CN102964459A - Lentinan calcium complex, and preparation method and application thereof - Google Patents
Lentinan calcium complex, and preparation method and application thereof Download PDFInfo
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- CN102964459A CN102964459A CN2012104308436A CN201210430843A CN102964459A CN 102964459 A CN102964459 A CN 102964459A CN 2012104308436 A CN2012104308436 A CN 2012104308436A CN 201210430843 A CN201210430843 A CN 201210430843A CN 102964459 A CN102964459 A CN 102964459A
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Abstract
The invention provides a lentinan calcium complex, and a preparation method and application thereof. The method comprises the steps of: adding a calcium chloride solution to an aqueous solution of lentinan with stirring, at 40-80 DEG C, regulating a pH value to 7-10 with sodium hydroxide, and continuously stirring for 1 h for a complex reaction; removing free calcium ions by a dialysis method; and concentrating and freeze-drying the lentinan compound solution to obtain the lentinan calcium complex with calcium content of 5.2mg / g. Compared with a blank group, the lentinan calcium complex can obviously promote mouse macrophage RAW264.7 to release immune factors, such as a tumor necrosis factor TNF-alpha and a cell factor IL-6. The preparation method of the lentinan calcium complex provided by the invention is simple and easy for practice. Besides, as an organic calcium, the lentinan calcium complex can also promote calcium absorption and improve immunity.
Description
Technical field
The present invention relates to bio-pharmaceuticals engineering and field of health care food, be specifically related to lentinan calcium complex and preparation method thereof and application.
Background technology
Mushroom belongs to Basidiomycetes Pleurotaceae fungi, be famous and precious edible one of the medicinal fungus of holding concurrently in the world, it contains multiple effective medicinal ingredients, and its main effective constituent lentinan is the polarity macromolecular cpd, be mainly derived from the cell walls of fungi, also have the parts of fine intracellular polyse.Studies confirm that lentinan have antiviral, antitumor, radioprotective, anti-ageing, anti-stress, anti-oxidant, to improve the various biological such as immunizing power active, wherein antitumor, anti-oxidant and to improve the immunizing power activity research more.
Calcium is one of the abundantest mineral element of people's in-vivo content, and have extremely important physiological action, medical research shows that calcium deficiency easily causes osteoporosis and the alienation of calcium position, the pathologies such as carotid artery stenosis, apoplexy, stenocardia, myocardial infarction appear, the serious harm human health.Yet because the diet formula of Chinese residents is take the cereal fruits and vegetables as main, China resident calcium intake is generally on the low side.Common calcium supplementing product mostly is inorganic calcium in the market, and its calcium source absorpting form is ionized calcium, and calcium ion often reacts with other negatively charged ion easily in the human intestines and stomach, produces and precipitates, poorly water-soluble, the defective such as bioavailability is low.
Summary of the invention
For above-mentioned situation, the present invention's purpose just provides a kind of preparation method and application of lentinan calcium complex.
For realizing the object of the invention, adopt following technical scheme:
A kind of preparation method of lentinan calcium complex, it comprises the steps:
(1) lentinan is dissolved in the distilled water preparation lentinan aqueous solution;
(2) dripping sodium hydroxide solution to pH value in the mushroom polysaccharide solution is 7 ~ 10; Under agitation, drip calcium chloride solution, wherein the mass ratio of lentinan and calcium chloride is 1:2 ~ 1:7; Continuation is stirred under 50 ~ 80 ℃ of water-baths, gets lentinan calcium complex solution;
(3) the lentinan calcium complex solution that step (2) is made is dialysed with dialysis tubing, and the solution freeze-drying in the dialysis tubing obtains the lentinan calcium complex.
Further optimize, described calcium chloride solution adopts the distilled water preparation, and concentration is 0.5-4mol/L; Described sodium hydroxide solution adopts the distilled water preparation, and concentration is 0.5-5mol/L.
Further optimize, step (2) is described to be 60-120min in the stirred in water bath time.
Further optimize, the concentration of rapid (1) described lentinan aqueous solution is 5 ~ 40mg/mL.
Further optimize, the time of the described dialysis of step (3) is 1 ~ 6 day, and the molecular weight that dialysis is held back is 1000 ~ 8000Da.
The present invention also provides the lentinan that is made by above-mentioned preparation method calcium complex, and this lentinan calcium complex can be in preparation calsium supplement, the medicine that improves immunizing power or the application in the healthcare products.
