application for sea grass polysaccharide and lentinan, through retrieval, does not still have bibliographical information at present.
Summary of the invention
The objective of the invention is in order to overcome the shortcoming and defect that prior art exists; And provide a kind of sea grass polysaccharide and lentinan are carried out Application of composite; And the biological activity of the complex that makes further strengthens, and plays the Entermorpha mushroom polysaccharide compound of synergic effect.
Another object of the present invention provides a kind of method for preparing of Entermorpha mushroom polysaccharide compound.
Another object of the present invention provides and can improve the human body immunity and can effectively suppress the application of the Entermorpha mushroom polysaccharide compound of tumor as health care medicine.
For realizing first purpose of the present invention, technical scheme of the present invention is that it is made up of sea grass polysaccharide and lentinan.
Further being provided with is sea grass polysaccharide, 30～50%; Lentinan, 50～70%.
Further be provided with is that preferred this polysaccharides compound is mixed by following components in weight percentage: sea grass polysaccharide, 50%; Lentinan, 50%.
For realizing second purpose of the present invention, its technical scheme is to prepare sea grass polysaccharide and lentinan respectively, and sea grass polysaccharide and lentinan are carried out the mixed Entermorpha mushroom polysaccharide compound that gets, and wherein the preparation process of sea grass polysaccharide comprises following operation:
(1) extract, air-dry after the Entermorpha sample cleans, chopping is that 1:15 adds distilled water with the mass ratio of Entermorpha and water, and 90 ℃ are extracted 2 h, filtration, and filtering residue repeats to extract three times.Merging filtrate concentrates, and adds 95% ethanol of 3 times of volumes, 4 ℃ of hold over night, centrifuging and taking precipitate the Entermorpha crude polysaccharides;
(2) freeze thawing, with Entermorpha crude polysaccharides solution ,-20 ℃ of freeze overnight, room temperature is slowly melted, and high speed centrifugation is removed precipitate;
(3) alcohol grading in the Entermorpha crude polysaccharides solution after freeze thawing, adds ethanol and makes its final concentration reach 50%, 4 ℃ to spend the night, centrifugal removal deposition, and supernatant continues to add ethanol to be made its concentration reach 70%, 4 ℃ to spend the night the centrifuging and taking deposition;
(4) deproteinization is removed albumen with the polysaccharide that 70% ethanol precipitation obtains with protease, is added in the polysaccharide solution than for 1:200 gets protease by enzyme and albumen quality, and the limit edged stirs, the mixed liquor bag filter of packing into, and 1%NaCl does activator, 37 ℃ of insulation 24 h.Solution after enzyme is handled concentrates, dialysis, and lyophilizing gets sea grass polysaccharide;
Wherein lentinan is prepared by following process:
(1) extract, air-dry after mushroom fruiting body cleans, pulverize, be that 1:20 adds distilled water with the mass ratio of Lentinus Edodes and water, 95 ℃ are extracted 2 h, filtration, filtering residue repeats to extract three times.Merging filtrate concentrates, and adds 95% ethanol of 3 times of volumes, 4 ℃ of hold over night, centrifuging and taking precipitate the Lentinus Edodes crude polysaccharides;
(2) alcohol grading purification in Lentinus Edodes crude polysaccharides solution, adds ethanol and makes its final concentration reach 30%, 4 ℃ to spend the night, centrifugal removal deposition, supernatant continues to add ethanol to be made its concentration reach 70%, 4 ℃ to spend the night, centrifuging and taking precipitate lentinan.
For realizing the 3rd purpose of the present invention, Entermorpha sea grass polysaccharide complex is applied to prepare immunostimulant health promoting product and antitumour auxiliary drug.Preferably, should, Entermorpha sea grass polysaccharide complex accounts for 50% (weight) and lentinan with sea grass polysaccharide and accounts for 50% (weight) and mix and use.
The present invention adopts modern scientific method, described sea grass polysaccharide is separated, purifies with lentinan, and they are mixed with polysaccharides compound according to a certain percentage.Prepared polysaccharides compound safety non-toxic, raw material is easy to get, and reliable preparation process is fit to large-scale industrial production.
Result according to pharmacology, pharmacodynamic study shows that the prepared Entermorpha mushroom polysaccharide compound of the present invention has tangible effect to improving immunity, to suppressing tumor certain effect is arranged also, can be developed into immunostimulant health promoting product and antineoplastic ancillary drug.
Test of pesticide effectiveness result
The Entermorpha mushroom polysaccharide compound is made up of 50% sea grass polysaccharide and 50% lentinan.
