CN113880960B - Anti-hypoxia active dendrobium officinale polysaccharide and steam explosion preparation method and application thereof - Google Patents
Anti-hypoxia active dendrobium officinale polysaccharide and steam explosion preparation method and application thereof Download PDFInfo
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- CN113880960B CN113880960B CN202111253620.2A CN202111253620A CN113880960B CN 113880960 B CN113880960 B CN 113880960B CN 202111253620 A CN202111253620 A CN 202111253620A CN 113880960 B CN113880960 B CN 113880960B
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- polysaccharide
- dendrobium officinale
- steam explosion
- hypoxia
- acute
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Abstract
The invention provides an anti-hypoxia active dendrobium officinale polysaccharide and a steam explosion preparation method and application thereof, belonging to the technical field of medicines. The method comprises the steps of subjecting the dendrobium officinale raw material to steam explosion treatment with specific parameters, performing water extraction and alcohol precipitation extraction, removing protein and micromolecular impurities, further purifying by using DEAE-Cellulose anion exchange column chromatography and sephadex column chromatography, and collecting the micromolecular (6.2-8.4 kDa) polysaccharide. The method degrades macromolecular polysaccharide in Dendrobium officinale into micromolecular polysaccharide under steam explosion effect, changes chemical groups and monosaccharide chemical compositions of polysaccharide, and improves uronic acid content in polysaccharide component with anti-anoxia activity, so that the prepared polysaccharide has activity of remarkably improving acute anoxia tolerance of organism, and can be used for preparing medicines or functional foods for resisting acute anoxia injury.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to an anti-hypoxia active dendrobium officinale polysaccharide, and a steam explosion preparation method and application thereof.
Background
Acute altitude sickness (AMS) refers to various clinical syndromes occurring within hours to days when entering a plateau from a plain or entering a higher altitude area from a plateau, including acute mild altitude sickness, high altitude pulmonary edema and high altitude cerebral edema, high altitude acute hypoxia being a major cause of AMS.
China is the world with the largest plateau area and the largest plateau population. The areas not only have rich energy, mineral resources, medicinal materials and animal husbandry resources, but also are important national defense sentinels and minority nationality habitats. With implementation of major development strategy in western regions and opening of Qinghai-Tibet railways, the highland tourism industry is increasingly prosperous, and according to data published by the Tibet autonomous region statistical office, the number of 4000 thousands of visitors who take care of tourists at home and abroad in the whole region in 2019 is increased by 19.1% compared with the number of visitors in the same period in the last year, and the Tibetan visitors generally face the problem of plateau compensation. Meanwhile, in the process of rapidly advancing to the plateau, high AMS (automatic maintenance system) is also a main factor which causes a large number of non-combat personnel reduction and seriously influences the fighting capacity of troops. In addition, in recent years, in the rescue operation of plateau natural disasters such as a cajuput earthquake, a navicular debris flow and the like, due to the lack of knowledge on AMS prevention and control knowledge and factors such as fatigue, cold, coldness and the like, a large number of AMS patients appear in rescuers, and the AMS patients become one of the biggest difficulties in the initial stage of disaster relief. AMS has become a more and more serious public health problem in the plateau areas of China, and brings great burden to the development of economy, medical treatment, health and various careers in the plateau areas. Therefore, the active measures for preventing and treating the injury caused by the acute hypoxia of the organism have important significance for ensuring the health of the human body.
At present, the anti-acute hypoxia drugs are mainly divided into chemical drugs and Chinese patent drugs, wherein the chemical drugs such as acetazolamide, dexamethasone, amifostine, nilestriol and the like have better anti-hypoxia effect but are limited due to obvious side effect; the anti-hypoxia Chinese patent medicines including ginseng, rhodiola rosea, cordyceps sinensis and the like can effectively improve the hypoxia tolerance of the organism, but are difficult to popularize and use in a large range due to limited resources and high price. Therefore, there is a need to develop a pharmaceutical or functional food with definite efficacy, high efficiency and economy against acute hypoxic injury.
Dendrobium officinale is a fresh or dry stem of an orchid plant, is the first of the "Jiudodan Mesona chinensis Benth", is listed as a new resource for developing health-care food by the "Chinese pharmacopoeia", and is also listed as a medicine and food homologous substance management test point list by the national Weijian Commission. The herba Dendrobii has effects of benefiting stomach, promoting fluid production, nourishing yin, clearing heat, relieving fatigue, eliminating phlegm, relieving cough, etc. The polysaccharide is used as a main active ingredient of the dendrobium officinale, and with the wide development of the efficacy research of the dendrobium officinale, the pharmacological activity and action mechanism research results of the dendrobium officinale polysaccharide are rapidly increased, however, no report on the anti-hypoxia aspect of the dendrobium officinale polysaccharide is found at present, and no report on how to extract the anti-hypoxia polysaccharide is found.
