CN108840956A - A kind of method of Bi-aqueous extraction purifying Dendrobium officinale polysaccharide - Google Patents
A kind of method of Bi-aqueous extraction purifying Dendrobium officinale polysaccharide Download PDFInfo
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- CN108840956A CN108840956A CN201810418532.5A CN201810418532A CN108840956A CN 108840956 A CN108840956 A CN 108840956A CN 201810418532 A CN201810418532 A CN 201810418532A CN 108840956 A CN108840956 A CN 108840956A
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- polysaccharide
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
Abstract
The invention belongs to active components of plants extractive technique fields, to solve the problems, such as that dendrobium polysaccharide exists in extracting and makees extractant purifying using the organic solvent that has security risk and be mingled in protein in polysaccharide, a kind of method of Bi-aqueous extraction purifying Dendrobium officinale polysaccharide is provided.The drying of dendrobium candidum stalk, crushes；By solid-liquid ratio 1:50 are dissolved in active water, and ultrasonication solution 2 times；2 times ultrasonic leaching liquor merges, leaching liquor ethanol precipitation；The precipitating concentration of acquisition is 80% ethyl alcohol heating for dissolving, and the protein being mixed in Thick many candies is removed with PEG/ ammonium sulfate mixed solution.It avoids purifying Thick many candies using toxic chloroform, purity is up to 90-95%.Recovery rate is the 177-187% of conventional hot water's extraction and ultrasonic extraction method rate.Extracting obtained polysaccharide has apparent scavenging ability of DPPH free radical, removes hydroxyl radical free radical ability, has significant oxidation resistance.Preparation method for other plant polyoses is offered reference.
Description
Technical field
The invention belongs to active components of plants extractive technique fields, and in particular to a kind of Bi-aqueous extraction purifying iron sheet
The method of dendrobium polysaccharide.
Background technique
Dendrobium nobile is orchid family（Orchidaceae）DendrobiumDendrobiumHerbaceos perennial is a kind of rare rare
Chinese medicine, be known as the title of " human world mesona ".Medicine is classical《Taoist Scriptures》Middle dendrobium candidum is referred to as first of Chinese nine big mesonas, civil
It is referred to as help mesona.Dendrobium candidumDendrobium officinale,It is commonly called as Tiepi Fengdou, is that China is distinctive rare
Medicinal material.《Compendium of Materia Medica》,《Chinese medicine dictionary》There is the record that dendrobium candidum is used as medicine in equal multi-sections ancient Chinese medical literature.It is existing
Show dendrobium polysaccharide for biochemical analysis and alkaloid is most important effective medicinal components in dendrobium.
Dendrobium polysaccharide has preferable healthcare function, have it is anti-oxidant, relieve fatigue, improve immunity and other effects.Sun Jingping
Mouse radioactive damage is caused Deng using radioactive ray irradiation mouse, dendrobium candidum is probed by feeding dendrobium candidum particle
Grain repairs the mechanism of acute radiation damage.The result shows that can be improved content and lymph of the DNA in marrow thin for dendrobium nobile particle
The conversion capability of born of the same parents.Promote to be generated IL-2 by radiation exposure mouse lymphocyte, adjusts CD4+、CD8+Cell subsets protects T leaching
Bar cell.To improve the immunity of mouse.Li Lina probes into dendrobium candidum by building galactolipin inducing mouse aging model
The anti-aging effects of polysaccharide are administered for continuous 5 weeks model mice being administered in a manner of stomach-filling Dendrobium officinale polysaccharide solution
Afterwards, the learning ability of model mice is detected, the biochemical indicator of mouse is detected after administration in continuous 6 weeks.Iron sheet as the result is shown
Dendrobium polysaccharide can improve the memory function of mouse, and liver organization, brain tissue, SOD and GSH-PX activity increases in blood, thus
Illustrate that Dendrobium officinale polysaccharide has the function of anti-aging.Guo Jie etc. is given using the dendrobium officinale tea preparation solution of various concentration gradient
Intragastric administration on mice measures every biochemical indicator of mouse after administration 30 days, dendrobium officinale tea has the function relieved fatigue as the result is shown
Effect.
