CN108250315B - Dendrobium officinale extract and preparation method thereof - Google Patents
Dendrobium officinale extract and preparation method thereof Download PDFInfo
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- CN108250315B CN108250315B CN201810040735.5A CN201810040735A CN108250315B CN 108250315 B CN108250315 B CN 108250315B CN 201810040735 A CN201810040735 A CN 201810040735A CN 108250315 B CN108250315 B CN 108250315B
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- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
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Abstract
The invention discloses a dendrobium officinale extract and a preparation method thereof. The preparation method comprises the following steps: (1) extracting herba Dendrobii with ethanol, and filtering to obtain extractive solution and residue; (2) and (3) carrying out enzymolysis on the feed liquid consisting of the filter residue and the water by using cellulase, amylase and protease to obtain an enzymolysis liquid. The preparation method can synchronously extract the dendrobium officinale polysaccharide and the polyphenol substances, and has simple and convenient operation steps and reasonable process.
Description
Technical Field
The invention relates to a dendrobium officinale extract and a preparation method thereof.
Background
Dendrobium officinale (Dendrobium candidum wall ex lindl) is a perennial herb of the genus Dendrobium of the family Orchidaceae and is a traditional and rare traditional Chinese medicine in China. There are over 1100 kinds of dendrobium plants in the world, mainly distributed in east asia, southeast asia, new guinea and australia. Dendrobium officinale is abundant in southern areas of Yunnan China, belongs to the dendrobium plant recorded in pharmacopoeia of the people's republic of China with dendrobium devoninum and dendrobium congolense, and has the effects of nourishing yin, promoting the production of body fluid, moistening throat, protecting throat, warming stomach, improving eyesight, tonifying kidney, benefiting strength and prolonging life. The classic dao home (Taoist Collection) of Tang Kaiyuan years lists Dendrobium officinale as the head of the nine-large Chinese immortal grass.
At present, healthy food or medicine related to the concept of dendrobium officinale in the market is favored by consumers, the product form is mainly dendrobium officinale bucket or dendrobium officinale ultra-fine powder obtained by baking and drying dendrobium officinale stems, but the dendrobium officinale is inconvenient to eat, is easy to damp and mildew, and has the problem of edible safety. In addition, some dendrobium wine can only extract low-polarity substances in dendrobium, and cannot effectively utilize polysaccharide substances which are main functional components of dendrobium officinale.
Chinese patent document CN104740337A provides a dendrobium officinale extract, which specifically comprises the following four steps: degreasing by using an organic solvent, soaking in an ethanol solution, carrying out flash extraction, removing protein by using a Sevage reagent, and decoloring by using activated carbon. Chinese patent document CN100497396C relates to a dendrobium officinale polysaccharide extract and a preparation method thereof, which comprises the steps of degreasing with organic solvent petroleum ether and ethyl acetate, extracting with water, precipitating with ethanol, washing with organic solvent petroleum ether, acetone, ethyl ether and the like, and drying to obtain polysaccharide; here, the purpose of the column adsorption in this patent document is only to purify the polysaccharide, and components other than the polysaccharide cannot be separated and retained. Chinese patent document CN103190623A provides a dendrobium officinale extract, wherein the content of dendrobium officinale polysaccharide in the extract is 70.0% -90.0%, and the content of total flavone is 0.5-1.0%, and also provides a preparation method of the extract, which specifically comprises the following four steps: defatting with organic solvent (such as petroleum ether), soaking in ethanol solution, flash extracting, removing protein with Sevage reagent, and decolorizing with active carbon.
The processes of the invention relate to the processes of organic solvent degreasing and Sevage reagent protein removal, toxic and harmful chemical reagents are used, the production process has safety problems, the industrial production is not facilitated, and serious environmental pollution is caused.
In addition, all processes in the prior art can only extract the dendrobium officinale polysaccharide, and none of the processes can extract polyphenol components in the dendrobium officinale.
Disclosure of Invention
The invention aims to overcome the defects that a preparation method of the dendrobium officinale extract in the prior art adopts a dangerous organic solvent, is not suitable for industrial production, pollutes the environment and only extracts one component of polysaccharide substances, and provides a method which does not use the dangerous organic solvent, is suitable for industrial mass production and can extract polysaccharide and polyphenol components in the dendrobium officinale. The preparation method can synchronously extract the dendrobium officinale polysaccharide and the polyphenol substances, and has simple and convenient operation steps and reasonable process. The preparation method of the invention can also obtain the dendrobium officinale polysaccharide and the dendrobium officinale polyphenol with high yield, and has certain economic and social benefits.
The invention provides a preparation method of a dendrobium officinale extract, which comprises the following steps:
(1) extracting herba Dendrobii with ethanol, and filtering to obtain extractive solution and residue;
(2) and (3) carrying out enzymolysis on the feed liquid consisting of the filter residue and the water by using cellulase to obtain an enzymolysis liquid.
In the step (1), the production place of the raw material of the dendrobium officinale can be Yunnan.
In the step (1), the operation and condition of extracting dendrobium officinale with ethanol can be the operation and condition of extracting polyphenol in the plant field.
In the step (1), the whole herb (fresh or dried herb) of the dendrobium officinale is preferably crushed to 1-3cm before the dendrobium officinale is extracted by ethanol.
In the step (1), the ethanol used for extraction is generally an ethanol aqueous solution, the volume fraction of ethanol in the ethanol aqueous solution may be 30% to 95%, for example, 50%, 70%, 85% or 90%, and the ratio of the mass of the ethanol aqueous solution to the mass of the dendrobium officinale may be (10-40): 1, for example, can be 10:1, 12.5:1, 15:1, or 20: 1.