Disclosure of Invention
In view of the above, the invention aims to provide a dendrobium officinale polysaccharide with anti-hypoxia activity, and a steam explosion preparation method and application thereof.
The invention provides a method for preparing anti-hypoxia active dendrobium officinale polysaccharide by steam explosion, which comprises the following steps:
1) Performing steam explosion treatment on the dendrobium officinale to obtain pretreated dendrobium officinale;
the conditions of the steam explosion treatment were as follows:
the charging coefficient of the blasting cavity is 0.7-0.9, the blasting steam pressure is 0.8-1.5 MPa, and the maintaining time is 60-180 s;
2) Refining the pretreated dendrobium officinale in the step 1), then carrying out reflux extraction by using water, precipitating the obtained leaching solution by using an ethanol solution, and collecting precipitates to obtain crude polysaccharide;
3) Dissolving the crude polysaccharide obtained in the step 2) in water, removing protein, dialyzing with a dialysis bag with molecular cut-off of 2000Da, and concentrating the polysaccharide solution in the dialysis bag;
4) Redissolving the polysaccharide solution concentrated in the step 3), loading the redissolved polysaccharide solution into a DEAE-Cellulose anion exchange column, continuously and linearly eluting by using 0-2 mol/L NaCl solution, detecting the polysaccharide content in each tube of eluent, drawing an elution curve, merging flow parts according to the peak condition on the elution curve, and collecting the polysaccharide with the weight-average molecular weight of 6.2-8.4 kDa as the anti-hypoxia active dendrobium officinale polysaccharide.
Preferably, the conditions of the steam explosion treatment are as follows:
the charging coefficient of the blasting cavity is 0.78, the blasting steam pressure is 1.0-1.3 MPa, and the pressure maintaining time is 80-160 s.
Preferably, the temperature of the reflux extraction in the step 2) is 70-100 ℃, the time of the reflux extraction is 2-3 h, and the times of the reflux extraction are 2-3 times;
the mass percentage of the ethanol solution is 70-100%.
Preferably, the method for removing protein in the step 3) is to use Sevage reagent to mix with the obtained crude polysaccharide solution in a vortex mode to remove protein precipitate;
the Sevage reagent is prepared by mixing chloroform and n-butanol according to the weight ratio of (3.5-4.5): 1 by volume ratio;
the volume ratio of the obtained crude polysaccharide solution to Sevage reagent is 1 (0.5-2), and the vortex mixing time is 5-10 min.
Preferably, the flow rate of elution in step 4) is 0.5 to 0.8mL/min.
Preferably, after combining the fractions in step 4), purifying the combined fractions;
the purification method adopts Sephadex G-100 Sephadex column chromatography to separate each flow component, and uses deionized water to elute, so as to obtain the refined polysaccharide.
The invention provides the anti-hypoxia active dendrobium candidum polysaccharide prepared by the method, which comprises mannose, glucose, arabinose, galacturonic acid and glucuronic acid, and the content of total glucuronic acid is more than 3wt%; the purity of the polysaccharide is 90wt% -95 wt%,
the molar ratio of the mannose to the glucose to the arabinose to the galacturonic acid to the glucuronic acid is (5.5-5.7), (2.1-2.15), (1.8-1.9), (0.26-0.28) and (0.08-0.1).
The invention provides application of the anti-hypoxia active dendrobium officinale polysaccharide in preparation of a medicine for resisting acute hypoxia injury.
Preferably, the acute hypoxic injury condition comprises acute altitude sickness;
the acute altitude disease comprises one or more of the following: acute mild altitude disease, high altitude pulmonary edema, high altitude cerebral edema.
The invention provides application of the anti-hypoxia active dendrobium officinale polysaccharide in functional food for relieving acute hypoxia injury.
The invention provides a method for preparing anti-hypoxia active dendrobium officinale polysaccharide by steam explosion, which comprises the steps of subjecting a dendrobium officinale raw material to steam explosion treatment with specific parameters, extracting with water and precipitating with ethanol, removing protein and micromolecular impurities, further separating polysaccharide by using a DEAE-Cellulose anion exchange column, and collecting the small molecular weight (6.2-8.4 kDa) polysaccharide which is the anti-hypoxia active dendrobium officinale polysaccharide. Experiments prove that compared with a treatment scheme without steam explosion treatment or with too high or too low explosion treatment degree, the treatment scheme of steam explosion treatment with specific parameters is favorable for obviously improving the anti-acute hypoxia activity of the polysaccharide. Meanwhile, structural analysis further shows that compared with the polysaccharide in the dendrobium officinale without steam explosion treatment, the polysaccharide microstructure of the dendrobium officinale with steam explosion treatment can be obviously changed, so that the low-molecular-weight polysaccharide with new conformation is obtained, the weight-average molecular weight is 6.2-8.4 kDa, the polysaccharide consists of mannose, glucose, arabinose, a small amount of galacturonic acid and glucuronic acid, the total content of the glucuronic acid is more than 3 percent (g/g), and experiments prove that the polysaccharide is the most important anti-hypoxia active polysaccharide. Wherein the uronic acid content of the polysaccharide fraction having anti-hypoxia activity is significantly increased. The extracted dendrobium officinale polysaccharide has remarkable acute hypoxia resisting activity, comprises the steps of remarkably improving survival time of mice in a normal pressure hypoxia resisting experiment and a sodium nitrite poisoning experiment, reduces dPC12 cell damage caused by acute hypoxia, has no obvious toxic or side effect, and can be used for preparing medicines or functional foods for resisting acute hypoxia injury.
