CN113621085A - Preparation method of dandelion root polysaccharide sulfation modification product and application of dandelion root polysaccharide sulfation modification product - Google Patents
Preparation method of dandelion root polysaccharide sulfation modification product and application of dandelion root polysaccharide sulfation modification product Download PDFInfo
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
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- 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
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A61K31/00—Medicinal preparations containing organic active ingredients
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- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
- A61K36/288—Taraxacum (dandelion)
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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Abstract
The invention discloses a preparation method of a dandelion root polysaccharide sulfation modification product and application of the dandelion root polysaccharide sulfation modification product, wherein the polysaccharide content in the polysaccharide sulfation modification product is improved by 60 percent compared with the polysaccharide content before modification. The invention selects an optimal extraction process through a plurality of groups of tests, obtains crude polysaccharide by adopting a water extraction and alcohol precipitation method, deproteinizes by adopting a Sevag method, then separates by adopting DEAE Sepharose Fast Flow, purifies by using glucan gel to prepare dandelion root polysaccharide with high purity, and then modifies by adopting a chlorosulfonic acid-dimethylformamide method to obtain a dandelion root polysaccharide sulfated modified product. The method fully utilizes the waste dandelion root resources, changes waste into valuable, obtains the dandelion root polysaccharide with immunoregulation activity and the sulfation modification product thereof, and realizes sustainable application of the dandelion medicinal resources.
Description
Technical Field
The invention relates to a plant polysaccharide, in particular to dandelion root polysaccharide with immunoregulatory activity, a preparation method of a sulfation modification product of the dandelion root polysaccharide and application of the sulfation modification product of the dandelion root polysaccharide.
Background
The dandelion is a perennial herb of the family Compositae, widely distributed in various regions of China, and has various varieties and high yield. The medicinal value of dandelion is loaded into various medical classics, and the records about dandelion are firstly found in Tang Ben Cao: sweet, mild and nontoxic. Housewife has a superficial infection of human milk. 'and' Ben Cao gang mu 'records that dandelion is mainly used for treating women's human milk carbuncle and swelling, relieving dyspepsia, dissipating stagnation of qi, removing heat toxin, eliminating swelling, tuberculosis and spur swelling. The roots, stems and leaves of the dandelion have high medicinal value, and the dandelion has complex chemical components, wherein the polysaccharide accounts for 30-50% of the dry weight, the part with the highest polysaccharide content is the roots of the dandelion, but the roots of the dandelion are mostly discarded in the harvesting process, so that the great waste of resources is caused.
The polysaccharide is formed by combining monosaccharide molecules through glycosidic bonds, is widely existed in plants, animals and microorganisms, and has pharmacological activities of improving the immunity of organisms, reducing blood sugar, resisting bacteria, enhancing the immunity, resisting tumors, protecting liver, delaying senescence and the like. The sulfation modification is that polyanionic sulfate radicals are introduced to change the spatial conformation of polysaccharide molecules, so that the activity is greatly changed, and the sulfated polysaccharide not only has the effects of anticoagulation and immunity enhancement, but also has the effects of reducing blood fat, reducing blood sugar and the like, prolongs the action time of the medicament, enhances the medicinal effect, reduces the toxic or side effect of the medicament and the like.
At present, the research on dandelion root polysaccharide at home and abroad mainly focuses on the analysis of chemical components of dandelion root polysaccharide, and the research on the activity of dandelion root polysaccharide is less. Therefore, the structure of dandelion root polysaccharide is changed by utilizing a sulfation modification technology, the immunocompetence of the dandelion root polysaccharide is enhanced, the dandelion root resource is efficiently utilized, and the method has great significance for changing waste into valuable.
Disclosure of Invention
To solve the problems set forth in the background art described above. The invention provides a preparation method of dandelion root polysaccharide with improved immunocompetence and a sulfated modified product thereof, wherein crude dandelion root polysaccharide is prepared by an optimal method, and then the structure of the dandelion root polysaccharide is changed by utilizing a sulfation modification technology to obtain the sulfated modified product of the dandelion root polysaccharide with immunoregulatory activity, so that the dandelion root resource is efficiently utilized, waste is turned into wealth, and the sustainable application of dandelion medicinal resources is realized.
