CN112250772A - Dendrobium officinale polysaccharide and preparation method thereof - Google Patents
Dendrobium officinale polysaccharide and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a dendrobium candidum polysaccharide and a preparation method thereof, which comprises the root of dendrobium candidum, wherein the dendrobium candidum rhizome is subjected to water extraction and alcohol precipitation to extract crude polysaccharide, separation and purification methods such as repeated freeze thawing, H2O2 decoloration, ultrafiltration, dialysis and the like are combined to respectively obtain two dendrobium candidum polysaccharides DOP-1 and DOP-2 with uniform molecular weight, because the viscosity of the DOP-2 is lower than that of the DOP-1, the relative peak response of 2D nuclear magnetic detection is better, after multiple measurement, the DOP-1 and the DOP-2 are crushed, water with the temperature of about 220 ℃ is added for soaking and heating, when the DOP-1 and the DOP-2 are dispersed, the heating is stopped, residues in the mixture are filtered, concentrated solution is extracted, supernatant fluid floating on the concentrated solution is removed by adopting a water extraction method, then precipitation and drying are carried out at high temperature, finally the dendrobium candidum polysaccharide is obtained, the sugar content of the dendrobium officinale is improved, raw materials are saved in the preparation process, and the reaction rate is effectively accelerated through hydroxyl free radicals and superoxide anions.
Description
Technical Field
The invention belongs to the technical field of dendrobium officinale polysaccharide, and particularly relates to dendrobium officinale polysaccharide and a preparation method thereof.
Background
Dendrobium officinale is slightly cold in nature and sweet and bland in taste. Has effects in nourishing yin, clearing away heat, nourishing stomach, and promoting salivation. Can be used for treating acute and chronic pharyngolaryngitis with yin deficiency syndrome, chronic bronchitis, tuberculosis, hyperthyroidism, diabetes, chronic hepatitis, chronic atrophic gastritis, immune function syndrome and regulating cancer radiotherapy and chemotherapy, and can enhance disease resistance of middle-aged and elderly people.
However, when the sugar in the dendrobium officinale is extracted by the prior art, the efficiency is low, the material loss is easily caused in the extraction process, and the cost is increased. Therefore, the preparation method of the dendrobium officinale polysaccharide needs to be improved, and the dendrobium officinale polysaccharide and the preparation method thereof are provided, so that the problems are better solved.
Disclosure of Invention
The invention aims to: in order to solve the problems, the dendrobium officinale polysaccharide and the preparation method thereof are provided.
The technical scheme adopted by the invention is as follows:
a dendrobium officinale polysaccharide and a preparation method thereof comprise dendrobium officinale roots, and the dendrobium officinale polysaccharide and the preparation method thereof comprise the following steps:
the method comprises the following steps: extracting polysaccharide, extracting crude polysaccharide from the rhizome of the dendrobium officinale by adopting a water extraction and alcohol precipitation method, and respectively obtaining two dendrobium officinale polysaccharides DOP-1 and DOP-2 with uniform molecular weight by combining separation and purification methods such as repeated freeze thawing, H2O2 decoloration, ultrafiltration, dialysis and the like;
step two: substance analysis, wherein sugar contents of DOP-1 and DOP-2 are respectively 99.47% and 97.46% measured by a phenol-sulfuric acid method, molecular weights of the two polysaccharides are respectively 4.929 × 105Da and 3.339 × 104Da measured by a high-efficiency gel permeation chromatography, the two dendrobium officinale polysaccharides are found to be basically free of nucleic acid and protein through ultraviolet spectrum qualitative scanning and protein content quantitative detection, the DOP-1 and the DOP-2 are determined to be free of uronic acid by an m-hydroxybiphenyl method, the total phenol content of the dendrobium officinale polysaccharides is determined by a Folin-Ciocalteu method, and results show that the total phenol content of the DOP-1 and the DOP-2 is respectively 147.25mg/kg and 918.67mg/kg, and the purity identification of the two polysaccharides shows that the components of the DOP-1 and the DOP-2 are uniform and high in purity, so that the requirement for polysaccharide substance structure identification is met;