In step (1), the temperature of the extraction may be 40 ℃ to 75 ℃, for example, 40 ℃, 50 ℃, 55 ℃, 60 ℃ or 75 ℃; the extraction time may be 1-3 hours, for example, 1 hour, 1.5 hours, 2 hours, 2.5 hours, or 3 hours.
In the step (1), the main component of the extracting solution is dendrobium officinale polyphenol substances.
In the step (2), the enzymolysis refers to decomposing substantially all of the cellulose in the filter residue.
In the step (2), the ratio of the mass of the water to the mass of the filter residue may be (5-40): 1, for example, can be 5:1, 10:1, 12.5:1, 15:1, or 20: 1.
In step (2), the cellulase may be a commercially available cellulase. Preferably, the tolerant temperature of the cellulase is 40-60 ℃, and the tolerant pH value is 4.0-6.0; or the tolerant temperature of the cellulase is 40-60 ℃, the tolerant pH value is 4.0-6.0, and the activity unit is more than or equal to 300000U/g. More preferably, the pH of the enzymatic hydrolysis is 4.0-6.0, and may be, for example, 4.0, 4.5, 5.0, 5.5, or 6.0; the temperature of the enzymolysis is 40-60 ℃, for example, 40 ℃, 45 ℃, 50 ℃, 55 ℃ or 60 ℃; the enzymolysis time is 2-6 hours, for example, 2 hours, 3 hours, 4 hours, 5 hours or 6 hours; the cellulase is present in an amount of 0.05% to 10% by mass of the water, for example 0.05%, 2%, 4%, 7% or 10% by mass.
In the step (2), preferably, the fiber residue is dried and superfine-pulverized to obtain the dendrobium officinale fiber powder, and the dendrobium officinale fiber powder can be used for functional foods.
After the step (2), preferably, the step (3) is carried out, wherein in the step (3), the enzymatic hydrolysate is subjected to enzymolysis by alpha-amylase, and after enzyme deactivation, the enzymatic hydrolysate is filtered to obtain fiber residue and polysaccharide extracting solution.
In the step (3), the enzymolysis refers to decomposing substantially all the alpha-starch in the enzymolysis liquid.
In step (3), the α -amylase may be a commercially available α -amylase. Preferably, the tolerance temperature of the alpha-amylase is 70-85 ℃, and the tolerance pH value is 5.0-6.0; or the tolerance temperature of the alpha-amylase is 70-85 ℃, the tolerance pH value is 5.0-6.0, and the activity unit is more than or equal to 13000 AAU/g. More preferably, the temperature of the enzymolysis is 70-85 ℃, for example, 70 ℃, 75 ℃, 80 ℃, 82 ℃ or 85 ℃; the enzymolysis time is 1-3 hours, for example, 1 hour, 1.5 hours, 2 hours, 2.5 hours or 3 hours; the mass of the alpha-amylase is 0.1-10% of the mass of the enzymolysis liquid, and can be 0.1%, 2%, 6%, 8% or 10%, for example.
In the step (3), the enzyme deactivation refers to deactivating the cellulase and the alpha-amylase, and the enzyme deactivation temperature is higher than the deactivation temperature of the cellulase and the alpha-amylase, for example, may be 90 ℃ to 95 ℃, and the enzyme deactivation time lasts until the cellulase and the alpha-amylase are completely deactivated, for example, may be 30 minutes.
More preferably, step (3) is followed by step (4), in step (4), after the polysaccharide extracting solution passes through macroporous adsorption resin, effluent liquid is collected until no polysaccharide exists in the effluent liquid; wherein, the macroporous absorption resin is nonpolar or weak polar resin.
In step (4), the type of the macroporous adsorption resin is preferably D101, AB8, H-60, H103 or XDA-1B, and the flow rate of the polysaccharide extracting solution can be 1BV/H-3BV/H, for example, 1BV/H, 1.5BV/H, 2BV/H, 2.5BV/H or 3 BV/H.
In the step (4), preferably, the effluent is subjected to primary fine filtration to obtain a fine filtrate; carrying out nanofiltration on the fine filtrate to obtain nanofiltration trapped fluid; nano-filtering and washing the nano-filtered trapped fluid by using water, and then carrying out nano-filtering concentration to obtain a nano-filtered concentrated solution; and (3) after the nanofiltration concentrated solution is subjected to enzymolysis by protease, performing secondary fine filtration to obtain polysaccharide fine filtrate.
Wherein, the pore diameter of the filter membrane adopted by the first microfiltration is preferably 0.22-0.45 μm.
Wherein the molecular weight cut-off of the nanofiltration membrane used for nanofiltration and nanofiltration washing is preferably 300-500.
Wherein, the ratio of the volume of water used for nanofiltration washing to the volume of the nanofiltration retentate is preferably 2-10 times, for example, 2 times, 4 times, 6 times, 8 times or 10 times.
The volume of the nanofiltration concentrate is preferably 2-5 times, for example, 2 times, 3 times, 3.5 times, 4 times or 5 times of the volume of the dendrobium officinale.
Wherein, the protease can be a commercially available protease. Preferably, the tolerant temperature of the protease is 40-60 ℃, and the tolerant pH value is 4.0-6.0; or the tolerant temperature of the protease is 40-60 ℃, the tolerant pH value is 4.0-6.0, and the activity unit is more than or equal to 500000 HUT/g. More preferably, the pH value of the enzymolysis is 4.0-6.0, for example; the temperature of the enzymolysis is 40-60 ℃, for example, 40 ℃, 45 ℃, 50 ℃, 55 ℃ or 60 ℃; the enzymolysis time is 2-6 hours, for example, 2 hours, 3 hours, 4 hours, 5 hours or 6 hours; the protease may be present in an amount of 0.05% to 2% by mass, for example 0.05%, 0.5%, 1.0%, 1.5% or 2.0% by mass, based on the mass of the nanofiltration concentrate.