Above operation steps if no special instructions, all by this area routine operation.
Compared with prior art, the present invention has following advantage and technique effect: lentinan calcium of the present invention, as a kind of organic calcium, overcome in the existing inorganic calcium calcium ion in the human intestines and stomach often easily and other negatively charged ion react, produce precipitation, affect calcium supplementing product and take the problem of effect, thereby developed a kind of good water solubility, specific absorption is high, have simultaneously the biomacromolecule calcium-supplementing preparation that can comprehensively improve from many aspects the advantages such as Organism immunoregulation ability, and its preparation method is simple.
Description of drawings
The infrared spectrogram of Fig. 1 lentinan.
The infrared spectrogram of lentinan calcium in Fig. 2 example.
Embodiment
The present invention is described in further detail below in conjunction with embodiment, but embodiment should not be understood as the restriction to protection domain of the present invention.
Embodiment 1
The present invention is realized by following steps in specific implementation process:
(1) preparation calcium chloride solution and sodium hydroxide solution: with the calcium chloride solution of distilled water preparation 2mol/L; Sodium hydroxide solution with distilled water preparation 2mol/L;
(2) take by weighing lentinan 100mg, be dissolved in the deionized water of 5mL, dropwise dripping sodium hydroxide to pH value in the solution is 8.Under agitation, splashing into the polysaccharide mass ratio is the calcium chloride of 5:1, continues at 60 ℃ of stirred in water bath 1h, and dialysis is 2 days in the dialysis tubing of molecular weight cut-off 3500Da, the calcium ion that amputation is free.Namely get lentinan calcium complexes L2-Ca80.2mg after the freeze-drying.
(3) get lentinan and each 2.0mg of lentinan calcium complex, with KBr mixing, the rear compressing tablet of grinding, use Nicolet Nexus 5DXC FT-IR at 500-4000cm respectively
-1Scan in the scope, its infrared absorption spectrum is respectively such as Fig. 1 and Fig. 2.By the contrast of Fig. 2 and Fig. 1, the basic framework of lentinan calcium complex spectrogram does not change.But compare with lentinan, each absorption peak of title complex all weakens to some extent, and wherein lentinan is at 3381cm
-1Near broad peak is that the stretching vibration of O-H key produces, and moves to high band when this absorption peak weakens, and illustrates that O-H associates to some extent; 2918 cm
-1About weak peak be the stretching vibration of c h bond, the lentinan calcium complexes also weakens at this regional absorption peak; Lentinan is positioned at 1414 cm
-1The c h bond angle at place vibrates 1045 cm
-1The C-O stretching vibration absorption peak at place weakens particularly remarkable, and the former shifts to high wave number 1421 cm
-1The complexing of inferring thus lentinan and calcium may be take the C-O coordination as main, because space behavior makes the angle vibration weakening of c h bond.
(4) take by weighing lentinan calcium 50mg, put into beaker and add mixing acid 10mL(nitric acid: hydrochloric acid=1:1), digestion is spent the night, and heating waits to emit the not dried front stopped heating of white cigarette solution, continues to be heated to solution behind the additional mixing acid and is the clear, colorless shape.The harmless lost territory of solution is transferred in the volumetric flask, be settled to scale (25mL) mixing with redistilled water, use Z-2000 series atomic absorption spectrophotometer, the calcium contents that adopts marker method to record in the complex compound is 5.2 mg/g.
Embodiment 2
The present invention also can be realized by following steps in specific implementation process:
(1) preparation calcium chloride solution and sodium hydroxide solution: with the calcium chloride solution of distilled water preparation 3mol/L; Sodium hydroxide solution with distilled water preparation 3mol/L;
(2) accurately take by weighing lentinan 50mg, be dissolved in the deionized water of 5mL, dropwise dripping sodium hydroxide to pH value in the solution is 8.5.Under agitation, splash into the calcium chloride that is about 4:1 with the polysaccharide mass ratio, continue at 70 ℃ of stirred in water bath 90min, dialysis is 3 days in the dialysis tubing of molecular weight cut-off 8000Da, goes out free calcium ion.Namely get the lentinan calcium complexes after the freeze-drying, detecting step and result are substantially with embodiment 1.