step three: structural unit determination, monosaccharide composition results show that both polysaccharides contain glucose and mannose, the composition ratio is 1.00:1.38 and 1.00:4.06 respectively, analysis results of a periodate oxidation-Smith degradation method show that the types of terminal-linked or 1.6-linked sugar residues of the two polysaccharides are low, the polysaccharides mainly comprise 1.4-linked or 1.4.6-linked sugar residues which are subjected to oxidative degradation into erythritol, and methylation analysis results show that the main linkage types of DOP-1 are 1, 4-linked glucose and 1.4-linked mannose, and the sugar residue ratio is 1.00: 1.78; DOP-2 is composed mainly of 1.4-linked glucose, 1.4-linked mannose and 1.6-linked glucose, the ratio of sugar residues is 1.90:14.10:1.00, and analysis combining the results of infrared scanning, Smith degradation GC and NMR spectra shows that the acetyl groups of both polysaccharides are attached to the 2-position or 3-position of 1.4-. beta. -D-Manp, and the acetylation ratios of DOP-1 and DOP-2 are about 50% and 30%, respectively. Because DOP-2 has smaller molecular weight, high solubility and lower viscosity compared with DOP-1, the 2D nuclear magnetic detection has better response of related peaks, the structure of the DOP-2 can be further determined by 1D and 2D NMR spectra, and the repeating structural unit of the DOP-2 is estimated;
step four: in-vitro antioxidant activity determination adopts three antioxidant systems, including a DPPH free radical scavenging system, a hydroxyl free radical scavenging system and a superoxide anion free radical scavenging system, and results show that DOP-1 and DOP-2 have higher in-vitro antioxidant capacity for three free radicals, and the hydroxyl free radical and the superoxide anion free radical scavenging capacity of the DOP-2 are higher than that of the DOP-1 and are possibly related to lower molecular weight, higher solubility, higher mannose content, lower acetylation degree and higher polyphenol content. In addition, an oxidation damage cell model is established by selecting H2O2, and the influence of dendrobium officinale polysaccharides DOP-1 and DOP-2 on the survival rate of oxidation damage Hep G2 cells is researched, and the results show that the DOP-1 and the DOP-2 have a certain protection effect on the oxidation damage Hep G2 cells, and the survival rate of the cells reaches over 90% when the concentration is 800 mug/m L. Meanwhile, both DOP-1 and DOP-2 can reduce the intensity of active oxygen in oxidative damage Hep G2 cells.
Step five: the combined preparation is that after the steps are completed, DOP-1 and DOP-2 are crushed, then water with the temperature of about 220 ℃ is added for soaking and heating, when the DOP-1 and DOP-2 are dispersed, the heating is stopped, the residue is filtered, and concentrated solution is extracted;
step six: and D, drying the precipitate, removing the supernatant of the concentrated solution by adopting a water extraction and alcohol precipitation method after the fifth step is finished, and then performing precipitation drying at high temperature to finally obtain the dendrobium officinale polysaccharide.
In a preferred embodiment, the water extraction and alcohol precipitation method refers to a method for refining an aqueous extract after solid-liquid separation by adding ethanol into an aqueous extract concentrated solution of traditional Chinese medicines to reach different alcohol contents, reducing the solubility of certain medicinal ingredients in the alcohol solution to precipitate precipitates, and generally operating the following steps: concentrating the Chinese medicinal water extract to 1: 1-1: 2 (ml: g), cooling the medicinal liquid, slowly adding ethanol under stirring to reach specified alcohol content, sealing, refrigerating for 24-48 h, filtering, and recovering ethanol from the filtrate to obtain refined solution. The following problems should be noted in operation:
the medicine liquid should be properly concentrated to reduce the dosage of ethanol. But the concentration degree should be controlled, if the concentration is too high, the effective components are easy to wrap in the sediment to cause loss;
② after cooling the concentrated liquid medicine, adding ethanol to avoid ethanol volatilization loss by heating;
thirdly, selecting proper alcohol precipitation concentration, removing impurities such as starch and the like when the alcohol content in the general liquid medicine reaches 50-60%, and removing most of the impurities with the alcohol content of more than 75% by precipitation;
stirring is accelerated slowly, the liquid medicine is stirred rapidly, and ethanol is added slowly to avoid the loss of the effective components caused by overhigh local alcohol concentration;
the cold storage is closed, so that the ethanol can be prevented from volatilizing, the precipitation of separated precipitates is promoted, and the filtration operation is convenient;
sixthly, washing the precipitate by using ethanol (the concentration of the ethanol is the same as that of the liquid medicine) to reduce the wrapping loss of the effective components in the precipitate.