Wherein, the pore diameter of the filter membrane adopted by the secondary microfiltration is preferably 0.22-0.45 μm.
Wherein, preferably, the polysaccharide fine filtration liquid is concentrated under reduced pressure to obtain polysaccharide concentrated liquid. Preferably, the polysaccharide concentrated solution is mixed with a mixed solution of ethanol and inorganic salt, and precipitates are separated out through stirring to obtain a dendrobium officinale polysaccharide crude product; in the mixed solution, the mass fraction of ethanol is 50% to 90%, for example, 50%, 60%, 70%, 80%, 90%, and the mass fraction of inorganic salt is 0.5% to 5%, for example, 0.5%, 2%, 3%, 4%, 5%, and the inorganic salt may be sodium chloride and/or sodium acetate. Further preferably, the dendrobium officinale polysaccharide wet product is obtained after centrifugal separation and filtration of the dendrobium officinale polysaccharide crude product. Drying the wet dendrobium officinale polysaccharide product to obtain the dendrobium officinale polysaccharide, wherein the drying can be freeze drying, vacuum drying or hot air drying.
More preferably, step (4) is followed by step (5), in step (5), the macroporous adsorbent resin is eluted by an ethanol aqueous solution with a volume fraction of 60% -80%, and the eluate is collected until no flavone reaction occurs in the eluate. In the technical scheme, through the matching of various technical characteristics and other characteristics, the polysaccharide extract can effectively separate polysaccharide and polyphenol components after passing through macroporous adsorption resin, and simultaneously remove heavy metals.
In the step (5), the volume fraction of ethanol in the ethanol aqueous solution may be, for example, 70%.
After the step (5), preferably, the eluent is combined with the extracting solution in the step (1), fine filtration is carried out to obtain fine filtration solution, and then the fine filtration concentrated solution is obtained after decompression and concentration until no ethanol exists. Wherein, the aperture of the filter membrane adopted by the precise filtration can be 0.22-0.45 μm; the vacuum degree of the reduced pressure concentration can be 0.08pa to 0.1pa, and the temperature of the reduced pressure concentration can be 50 ℃ to 70 ℃, for example, 50 ℃, 55 ℃, 60 ℃, 65 ℃ or 70 ℃.
Wherein the fine filtration concentrated solution is mixed with polyhydric alcohol, a preservative and water to obtain a dendrobium officinale polyphenol solution; the polyhydric alcohol can be propylene glycol and/or butylene glycol, and the preservative is a preservative allowed to be used in cosmetics, and can be one or more of phenoxyethanol, 9010 and A631. Further preferably, in the dendrobium officinale polyphenol solution, the mass fraction of dendrobium officinale polyphenol is 1% -10%, and the mass fraction of polyalcohol is 5% -50%.
Drying the fine filtration concentrated solution to obtain solid powder of polyphenol of dendrobium officinale, wherein the solid powder of polyphenol of dendrobium officinale is rich in polyphenol of dendrobium officinale and dendrobine; the drying may be, for example, freeze drying or vacuum drying.
The invention also provides the dendrobium officinale fiber powder prepared by the preparation method.
The invention also provides the dendrobium officinale polysaccharide prepared by the preparation method.
The invention also provides the dendrobium officinale polyphenol solution prepared by the preparation method.
The invention also provides the dendrobium officinale polyphenol solid powder prepared by the preparation method.
The above preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention without departing from the common general knowledge in the art.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows: the invention provides a dendrobium officinale extract and a preparation method thereof. The preparation method can synchronously extract the dendrobium officinale polysaccharide and the polyphenol substances, and has simple and convenient operation steps and reasonable process. The preparation method of the invention can also obtain the dendrobium officinale polysaccharide and the dendrobium officinale polyphenol with high yield, and has certain economic and social benefits.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
In the following embodiments, the raw material producing area of dendrobium officinale is Yunnan; the cellulase is commercially available cellulase with tolerance temperature of 40-60 ℃ and tolerance pH value of 4.0-6.0, and the activity unit is more than or equal to 300000U/g; the alpha-amylase is commercially available alpha-amylase with tolerance temperature of 70-85 ℃ and tolerance pH value of 5.0-6.0, and the activity unit is more than or equal to 13000 AAU/g; the protease is commercially available, has tolerance temperature of 40-60 deg.C and tolerance pH of 4.0-6.0, and activity unit is greater than or equal to 500000 HUT/g.
In the following examples, in the step (2), the mass percentage of the cellulase refers to the ratio of the mass of the cellulase to the mass of the water; in the step (3), the mass percentage of the alpha-amylase refers to the ratio of the mass of the alpha-amylase to the mass of the enzymolysis solution; in the preparation method of the dendrobium officinale polysaccharide, the mass percentage of the protease refers to the ratio of the mass of the protease to the mass of the nanofiltration concentrated solution.
In the following examples, the yield of dendrobium officinale polysaccharide is calculated by the following formula: dividing the mass of the dendrobium officinale polysaccharide by the mass of the raw material dendrobium officinale; the calculation formula of the yield of the dendrobium officinale polyphenol solution is as follows: dividing the mass of the dendrobium officinale polyphenol solution by the mass of the raw material dendrobium officinale; the calculation formula of the yield of the dendrobium officinale polyphenol solid powder is as follows: dividing the mass of the solid powder of the polyphenol of the dendrobium officinale by the mass of the raw material dendrobium officinale.