Meanwhile, after the dendrobium officinale raw material is treated by the steam explosion technology, the dendrobium officinale polysaccharide can be extracted quickly and efficiently, and the polysaccharide yield is improved by more than 60%.
Drawings
FIG. 1 shows the survival time of mice in each group in the normal pressure hypoxia tolerance (A) and sodium nitrite poisoning (B) experiments (marked difference compared with the blank control group, p is less than 0.05);
FIG. 2 is electron microscope scan of polysaccharides ((1) S-DOP1, (1) S-DOP2, (2) S-DOP1, (2) S-DOP2 and (5) DOP) of Dendrobium officinale before and after steam explosion, wherein FIG. 2A: (1) S-DOP1 polysaccharide; FIG. 2B: (1) S-DOP2 polysaccharide; FIG. 2C: (2) S-DOP1 polysaccharide; FIG. 2D: (2) S-DOP2 polysaccharide; FIG. 2E: (5) a DOP polysaccharide;
FIG. 3 is an infrared spectrum of a polysaccharide ((1) S-DOP1, (1) S-DOP2, (2) S-DOP1, (2) S-DOP2 and (5) DOP) of Dendrobium officinale before and after steam explosion;
FIG. 4 is an HPLC-GPC chromatogram of antihypoxic active polysaccharides (1), S-DOP1 (A) and (2) and S-DOP1 (B);
FIG. 5 is a monosaccharide composition HPLC analysis of antihypoxic active polysaccharide (1) S-DOP1, wherein FIG. 5A: a standard substance; FIG. 5B: (1) S-DOP1 polysaccharide.
Detailed Description
The invention provides a method for preparing anti-hypoxia active dendrobium officinale polysaccharide by steam explosion, which comprises the following steps:
1) Performing steam explosion treatment on the dendrobium officinale to obtain pretreated dendrobium officinale;
the conditions of the steam explosion treatment were as follows:
the charging coefficient of the blasting cavity is 0.7-0.9, the blasting steam pressure is 0.8-1.5 MPa, and the maintaining time is 60-180 s;
2) Refining the pretreated dendrobium officinale in the step 1), then carrying out reflux extraction by using water, precipitating the obtained leaching solution by using an ethanol solution, and collecting precipitates to obtain crude polysaccharide;
3) Dissolving the crude polysaccharide obtained in the step 2) in water, removing protein, dialyzing by using a dialysis bag with molecular cut-off of 2000Da, and concentrating the polysaccharide solution in the dialysis bag;
4) Redissolving the polysaccharide solution concentrated in the step 3), loading the redissolved solution on a DEAE-Cellulose anion exchange column, continuously and linearly eluting by using 0-2 mol/L NaCl solution, detecting the polysaccharide content in each tube of eluent, drawing an elution curve, merging fractions, and collecting polysaccharide with the weight-average molecular weight of 6.2-8.4 kDa as the anti-hypoxia active dendrobium officinale polysaccharide.
The invention obtains the pretreated dendrobium officinale by performing steam explosion treatment on the dendrobium officinale.
In the invention, before the steam explosion treatment, the dendrobium officinale is preferably dried. The conditions of the steam explosion treatment are preferably as follows: the charging coefficient of the blasting cavity is 0.78, the pressure of the blasting steam is 1.0-1.3 MPa, and the pressure maintaining time is 80-160 s. The steam explosion treatment is favorable for causing the cell walls of the raw materials to be broken to form multiple pores, greatly improves the accessibility of enzyme and chemical extracting agent in the materials, increases the dissolution rate of target substances from cells, and realizes the high-efficiency extraction of polysaccharide. Meanwhile, the high temperature and the high pressure in the steam explosion treatment process can also cause the synergistic effects like acid hydrolysis, thermal degradation, similar mechanical fracture, hydrogen bond damage, structural rearrangement and the like in the materials, so as to cause the degradation or reconstruction of the chemical components of the dendrobium officinale. The macromolecular polysaccharide is degraded into micromolecular polysaccharide under the action of steam explosion, and the chemical groups of the polysaccharide and the chemical compositions of monosaccharide are changed. Compared with the polysaccharide in the dendrobium officinale without steam explosion treatment, the polysaccharide microstructure of the dendrobium officinale with steam explosion treatment can be obviously changed, and the uronic acid content in the polysaccharide component exerting the anti-hypoxia activity is obviously increased.