In a preferred embodiment, the phenol-sulfuric acid method is to utilize polysaccharide to hydrolyze into monosaccharide under the action of sulfuric acid, and dehydrate rapidly to generate furfural derivatives, then generate orange yellow compounds with phenol, and determine by colorimetry. The optical density value is preferably between 0.1 and 0.3. For example, less than 0.1 may be considered to take 2 g of sample, and 0.2ml of sample solution may be taken, for example, more than 0.3 may be measured by halving 0.1ml of sample solution.
In a preferred embodiment, the DPPH is 1.1-diphenyl-2-trinitrophenylhydrazine, also known as 1.1-diphenyl-2-picrylhydrazino (radical), 1.2-ditalloyl-2-trinitrophenylhydrazine, 1.2-diphenyl-2-picrylhydrazino, is a stable radical, is dark purple prism-like crystalline, has a melting point of 127-129 ℃, a maximum absorption wavelength of 528nm, is an analytical reagent for photometric determination of tocopherol and aliphatic thiols, reducing substances, is also a commonly used polymerization inhibitor, PPH has several different crystal forms, and has differences in lattice symmetry and melting point (m.p.), and commercial powders are mixtures of different crystal phases, and have a melting point around 130 ℃. DPPH-I (m.p.106 ℃) is an orthorhombic system, DPPH-II (m.p.137 ℃) is an amorphous state, DPPH-III (m.p.128-129 ℃) is a triclinic system, DPPH is a very stable nitrogen-centered radical, the stability of DPPH is mainly from the pi-pi co-conjugation of 3 benzene rings and steric hindrance, so that an unpaired electron on the nitrogen atom sandwiched in the middle cannot exert its own electron pairing effect, and DPPH can capture ("scavenge") other radicals as a stable radical, therefore whether a chemical reaction has an intrinsic radical reaction is determined by observing whether the rate of the reaction is slowed down after DPPH is added, and DPPH radicals have strong absorption at the center of 300-400, therefore, the DPPH radical in a solution is dark purple and becomes colorless or pale yellow after being neutralized. By using the characteristic, the reaction process can be visually detected, and the initial free radical quantity can be obtained by recording the change of DPPH at the absorbance value of 520nm or EPR signal.
In a preferred embodiment, the hydroxyl radical is an important active oxygen, and is formed by losing one electron from hydroxyl radical in terms of formula, the hydroxyl radical has extremely strong electron-gaining ability, namely oxidation ability, the oxidation potential is 2.8V, and is an oxidizing agent second to fluorine in nature, when the hydroxyl free radical adopts a three-dimensional electrode method, the excellent water treatment effect can be achieved within a short time, and the determination result of a colorimetric method shows that the stainless steel electrode material generates OH with extremely strong oxidizing capability in the electrocatalytic degradation process, in the electrochemical oxidation process of a bipolar three-dimensional electrolytic cell under the conditions of filling particles and introducing air, utilizes the direct oxidation of the anode, the indirect oxidation of the anode OH and the cathode to generate H2O2, therefore, under the condition of lower energy consumption, the utilization rate of the filling particles is fully improved, and a better degradation effect is achieved.
In a preferred embodiment, the superoxide anion is oxygen molecules in aerobic organisms, which serve as the most important electron acceptor and are reduced into water in the process of material metabolism, wherein O2+4e → 2H2O, the oxygen used in the method accounts for about 95% of the total oxygen consumption of tissues, the rest 5% of the oxygen can form active oxygen with various active properties due to unequal electron accepting number in the reduction process, the product of the oxygen molecules reduced by single electron is called superoxide anion O2+ e → O2, O2 is anion and free radical, is active in property and has strong oxidizing property and reducing property, and is an oxidizing agent and a reducing agent.
In conclusion, due to the adoption of the technical scheme, the dendrobium officinale kimura et migo sugar content improving agent has the beneficial effects that the sugar content of the dendrobium officinale kimura et migo is improved, raw materials are saved in the preparation process, and the reaction rate is effectively accelerated through hydroxyl radicals and superoxide anions.