Example 1
First, separation method
(1) Taking whole herb (fresh herb) of Dendrobium officinale, pre-crushing by using a traditional Chinese medicine coarse crusher, and crushing into short fibers of 1-3 cm; putting 200g of pre-crushed dendrobium officinale into an extraction tank, adding 2000mL of 70 v% ethanol, performing heat preservation extraction at 50 ℃ for 1 hour, and filtering to obtain an extracting solution and filter residues; the extract mainly contains Dendrobium officinale polyphenol substances.
(2) 4000mL of pure water is added into the filter residue to obtain feed liquid; adjusting the pH value of the feed liquid to 5.0, heating the feed liquid to 40 ℃, adding 7% of cellulase, and preserving the heat for 3 hours to obtain enzymatic hydrolysate.
(3) Adjusting the pH value of the enzymolysis liquid to 5.0, heating to 75 ℃, adding 2% high-temperature alpha-amylase, preserving heat for 3 hours, after extraction, heating to 95 ℃, inactivating enzyme for 30 minutes, and filtering while hot to obtain fiber residue and polysaccharide extract.
(4) And (3) carrying out column chromatography separation on the polysaccharide extracting solution, carrying out macroporous adsorption resin model D101, carrying out adsorption flow rate of 1BV/h, washing the resin by using pure water after adsorption is finished, washing until effluent liquid has no polysaccharide reaction when detection is carried out, and collecting all effluent liquid in the process.
(5) Eluting the macroporous adsorption resin by 70 v% ethanol water, and collecting the eluent until the eluent has no flavone reaction.
Second, preparation method of dendrobium officinale fiber powder
And (4) drying and superfine grinding the fiber residue obtained in the step (3) to obtain dendrobium officinale fiber powder which can be used for functional food.
Preparation method of dendrobium officinale polysaccharide
Performing fine filtration (0.22-0.45 μm) on the effluent liquid in the step (4), and collecting fine filtrate; filtering the fine filtrate by using a nanofiltration membrane (the cut-off molecular weight is 300-500) to obtain nanofiltration cut-off liquid; and mixing the nanofiltration trapped fluid with 2 times of pure water, performing nanofiltration washing, performing nanofiltration concentration, and concentrating until the volume of the nanofiltration concentrated fluid is 2 times of that of the dendrobium officinale.
Adjusting pH of the nanofiltration concentrated solution to 4.0, heating to 40 deg.C, adding 0.05% protease, keeping the temperature for 2 hr, fine filtering (0.22-0.45 μm), and collecting fine filtrate to obtain polysaccharide fine filtrate.
And (3) concentrating the polysaccharide fine filtrate under reduced pressure to obtain a polysaccharide concentrated solution, adding ethanol into the polysaccharide concentrated solution until the volume fraction of the ethanol reaches 50%, adding 0.5% of sodium chloride, and stirring to separate out a precipitate to obtain a dendrobium officinale polysaccharide crude product.
And carrying out centrifugal separation and filtration on the dendrobium officinale polysaccharide crude product to obtain a dendrobium officinale polysaccharide wet product.
And (3) freeze-drying the dendrobium officinale polysaccharide wet product to obtain the dendrobium officinale polysaccharide.
Effect data: the yield of the dendrobium officinale polysaccharide is 25.67%.
Preparation method of dendrobium officinale polyphenol solution
Mixing the eluate of step (5) with the extractive solution of step (1), fine filtering (filter membrane aperture 0.22-0.45 μm), and concentrating under reduced pressure at vacuum degree of 0.08-0.1 pa and 65 deg.C until ethanol is removed to obtain fine filtrate concentrate;
adding propylene glycol, antiseptic (phenoxyethanol) and pure water into the concentrate to prepare a dendrobium officinale polyphenol solution, wherein: 1 wt% of dendrobium officinale polyphenol, 5 wt% of propylene glycol and 0.4 wt% of phenoxyethanol.
Effect data: the yield of the dendrobium officinale polyphenol solution is as follows: 153.5 percent.
Example 2
First, separation method
(1) Taking whole herb (fresh herb) of Dendrobium officinale, pre-crushing by using a traditional Chinese medicine coarse crusher, and crushing into short fibers of 1-3 cm; putting 200g of pre-crushed dendrobium officinale into an extraction tank, adding 4000mL of 30 v% ethanol, performing heat preservation extraction at 75 ℃ for 2 hours, and filtering to obtain an extracting solution and filter residues; the extract mainly contains Dendrobium officinale polyphenol substances.
(2) Adding 1000mL of pure water into the filter residue to obtain a feed liquid; adjusting the pH value of the feed liquid to 5.5, heating the feed liquid to 60 ℃, adding 10% of cellulase, and preserving the heat for 5 hours to obtain enzymatic hydrolysate.
(3) Adjusting the pH value of the enzymolysis liquid to 6.0, heating to 82 ℃, adding 0.1% high-temperature alpha-amylase, preserving heat for 2.5h, after extraction, heating to 95 ℃, inactivating enzyme for 30 min, and filtering while hot to obtain fiber residue and polysaccharide extract.
(4) And (3) carrying out column chromatography separation on the polysaccharide extracting solution, carrying out macroporous adsorption resin model AB8, carrying out adsorption flow rate of 3BV/h, washing the resin by using pure water after adsorption is finished, washing until effluent liquid has no polysaccharide reaction when detection is carried out, and collecting all effluent liquid in the process.
(5) Eluting the macroporous adsorption resin by 70 v% ethanol water, and collecting the eluent until the eluent has no flavone reaction.
Second, preparation method of dendrobium officinale fiber powder
And (4) drying and superfine grinding the fiber residue obtained in the step (3) to obtain dendrobium officinale fiber powder which can be used for functional food.