, the Entermorpha mushroom polysaccharide compound is to the result that influences of mice S-180 cell sarcoma growth
The S-180 cell of recovery is injected the cultivation of going down to posterity in mouse peritoneal, 0.3 mL/ only.After seven days, the ascites of S-180 mice has been inoculated in aseptic absorption, is diluted to l * 10 with normal saline
7The cell suspension of individual/mL concentration, 0.2 mL/ (2 * 106 cells) are inoculated in mice, and (18～22g) right fore axillary fossas are subcutaneous.Behind 24 h mice is divided into negative control group, polysaccharide group, positive controls immediately, 5 every group.Entermorpha mushroom polysaccharide compound sample is dissolved in normal saline, carried out gastric infusion (0.2 ml/ only) in continuous 10 days, negative control group is irritated stomach with the normal saline of equivalent every day, and positive controls is a cyclophosphamide.The next day of last administration, mice to be weighed, the cervical vertebra dislocation is put to death, and peels off the tumor piece, and by formula calculates tumour inhibiting rate:
Tumour inhibiting rate (%)=(negative control group tumor weight-treatment group tumor is heavy)/negative control group tumor heavy * 100%
Cut open and get thymus and spleen, claim its weight, be calculated as follows spleen index and thymus index:
Spleen index SI=spleen weight/body weight
Thymus index TI=thymus weight/body weight
Experimental result is seen table 1, compares with sea grass polysaccharide effect group separately, and the anti-tumor activity of Entermorpha mushroom polysaccharide compound obviously strengthens, and also presents dose-dependence.When low dosage (50mg/kg) administration, the Entermorpha mushroom polysaccharide compound can significantly suppress the growth of S-180 sarcoma; When administration was measured at 100mg/kg, anti-tumor activity promptly reached utmost point significant level.
The thymus index of the S-180 sarcoma mice that gives the Entermorpha mushroom polysaccharide compound and spleen index are measured the result show that the Entermorpha mushroom polysaccharide compound can recover the immunologic function of mice with tumor.When the low concentration administration was handled, the Entermorpha mushroom polysaccharide compound can strengthen spleen index significantly, and the enhancing ability and the dosage of spleen index present positive correlation.Compare for individually dosed group with sea grass polysaccharide, activity also obviously improves.For the recovery capability of TI, individually dosed group of sea grass polysaccharide and Entermorpha mushroom polysaccharide compound administration group be in the level that can the thymus index of mice with tumor be returned to the normal saline matched group than low dosage, and dosage increases TI is not made significant difference.
Table 1: the Entermorpha mushroom polysaccharide compound suppresses the effect of growth of tumour cell
B, Entermorpha mushroom polysaccharide compound are to the lymphopoietic result that influences
Male ICR mouse (18～22g), 5 every group.Standard environment is raised to shake down.Lumbar injection polysaccharide (various dose, 0.2ml/ days), matched group injecting normal saline, successive administration 10 days.Put to death mice in the dislocation of last administration cervical vertebra next day, 75% alcohol-pickled sterilization 2 minutes, the aseptic spleen of getting, the lymphocyte separation medium separation obtains lymphocyte, microscopic counting, adjustment cell concentration to 5 * 10
6/ ml spreads 96 orifice plates, the 100ul/ hole.The polysaccharide group adds 10%NBCS-RPMI-1640 complete medium 100ul, and ConA induces group to add ConA (final concentration 5 μ g/mL) 100ul, and LPS induces group to add LPS (final concentration 10 μ g/mL) 100ul, and each experimental port final volume 200ul all establishes 3 multiple holes.37 ℃, 5%CO2 incubator are cultivated 48 h.Mtt assay detects lymphocyte proliferation activity.
Entermorpha mushroom polysaccharide compound vivo medicine-feeding shows that to the result's (table 2) that influences of normal mouse lymphocyte propagation the synergism ability of two kinds of polysaccharide is superior to the independent ability to function of sea grass polysaccharide, and concentration relationship is consistent with individually dosed group.When ConA and LPS exist down as the mitogen of T, bone-marrow-derived lymphocyte respectively, the Entermorpha mushroom polysaccharide compound still has activation to T, bone-marrow-derived lymphocyte, also is better than individually dosed group of sea grass polysaccharide.