In order to achieve the purpose, the invention adopts the following technical scheme:
a sulfated product of dandelion root polysaccharide has a polysaccharide content increased by 60% compared with that before modification.
Preferably, the preparation method of the sulfated modified dandelion root polysaccharide product comprises the following steps:
(1) pulverizing the raw material dandelion root, placing into an extraction tank, adding 10-30 times of water, extracting with boiling water for 3-5 hr, and repeating for 2-3 times. Removing impurities from the supernatant by a filter screen, appropriately concentrating, centrifuging by adopting Sevag reagent to remove protein, centrifuging, taking the supernatant, adding 2-4 times of anhydrous ethanol, uniformly stirring, and precipitating overnight. Suction filtration was performed using a buchner funnel and filter paper to obtain a precipitate. Dissolving the precipitate with distilled water again, volatilizing ethanol in 50-60 deg.C water bath, and lyophilizing with a lyophilizer to obtain crude polysaccharide of radix Taraxaci;
(2) separation of crude Dandelion root polysaccharide
Weighing the crude dandelion root polysaccharide prepared in the step (1), adding distilled water for dissolving, centrifuging at the rotation speed of 12000rpm of 8000-. Then the elution is carried out by gradient elution with 0.0, 0.2, 0.5 and 1.0mol/L NaCl solution. Tracking and detecting the polysaccharide content by adopting a phenol-sulfuric acid method, respectively collecting different elution peaks, carrying out reduced pressure concentration, dialyzing by using a 2000-4000Da dialysis bag, and carrying out freeze drying to obtain dandelion root polysaccharide;
(3) sulfated modification of dandelion root polysaccharide
Taking the dandelion root polysaccharide prepared in the step (2), and modifying by adopting a chlorosulfonic acid-dimethylformamide method: accurately weighing dandelion root polysaccharide, adding pyridine, adding 2mL chlorosulfonic acid solution under ice bath condition, stirring at room temperature for 10-20min, gradually melting the mixed solvent to obtain light yellow clear liquid, adding 3mL formamide, and heating to dissolve completely. And (3) carrying out ice bath to 0 ℃, adding the mixture into a sulfuric acid derivatization reagent, stirring and dissolving the mixture, reacting the mixture in the ice bath for 1-2h to normal temperature, regulating the pH value to 7-8 by using NaOH, dialyzing the mixture for 2 days, taking dialysate, concentrating and freeze-drying the dialysate to obtain the dandelion root polysaccharide sulfation modification product.
Preferably, in the preparation method of the sulfated modified dandelion root polysaccharide, 100mg of dandelion root polysaccharide is accurately weighed in the step (3), 1mL of pyridine is added, 2mL of chlorosulfonic acid solution is added under the ice bath condition, the mixture is stirred at room temperature for 10-20min, and after the mixed solvent is gradually melted to become light yellow clear liquid, 3mL of formamide is added, and the mixture is heated until the mixed solvent is completely dissolved. And (3) carrying out ice bath to 0 ℃, adding the mixture into a sulfuric acid derivatization reagent, stirring and dissolving the mixture, reacting the mixture in the ice bath for 1-2h to normal temperature, regulating the pH value to 7-8 by using NaOH, dialyzing the mixture for 2 days, taking dialysate, concentrating and freeze-drying the dialysate to obtain the dandelion root polysaccharide sulfation modification product.
Experimental research shows that the dandelion root polysaccharide product obtained through sulfation modification has the obvious effect of reducing the inflammatory reaction of mouse ulcerative colitis, and can be used for preparing health-care food or medicines for immunoregulation.
The sulfated modified dandelion root polysaccharide product may be prepared into capsule, granule, tablet, embedding agent, etc. with the material.