Preparation method of dendrobium officinale polysaccharide
Performing fine filtration (0.22-0.45 μm) on the effluent liquid in the step (4), and collecting fine filtrate; filtering the fine filtrate by using a nanofiltration membrane (the cut-off molecular weight is 300-500) to obtain nanofiltration cut-off liquid; and mixing the nanofiltration trapped fluid with pure water of 6 times, performing nanofiltration washing, performing nanofiltration concentration, and concentrating until the volume of the nanofiltration concentrated solution is 3.5 times of that of the dendrobium officinale.
Adjusting pH of the nanofiltration concentrated solution to 5.5, heating to 55 deg.C, adding 1.5% protease, keeping the temperature for 4 hr, fine filtering (0.22-0.45 μm), and collecting fine filtrate to obtain polysaccharide fine filtrate.
And (3) concentrating the polysaccharide fine filtrate under reduced pressure to obtain a polysaccharide concentrated solution, adding ethanol into the polysaccharide concentrated solution until the volume fraction of the ethanol reaches 80%, adding 3% sodium acetate into the polysaccharide concentrated solution, and stirring to separate out a precipitate to obtain a dendrobium officinale polysaccharide crude product.
And carrying out centrifugal separation and filtration on the dendrobium officinale polysaccharide crude product to obtain a dendrobium officinale polysaccharide wet product.
And (3) freeze-drying the dendrobium officinale polysaccharide wet product to obtain the dendrobium officinale polysaccharide.
Effect data: the yield of the dendrobium officinale polysaccharide is 29.28%.
Preparation method of dendrobium officinale polyphenol solution
Mixing the eluate of step (5) with the extractive solution of step (1), fine filtering (filter membrane aperture 0.22-0.45 μm), and concentrating under reduced pressure at vacuum degree of 0.08-0.1 pa and 55 deg.C until ethanol is removed to obtain fine filtrate concentrate;
adding butanediol, antiseptic 9010 and pure water into the concentrate to prepare a dendrobium officinale polyphenol solution, wherein: 10 wt% of dendrobium officinale polyphenol, 50 wt% of butanediol and 0.8 wt% of preservative 9010.
Effect data: the yield of the dendrobium officinale polyphenol solution is as follows: 216.7 percent.
Example 3
First, separation method
(1) Taking whole herb (fresh herb) of Dendrobium officinale, pre-crushing by using a traditional Chinese medicine coarse crusher, and crushing into short fibers of 1-3 cm; putting 200g of pre-crushed dendrobium officinale into an extraction tank, adding 2000mL of 90 v% ethanol, performing heat preservation extraction at 40 ℃ for 3 hours, and filtering to obtain an extracting solution and filter residues; the extract mainly contains Dendrobium officinale polyphenol substances.
(2) Adding 3000mL of pure water into the filter residue to obtain a feed liquid; adjusting the pH value of the feed liquid to 4.0, heating the feed liquid to 50 ℃, adding 0.05% of cellulase, and preserving the heat for 6 hours to obtain enzymatic hydrolysate.
(3) Adjusting the pH value of the enzymolysis liquid to 5.5, heating to 85 ℃, adding 8% high-temperature alpha-amylase, preserving heat for 1h, after extraction, heating to 95 ℃, inactivating enzyme for 30 min, and filtering while hot to obtain fiber residue and polysaccharide extract.
(4) And (3) carrying out column chromatography separation on the polysaccharide extracting solution, carrying out macroporous adsorption resin model H103, carrying out adsorption flow rate of 2BV/H, washing the resin by using pure water after adsorption is finished, washing until effluent liquid has no polysaccharide reaction when detection is carried out, and collecting all effluent liquid in the process.
(5) Eluting the macroporous adsorption resin by 70 v% ethanol water, and collecting the eluent until the eluent has no flavone reaction.
Second, preparation method of dendrobium officinale fiber powder
And (4) drying and superfine grinding the fiber residue obtained in the step (3) to obtain dendrobium officinale fiber powder which can be used for functional food.
Preparation method of dendrobium officinale polysaccharide
Performing fine filtration (0.22-0.45 μm) on the effluent liquid in the step (4), and collecting fine filtrate; filtering the fine filtrate by using a nanofiltration membrane (the cut-off molecular weight is 300-500) to obtain nanofiltration cut-off liquid; and mixing the nanofiltration trapped fluid with 10 times of pure water, performing nanofiltration washing, performing nanofiltration concentration, and concentrating until the volume of the nanofiltration concentrated solution is 5 times of that of the dendrobium officinale.
Adjusting pH of the nanofiltration concentrated solution to 6.0, heating to 45 deg.C, adding 0.5% protease, keeping the temperature for 6 hr, fine filtering (0.22-0.45 μm), and collecting fine filtrate to obtain polysaccharide fine filtrate.
And (3) concentrating the polysaccharide fine filtrate under reduced pressure to obtain a polysaccharide concentrated solution, adding ethanol into the polysaccharide concentrated solution until the volume fraction of the ethanol reaches 90%, adding 2% sodium acetate into the polysaccharide concentrated solution, and stirring to separate out a precipitate to obtain a dendrobium officinale polysaccharide crude product.
And carrying out centrifugal separation and filtration on the dendrobium officinale polysaccharide crude product to obtain a dendrobium officinale polysaccharide wet product.
And (3) freeze-drying the dendrobium officinale polysaccharide wet product to obtain the dendrobium officinale polysaccharide.
Effect data: the yield of the dendrobium officinale polysaccharide is 31.42%.
Preparation method of dendrobium officinale polyphenol solution
Mixing the eluate of step (5) with the extractive solution of step (1), fine filtering (filter membrane aperture 0.22-0.45 μm), and concentrating under reduced pressure at 50 deg.C under vacuum degree of 0.08-0.1 pa until ethanol is removed to obtain fine filtrate concentrate;
adding butanediol, preservative A631 and pure water into the concentrate to prepare a dendrobium officinale polyphenol solution, wherein: 5 wt% of dendrobium officinale polyphenol, 20 wt% of butanediol and 1.5 wt% of preservative A631.