Table 2:
The Entermorpha mushroom polysaccharide compound is to lymphopoietic effect
C, Entermorpha mushroom polysaccharide compound are to the result that influences of NK cytotoxic activity
With YAC-1 (ATCC, Rochville, MD, USA) cell is as the target cell that detects the NK cytotoxic activity.Add 96 orifice plates according to imitating target ratio 1:50, that is: splenocyte (1 * 10
6/ hole), the YAC-1 cell (5.0 * 10
4/ hole).After the CO2 incubator is cultivated 6 hours.Mtt assay detects the NK cytotoxic activity.The cytotoxic activity computing formula is following:
(OD
E: individually dosed group of light absorption value of effector lymphocyte; OD
T: target cell single culture group light absorption value; OD
(E+T): effector lymphocyte and target cell be the culture group light absorption value altogether)
The Entermorpha mushroom polysaccharide compound is acted in the normal mouse body, detect the toxic action of NK cell to the YAC-1 cell, normal saline group, WEP group are as contrast.Compare (table 3) with the normal saline matched group, the Entermorpha mushroom polysaccharide compound has the toxic ability of the NK cells in mice of enhancing, and activity strengthens along with the increase of concentration; Compare for individually dosed group with sea grass polysaccharide, the NK cytotoxic activity of Entermorpha mushroom polysaccharide compound also has enhancing, has been better than the activity under the individually dosed concentration 100mg/kg of sea grass polysaccharide in the activity under the 50mg/kg administration concentration.
Table 3:
The Entermorpha mushroom polysaccharide compound is to the influence of NK cytotoxic activity
D, Entermorpha mushroom polysaccharide compound are secreted the result that influences of NO and TNF-α to macrophage phagocytic dimethyl diaminophenazine chloride ability, macrophage cytotoxic activity, macrophage
Sea grass polysaccharide and Entermorpha mushroom polysaccharide compound are acted on normal mouse respectively, and the vivo medicine-feeding mice is put to death in the cervical vertebra dislocation.D-Hanks cleans the abdominal cavity, and liquid collecting in the abdominal cavity is gone in the centrifuge tube, and 4 ℃ centrifugal (1000rpm/min, 10 min) remove supernatant, with 1640 complete medium re-suspended cells.The adjustment cell concentration is 2 * 106/ml, and every hole 100 μ l add in 96 orifice plates, after 5 % CO2,37 ℃ are incubated 4h in advance, washes 2-3 time with 37 ℃ of warm in advance PBS (pH=7.4), removes not attached cell, promptly obtains the macrophage of purification.Behind the macrophage purification; Adding concentration is 0.075% neutral red solution, and every hole 100 μ l continue to cultivate 1 h; After the remaining dimethyl diaminophenazine chloride of PBS (PH=7.4) flush away; Add 100 μ l cell pyrolysis liquid (dehydrated alcohol: 0.1mol/L acetic acid=1:1, v/v), detect the 540nm light absorption value with ELIASA behind the 24h.
Table 4 shows that the Entermorpha mushroom polysaccharide compound is better than individually dosed group of sea grass polysaccharide to the phagocytic activity of macrophage, and the concentration relationship of effect is similar with the NK cytotoxic activity.
Table 4:
The Entermorpha mushroom polysaccharide compound is to the influence of macrophage phagocytic dimethyl diaminophenazine chloride ability
With the peritoneal macrophage that makes as stated above (1.0 * 10
5/ hole), add complete medium or target cell B16 lymphoma cell (1.0 * 104/hole then; Imitate target than being 10:1) continue to cultivate 16 hours. detect with mtt assay pair cell density at last.The cytotoxic activity computing formula is following:
(OD
E: individually dosed group of light absorption value of effector lymphocyte; OD
T: target cell single culture group light absorption value; OD
(E+T): effector lymphocyte and target cell be the culture group light absorption value altogether)
Macrophage cytotoxic activity result (table 5) shows that the effect highly significant of Entermorpha lentinan can reach utmost point significant level under low concentration, and all is superior to individually dosed group of sea grass polysaccharide under each concentration administration condition.
Table 5:
The Entermorpha mushroom polysaccharide compound is to the influence of macrophage cytotoxic activity
The Griess method is measured the level of the NO of macrophage generation.Cell is with 5 * 10
5The concentration in individual/hole adds in 24 orifice plates.After cultivating 48 h; Draw cell culture fluid supernatant (50 μ l) and add in another 96 well culture plate, and add 100 μ l Griess reagent, detect 540nm place light absorption value with ELIASA behind the 30min; According to the standard curve that NaNO2 solution is drawn, calculate the output of NO.Peritoneal macrophage and testing sample are cultivated altogether, got supernatant and detect TNF-α with the ELISA test kit.
Macrophage mostly is because the excretory factor of macrophage to the toxic action of tumor cell.From the ability of macrophage secretion NO (table 6) and TNF-α (table 7), the effect of WEP-WLDP will significantly be better than WEP effect group separately.The above results is consistent with macrophage-mediated cytotoxicity result.