Embodiment 3
The present invention also can be realized by following steps in specific implementation process:
(1) preparation calcium chloride solution and sodium hydroxide solution: with the calcium chloride solution of distilled water preparation 4mol/L; Sodium hydroxide solution with distilled water preparation 4mol/L;
(2) accurately take by weighing lentinan 100mg, be dissolved in the deionized water of 30mL, dropwise dripping sodium hydroxide to pH value in the solution is 9.Under agitation, splash into the calcium chloride that is about 2:1 with the polysaccharide mass ratio, continue at 75 ℃ of stirred in water bath 80min, dialysis is 4 days in the dialysis tubing of molecular weight cut-off 1000Da, goes out free calcium ion.Namely get the lentinan calcium complexes after the freeze-drying, detecting step and result are substantially with embodiment 1.
Embodiment 4
The present invention also can be realized by following steps in specific implementation process:
(1) preparation calcium chloride solution and sodium hydroxide solution: with the calcium chloride solution of distilled water preparation 1mol/L; Sodium hydroxide solution with distilled water preparation 1mol/L;
(2) accurately take by weighing lentinan 80mg, be dissolved in the deionized water of 5mL, dropwise dripping sodium hydroxide to pH value in the solution is 10.Under agitation, splash into the calcium chloride that is about 3:1 with the polysaccharide mass ratio, continue at 80 ℃ of stirred in water bath 110min, dialysis is 5 days in the dialysis tubing of molecular weight cut-off 8000Da, goes out free calcium ion.Namely get the lentinan calcium complexes after the freeze-drying, detecting step and result are substantially with embodiment 1.
Embodiment 5
The immunocompetence of lentinan calcium complex is measured
Experimental cell: mouse macrophage RAW264.7
Experimental technique: take the logarithm vegetative period mouse macrophage RAW264.7 with trysinization after, and piping and druming is prepared into single cell suspension.Behind the tally counting, by dilution the cell concn adjustment is approximately 1 * 10
6Individual/mL, be inoculated in 96 orifice plates.Test is made as zeroing group, experimental group, control group and positive controls.Wherein zeroing group does not add cell, adds 100 μ l nutrient solutions, and experimental group, control group add and positive controls, and every hole adds 100 μ L cell suspending liquids.Dull and stereotyped outermost edge adds the PBS solution that bacterium is crossed in death of monks or nuns, and every hole adds 200 μ L.
96 orifice plates are put 37 ℃, 5%CO
2In the incubator 24 hours, make cell attachment.Discard old nutrient solution in zeroing Zu ﹑ Dui Zhao Zu ﹑ experimental group and the positive controls behind the 24h, zeroing group and control group still change 100 μ l fresh mediums, experimental group adds respectively lentinan or the lentinan calcium solution 100 μ l of 250 μ g/mL, lipopolysaccharides (LPS) the 100 μ l(liquids and the positive control that add 50 μ g/ml in the positive controls are all prepared with not containing blood serum medium), each different components sample is established 6 parallel holes.For preventing microbiological contamination, use sealed membrane after alcohol wipe to seal flat board and put into again 37 ℃, 5%CO
2Hatched in the incubator 24 hours.Use respectively the secretory volume of TNF-α, IL-6 in Griess method, the Elisa kit detection cell supernatant liquor.
Experimental result: the lentinan of table 1 explanation different concns and lentinan calcium complex are on the impact of mouse macrophage TNF secretion-α.The lentinan of table 2 explanation different concns and lentinan calcium complex are on the impact of mouse macrophage secretion IL-6.
As shown in Table 1, compare with the blank group, the lentinan calcium complex of different concns can obviously promote mouse macrophage RAW264.7 release tumor necrosis factor TNF-α, and on the impact of TNF-α burst size and with the lentinan of concentration without significant difference.
Such as table 2, to compare with the blank group, the lentinan calcium complex of different concns also has remarkable promoter action to immune factor IL-6 burst size, but during high density, the increasing amount of IL-6 decreases with comparing with the lentinan of concentration.
Table 1
| Dosage (μg/mL） | Lentinan（pg/mL） | Lentinan calcium（pg/mL） |
| The blank group | 394.1053±3.7216 | 394.1053±3.7216 |
| 31.5 | 380.5965±24.4968 | 451.4737±27.8152 A** |
| 62.5 | 422.0000±6.7196 | 417.0175±11.9678 A** |
| 125 | 428.8421±21.4373 | 416.5789±24.3825 A** |
| 250 | 450.9474±18.7046 | 414.1404±13.4804 A** |
| 500 | 432.3509±20.0438 | 1391.2982±85.29131 A** |
In the table 1: * represents P＜0.05, significant difference, and * * represents P＜0.01, difference is (the p value is that observations is thought effectively namely have the overall representational probability of making a mistake) extremely significantly
A: compare B with control group: the lentinan with concentration is compared.