After the pretreated dendrobium officinale is obtained, the pretreated dendrobium officinale is refined and then is extracted by pure water under reflux, the obtained leaching liquor is precipitated by ethanol solution, and the precipitate is collected to obtain crude polysaccharide.
In the present invention, the method of the refining is preferably pulverization. The particle size of the crushed dendrobium officinale is preferably not higher than 60 meshes, and more preferably 70-100 meshes. The pulverization is favorable for improving the extraction efficiency of the polysaccharide.
In the invention, during reflux extraction, the mass ratio of the dendrobium officinale powder to water is preferably 1. The temperature for reflux extraction is preferably 70 to 100 deg.C, more preferably 80 to 100 deg.C. The time for reflux extraction is preferably 2 to 3 hours, more preferably 2.5 hours. The number of reflux extractions is preferably 2. After reflux extraction, decompression concentration is carried out to 1/2-1/5, and centrifugation is carried out to remove sediment to obtain leaching liquor. The rotating speed of the centrifugal sediment removal is preferably 6000 to 8000rpm, and more preferably 7000rpm. The centrifugation time is preferably 5 to 15min, more preferably 10min.
In the invention, the mass percentage content of the ethanol solution is preferably 70-100%. The solvent in the ethanol solution is preferably water. The time for precipitating the polysaccharide by the ethanol solution is preferably 12 to 24 hours, more preferably 15 to 20 hours, and most preferably 18 hours. The addition amount of the ethanol solution is preferably 3-5 times of the volume of the leaching solution. The ethanol solution is preferably precooled ethanol solution.
After obtaining the crude polysaccharide, the invention dissolves the crude polysaccharide in water to remove protein, dialyzes the crude polysaccharide by a dialysis bag with molecular cut-off of 2000Da, and concentrates the polysaccharide solution in the dialysis bag.
In the present invention, the method for removing protein is preferably to remove protein precipitate by vortex mixing Sevage reagent with the obtained crude polysaccharide solution. The Sevage reagent is preferably prepared by mixing chloroform and n-butanol according to the weight ratio of (3.5-4.5): 1, and the volume ratio of chloroform to n-butanol is more preferably 4. The volume ratio of the obtained crude polysaccharide solution to the Sevage reagent is preferably 1 (0.5-2), more preferably 1. The time for vortex mixing is preferably 5 to 10min, more preferably 8min.
In the present invention, the crude polysaccharide after protein removal is dialyzed with a dialysis bag having a molecular cut-off of 2000Da to remove small molecular impurities. The dialysis is preferably carried out at 2 to 6 ℃. The dialysis time is preferably 24 to 48 hours, more preferably 36 hours.
In the invention, after dialysis, the crude polysaccharide in the dialysis bag is subjected to vacuum concentration and freeze drying to obtain the polysaccharide with the purity of more than 60wt% (measured by a phenol-sulfuric acid method).
After concentration, the concentrated polysaccharide solution is redissolved and loaded on a DEAE-Cellulose anion exchange column, continuous linear elution is carried out by using 0-2 mol/L NaCl solution, the polysaccharide content in each tube of eluent is detected, an elution curve is drawn, fractions are merged, and the polysaccharide with the weight-average molecular weight of 6.2-8.4 kDa is collected as the anti-hypoxia active dendrobium officinale polysaccharide.
In the present invention, the concentrated polysaccharide solution is preferably reconstituted with 10mmol/L Tris-HCl (pH = 7.4) buffer. The flow rate of elution is preferably 0.5 to 0.8mL/min. The procedure for continuous linear elution of NaCl solution was: the gradient was increased from 0 to 2mol/L in 0.2mol/L increments eluting 5 column volumes per concentration. The method for detecting the polysaccharide content in the eluate is preferably carried out by the phenol-sulfuric acid method. The horizontal axis of the elution curve is the polysaccharide collected at different times, and the vertical axis is the polysaccharide content. The method for combining fractions is to combine polysaccharides belonging to one peak into one tube according to the peak trend of the elution curve, and the tube is used for determining the weight average molecular weight.
In the present invention, in order to make the prepared polysaccharide have better therapeutic effect in preparing the medicament, the present invention preferably further comprises purifying the combined fractions after combining the fractions. The purification method preferably adopts Sephadex G-100 Sephadex column chromatography for each fraction, and uses deionized water for elution to obtain refined polysaccharide. The elution rate is preferably 0.5mL/min.