The effective effect is as follows: compared with the prior art, the dandelion root polysaccharide with immunoregulation activity and the sulfation modification product thereof provided by the invention have the advantages that:
the invention selects an optimal extraction process through a plurality of groups of tests, obtains crude polysaccharide by adopting a water extraction and alcohol precipitation method, deproteinizes by adopting a Sevag method, then separates by adopting DEAE Sepharose Fast Flow, purifies by using glucan gel to prepare dandelion root polysaccharide with high purity, and then modifies by adopting a chlorosulfonic acid-dimethylformamide method to obtain a dandelion root polysaccharide sulfated modified product. The method fully utilizes the waste dandelion root resources, changes waste into valuable, obtains the dandelion root polysaccharide with immunoregulation activity and the sulfation modification product thereof, and realizes sustainable application of the dandelion medicinal resources.
Drawings
FIG. 1 is an infrared spectrum of crude Dandelion root polysaccharide;
FIG. 2 is an infrared spectrum of the sulfated modified dandelion root polysaccharide;
FIG. 3 is a comparison graph of infrared spectra before and after sulfated modification of dandelion root polysaccharide.
In the figure, when the figure is rotated 90 ° to the left, the vertical axis represents transmittance (%), and the horizontal axis represents wave number (cm)-1)。
Detailed Description
The invention will be better understood from the following examples. However, one skilled in the art will readily appreciate that the specific material proportions, process conditions, and results thereof described in the examples are illustrative only and should not be taken as limiting the invention as detailed in the claims.
Example 1
The preparation method of the sulfated modified product of dandelion root polysaccharide comprises the following steps:
(1) pulverizing the raw material dandelion root, placing in an extraction tank, adding 10 times of water, extracting with boiling water for 3 hr, and repeating for 2 times. Removing impurities from the supernatant through a filter screen, appropriately concentrating, centrifuging to remove protein by adopting Sevag reagent, centrifuging, taking the supernatant, adding 2 times of volume of absolute ethyl alcohol, uniformly stirring, and precipitating overnight. Suction filtration was performed using a buchner funnel and filter paper to obtain a precipitate. Dissolving the precipitate with distilled water again, volatilizing ethanol in 50 deg.C water bath, and lyophilizing with a lyophilizer to obtain crude polysaccharide of radix Taraxaci;
(2) separation of crude Dandelion root polysaccharide
Weighing the crude dandelion root polysaccharide prepared in the step (1), adding distilled water to dissolve, centrifuging at 8000rpm, taking a supernatant, and eluting with distilled water at a flow rate of 10 ml/min. Then the elution is carried out by gradient elution with 0.0, 0.2, 0.5 and 1.0mol/L NaCl solution. Tracking and detecting polysaccharide content by adopting a phenol-sulfuric acid method, respectively collecting different elution peaks, concentrating under reduced pressure, dialyzing by using a 2000Da dialysis bag, and freeze-drying to obtain dandelion root polysaccharide;
(3) sulfated modification of dandelion root polysaccharide
Taking the dandelion root polysaccharide prepared in the step (2), and modifying by adopting a chlorosulfonic acid-dimethylformamide method: accurately weighing 100mg of dandelion root polysaccharide, adding 1mL of pyridine, adding 2mL of chlorosulfonic acid solution under the ice bath condition, stirring for 10min at room temperature until the mixed solvent is gradually melted to become light yellow clear liquid, adding 3mL of formamide, and heating until the mixed solvent is completely dissolved. And (3) carrying out ice bath to 0 ℃, adding the mixture into a sulfuric acid derivatization reagent, stirring and dissolving the mixture, reacting the mixture in the ice bath for 1h to normal temperature, regulating the pH value to 7 by using NaOH, dialyzing the mixture for 2 days, taking dialysate, concentrating and freeze-drying the dialysate to obtain the dandelion root polysaccharide sulfated modified product.