The extracting method of Dendrobium officinale polysaccharide mainly has at present：It is conventional hot water's extraction, ultrasonic wave assisted extraction method, micro-
Wave assisted extraction method, enzyme assisted extraction method, sudden strain of a muscle formulation, soybean dietary fiber assisted extraction method, overcritical assisted extraction method etc..It is raw
Common method in production is first is that conventional hot water's extraction, second is that ultrasonic wave assisted water extraction's method.The operation of conventional hot water's extraction
Simply, of less demanding to relevant operation personnel and equipment and instrument.Li Chengcheng etc. optimizes conventional hot water's extraction by orthogonal experiment
Extraction proposes skin dendrobium polysaccharide technique, is by optimizing obtained best hot water extraction technique：Hot water extraction temperature is 90 DEG C, iron sheet
Dendrobium nobile quality and the volume ratio of water are 1:40, it repeats to extract 3 times, the recovery rate of Dendrobium officinale polysaccharide is 24.6%.Ultrasonic wave conduct
A kind of easy, effective assisted extraction method is used widely in active skull cap components extraction, by the cavitation of ultrasonic wave
Effect and mechanism promote the dissolution of effective component.Ye Yuyuan is by the method with ultrasonic wave assisted extraction to dendrobium candidum
The extraction process of polysaccharide optimizes, and obtains the optimised process of ultrasonic wave assisted extraction Dendrobium officinale polysaccharide：Best ultrasound wave frequency
Rate is 45 KHz, and it is 50 DEG C that best ultrasonic water, which mentions Extracting temperature, and extraction time is 1.5 h, the body of dendrobium candidum quality and water
Product is than being 1:30, the recovery rate of Dendrobium officinale polysaccharide is 15.3% under this condition.During dendrobium polysaccharide extracts, holosaccharide in order to obtain,
It needs to remove protein impurities with Sevag method after Thick many candies are made.
Summary of the invention
The present invention in order to solve existing dendrobium polysaccharide extract in there is use the organic solvent of security risk as extracting
The problem of taking agent purifying to be mingled in protein in polysaccharide provides a kind of side of Bi-aqueous extraction purifying Dendrobium officinale polysaccharide
Method.
The present invention is realized by following technical solution：A kind of method of Bi-aqueous extraction purifying Dendrobium officinale polysaccharide,
30-41 DEG C of dendrobium candidum stalk drying, is crushed to 50-100 mesh；Dendrobium candidum powder is 1 according to solid-liquid ratio:50 are dissolved in activation
In water, control ultrasonication temperature be 35-41 DEG C, 250W ultrasonication dendrobium candidum solution 2 times, each 20-30min；2
Secondary ultrasonic extraction liquid merges, with ethanol precipitation, final concentration of 80%；The precipitating concentration of acquisition be 80% ethanol washing twice, and
It is dissolved by heating with activated water, the protein being mixed in Thick many candies is removed with PEG/ ammonium sulfate mixed solution；Wherein PEG selects molecule
Amount is 2000-6000, and the additional amount of PEG is 3-5 g/20 mL, and the additional amount of ammonium sulfate is 3-7 g/20 mL.
Precipitate the final concentration of 70%-90% of ethyl alcohol of leaching liquor；Specifically intermediate processing is：It is added in leaching liquor in proportion dense
Degree shakes up for 95% ethyl alcohol, 4 DEG C of refrigeration 12h, and 4000 r/min are centrifuged 20 min after taking-up, stays precipitating, precipitates 80% ethyl alcohol
Washing 2 times.The temperature for the protein that the PEG/ ammonium sulfate mixed solution removal is mixed in Thick many candies is 35-41 DEG C, and stirring makes
Solid dissolution, postposition vortex oscillator mix, and stand 40 min, set 6000 r/min of supercentrifuge and are centrifuged 6 min.It is described
In the protein that the removal of PEG/ ammonium sulfate mixed solution is mixed in Thick many candies, preferably：PEG molecular weight is the additive amount of 6000, PEG
For 3 g/20ml, the additive amount of ammonium sulfate is 5 g/20ml.