In the invention, after the dendrobium officinale raw material is treated by the steam explosion technology, the dendrobium officinale polysaccharide can be extracted quickly and efficiently, and the polysaccharide yield is improved by more than 60%. After the dendrobium officinale raw material is treated by using a steam explosion technology, a low molecular weight polysaccharide with a new conformation is obtained, the weight average molecular weight is 6.2-8.4 kDa, the polysaccharide consists of mannose, glucose, arabinose, a small amount of galacturonic acid and glucuronic acid, the total content of the glucuronic acid is more than 3% (g/g), and experiments prove that the polysaccharide is the most main anti-hypoxia active polysaccharide. Compared with the polysaccharide which is not subjected to the steam explosion technology or is not subjected to the limited steam explosion method, the polysaccharide prepared by the preparation method has the advantages that the anti-acute hypoxia activity is remarkably improved, the survival time of mice in a normal-pressure hypoxia-resistant experiment and a sodium nitrite poisoning experiment is remarkably prolonged, the dPC12 cell damage caused by acute hypoxia is reduced, and the polysaccharide can be used for preparing medicines or functional foods for resisting acute hypoxia damage.
The invention provides the anti-hypoxia active dendrobium candidum polysaccharide prepared by the method, which comprises mannose, glucose, arabinose, galacturonic acid and glucuronic acid, and the content of total glucuronic acid is more than 3wt%; the purity of the polysaccharide is 90wt% -95 wt%; the molar ratio of the mannose to the glucose to the arabinose to the galacturonic acid to the glucuronic acid is (5.5-5.7), (2.1-2.15), (1.8-1.9), (0.26-0.28) and (0.08-0.1).
The invention provides application of the anti-hypoxia active dendrobium officinale polysaccharide in preparation of a medicine for resisting acute hypoxia injury. The disease of acute hypoxic injury preferably comprises an acute altitude disease. The acute altitude disease comprises one or more of the following: acute mild altitude disease, high altitude pulmonary edema, high altitude cerebral edema.
The invention provides application of the anti-hypoxia active dendrobium officinale polysaccharide in functional food for relieving acute hypoxia injury.
The invention firstly develops the new effect of the dendrobium officinale polysaccharide on resisting acute anoxia by applying the steam explosion technology and expands the new application of the dendrobium officinale polysaccharide.
The preparation method and application of the anti-hypoxia active dendrobium officinale polysaccharide provided by the invention by steam explosion are described in detail in the following embodiments, but the invention is not limited to the scope of the invention.
Example 1
Preparation method of dendrobium officinale polysaccharide
(1) Taking fresh stem of Dendrobium officinale Kimura et Migo of Orchidaceae produced from Yandangshan mountain in Zhejiang, drying at 65 deg.C, and treating with QBS-80B steam explosion test bench (Henan Crane wall Zhengdao bioenergy Co., ltd.). The following four groups of steam blasting with different parameters are respectively adopted for processing the dendrobium officinale:
(1) and (3) treatment: the pressure is 1.2MPa, and the processing time is 120 seconds;
(2) and (3) processing: the pressure is 1.5MPa, and the processing time is 180 seconds;
(3) and (3) processing: the pressure is 0.8MPa, and the processing time is 60 seconds;
(4) and (3) treatment: the pressure is 0.6MPa, and the processing time is 240 seconds;
(5) and (3) treatment: dendrobium officinale without steam explosion treatment.
After the treatment of each group is finished, drying at 65 ℃ for later use.
(2) And respectively cutting the obtained steam explosion treatment and untreated dendrobium officinale samples into sections, crushing, sieving by a 60-mesh sieve, and collecting undersize products. Taking 20g of each of the dry powders treated in the steps (1) to (5), carrying out reflux extraction for 2 times and 2 hours each time at 100 ℃ by using distilled water in an amount which is 25 times (by mass) that of the dry powder treated in the step (1) to (5) to obtain leaching liquor, combining the leaching liquor obtained in the step (1) and the leaching liquor obtained in the step (5), concentrating the leaching liquor to 1/4 of the volume of the leaching liquor, and centrifuging the leaching liquor at 7000rpm for 10min to remove insoluble substances to obtain concentrated extracting liquor treated in the step (1) to (5);
(3) Adding 4 times volume of 95% precooled ethanol solution into the concentrated extracting solution treated in the steps (1) to (5) respectively under continuous stirring, standing at 4 ℃ for 18h, precipitating polysaccharide in the concentrated extracting solution, centrifuging and collecting precipitates, and freeze-drying the precipitates to obtain crude polysaccharide treated in the steps (1) to (5) respectively;
(4) Dissolving the crude polysaccharides obtained in the step (3) by using deionized water, then performing protein removal operation on the polysaccharides by using a Sevage (chloroform: n-butyl alcohol =4:1, v/v) reagent, repeating the operation for 6 times until the proteins are completely removed (no opalescence precipitate is separated out), removing the Sevage reagent by concentrating under reduced pressure, adding a proper amount of deionized water, heating to 70 ℃, filtering out insoluble substances while hot, cooling the filtrate, then placing the filtrate in a dialysis bag with molecular cut-off of 2000Da, dialyzing by using the deionized water at 4 ℃ to remove small molecular impurities, and drying to obtain the total polysaccharides. The purity of each polysaccharide was determined by phenol-sulfuric acid method and the yield of polysaccharide in each sample was calculated, the results are shown in table 1.