Example 2
(1) Pulverizing the raw material dandelion root, placing into an extraction tank, adding 10-30 times of water, extracting with boiling water for 5 hr, and repeating for 3 times. Removing impurities from the supernatant through a filter screen, appropriately concentrating, centrifuging to remove protein by adopting Sevag reagent, centrifuging, taking the supernatant, adding 4 times of volume of absolute ethyl alcohol, uniformly stirring, and precipitating overnight. Suction filtration was performed using a buchner funnel and filter paper to obtain a precipitate. Dissolving the precipitate with distilled water again, volatilizing ethanol in water bath at 60 deg.C, and lyophilizing with a lyophilizer to obtain crude polysaccharide of radix Taraxaci;
(2) separation of crude Dandelion root polysaccharide
Weighing the crude dandelion root polysaccharide prepared in the step (1), adding distilled water to dissolve, centrifuging at the rotating speed of 12000rpm, taking a supernatant, and eluting with distilled water at the flow rate of 20 ml/min. Then the elution is carried out by gradient elution with 0.0, 0.2, 0.5 and 1.0mol/L NaCl solution. Tracking and detecting polysaccharide content by adopting a phenol-sulfuric acid method, respectively collecting different elution peaks, concentrating under reduced pressure, dialyzing by using a 4000Da dialysis bag, and freeze-drying to obtain dandelion root polysaccharide;
(3) sulfated modification of dandelion root polysaccharide
Taking the dandelion root polysaccharide prepared in the step (2), and modifying by adopting a chlorosulfonic acid-dimethylformamide method: accurately weighing 100mg of dandelion root polysaccharide, adding 1mL of pyridine, adding 2mL of chlorosulfonic acid solution under the ice bath condition, stirring for 20min at room temperature until the mixed solvent is gradually melted to become light yellow clear liquid, adding 3mL of formamide, and heating until the mixed solvent is completely dissolved. And (3) carrying out ice bath to 0 ℃, adding the mixture into a sulfuric acid derivatization reagent, stirring and dissolving the mixture, reacting the mixture in the ice bath for 2 hours to normal temperature, regulating the pH value to 8 by using NaOH, dialyzing the mixture for 2 days, taking dialysate, concentrating and freeze-drying the dialysate to obtain the dandelion root polysaccharide sulfated modified product.
(4) Infrared spectroscopic analysis
Taking 1mg of each of dried dandelion root crude polysaccharide and dandelion root polysaccharide sulfation modification product samples, and uniformly grinding the dried dandelion root crude polysaccharide and dandelion root polysaccharide sulfation modification product samples and 100mg of dried potassium bromide powder in an agate mortar; pressing into thin sheets, and measuring 4000-400cm on infrared spectrometer-1Infrared spectrum of (D). As shown in FIG. 1, FIG. 2 and FIG. 3, the crude polysaccharide of dandelion root is 1240cm-1The absorption peaks are S ═ O stretching vibration, obviously enhanced after sulfation, and the crude polysaccharide of dandelion root is 808cm-1The absorption peaks at the left and right are the tensile oscillations of C-O-S, which are significantly enhanced after sulfation, indicating the success of sulfation.
Claims (4)
1. A dandelion root polysaccharide sulfation modification product is characterized in that the polysaccharide content in the dandelion root polysaccharide sulfation modification product is improved by 60 percent compared with the polysaccharide content before modification;
the preparation method of the dandelion root polysaccharide sulfation modification product comprises the following steps:
(1) crushing a raw material dandelion root, placing the crushed dandelion root into an extraction tank, adding water for extraction with boiling water, removing impurities and concentrating a supernatant, centrifuging the supernatant to remove protein by adopting a Sevag reagent, centrifuging the supernatant, adding absolute ethyl alcohol into the supernatant, uniformly mixing the supernatant and the absolute ethyl alcohol, precipitating the mixture overnight, performing suction filtration to obtain a precipitate, dissolving the precipitate in distilled water again, volatilizing the ethyl alcohol in water bath, and performing freeze drying to obtain crude dandelion root polysaccharide;
(2) separation of crude Dandelion root polysaccharide
Dissolving the crude dandelion root polysaccharide prepared in the step (1) with distilled water, centrifuging, taking a supernatant for sampling, eluting with distilled water, performing gradient elution with NaCl solutions with different concentrations, tracking and detecting the polysaccharide content by adopting a phenol-sulfuric acid method, respectively collecting different elution peaks, performing reduced pressure concentration, dialyzing in a dialysis bag, and performing freeze drying to obtain dandelion root polysaccharide;
(3) sulfated modification of dandelion root polysaccharide
Taking the dandelion root polysaccharide prepared in the step (2), and modifying by adopting a chlorosulfonic acid-dimethylformamide method: accurately weighing dandelion root polysaccharide, adding pyridine, adding chlorosulfonic acid solution under the ice bath condition, stirring at room temperature, gradually melting the mixed solvent to obtain a light yellow clear liquid, adding formamide, heating until the mixed solvent is completely dissolved, adding the mixed solvent into a sulfuric acid derivatization reagent at 0 ℃, stirring and dissolving, reacting in an ice bath to normal temperature, adjusting the pH value to 7-8 with NaOH, dialyzing, taking dialysate, concentrating, and freeze-drying to obtain the dandelion root polysaccharide sulfated modification product.