1. In the invention, hydroxyl free radical is important active oxygen, is formed by losing one electron from hydroxyl radical in terms of molecular formula, has extremely strong electron obtaining capability, namely oxidation capability, oxidation potential of 2.8V, is an oxidant next to fluorine in nature, when the hydroxyl free radical adopts a three-dimensional electrode method, the excellent water treatment effect can be achieved within a short time, and the determination result of a colorimetric method shows that the stainless steel electrode material generates OH with extremely strong oxidizing capability in the electrocatalytic degradation process, in the electrochemical oxidation process of a bipolar three-dimensional electrolytic cell under the conditions of filling particles and introducing air, utilizes the direct oxidation of the anode, the indirect oxidation of the anode OH and the cathode to generate H2O2, therefore, under the condition of lower energy consumption, the utilization rate of the filling particles is fully improved, and a better degradation effect is achieved.
2. In the invention, superoxide anion is oxygen molecules in aerobic organisms and is used as the most important electron acceptor to be reduced into water in the process of material metabolism, O2+4e → 2H2O, the oxygen utilized in the method accounts for about 95 percent of the total oxygen consumption of tissues, the rest 5 percent of oxygen can form active oxygen with various active properties due to unequal electron accepting number in the reduction process, the product of oxygen molecules reduced by single electron is called superoxide anion O2+ e → O2, O2 is anion and free radical, the product has active properties and strong oxidizability and reducibility, is an oxidant and a reducer, the oxidation rate is effectively improved, and raw materials are saved.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
A Dendrobium officinale polysaccharide and a preparation method thereof, comprising the root of Dendrobium officinale, the Dendrobium officinale polysaccharide and the preparation method thereof comprise the following steps:
the method comprises the following steps: extracting polysaccharide, extracting crude polysaccharide from the rhizome of the dendrobium officinale by adopting a water extraction and alcohol precipitation method, and respectively obtaining two dendrobium officinale polysaccharides DOP-1 and DOP-2 with uniform molecular weight by combining separation and purification methods such as repeated freeze thawing, H2O2 decoloration, ultrafiltration, dialysis and the like;
step two: substance analysis, wherein sugar contents of DOP-1 and DOP-2 are respectively 99.47% and 97.46% measured by a phenol-sulfuric acid method, molecular weights of the two polysaccharides are respectively 4.929 × 105Da and 3.339 × 104Da measured by a high-efficiency gel permeation chromatography, the two dendrobium officinale polysaccharides are found to be basically free of nucleic acid and protein through ultraviolet spectrum qualitative scanning and protein content quantitative detection, the DOP-1 and the DOP-2 are determined to be free of uronic acid by an m-hydroxybiphenyl method, the total phenol content of the dendrobium officinale polysaccharides is determined by a Folin-Ciocalteu method, and results show that the total phenol content of the DOP-1 and the DOP-2 is respectively 147.25mg/kg and 918.67mg/kg, and the purity identification of the two polysaccharides shows that the components of the DOP-1 and the DOP-2 are uniform and high in purity, so that the requirement for polysaccharide substance structure identification is met;
step three: structural unit determination, monosaccharide composition results show that both polysaccharides contain glucose and mannose, the composition ratio is 1.00:1.38 and 1.00:4.06 respectively, analysis results of a periodate oxidation-Smith degradation method show that the types of terminal-linked or 1.6-linked sugar residues of the two polysaccharides are low, the polysaccharides mainly comprise 1.4-linked or 1.4.6-linked sugar residues which are subjected to oxidative degradation into erythritol, and methylation analysis results show that the main linkage types of DOP-1 are 1, 4-linked glucose and 1.4-linked mannose, and the sugar residue ratio is 1.00: 1.78; DOP-2 is composed mainly of 1.4-linked glucose, 1.4-linked mannose and 1.6-linked glucose, the ratio of sugar residues is 1.90:14.10:1.00, and analysis combining the results of infrared scanning, Smith degradation GC and NMR spectra shows that the acetyl groups of both polysaccharides are attached to the 2-position or 3-position of 1.4-. beta. -D-Manp, and the acetylation ratios of DOP-1 and DOP-2 are about 50% and 30%, respectively. Because DOP-2 has smaller molecular weight, high solubility and lower viscosity compared with DOP-1, the 2D nuclear magnetic detection has better response of related peaks, the structure of the DOP-2 can be further determined by 1D and 2D NMR spectra, and the repeating structural unit of the DOP-2 is estimated;
step four: in-vitro antioxidant activity determination adopts three antioxidant systems, including a DPPH free radical scavenging system, a hydroxyl free radical scavenging system and a superoxide anion free radical scavenging system, and results show that DOP-1 and DOP-2 have higher in-vitro antioxidant capacity for three free radicals, and the hydroxyl free radical and the superoxide anion free radical scavenging capacity of the DOP-2 are higher than that of the DOP-1 and are possibly related to lower molecular weight, higher solubility, higher mannose content, lower acetylation degree and higher polyphenol content. In addition, an oxidation damage cell model is established by selecting H2O2, and the influence of dendrobium officinale polysaccharides DOP-1 and DOP-2 on the survival rate of oxidation damage Hep G2 cells is researched, and the results show that the DOP-1 and the DOP-2 have a certain protection effect on the oxidation damage Hep G2 cells, and the survival rate of the cells reaches over 90% when the concentration is 800 mug/m L. Meanwhile, both DOP-1 and DOP-2 can reduce the intensity of active oxygen in oxidative damage Hep G2 cells.