TABLE 1 purity and yield of polysaccharides extracted from Dendrobium officinale samples
As can be seen from Table 1, the steam explosion pretreatment is beneficial to increasing the polysaccharide yield of Dendrobium officinale.
(5) Dissolving the dendrobium officinale polysaccharide obtained in the step (4) in 10mmol/L Tris-HCl (pH = 7.4) buffer solution, loading the solution on a DEAE-Cellulose anion exchange column, performing linear elution by using 0-2 mol/L NaCl solution at the elution flow rate of 0.5mL/min, tracking and monitoring the polysaccharide content in each tube of eluent by using a phenol-sulfuric acid method, preparing an elution curve of the polysaccharide in a chromatographic column, and combining the polysaccharide solutions belonging to one peak with the flow according to the curve rule. Wherein the polysaccharides extracted from samples (1) - (3) all obtained two fractions of different molecular weight, designated as (1). While samples (4) and (5) had only a fraction of the extracted polysaccharide.
(6) And (4) respectively carrying out Sephadex G-100 Sephadex column chromatography on each flow part in the step (5) for further purification, eluting with deionized water, and carrying out freeze drying to obtain the refined polysaccharide. The purity of each purified polysaccharide was measured by the phenol-sulfuric acid method, and the results are shown in Table 2.
TABLE 2 purity of the refined polysaccharides obtained from each Dendrobium officinale sample
Example 2
Pharmacodynamic experiment of acute anoxia resistance
(1) Evaluation of animal experiments
180 healthy male ICR mice with the weight of 20 +/-2 g are taken, are provided by the animal center of Zhejiang Chinese medicine university (ethical approval number: IACUC-20210412-07, experimental animal management and ethical committee of Zhejiang Chinese medicine university), are randomly divided into 9 groups according to the weight, 20 mice in each group (the specific grouping condition is shown in Table 3), and are subjected to the experiment after 5 days of adaptive culture. The evaluation of the acute hypoxia resistance comprises a normal pressure hypoxia tolerance experiment test and a sodium nitrite poisoning experiment test.
The refined polysaccharides prepared in example 1 were dispersed and dissolved in distilled water, and distilled water was used as a blank control group, and after administration for 15 days by gavage, 10 mice per group were randomly selected for the atmospheric hypoxia tolerance test, and another 10 mice were subjected to the sodium nitrite poisoning test. During feeding and administration, water and food are freely drunk, and the weight of the mice is weighed.
TABLE 3 grouping of experimental mice
(1) Testing the normal-pressure hypoxia-resistant experiment: reference [ Xie Y, et al. Composition analysis and anti-hypoxia activity of polysaccharide from Brassica rapa L. [ J ]. International Journal of biological Macromolecules,2010,47 (4): 528-533 ]. After the experimental mouse is subjected to last gastric lavage and administration, the experimental mouse is fasted and water is not forbidden, after the experimental mouse moves for 1 hour under a normal environment, the experimental mouse is respectively put into 250mL wide-mouth bottles (1 bottle) with plugs and ground openings, 5g of soda lime is filled in the wide-mouth bottles, vaseline is coated around the bottle caps for sealing, and the timing is carried out immediately after the bottles are capped. And (4) stopping timing by taking the respiratory arrest of the mice as an end point, recording the death time of the mice due to hypoxia, and calculating the survival time prolonging rate of the mice according to the formula I. Meanwhile, the organs were dissected and the water content of lung tissue was measured immediately after death of the mice.
Survival extension (%) = [ (dosing group survival-model group survival)/model group survival ] × 100% formula I.
(2) Sodium nitrite poisoning experiment test: reference [ Zhang C, et al, anti-hysoxica activity of a polysaccharide extracted from the lateral nucleus L [ J ]. International Journal of biological Macromolecules,2011,49 ]. After the experimental mouse is subjected to last gastric lavage administration, the experimental mouse is fasted and water is forbidden, after the mouse moves for 1 hour under a normal environment, sodium nitrite solution with the concentration of 200mg/kg.bw (the injection amount is 0.1g/10 g) is injected into the abdominal cavity, timing is started immediately, the survival time of the mouse is recorded, and the survival time prolonging rate of the mouse is calculated according to the formula I.
TABLE 4 survival time of mice in each group for normbaric hypoxia tolerance experiment (mean + -SD)
Note: * Indicating significant difference compared with the blank control group, p < 0.05.
TABLE 5 pulmonary Water content (mean + -SD) after acute hypoxic death in groups of mice
Note: * Indicating significant difference compared with the blank control group, p < 0.05.
TABLE 6 survival time (mean + -SD) of mice in each group for sodium nitrite poisoning experiment
Note: * Indicating significant difference compared with the blank control group, p < 0.05.