2. The method for producing a sulfated modified product of dandelion root polysaccharide according to claim 1, wherein the method for producing a sulfated modified product of dandelion root polysaccharide comprises the steps of:
(1) crushing raw material dandelion roots, placing the crushed dandelion roots into an extraction tank, adding water with the volume of 10-30 times of the crushed dandelion roots, extracting the dandelion roots for 3-5 hours in boiling water, repeating the steps for 2-3 times, removing impurities from the supernatant through a filter screen, properly concentrating the supernatant, centrifuging the supernatant by adopting Sevag reagent to remove protein, centrifuging the supernatant, taking the supernatant, adding absolute ethyl alcohol with the volume of 2-4 times of the supernatant, uniformly stirring the mixture, precipitating the mixture overnight, performing suction filtration by using a Buchner funnel and filter paper to obtain a precipitate, dissolving the precipitate again by using distilled water, volatilizing the ethyl alcohol in water bath at 50-60 ℃, and performing freeze-drying by using a freeze dryer to obtain crude dandelion root polysaccharides;
(2) separation of crude Dandelion root polysaccharide
Weighing the crude dandelion root polysaccharide prepared in the step (1), adding distilled water for dissolving, centrifuging at the rotation speed of 12000rpm of 8000-;
(3) sulfated modification of dandelion root polysaccharide
Taking the dandelion root polysaccharide prepared in the step (2), and modifying by adopting a chlorosulfonic acid-dimethylformamide method: accurately weighing dandelion root polysaccharide, adding pyridine, adding chlorosulfonic acid solution under the ice bath condition, stirring for 10-20min at room temperature, gradually melting the mixed solvent to obtain a light yellow clear liquid, adding formamide, heating to completely dissolve, performing ice bath to 0 ℃, adding the mixture into a sulfuric acid derivatization reagent, stirring for dissolving, reacting for 1-2h in the ice bath to normal temperature, adjusting pH to 7-8 with NaOH, dialyzing for 2 days, taking dialysate, concentrating, and freeze-drying to obtain the dandelion root polysaccharide sulfation modification product.
3. The preparation method of the sulfated modified product of dandelion root polysaccharide according to claim 2, wherein 100mg of dandelion root polysaccharide is accurately weighed in step (3), 1mL of pyridine is added, 2mL of chlorosulfonic acid solution is added under ice bath conditions, stirring is carried out for 10-20min at room temperature, after the mixed solvent is gradually melted to become a light yellow clear liquid, 3mL of formamide is added, heating is carried out until the mixed solvent is completely dissolved, the mixed solvent is ice-cooled to 0 ℃, added into a sulfuric acid derivatization reagent, stirred and dissolved, reacted in ice bath for 1-2h to room temperature, NaOH is used for adjusting pH to 7-8, dialysis is carried out for 2 days, and dialysate is taken, concentrated and freeze-dried to obtain the sulfated modified product of dandelion root polysaccharide.
4. The sulfated modified dandelion root polysaccharide product of any one of claims 1 to 3, which is used in health food or pharmaceutical preparations for regulating immunity.
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