Step five: the combined preparation is that after the steps are completed, DOP-1 and DOP-2 are crushed, then water with the temperature of about 220 ℃ is added for soaking and heating, when the DOP-1 and DOP-2 are dispersed, the heating is stopped, the residue is filtered, and concentrated solution is extracted;
step six: and D, drying the precipitate, removing the supernatant of the concentrated solution by adopting a water extraction and alcohol precipitation method after the fifth step is finished, and then performing precipitation drying at high temperature to finally obtain the dendrobium officinale polysaccharide.
The water extraction and alcohol precipitation method is a method for refining water extract after solid-liquid separation by adding ethanol into the water extract concentrated solution of traditional Chinese medicine to reach different alcohol contents, reducing the solubility of some medicine components in the alcohol solution to precipitate out precipitates, and the general operation process is as follows: concentrating the Chinese medicinal water extract to 1: 1-1: 2 (ml: g), cooling the medicinal liquid, slowly adding ethanol under stirring to reach specified alcohol content, sealing, refrigerating for 24-48 h, filtering, and recovering ethanol from the filtrate to obtain refined solution. The following problems should be noted in operation:
the medicine liquid should be properly concentrated to reduce the dosage of ethanol. But the concentration degree should be controlled, if the concentration is too high, the effective components are easy to wrap in the sediment to cause loss;
② after cooling the concentrated liquid medicine, adding ethanol to avoid ethanol volatilization loss by heating;
thirdly, selecting proper alcohol precipitation concentration, removing impurities such as starch and the like when the alcohol content in the general liquid medicine reaches 50-60%, and removing most of the impurities with the alcohol content of more than 75% by precipitation;
stirring is accelerated slowly, the liquid medicine is stirred rapidly, and ethanol is added slowly to avoid the loss of the effective components caused by overhigh local alcohol concentration;
the cold storage is closed, so that the ethanol can be prevented from volatilizing, the precipitation of separated precipitates is promoted, and the filtration operation is convenient;
sixthly, washing the precipitate by using ethanol (the concentration of the ethanol is the same as that of the liquid medicine) to reduce the wrapping loss of the effective components in the precipitate.
The phenol-sulfuric acid method is characterized in that polysaccharide is firstly hydrolyzed into monosaccharide under the action of sulfuric acid, the monosaccharide and the monosaccharide are quickly dehydrated to generate a furfural derivative, the furfural derivative and phenol are then generated into an orange-yellow compound, and the orange-yellow compound is measured by a colorimetric method. The optical density value is preferably between 0.1 and 0.3. For example, less than 0.1 may be considered to take 2 g of sample, and 0.2ml of sample solution may be taken, for example, more than 0.3 may be measured by halving 0.1ml of sample solution.