The preparation method of the activated water is：Distilled water Amada Co., Ltd. Off Network ロ ウ is produced at " Fuji crystal "
It manages 3 h and activated water is made.
Compared with traditional water extraction, the method for the invention significantly improves polysaccharide yield, and in entire extraction process
In avoid using virose organic solvent.Traditional dendrobium polysaccharide water extraction（Including ultrasonic extraction）All make in extracting method
The protein being mingled in polysaccharide is removed with Sevage method.In Sevage method, with n-butanol, chloroform mixed solution, removal is mixed
The miscellaneous protein impurities in polysaccharide solution.Chloroform is that one kind has moderately toxic chemical reagent, can be through alimentary canal, breathing
Road, skin contact enter body, and main acute toxic effect is that Central nervous system has anesthetic effect, to eye and skin spinosity
Swash effect, and heart, liver, kidney can be damaged etc..Chloroform is classified as human body suspecious carcinogen by International Cancer Research Center.Cause
This, security risk can be brought to dendrobium polysaccharide preparation by taking off albumen using Sevage method.It is molten with the aqueous two-phase that PEG/ inorganic salts form
Agent removes the protein impurities being mingled in dendrobium polysaccharide.The safety for realizing dendrobium polysaccharide is extracted.
The method of the invention isolates and purifies Dendrobium officinale polysaccharide, avoids using toxic chloroform purifying Thick many candies, no
Only recovery rate, purity increase substantially, and purity can achieve 90-95%.Recovery rate is that conventional hot water's extraction and supercritical ultrasonics technology mention
Take the 177-187% of rate.Antioxidation in vitro functional experiment proves that the polysaccharide that this method is extracted has apparent removing DPPH
Free radical ability removes hydroxyl radical free radical ability, has significant oxidation resistance.The present invention provide not only it is a kind of efficiently,
The dendrobium polysaccharide preparation method of safety, and can offer reference for the preparation method of other plant polyoses.
Detailed description of the invention
Fig. 1 is the external anti-removing hydroxyl radical free radical ability testing result figure of various concentration Dendrobium officinale polysaccharide.
Specific embodiment
The fresh item of dendrobium candidum used in the embodiment of the present invention is purchased from Wenzhou Kun Fu dendrobium candidum Co., Ltd.
Embodiment 1：A kind of method of Bi-aqueous extraction purifying Dendrobium officinale polysaccharide, 30-41 DEG C of dendrobium candidum stalk
Drying, is crushed to 50-100 mesh；Dendrobium candidum powder 0.3g is accurately weighed, according to 1:50 solid-liquid ratios, which are added, uses " Fuji crystal "
Processed activated water 15mL；Solution temperature is maintained at 35-41 DEG C, 250W ultrasonication 2 times, 20-30 minutes each；By 2
Secondary extracting solution merging is settled in 100 mL volumetric flasks, is shaken up, and accurate 2 mL of extracting solution that measures is placed in 10 mL centrifuge tubes, quasi-
95% ethyl alcohol, 8 mL is really added, shakes up, 4 DEG C of 12 h of refrigeration, 4000 r/min are centrifuged 20 min after taking-up, and it abandons supernatant and stays precipitating,
Precipitating is with 80% ethanol washing 2 times, and every time plus 8 mL, 80% ethyl alcohol is settled to 50 by precipitating heating for dissolving in activated water
In mL volumetric flask, shake up up to Thick many candies solution.Control is according to 1:Distilled water is added in 50 solid-liquid ratios, ultrasonication is not added, only
It is extracted 2 times with 35-41 DEG C of hot water.Remaining step is the same as step described in embodiment 1.