The results show that the effects of different steam blasting treatments on the refined polysaccharide in the dendrobium officinale through continuous gavage for 15 days have great difference on the hypoxia tolerance of mice. Wherein, the refined polysaccharide (number (1) S-DOP1, (2) S-DOP1 and (3) S-DOP1 can obviously prolong the survival time (p < 0.05) of the mouse in the normal pressure hypoxia-resistant and sodium nitrite poisoning experiments and simultaneously obviously reduce the water content (p < 0.05) of the lung tissue after the acute hypoxia death of the mouse) after the steam explosion treatment of the parameter (1) (pressure 1.2MPa +120 seconds), the parameter (2) (pressure 1.5MPa +180 seconds) and the parameter (3) (pressure 0.8MPa +60 seconds). The effect of the polysaccharide in the dendrobium officinale treated by steam explosion with the parameter (4) (pressure 0.6MPa +240 seconds) in the example 1 or without steam explosion is not shown (figure 1, tables 4-6). This shows that the steam explosion treatment of Dendrobium officinale can effectively improve the anti-hypoxia activity of the refined polysaccharide and alleviate acute hypoxic pulmonary edema, but is closely related to the steam explosion parameters which determine the reconstitution characteristics of the polysaccharide in Dendrobium officinale. In addition, the mice were in good health and none died, and the body weight and organ index of the mice were not significantly different between groups (p > 0.05) throughout the gavage administration (data not shown).
(2) Evaluation of cell experiments
The protective effect of each purified polysaccharide prepared in example 1 on acute hypoxic injury of cells was evaluated for a neuronal-like cell line, dPC 12. The specific experimental method is as follows:
1) dPC12 cells were cultured in RPMI 1640 containing 10% fetal bovine serum, 100U/mL penicillin and streptomycin at 37 ℃ with 5% CO 2 The culture medium is replaced every two days until the cells enter the logarithmic growth phase.
2) mu.L of dPC12 cell fluid (about 5X 10) was inoculated per well in a 96-well plate 3 And then culturing for 24 hours under normal conditions to ensure that the cells are attached to the wall and grow normally.
3) The medium was carefully removed and medium containing different refined polysaccharides were added, each polysaccharide being set separately in a low dose group (10.0. Mu.g/mL) and a high dose group (50.0. Mu.g/mL), while an anoxic model group and a normal control group were set, each group being 6 replicate wells. At 37 ℃ C, 5% CO 2 After incubation for 3h in a Normal incubator under the conditions, the plates of the remaining groups were transferred to 37 ℃ and 0.5% 2 +5％CO 2 +94.5％N 2 Culturing in a three-air incubator under the condition for 36h.
4) Carefully add 10. Mu.L of CCK-8 solution to each well in a clean bench in the dark and mix well. After incubation for 3h in an incubator at 37 ℃, absorbance at a wavelength of 450nm is measured by using a microplate reader.
TABLE 7 protective action of Dendrobium officinale refined polysaccharides on dPC12 cell damage under acute anoxia (mean + -SD)
Note: * Representing significant difference compared with the hypoxia model group, wherein p is less than 0.05; * P < 0.01. ## The significant difference of the hypoxia model group and the normal group is shown, and p is less than 0.01.
The results show that 36h of dPC12 cells cultured under acute hypoxia significantly reduced the cell viability (p < 0.01), which is only 49.6% of the normal culture group. The dPC12 cell activity can be remarkably improved (p is less than 0.05) by adopting the co-incubation culture of S-DOP1, (2) S-DOP1 and (3) S-DOP1 refined polysaccharide with the concentrations of 10.0 mu g/mL and 50.0 mu g/mL (1), and the good protection effect of acute hypoxic cell injury is shown; however, the purified polysaccharides of the remaining groups did not exhibit the effect against acute hypoxic injury (Table 7). The results were consistent with the "(1) animal experiment evaluation" results of example 2.
Example 3
Measurement of uronic acid content in each of the purified polysaccharides obtained in example 1
The uronic acid content of each purified polysaccharide obtained in example 1 was determined. The content of uronic acid in rhizoma Polygonati of different origins is compared by sulfuric acid-carbazole method, reference [ Huang Zhao just, et al, J, chinese pharmacist, 2004,7 (6): 433-434], galacturonic acid is used as standard, the measurement wavelength is 530nm, and each sample is measured three times.
As can be seen from Table 8, the uronic acid content of (1) S-DOP1 polysaccharide, (2) S-DOP1 polysaccharide and (3) S-DOP1 polysaccharide obtained by extraction, separation and purification from Dendrobium officinale subjected to steam explosion treatment according to the treatment parameters (1) to (3) was significantly higher than that of (5) DOP polysaccharide (p < 0.05) not subjected to steam explosion treatment, wherein the uronic acid content of (1) S-DOP1 polysaccharide (3.66%) was increased by 118.3% as compared with that of (5) DOP polysaccharide (1.68%). Whereas the treatment group with parameter (4) (pressure 0.6MPa, treatment time 240 seconds) showed no significant increase in uronic acid content.