Table 6:
The Entermorpha mushroom polysaccharide compound is to the influence of macrophage secretion NO ability
Table 7:
The Entermorpha mushroom polysaccharide compound is to the influence of macrophage TNF secretion-α ability
Below in conjunction with the specific embodiment the present invention is done further introduction.
The specific embodiment
Through embodiment the present invention is carried out concrete description below; Only be used for the present invention is further specified; Can not be interpreted as the qualification to protection domain of the present invention, the technician in this field can make some nonessential improvement and adjustment to the present invention according to the content of foregoing invention.
Embodiment 1: the sea grass polysaccharide preparation
After the Entermorpha sample cleaned, air-dry, 500g Entermorpha material was got in chopping, adds the 7.5L distilled water, and 90 ℃ are extracted 2 h, filter, and filtering residue repeats to extract three times.Merging filtrate is concentrated into 500mL, adds 95% ethanol of 3 times of volumes, 4 ℃ of hold over night, centrifugal (3000r/min) get precipitate the Entermorpha crude polysaccharides.Entermorpha crude polysaccharides solution with 2% ,-20 ℃ of freeze overnight, room temperature is slowly melted, and high speed centrifugation (7000r/min) is removed precipitate.
Sea grass polysaccharide after the freeze thawing is made into 5% solution, adds ethanol and make its final concentration reach 50%, 4 ℃ to spend the night, centrifugal (3000r/min) removes deposition, and supernatant continues to add ethanol to be made its concentration reach 70%, 4 ℃ to spend the night, and centrifugal (3000r/min) gets deposition.The polysaccharide that 70% ethanol precipitation obtains is removed albumen with protease, be added in the polysaccharide solution than for 1:200 gets protease by enzyme and albumen quality, the limit edged stirs, the mixed liquor bag filter of packing into, and 1%NaCl does activator, 37 ℃ of insulation 24 h.Solution after enzyme is handled is concentrated into 200mL, dialysis, and lyophilization gets sea grass polysaccharide 23.6g.
Embodiment 2: the sea grass polysaccharide preparation
After the Entermorpha sample cleaned, air-dry, 5000g Entermorpha material was got in chopping, adds the 75L distilled water, and 90 ℃ are extracted 2 h, filter, and filtering residue repeats to extract three times.Merging filtrate is concentrated into 1.5L, adds 95% ethanol of 3 times of volumes, 4 ℃ of hold over night, centrifugal (3000r/min) get precipitate the Entermorpha crude polysaccharides.Entermorpha crude polysaccharides solution with 2% ,-20 ℃ of freeze overnight, room temperature is slowly melted, and high speed centrifugation (7000r/min) is removed precipitate.
Sea grass polysaccharide after the freeze thawing is made into 5% solution, adds ethanol and make its final concentration reach 50%, 4 ℃ to spend the night, centrifugal (3000r/min) removes deposition, and supernatant continues to add ethanol to be made its concentration reach 70%, 4 ℃ to spend the night, and centrifugal (3000r/min) gets deposition.The polysaccharide that 70% ethanol precipitation obtains is removed albumen with protease, be added in the polysaccharide solution than for 1:200 gets protease by enzyme and albumen quality, the limit edged stirs, the mixed liquor bag filter of packing into, and 1%NaCl does activator, 37 ℃ of insulation 24 h.Solution after enzyme is handled is concentrated into 600mL, dialysis, and lyophilizing gets sea grass polysaccharide 272g.
Embodiment 3: the lentinan preparation
After mushroom fruiting body cleans, air-dry, pulverize, get the 200g Lentinus Edodes and add the 4L distilled water, 95 ℃ are extracted 2 h, filter, and filtering residue repeats to extract three times.Merging filtrate is concentrated into 500mL, adds 95% ethanol of 3 times of volumes, 4 ℃ of hold over night, centrifugal (3000r/min) get precipitate the Lentinus Edodes crude polysaccharides.In preparation 10% the Lentinus Edodes crude polysaccharides solution, add ethanol and make its final concentration reach 30%, 4 ℃ to spend the night; Centrifugal (3000r/min) removes deposition, and supernatant continues to add ethanol to be made its concentration reach 70%, 4 ℃ to spend the night; Centrifugal (3000r/min) gets deposition, and lyophilization gets lentinan 20.8g.