Effect data: the yield of the dendrobium officinale polyphenol solution is as follows: 185.7 percent.
Example 4
First, separation method
(1) Taking whole herb (fresh herb) of Dendrobium officinale, pre-crushing by using a traditional Chinese medicine coarse crusher, and crushing into short fibers of 1-3 cm; putting 200g of pre-crushed dendrobium officinale into an extraction tank, adding 3000mL of 85 v% ethanol, performing heat preservation extraction at 55 ℃ for 2.5 hours, and filtering to obtain an extracting solution and filter residues; the extract mainly contains Dendrobium officinale polyphenol substances.
(2) Adding 2000mL of pure water into the filter residue to obtain a feed liquid; adjusting the pH value of the feed liquid to 4.5, heating the feed liquid to 45 ℃, adding 2% of cellulase, and preserving the heat for 4 hours to obtain enzymatic hydrolysate.
(3) Adjusting the pH value of the enzymolysis liquid to 5.7, heating to 70 ℃, adding 10% high-temperature alpha-amylase, preserving heat for 1.5h, after extraction, heating to 95 ℃, inactivating enzyme for 30 min, and filtering while hot to obtain fiber residue and polysaccharide extract.
(4) Performing column chromatography separation on the polysaccharide extract, performing macroporous adsorption resin model XDA-1B at an adsorption flow rate of 2.5BV/h, washing the resin with pure water after adsorption is finished, washing until no polysaccharide reaction is detected in effluent, and collecting all effluent in the process.
(5) Eluting the macroporous adsorption resin by 70 v% ethanol water, and collecting the eluent until the eluent has no flavone reaction.
Second, preparation method of dendrobium officinale fiber powder
And (4) drying and superfine grinding the fiber residue obtained in the step (3) to obtain dendrobium officinale fiber powder which can be used for functional food.
Preparation method of dendrobium officinale polysaccharide
Performing fine filtration (0.22-0.45 μm) on the effluent liquid in the step (4), and collecting fine filtrate; filtering the fine filtrate by using a nanofiltration membrane (the cut-off molecular weight is 300-500) to obtain nanofiltration cut-off liquid; and mixing the nanofiltration trapped fluid with 4 times of pure water, performing nanofiltration washing, performing nanofiltration concentration, and concentrating until the volume of the nanofiltration concentrated solution is 3 times of that of the dendrobium officinale.
Adjusting pH of the nanofiltration concentrated solution to 4.5, heating to 60 deg.C, adding 2.0% protease, keeping the temperature for 3 hr, fine filtering (0.22-0.45 μm), and collecting fine filtrate to obtain polysaccharide fine filtrate.
And (3) concentrating the polysaccharide fine filtrate under reduced pressure to obtain a polysaccharide concentrated solution, adding ethanol into the polysaccharide concentrated solution until the volume fraction of the ethanol reaches 60%, adding 4% of sodium chloride, and stirring to separate out a precipitate to obtain a dendrobium officinale polysaccharide crude product.
And carrying out centrifugal separation and filtration on the dendrobium officinale polysaccharide crude product to obtain a dendrobium officinale polysaccharide wet product.
And (3) freeze-drying the dendrobium officinale polysaccharide wet product to obtain the dendrobium officinale polysaccharide.
Effect data: the yield of the dendrobium officinale polysaccharide is 27.85%.
Preparation method of dendrobium officinale polyphenol solid powder
Mixing the eluate of step (5) with the extractive solution of step (1), fine filtering (filter membrane aperture 0.22-0.45 μm), and concentrating under reduced pressure at 70 deg.C under vacuum degree of 0.08-0.1 pa until ethanol is removed to obtain fine filtrate concentrate; and (4) freeze-drying the fine-filtered concentrated solution to obtain solid powder of the dendrobium officinale polyphenol.
Effect data: the yield of the dendrobium officinale polyphenol solid powder is as follows: 2.45 percent.
Example 5
First, separation method
(1) Taking whole herb (fresh herb) of Dendrobium officinale, pre-crushing by using a traditional Chinese medicine coarse crusher, and crushing into short fibers of 1-3 cm; putting 200g of pre-crushed dendrobium officinale into an extraction tank, adding 2500mL of 50 v% ethanol, performing heat preservation extraction at 60 ℃ for 1.5 hours, and filtering to obtain an extracting solution and filter residues; the extract mainly contains Dendrobium officinale polyphenol substances.
(2) Adding 2500mL of pure water into the filter residue to obtain a feed liquid; adjusting the pH value of the feed liquid to 6.0, heating the feed liquid to 55 ℃, adding 4% of cellulase, and preserving the heat for 2 hours to obtain enzymatic hydrolysate.
(3) Adjusting the pH value of the enzymolysis liquid to 5.8, heating to 80 ℃, adding 6% high-temperature alpha-amylase, preserving the temperature for 2h, after extraction, heating to 95 ℃, inactivating the enzyme for 30 min, and filtering while hot to obtain fiber residue and polysaccharide extract.
(4) And (3) carrying out column chromatography separation on the polysaccharide extracting solution, carrying out macroporous adsorption resin model H-6, carrying out adsorption flow rate of 1.5BV/H, washing the resin by using pure water after adsorption is finished, washing until effluent liquid has no polysaccharide reaction when detection is carried out, and collecting all effluent liquid in the process.
(5) Eluting the macroporous adsorption resin by 70 v% ethanol water, and collecting the eluent until the eluent has no flavone reaction.
Second, preparation method of dendrobium officinale fiber powder
And (4) drying and superfine grinding the fiber residue obtained in the step (3) to obtain dendrobium officinale fiber powder which can be used for functional food.