DPPH is 1.1-diphenyl-2-trinitrophenylhydrazine, namely 1.1-diphenyl-2-picrylhydrazino (free radical), 1.2-biternary-2-trinitrophenylhydrazine and 1.2-diphenyl-2-picrylhydrazino, is a stable free radical, is dark purple prism-shaped crystal, has a melting point of 127-129 ℃, has a maximum absorption wavelength of 528nm, can be used for photometrically measuring analytical reagents of tocopherol, aliphatic thiols and reducing substances, is also a common polymerization inhibitor, PPH has several different crystal forms, has different lattice symmetries and melting points (m.p.), and is a mixture of different crystal phases, and the melting point is near 130 ℃. DPPH-I (m.p.106 ℃) is an orthorhombic system, DPPH-II (m.p.137 ℃) is an amorphous state, DPPH-III (m.p.128-129 ℃) is a triclinic system, DPPH is a very stable nitrogen-centered radical, the stability of DPPH is mainly from the pi-pi co-conjugation of 3 benzene rings and steric hindrance, so that an unpaired electron on the nitrogen atom sandwiched in the middle cannot exert its own electron pairing effect, and DPPH can capture ("scavenge") other radicals as a stable radical, therefore whether a chemical reaction has an intrinsic radical reaction is determined by observing whether the rate of the reaction is slowed down after DPPH is added, and DPPH radicals have strong absorption at the center of 300-400, therefore, the DPPH radical in a solution is dark purple and becomes colorless or pale yellow after being neutralized. By using the characteristic, the reaction process can be visually detected, and the initial free radical quantity can be obtained by recording the change of DPPH at the absorbance value of 520nm or EPR signal.
The hydroxyl radical is important active oxygen, is formed by losing one electron from a hydroxyl radical in terms of molecular formula, has extremely strong electron obtaining capability, namely oxidation capability, has an oxidation potential of 2.8V, and is an oxidant second to fluorine in nature, and can achieve excellent water treatment effect in a short time when a three-dimensional electrode method is adopted, and the determination result of a colorimetric method shows that the stainless steel electrode material generates OH with extremely strong oxidation capability in the electrocatalytic degradation process, and the direct oxidation of an anode, the indirect oxidation of H2O2 generated by the anode OH and a cathode in the electrochemical oxidation process of filling particles and introducing air in a bipolar three-dimensional electrolytic cell, so that the utilization rate of the filling energy consumption particles is fully improved and better degradation effect is achieved under the condition of lower energy consumption.
The superoxide anion is oxygen molecule in aerobic organism as the most important electron acceptor to be reduced into water in the process of material metabolism, O2+4e → 2H2O, the oxygen used in the process accounts for about 95% of the total oxygen consumption of the tissue, the rest 5% of oxygen can form active oxygen with various active properties due to the unequal number of the accepted electrons in the reduction process, the product of oxygen molecule reduced by single electron is called superoxide anion O2+ e → O2, O2 is anion and free radical, and has active properties, strong oxidizability and reducibility, and is an oxidizing agent and a reducing agent.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (6)
1. A dendrobium officinale polysaccharide and a preparation method thereof, comprising dendrobium officinale roots, and is characterized in that: the dendrobium officinale polysaccharide and the preparation method thereof comprise the following steps:
the method comprises the following steps: extracting polysaccharide, extracting crude polysaccharide from the rhizome of the dendrobium officinale by adopting a water extraction and alcohol precipitation method, and respectively obtaining two dendrobium officinale polysaccharides DOP-1 and DOP-2 with uniform molecular weight by combining separation and purification methods such as repeated freeze thawing, H2O2 decoloration, ultrafiltration, dialysis and the like;
step two: substance analysis, wherein sugar contents of DOP-1 and DOP-2 are respectively 99.47% and 97.46% measured by a phenol-sulfuric acid method, molecular weights of the two polysaccharides are respectively 4.929 × 105Da and 3.339 × 104Da measured by a high-efficiency gel permeation chromatography, the two dendrobium officinale polysaccharides are found to be basically free of nucleic acid and protein through ultraviolet spectrum qualitative scanning and protein content quantitative detection, the DOP-1 and the DOP-2 are determined to be free of uronic acid by an m-hydroxybiphenyl method, the total phenol content of the dendrobium officinale polysaccharides is determined by a Folin-Ciocalteu method, and results show that the total phenol content of the DOP-1 and the DOP-2 is respectively 147.25mg/kg and 918.67mg/kg, and the purity identification of the two polysaccharides shows that the components of the DOP-1 and the DOP-2 are uniform and high in purity, so that the requirement for polysaccharide substance structure identification is met;