Embodiment 4: the lentinan preparation
After mushroom fruiting body cleans, air-dry, pulverize, get the 5000g Lentinus Edodes and add the 100L distilled water, 95 ℃ are extracted 2 h, filter, and filtering residue repeats to extract three times.Merging filtrate is concentrated into 2000mL, adds 95% ethanol of 3 times of volumes, 4 ℃ of hold over night, centrifugal (3000r/min) get precipitate the Lentinus Edodes crude polysaccharides.In preparation 10% the Lentinus Edodes crude polysaccharides solution, add ethanol and make its final concentration reach 30%, 4 ℃ to spend the night; Centrifugal (3000r/min) removes deposition, and supernatant continues to add ethanol to be made its concentration reach 70%, 4 ℃ to spend the night; Centrifugal (3000r/min) gets deposition, and lyophilization gets lentinan 537g.
Claims (2)
1. the method for preparing of an Entermorpha mushroom polysaccharide compound is characterized in that preparing respectively sea grass polysaccharide and lentinan, and sea grass polysaccharide and lentinan are carried out the mixed Entermorpha mushroom polysaccharide compound that gets,
Wherein the preparation process of sea grass polysaccharide comprises following operation:
(1) extract, air-dry after Entermorpha cleans, chopping is a 1:15 adding distilled water with the mass ratio of Entermorpha and water; 90 ℃ are extracted 2 h, filter, and filtering residue repeats to extract three times, merging filtrate; Concentrate, add 95% ethanol of 3 times of volumes, 4 ℃ of hold over night, centrifuging and taking precipitate the Entermorpha crude polysaccharides;
(2) freeze thawing, with Entermorpha crude polysaccharides solution, subzero 20 ℃ of freeze overnight, room temperature is slowly melted, and high speed centrifugation is removed precipitate;
(3) alcohol grading in the Entermorpha crude polysaccharides solution after freeze thawing, adds ethanol and makes its final concentration reach 50%, 4 ℃ to spend the night, centrifugal removal deposition, and supernatant continues to add ethanol to be made its concentration reach 70%, 4 ℃ to spend the night the centrifuging and taking deposition;
(4) deproteinization is removed albumen with the polysaccharide that 70% ethanol precipitation obtains with protease, is added in the polysaccharide solution than for 1:200 gets protease by enzyme and albumen quality, and the limit edged stirs, the mixed liquor bag filter of packing into, and 1%NaCl does activator, 37 ℃ of insulation 24 h; Solution after enzyme is handled concentrates, dialysis, and lyophilizing gets sea grass polysaccharide;
Wherein the lentinan preparation process comprises following operation:
(1) extract, air-dry after mushroom fruiting body cleans, pulverize, be that 1:20 adds distilled water with the mass ratio of Lentinus Edodes and water, 95 ℃ are extracted 2 h, filtration, filtering residue repeats to extract three times; Merging filtrate concentrates, and adds 95% ethanol of 3 times of volumes, 4 ℃ of hold over night, centrifuging and taking precipitate the Lentinus Edodes crude polysaccharides;
(2) alcohol grading purification in Lentinus Edodes crude polysaccharides solution, adds ethanol and makes its final concentration reach 30%, 4 ℃ to spend the night, centrifugal removal deposition, supernatant continues to add ethanol to be made its concentration reach 70%, 4 ℃ to spend the night, centrifuging and taking precipitate lentinan.
2. the method for preparing of Entermorpha mushroom polysaccharide compound according to claim 1 is characterized in that: described sea grass polysaccharide and lentinan are by 50% mass ratio mixing separately.
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| CN102144743B (en) * | 2011-01-11 | 2012-08-22 | 福建农林大学 | Enteromorpha prolifera jelly and preparation method thereof |
| CN102166015B (en) * | 2011-03-25 | 2012-12-19 | 宁波大学 | Preparation method of probiotics agent capable of lowering blood fat |
| CN103333267A (en) * | 2013-07-12 | 2013-10-02 | 青岛王牌动物健康产品有限公司 | Enteromorpha se-polysaccharide with biological activity as well as preparation method and application of enteromorpha se-polysaccharide |
| CN103910806A (en) * | 2013-10-22 | 2014-07-09 | 上海海洋大学 | Method for simply preparing crude enteromorpha prolifera polysaccharide |
| CN105294871B (en) * | 2015-09-25 | 2018-06-22 | 广东医学院 | A kind of preparation method and application with enhancing immune function sea grass polysaccharide |
| CN109393451A (en) * | 2017-08-18 | 2019-03-01 | 青岛瑞思德生物科技有限公司 | A kind of health food that immunity can be improved containing Enteromorpha |
| CN109867731A (en) * | 2019-04-12 | 2019-06-11 | 无限极（中国）有限公司 | A method of extracting lentinan |
| CN115975061A (en) * | 2022-11-30 | 2023-04-18 | 福建卫生职业技术学院 | Enteromorpha polysaccharide purification method and application thereof |
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