Preparation method of dendrobium officinale polysaccharide
Performing fine filtration (0.22-0.45 μm) on the effluent liquid in the step (4), and collecting fine filtrate; filtering the fine filtrate by using a nanofiltration membrane (the cut-off molecular weight is 300-500) to obtain nanofiltration cut-off liquid; and mixing the nanofiltration trapped fluid with 8 times of pure water, performing nanofiltration washing, performing nanofiltration concentration, and concentrating until the volume of the nanofiltration concentrated solution is 4 times of that of the dendrobium officinale.
Adjusting pH of the nanofiltration concentrated solution to 5.0, heating to 50 deg.C, adding 1.0% protease, keeping the temperature for 5 hr, fine filtering (0.22-0.45 μm), and collecting fine filtrate to obtain polysaccharide fine filtrate.
And (3) concentrating the polysaccharide fine filtrate under reduced pressure to obtain a polysaccharide concentrated solution, adding ethanol into the polysaccharide concentrated solution until the volume fraction of the ethanol reaches 70%, adding 5% of sodium chloride, and stirring to separate out a precipitate to obtain a dendrobium officinale polysaccharide crude product.
And carrying out centrifugal separation and filtration on the dendrobium officinale polysaccharide crude product to obtain a dendrobium officinale polysaccharide wet product.
And (3) freeze-drying the dendrobium officinale polysaccharide wet product to obtain the dendrobium officinale polysaccharide.
Effect data: the yield of the dendrobium officinale polysaccharide is 28.87%.
Preparation method of dendrobium officinale polyphenol solid powder
Mixing the eluate of step (5) with the extractive solution of step (1), fine filtering (filter membrane aperture 0.22-0.45 μm), and concentrating under reduced pressure at vacuum degree of 0.08-0.1 pa and 60 deg.C until ethanol is removed to obtain fine filtrate concentrate;
and (4) freeze-drying the fine-filtered concentrated solution to obtain solid powder of the dendrobium officinale polyphenol.
Effect data: the yield of the dendrobium officinale polyphenol solid powder is as follows: 2.08 percent.
Example 6
Step (1), (2) and (3) are the same as example 1 and step (4), the extracting solution is concentrated to remove ethanol, then the extracting solution is mixed with the polysaccharide extracting solution, the mixture is continuously concentrated to a certain volume, and spray drying is carried out to obtain the dendrobium officinale extract dry product.
Effect data: the yield is as follows: 32.21 percent.
Example 7
The protease in the step (5) in example 3 was not used for the enzymatic hydrolysis, and the other steps were the same as in example 3.
Effect data: the yield of the dendrobium officinale polysaccharide is 30.87%; the yield of the dendrobium officinale polyphenol solution is 174.5%. However, the increased protein content in the dendrobium officinale polysaccharide increases the risk of foreign protein allergy of the product in use.
Comparative example 1
Step (1) and example 2, step (2) and step (3) are combined, and high-temperature extraction is carried out only by adopting pure water to obtain extracting solution and filter residue; the extract was subjected directly to step (4) and subsequent steps in example 2 without enzymatic hydrolysis with cellulase and high temperature α -amylase.
Effect data: the yield of the dendrobium officinale polysaccharide is as follows: 18.59 percent. Compared with the examples, the yield of the dendrobium officinale polysaccharide is obviously reduced, and the total polysaccharide and mannose content are reduced (the specific numerical values are shown in table 1). The yield of the dendrobium officinale polyphenol solution is 200.3 percent.
TABLE 1 quality index of Dendrobium officinale polysaccharide
Note: the method for measuring the content of mannose and the content of total polysaccharides comprises the following steps: dendrobium officinale in first pharmacopoeia of China, 2015 edition; the nitrogen content determination method comprises the following steps: kjeldahl method; the molecular weight determination method comprises the following steps: performing Sephadex chromatography (with pullulan as standard).
TABLE 2 Polyphenol quality index of Dendrobium officinale
Note: the method for measuring the total polyphenol content comprises the following steps: folin & Clocalteu's pHenol reagent, brewing science 2011,2011(4) 29-32.
The polyphenol products obtained in examples 1,2, 3, 7 and comparative example 1 are dendrobium officinale polyphenol solutions, and then the total polyphenol content in table 2 refers to the polyphenol content in dry dendrobium officinale polyphenol solid powder.
The polyphenol products obtained in examples 4 and 5 are dendrobium officinale polyphenol solid powder, and the total polyphenol content in table 2 refers to the polyphenol content in the dendrobium officinale polyphenol solid powder.
While specific embodiments of the invention have been described above, it will be appreciated by those skilled in the art that this is by way of example only, and that the scope of the invention is defined by the appended claims. Various changes and modifications to these embodiments may be made by those skilled in the art without departing from the spirit and scope of the invention, and these changes and modifications are within the scope of the invention.