step three: structural unit determination, monosaccharide composition results show that both polysaccharides contain glucose and mannose, the composition ratio is 1.00:1.38 and 1.00:4.06 respectively, analysis results of a periodate oxidation-Smith degradation method show that the types of terminal-linked or 1.6-linked sugar residues of the two polysaccharides are low, the polysaccharides mainly comprise 1.4-linked or 1.4.6-linked sugar residues which are subjected to oxidative degradation into erythritol, and methylation analysis results show that the main linkage types of DOP-1 are 1, 4-linked glucose and 1.4-linked mannose, and the sugar residue ratio is 1.00: 1.78; DOP-2 mainly comprises 1.4-linked glucose, 1.4-linked mannose and 1.6-linked glucose, the proportion of sugar residues is 1.90:14.10:1.00, analysis is carried out by combining an infrared scanning spectrum, a Smith degradation GC result and an NMR spectrum result, the acetyl groups of the two polysaccharides are connected at the 2 position or the 3 position of 1.4-beta-D-Manp, the acetylation proportion of the DOP-1 and the DOP-2 is respectively about 50 percent and 30 percent, and as the molecular weight of the DOP-2 is smaller, the solubility is high, the viscosity is lower than that of the DOP-1, the 2D nuclear magnetic detection related peak response is better, the structure of the DOP-2 can be further determined by the 1D and 2D NMR spectrums, and the repeated structural unit of the DOP-2 is estimated;
step four: in vitro antioxidant activity determination, three antioxidant systems are adopted for in vitro antioxidant activity determination, including a DPPH free radical scavenging system, a hydroxyl free radical scavenging system and a superoxide anion free radical scavenging system, results show that DOP-1 and DOP-2 have higher in vitro antioxidant capacity for three free radicals, the hydroxyl free radical and the superoxide anion free radical scavenging capacity of DOP-2 are higher than that of DOP-1 and are probably related to lower molecular weight, higher solubility, higher mannose content, lower acetylation degree and higher polyphenol content, in addition, H2O2 is selected to establish an oxidative damage cell model to research the influence of dendrobium candidum polysaccharides DOP-1 and DOP-2 on the survival rate of oxidative damage Hep G2 cells, and results show that DOP-1 and DOP-2 have certain protective effect on oxidative damage Hep G2 cells, when the concentration is 800 mug/m L, the cell survival rate reaches more than 90%, and meanwhile, DOP-1 and DOP-2 can reduce the strength of active oxygen in Hep G2 cells damaged by oxidation;
step five: the combined preparation is that after the steps are completed, DOP-1 and DOP-2 are crushed, then water with the temperature of about 220 ℃ is added for soaking and heating, when the DOP-1 and DOP-2 are dispersed, the heating is stopped, the residue is filtered, and concentrated solution is extracted;
step six: and D, drying the precipitate, removing the supernatant of the concentrated solution by adopting a water extraction and alcohol precipitation method after the fifth step is finished, and then performing precipitation drying at high temperature to finally obtain the dendrobium officinale polysaccharide.
2. The dendrobium officinale polysaccharide and the preparation method thereof of claim 1, wherein the dendrobium officinale polysaccharide comprises the following components in percentage by weight: the water extraction and alcohol precipitation method is a method for refining a water extract after solid-liquid separation by adding ethanol into a water extraction concentrated solution of a traditional Chinese medicine to reach different alcohol contents, reducing the solubility of certain medicinal components in the alcohol solution to precipitate out precipitates, and generally comprising the following operation processes: concentrating the Chinese medicinal water extract to 1: 1-1: 2 (ml: g), cooling the liquid medicine, slowly adding ethanol while stirring to reach the specified alcohol content, refrigerating for 24-48 h in a sealed manner, filtering, and recovering ethanol from the filtrate to obtain a refined liquid, wherein the following problems should be taken during operation:
firstly, the liquid medicine should be properly concentrated to reduce the dosage of ethanol, but the concentration degree should be controlled, if the concentration is too high, the effective components are easy to wrap in the sediment to cause loss;
② after cooling the concentrated liquid medicine, adding ethanol to avoid ethanol volatilization loss by heating;
thirdly, selecting proper alcohol precipitation concentration, removing impurities such as starch and the like when the alcohol content in the general liquid medicine reaches 50-60%, and removing most of the impurities with the alcohol content of more than 75% by precipitation;
stirring is accelerated slowly, the liquid medicine is stirred rapidly, and ethanol is added slowly to avoid the loss of the effective components caused by overhigh local alcohol concentration;
the cold storage is closed, so that the ethanol can be prevented from volatilizing, the precipitation of separated precipitates is promoted, and the filtration operation is convenient;
sixthly, washing the precipitate by using ethanol (the concentration of the ethanol is the same as that of the liquid medicine) to reduce the wrapping loss of the effective components in the precipitate.