Control 2：According to 1:Distilled water is added in 50 solid-liquid ratios, is directly extracted with supercritical ultrasonics technology, and ultrasound condition is the same as implementation
Ultrasound condition described in example 1, then extracting solution accurately measures 2 mL of extracting solution and is placed in 10 mL centrifuge tubes, and deionization is accurately added
8 mL of water, shakes up, 4 DEG C of 12 h of refrigeration, and 4000 r/min are centrifuged 20 min after taking-up, abandons supernatant and stays precipitating, precipitates 80% second
Alcohol washs 2 times, and every time plus precipitating heating for dissolving is settled in 50 mL volumetric flasks by 8 mL, 80% ethyl alcohol, shakes up up to thick more
Sugar juice.
The formulation of standard curve and the measurement of coarse polysaccharide extractive rate：Precision measures the above-mentioned three kinds of preparation methods of 1 mL and obtains
Polysaccharide solution, set respectively in 15 mL tool plug test tubes according to sulfuric acid-phynol method measure solution absorbance value, calculate dendrobium candidum
The recovery rate of Thick many candies, calculation formula are：Dendrobium candidum coarse polysaccharide extractive rate %（g/g）=Dendrobium officinale polysaccharide quality/iron sheet stone
Dry measure used in former times powder quality × 100%.
Sulfuric acid-phynol method measures the content of polysaccharide, and grape standard items are set baking oven and are dried to constant weight, 0.09 g is accurately weighed,
It is configured to 0.9 mg/mL glucose solution, 10 times is then diluted and obtains 90 μ g/mL glucose standards solutions.It is accurate to measure glucose
0.2 mL of titer, 0.4 mL, 0.6 mL, 0.8 mL, 1.0 mL set in 10 mL tool plug test tubes respectively, each tool plug test tube are mended
5% phenol solution is added into each tool plug test tube to 1.0 mL for water（It is ready-to-use）, shake up, add the 5 mL concentrated sulfuric acids, shake
Even, 90 DEG C of 20 min of water-bath then take out cooling 5 min of rear ice bath, measure absorbance value, at 488 nm wavelength with grape
Sugar juice concentration is ordinate, and absorbance value is abscissa, draws standard curve.Control group is carried out at aforesaid operations with distilled water
Reason.
Absorbance value of the dendrobium candidum Thick many candies at 488 nm wavelength is measured with same method, according to the standard of drafting
Curve determines Dendrobium officinale polysaccharide quality, and calculates polysaccharide extract rate.
According to above-mentioned dendrobium candidum Thick many candies extracting method, extraction obtains dendrobium candidum Thick many candies, the results are shown in Table 1.From table 1
As can be seen that the method for the present invention compared with traditional water extraction and ultrasonic extraction, can significantly improve polysaccharide extract rate.
The dendrobium candidum recovery rate % that 1 Different Extraction Method of table obtains
Aqueous two-phase extraction purifies Dendrobium officinale polysaccharide：Sieving 5 g of dendrobe powder is taken, by solid-liquid ratio 1：50 are added activated water, and stirring makes
Powder is evenly dispersed, 250 w ultrasonication, 30 min at 35-41 DEG C, and centrifuging and taking supernatant simultaneously repeats above operation primary.It closes
And it the supernatant that obtains twice and is settled to spare in 500 ml volumetric flasks.Removing protein is removed using two aqueous phase extraction method
The impurity such as matter, pigment.20 ml polysaccharide solutions are taken, according to the design of table 2, the PEG and ammonium sulfate of different quality is weighed respectively, stirs
Mixing dissolves solid, and postposition vortex oscillator mixes, and stands 40 min, sets 6000 r/min of supercentrifuge and is centrifuged 6 min, point
Phase volume above and below accurate measuring, draws mutually each 1 ml up and down respectively after phase, ten times is diluted, in 490 after phenolsulfuric acid reacts
Light absorption value is measured under nm.Wherein PEG molecular weight, the quality of PEG, the quality of ammonium sulfate and extraction yield are shown in Table 2.
2 aqueous two-phase of table composition and extraction yield
Purify dendrobium candidum Thick many candies with the aqueous two-phase that the PEG/ ammonium sulfate of different quality forms, the results are shown in Table 2.It can be with from table 2
Find out, the aqueous two-phase of the PEG/ ammonium sulfate composition of different quality containing, extraction yield is different.Extraction yield is arrived 80.08 in this experiment
Between 93.3.As addition 3 g of PEG6000 in every 20 ml system；When 5 g of ammonium sulfate, extraction yield is up to 93.30%.