Claims (20)
1. The preparation method of the dendrobium officinale extract is characterized by comprising the following steps:
(1) extracting herba Dendrobii with ethanol, and filtering to obtain extractive solution and residue;
(2) the feed liquid consisting of the filter residue and the water is subjected to enzymolysis by cellulase to obtain an enzymolysis liquid;
(3) carrying out enzymolysis on the enzymolysis liquid by alpha-amylase, inactivating enzymes, and filtering to obtain fiber residues and a polysaccharide extracting solution; wherein the tolerance temperature of the alpha-amylase is 70-85 ℃, the tolerance pH value is 5.0-6.0, and the activity unit is more than or equal to 13000 AAU/g; the temperature of the enzymolysis is 70-85 ℃, the time of the enzymolysis is 1-3 hours, and the mass of the alpha-amylase is 0.1-10% of that of the enzymolysis liquid;
(4) after the polysaccharide extracting solution passes through macroporous adsorption resin, collecting effluent liquid until no polysaccharide exists in the effluent liquid; wherein the macroporous adsorption resin is nonpolar or weakly polar resin, the type of the macroporous adsorption resin is D101, AB8, H-60, H103 or XDA-1B, and the flow rate of the polysaccharide extracting solution is 1BV/H-3 BV/H;
the effluent liquid is subjected to primary fine filtration to obtain fine filtrate; wherein the aperture of the filter membrane adopted by the first-time precise filtration is 0.22-0.45 μm;
carrying out nanofiltration on the fine filtrate to obtain nanofiltration trapped fluid;
nano-filtering and washing the nano-filtered trapped fluid by using water, and then carrying out nano-filtering concentration to obtain a nano-filtered concentrated solution;
after the nanofiltration concentrated solution is subjected to enzymolysis by protease, performing secondary fine filtration to obtain polysaccharide fine filtrate; wherein the aperture of the filter membrane adopted by the secondary microfiltration is 0.22-0.45 μm;
(5) eluting the macroporous adsorption resin by using 60-80% ethanol water solution by volume fraction, and collecting eluent until the eluent has no flavone reaction.
2. The preparation method according to claim 1, wherein in the step (1), the whole plant of Dendrobium officinale is pulverized to 1-3cm before the extraction with ethanol;
and/or in the step (1), the ethanol adopted for extraction is an ethanol aqueous solution, the volume fraction of the ethanol in the ethanol aqueous solution is 30-95%, and the ratio of the mass of the ethanol aqueous solution to the mass of the dendrobium officinale is (10-40): 1;
and/or, in the step (1), the extraction temperature is 40-75 ℃, and the extraction time is 1-3 hours.
3. The method according to claim 1, wherein in the step (2), the ratio of the mass of the water to the mass of the residue is (10-40): 1.
4. the method according to claim 3, wherein in the step (2), the cellulase has a tolerance temperature of 40 ℃ to 60 ℃ and a tolerance pH of 4.0 to 6.0; or the tolerant temperature of the cellulase is 40-60 ℃, the tolerant pH value is 4.0-6.0, and the activity unit is more than or equal to 300000U/g; the pH value of the enzymolysis is 4.0-6.0, the enzymolysis temperature is 40-60 ℃, the enzymolysis time is 2-6 hours, and the mass of the cellulase is 0.05-10% of the mass of the water.
5. The preparation method according to claim 4, wherein in the step (3), the fiber residue is dried and superfine-pulverized to obtain the dendrobium officinale fiber powder.
6. The method as claimed in claim 1, wherein the nanofiltration membrane used in the nanofiltration and the nanofiltration washing has a molecular weight cut-off of 300-500.
7. The method of claim 1, wherein the ratio of the volume of water used for nanofiltration washing to the volume of the nanofiltration retentate is 2 to 10 times.
8. The method of claim 1, wherein the protease is resistant at a temperature of 40 ℃ to 60 ℃ and at a pH of 4.0 to 6.0; or the tolerant temperature of the protease is 40-60 ℃, the tolerant pH value is 4.0-6.0, and the activity unit is more than or equal to 500000 HUT/g; the pH value of the enzymolysis is 4.0-6.0, the enzymolysis temperature is 40-60 ℃, the enzymolysis time is 2-6 hours, and the mass of the protease is 0.05-2% of the mass of the nanofiltration concentrated solution.
9. The method of claim 1, wherein the polysaccharide concentrate is obtained by concentrating the fine polysaccharide filtrate under reduced pressure.
10. The preparation method of claim 9, wherein the polysaccharide concentrated solution is mixed with a mixture of ethanol and inorganic salt, and the mixture is stirred to precipitate out and obtain a crude dendrobium officinale polysaccharide product; in the mixed solution, the mass fraction of the ethanol is 50-90%, the mass fraction of the inorganic salt is 0.5-5%, and the inorganic salt is sodium chloride and/or sodium acetate.
11. The preparation method of claim 10, wherein the crude dendrobium officinale polysaccharide product is centrifuged and filtered to obtain a wet dendrobium officinale polysaccharide product; and drying the wet dendrobium officinale polysaccharide product to obtain the dendrobium officinale polysaccharide.
12. The method according to claim 1, wherein in the step (5), the volume fraction of ethanol in the ethanol aqueous solution is 70%;
and/or after the step (5), mixing the eluent and the extracting solution in the step (1), performing precision filtration to obtain a fine filtrate, and performing reduced pressure concentration until no ethanol exists to obtain a fine filtration concentrated solution.
13. The method according to claim 12, wherein the pore size of the filter membrane used for the microfiltration is 0.22 μm to 0.45 μm; the vacuum degree of the reduced pressure concentration is 0.08pa to 0.1pa, and the temperature of the reduced pressure concentration is 50 ℃ to 70 ℃.
14. The method of claim 12, wherein the concentrate is mixed with a polyol, a preservative and water to obtain a dendrobium officinale polyphenol solution.
15. The method of claim 14, wherein the polyol is propylene glycol and/or butylene glycol and the preservative is one or more of phenoxyethanol, 9010 and a 631.
16. The method according to claim 15, wherein the mass fraction of the dendrobium officinale polyphenol in the dendrobium officinale polyphenol solution is 1% -10%, and the mass fraction of the polyol is 5% -50%.
17. The method of claim 12, wherein the concentrate is dried to obtain a solid powder of the polyphenols from Dendrobium officinale.
18. The dendrobium officinale polysaccharide prepared by the preparation method of claim 11.
19. The dendrobium officinale polyphenol solution prepared by the preparation method of claim 14.
20. The dendrobium officinale polyphenol solid powder prepared by the preparation method of claim 17.
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