3. The dendrobium officinale polysaccharide and the preparation method thereof of claim 1, wherein the dendrobium officinale polysaccharide comprises the following components in percentage by weight: the phenol-sulfuric acid method is characterized in that polysaccharide is firstly hydrolyzed into monosaccharide under the action of sulfuric acid, the monosaccharide is quickly dehydrated to generate a furfural derivative, the furfural derivative and phenol generate an orange yellow compound, and then the orange yellow compound and phenol are measured by a colorimetric method, the method is simple, quick, sensitive and good in repeatability, only one standard curve is made for each kind of sugar, the color is durable, the sampling amount is determined according to the optical density value during measurement, the optical density value is preferably between 0.1 and 0.3, for example, 2 grams can be taken when the sample is considered to be taken under 0.1, 0.2ml of sample liquid is still taken, and 0.1ml of sample liquid can be half-subtracted to be taken for measurement when the optical density value is more than 0.3.
4. The dendrobium officinale polysaccharide and the preparation method thereof of claim 1, wherein the dendrobium officinale polysaccharide comprises the following components in percentage by weight: the DPPH is 1.1-diphenyl-2-trinitrophenylhydrazine, is also named as 1.1-diphenyl-2-picrylhydrazino (free radical), 1.2-bitertanyl-2-trinitrophenylhydrazine and 1.2-diphenyl-2-picrylhydrazino, is a stable free radical, is dark purple prism-shaped crystal, has a melting point of 127-129 ℃, has a maximum absorption wavelength of 528nm, can be used for photometrically measuring analytical reagents of tocopherol, aliphatic thiols and reducing substances, and is also a common polymerization inhibitor, PPH has several different crystal forms, has differences in lattice symmetry and melting point (m.p.), is a mixture of different crystal phases, has a melting point of about 130 ℃, is an orthorhombic system at DPPH-I (m.p.106 ℃), is amorphous at DPPH-II (m.p.137 ℃), is a triclinic system at DPPH-III (m.p.128-129 ℃), DPPH is a very stable nitrogen-centered radical, the stability of which is mainly due to the pi-pi co-conjugation of 3 benzene rings and steric hindrance, so that the unpaired electron on the nitrogen atom sandwiched in the middle cannot exert the corresponding electron pairing effect, and DPPH can capture (scavenge) other radicals as a stable radical, thus, whether a chemical reaction slows down after the addition of DPPH is an indicator of whether the reaction is of the nature of a free radical reaction, the DPPH free radical is strongly absorbed in the center of 300-400, so that the DPPH free radical is dark purple in the solution, and becomes colorless or pale yellow after being neutralized, the progress of the reaction can be visually detected by utilizing the characteristic, the amount of initial free radicals can be obtained by recording the change in DPPH at an absorbance value or EPR signal at 520 nm.
5. The dendrobium officinale polysaccharide and the preparation method thereof of claim 1, wherein the dendrobium officinale polysaccharide comprises the following components in percentage by weight: the hydroxyl radical is important active oxygen and is formed by losing one electron from a hydroxyl radical in terms of molecular formula, the hydroxyl radical has extremely strong electron obtaining capacity, namely oxidation capacity, the oxidation potential is 2.8V, and is an oxidant second to fluorine in nature, when the hydroxyl radical adopts a three-dimensional electrode method, the excellent water treatment effect can be achieved within a short time, and the determination result of a colorimetric method shows that the stainless steel electrode material generates OH with extremely strong oxidation capacity in the electrocatalytic degradation process, and in the electrochemical oxidation process under the conditions of filling particles and introducing air in a bipolar three-dimensional electrolytic cell, the direct oxidation of an anode, the indirect oxidation of H2O2 generated by the anode OH and a cathode are utilized, so that the utilization rate of the filling particles is fully improved under the condition of lower energy consumption, and the better degradation effect is achieved.
6. The dendrobium officinale polysaccharide and the preparation method thereof of claim 1, wherein the dendrobium officinale polysaccharide comprises the following components in percentage by weight: the superoxide anion is O2+4e → 2H2O, the oxygen used in the process accounts for about 95% of the total oxygen consumption of the tissue, the rest 5% of the oxygen can form active oxygen with various active properties due to unequal electron accepting number in the reduction process, the product of the oxygen molecule reduced by single electron is called superoxide anion O2+ e → O2, O2 is anion and free radical, and has active properties, strong oxidizability and reducibility, and is an oxidizing agent and a reducing agent.
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