The iron plate dendrobium polysaccharide that the method for the invention obtains removes the ability detection of hydroxyl radical free radical：It is reacted with Fenton
System generates OH, removes hydroxyl radical free radical ability with salicylic acid determination of color.0.2 mg/ml of accurate measuring respectively, 0.4
Mg/ml, 0.6 mg/ml, 0.8 mg/ml, 1.0 mg/ml Dendrobium officinale polysaccharides, 1.0 mL of night to be measured, then respectively successively
1.0 mL, 2 mmol/mL FeSO is added4Solution, 1.0 mL, 2 mmol/mL H2O2Solution mixes, and reacts at room temperature 10 min,
Then 1.0 mL, 2 mmol/mL salicylic acid solution is added, mixes, 37 DEG C of 30 min of isothermal reaction.Blank group is with distilled water generation
For Dendrobium officinale polysaccharide solution, control group replaces salicylic acid solution with distilled water, measures absorbance value at 510nm.Clearance rate meter
Calculating formula is：Clearance rate（%）={ [ A0-(ASample-AControl)]/A0}×100%。
The Dendrobium officinale polysaccharide that the method for the invention obtains removes hydroxyl free using various concentration Dendrobium officinale polysaccharide
The measurement of base ability, the result is shown in Figure 1.Dendrobium officinale polysaccharide has the stronger energy for removing hydroxyl radical free radical as can be seen from Figure 1
Power, and there is dosage effect.
Claims (4)
1. a kind of method of Bi-aqueous extraction purifying Dendrobium officinale polysaccharide, it is characterised in that：30-41 DEG C of dendrobium candidum stalk
Drying, is crushed to 50-100 mesh；Dendrobium candidum powder is 1 according to solid-liquid ratio:50 are dissolved in activated water, control ultrasonication
Temperature be 35-41 DEG C, 250W ultrasonication dendrobium candidum solution 2 times, each 20-30min；2 times ultrasonic extraction liquid merges, and uses
Ethanol precipitation, final concentration of 80%；The precipitating concentration of acquisition washes twice for 80%, and is dissolved by heating with activated water, uses PEG/
Ammonium sulfate mixed solution removes the protein being mixed in Thick many candies；Wherein PEG molecular weight is 2000-6000, and the additional amount of PEG is
3-5 g/20 mL, the additional amount of ammonium sulfate are 3-7 g/20 mL.
2. a kind of method of Bi-aqueous extraction purifying Dendrobium officinale polysaccharide according to claim 1, it is characterised in that：
Precipitate the final concentration of 70%-90% of ethyl alcohol of leaching liquor；Specifically intermediate processing is：It is 95% that concentration is added in leaching liquor in proportion
Ethyl alcohol shake up, 4 DEG C of refrigeration 12h, 4000 r/min are centrifuged 20 min after taking-up, stay precipitating, 80% ethanol washing 2 of precipitating
It is secondary.
3. a kind of method of Bi-aqueous extraction purifying Dendrobium officinale polysaccharide according to claim 1, it is characterised in that：
The temperature for the protein that the PEG/ ammonium sulfate mixed solution removal is mixed in Thick many candies is 35-41 DEG C, and stirring dissolves solid,
Postposition vortex oscillator mixes, and stands 40 min, sets 6000 r/min of supercentrifuge and is centrifuged 6 min.
4. a kind of method of Bi-aqueous extraction purifying Dendrobium officinale polysaccharide according to claim 1, it is characterised in that：
In the protein that the PEG/ ammonium sulfate mixed solution removal is mixed in Thick many candies, PEG molecular weight is the additive amount of 6000, PEG
For 3 g/20ml, the additive amount of ammonium sulfate is 5 g/20ml.