Table 2
In the table 2: * represents P＜0.05, significant difference, and * * represents P＜0.01, difference is (the p value is that observations is thought effectively namely have the overall representational probability of making a mistake) extremely significantly
A: compare B with control group: the lentinan with concentration is compared.
Claims (7)
1. the preparation method of a lentinan calcium complex is characterized in that comprising the steps:
(1) lentinan is dissolved in the distilled water preparation lentinan aqueous solution;
(2) dripping sodium hydroxide solution to pH value in the mushroom polysaccharide solution is 7 ~ 10; Under agitation, drip calcium chloride solution, wherein the mass ratio of lentinan and calcium chloride is 1:2 ~ 1:7; Continuation is stirred under 50 ~ 80 ℃ of water-baths, gets lentinan calcium complex solution;
(3) the lentinan calcium complex solution that step (2) is made is dialysed with dialysis tubing, and the solution freeze-drying in the dialysis tubing obtains the lentinan calcium complex.
2. the preparation method of a kind of lentinan calcium complex according to claim 1 is characterized in that described calcium chloride solution adopts the distilled water preparation, and concentration is 0.5-4mol/L; Described sodium hydroxide solution adopts the distilled water preparation, and concentration is 0.5-5mol/L.
3. the preparation method of a kind of lentinan calcium complex according to claim 1 is characterized in that the described time of stirring of step (2) is 1-2h under 50 ~ 80 ℃ of water-baths.
4. the preparation method of a kind of lentinan calcium complex according to claim 1 is characterized in that the concentration of the described lentinan aqueous solution of step (1) is 5 ~ 40mg/mL.
5. the preparation method of a kind of lentinan calcium complex according to claim 1 is characterized in that the time of the described dialysis of step (3) is 1 ~ 6 day, and the molecular weight that dialysis is held back is 1000 ~ 8000Da.
6. the lentinan calcium complex that is made by each described preparation method of claim 1～5.
7. the described lentinan calcium complex of claim 6 is in preparation calsium supplement, the medicine that improves immunizing power or the application in the healthcare products.
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Cited By (6)
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| CN104774277A (en) * | 2015-03-26 | 2015-07-15 | 广东省农业科学院蚕业与农产品加工研究所 | Flammulina velutipes polysaccharide chelated trace element calcium preparation method |
| CN105859902A (en) * | 2016-04-28 | 2016-08-17 | 武汉格罗宁根医药科技有限公司 | Water-soluble pachymaran metal complex and preparing method and application thereof |
| CN106279306A (en) * | 2015-05-13 | 2017-01-04 | 天津农学院 | A kind of O-is acylated Carrageenan sugar chelating calcium and preparation method thereof |
| CN107312108A (en) * | 2017-07-27 | 2017-11-03 | 福建农林大学 | A kind of preparation method and applications of lentinan chromic compound |
| CN110051647A (en) * | 2019-03-29 | 2019-07-26 | 常熟理工学院 | A kind of hericium erinaceum polysaccharide chelated zinc microcapsules and preparation method thereof |
| CN116874643A (en) * | 2023-05-27 | 2023-10-13 | 桂林理工大学 | Xylan calcium complex and application thereof in antioxidant products |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104774277A (en) * | 2015-03-26 | 2015-07-15 | 广东省农业科学院蚕业与农产品加工研究所 | Flammulina velutipes polysaccharide chelated trace element calcium preparation method |
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| CN105859902A (en) * | 2016-04-28 | 2016-08-17 | 武汉格罗宁根医药科技有限公司 | Water-soluble pachymaran metal complex and preparing method and application thereof |
| CN107312108A (en) * | 2017-07-27 | 2017-11-03 | 福建农林大学 | A kind of preparation method and applications of lentinan chromic compound |
| CN110051647A (en) * | 2019-03-29 | 2019-07-26 | 常熟理工学院 | A kind of hericium erinaceum polysaccharide chelated zinc microcapsules and preparation method thereof |
| CN110051647B (en) * | 2019-03-29 | 2021-06-08 | 常熟理工学院 | Hericium erinaceus polysaccharide chelated zinc microcapsule and preparation method thereof |
| CN116874643A (en) * | 2023-05-27 | 2023-10-13 | 桂林理工大学 | Xylan calcium complex and application thereof in antioxidant products |
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Application publication date: 20130313 |