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| CN109652479A (en) * | 2019-01-25 | 2019-04-19 | 华南农业大学 | A method of improving dendrobium polysaccharide oxidation resistance |
| CN110066313A (en) * | 2019-05-05 | 2019-07-30 | 中国科学院兰州化学物理研究所 | A kind of preparation method of high-purity Lycium chinense glycopeptide |
| CN111671711A (en) * | 2020-07-31 | 2020-09-18 | 广州荃智美肤生物科技研究院有限公司 | Bacteriostatic hand sanitizer for children and preparation method thereof |
| CN111825779A (en) * | 2020-08-21 | 2020-10-27 | 武汉理工大学 | Extraction method of dendrobium officinale polysaccharide |
| CN111909284A (en) * | 2020-08-21 | 2020-11-10 | 广州德谷个人护理用品有限公司 | Extraction and purification method of tea bran polysaccharide and scalp care composition containing tea bran polysaccharide |
| CN111978424A (en) * | 2020-08-27 | 2020-11-24 | 上海应用技术大学 | Method for purifying dendrobium officinale water-soluble polysaccharide in one step |
| CN112778435A (en) * | 2021-03-18 | 2021-05-11 | 广东工业大学 | Method for separating and purifying dendrobium officinale polysaccharide by aqueous two-phase extraction |
| CN113354748A (en) * | 2021-05-13 | 2021-09-07 | 浙江工业大学 | Dendrobium officinale leaf polysaccharide and preparation and application thereof |
| CN113577201A (en) * | 2021-08-17 | 2021-11-02 | 皖西学院 | Huoshan dendrobium officinale oral liquid and preparation method thereof |
| CN114045324A (en) * | 2021-12-13 | 2022-02-15 | 中南民族大学 | Method for extracting and separating protein from bamboo fungus |
| CN114957494A (en) * | 2021-02-23 | 2022-08-30 | 江苏农牧科技职业学院 | Process for extracting protein in polygonatum polysaccharide by adopting double aqueous phases |
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|---|---|---|---|---|
| CN109652479A (en) * | 2019-01-25 | 2019-04-19 | 华南农业大学 | A method of improving dendrobium polysaccharide oxidation resistance |
| CN109652479B (en) * | 2019-01-25 | 2020-12-15 | 华南农业大学 | Method for improving antioxidant capacity of dendrobe polysaccharide |
| CN110066313A (en) * | 2019-05-05 | 2019-07-30 | 中国科学院兰州化学物理研究所 | A kind of preparation method of high-purity Lycium chinense glycopeptide |
| CN111671711A (en) * | 2020-07-31 | 2020-09-18 | 广州荃智美肤生物科技研究院有限公司 | Bacteriostatic hand sanitizer for children and preparation method thereof |
| CN111825779A (en) * | 2020-08-21 | 2020-10-27 | 武汉理工大学 | Extraction method of dendrobium officinale polysaccharide |
| CN111909284A (en) * | 2020-08-21 | 2020-11-10 | 广州德谷个人护理用品有限公司 | Extraction and purification method of tea bran polysaccharide and scalp care composition containing tea bran polysaccharide |
| CN111978424A (en) * | 2020-08-27 | 2020-11-24 | 上海应用技术大学 | Method for purifying dendrobium officinale water-soluble polysaccharide in one step |
| CN114957494A (en) * | 2021-02-23 | 2022-08-30 | 江苏农牧科技职业学院 | Process for extracting protein in polygonatum polysaccharide by adopting double aqueous phases |
| CN112778435A (en) * | 2021-03-18 | 2021-05-11 | 广东工业大学 | Method for separating and purifying dendrobium officinale polysaccharide by aqueous two-phase extraction |
| CN113354748A (en) * | 2021-05-13 | 2021-09-07 | 浙江工业大学 | Dendrobium officinale leaf polysaccharide and preparation and application thereof |
| CN113577201A (en) * | 2021-08-17 | 2021-11-02 | 皖西学院 | Huoshan dendrobium officinale oral liquid and preparation method thereof |
| CN114045324A (en) * | 2021-12-13 | 2022-02-15 | 中南民族大学 | Method for extracting and separating protein from bamboo fungus |
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Application publication date: 20181120 |