TABLE 8 uronic acid content (mean + -SD) of refined polysaccharide of Dendrobium officinale after steam explosion treatment with different parameters
Note: * Shows significant difference compared with (5) DOP refined polysaccharide (without steam explosion treatment), p is less than 0.05; * P < 0.01.
Example 4
Structural analysis of antihypoxic active polysaccharide
(1) Scanning electron microscope characterization of polysaccharide microstructure
According to the results of functional evaluation of example 2, the purified polysaccharides (1) obtained in example 1, S-DOP1, (1) S-DOP2, (2) S-DOP1, (2) S-DOP2 and (5) DOP were used as subjects, and their microstructures were characterized by scanning electron microscopy, and the results are shown in FIG. 2. The marked difference between polysaccharide ((5) DOP) in dendrobium officinale without steam explosion treatment (figure 2E) and polysaccharide prepared after steam explosion treatment of parameter (1) (pressure 1.2MPa, treatment time 120 seconds) and parameter (2) (pressure 1.5MPa, treatment time 180 seconds) is shown (figures 2A-2D), fragmentation and disordering of polysaccharide in dendrobium officinale can be aggravated by steam explosion, and naked residues of polysaccharide are increased along with the steam explosion, which indicates that the action mechanism of steam explosion treatment of dendrobium officinale to improve the anti-hypoxia activity of polysaccharide is possible. Meanwhile, after the dendrobium officinale is treated by steam explosion with different parameters, the microstructure of the refined polysaccharide also has certain difference.
(2) Structure for contrastive analysis of polysaccharide by adopting infrared spectrometer
The purified polysaccharide (1) obtained in example 1 was compressed with potassium bromide to give a mixture of (1) S-DOP1, (1) S-DOP2, (2) S-DOP1, (2) S-DOP2 and (5) DOP, and the mixture was then compressed with potassium bromide to give a mixture of (4000-400) cm -1 Infrared spectral scanning was performed over the wavelength range and the results are shown in figure 3. Some basic groups in the polysaccharide have characteristic absorption peaks in the spectral region. 3425cm -1 The strong absorption at (B) represents the hydroxyl stretching vibration absorption peak of polysaccharide, 2924cm -1 The signal represents the C-H stretching and bending vibration of the polysaccharide; and 1736cm -1 Belongs to C = O asymmetric stretching vibration on acetyl or carboxyl in polysaccharide, and suggests that the dendrobium officinale polysaccharide contains uronic acid, and the abundance of uronic acid in (1) S-DOP1 and (2) S-DOP1 polysaccharide is obviously highCorresponding to the results of the measurement of the content of aldehyde acid in example 3 from (1) S-DOP2, (2) S-DOP2 and (5) DOP; 1640cm -1 The absorption peak of crystal water or hydrate is probably larger because polysaccharide absorbs water easily; 1378cm -1 Bending vibration with an absorption peak of O-H; 1250cm -1 、1150cm -1 、1060cm -1 And 1030cm -1 The four absorption peaks come from C-O-H stretching vibration of pyranose ring and asymmetric stretching vibration of C-O-C glycosidic bond; 811cm -1 Nearby is the characteristic absorption of mannose. The infrared spectrum absorption of Dendrobium officinale polysaccharide before and after steam explosion has large difference, especially 1736cm -1 、1378cm -1 、1250cm -1 、1060cm -1 、1030cm -1 And 811cm -1 The absorption peak at (b). The steam explosion treatment of the dendrobium officinale is prompted to have great influence on chemical groups and structures of polysaccharides of the dendrobium officinale.
(3) (1) weight average molecular weight analysis of S-DOP1 and (2) refined polysaccharides of S-DOP1
The anti-hypoxic active polysaccharides (1, s-DOP1 and (2) prepared in example 1 were subjected to weight average molecular weight measurement using Gel Permeation Chromatography (GPC), method reference [ chemical structures of tangvain. Turnip polysaccharide and maca polysaccharide and their anti-fatigue effects comparative study [ D ]. University doctor thesis of zhejiang, 2017, zhejiang: hangzhou ]. The standard curves prepared by combining dextran standards with different molecular weights and GPC chromatograph software are Log10 (Mw) = -0.8251x +10.788, (1) the retention time of S-DOP1 and (2) the retention time of S-DOP1 are respectively 8.411min and 8.476min, and the weight average molecular weights of the S-DOP1 polysaccharides (1) and (2) the S-DOP1 polysaccharides are respectively 7.048kDa and 6.229kDa (figure 4).
(4) (1) analysis of monosaccharide composition of S-DOP1 polysaccharide
The antihypoxic active polysaccharide (1) obtained in example 1 and S-DOP1 was hydrolyzed at 100 ℃ for 12 hours after charging nitrogen by weighing 10mg (to the nearest 0.1 mg) and adding 2mL of a 2mol/L trifluoroacetic acid solution. The PMP method was then used for monosaccharide derivatization. The monosaccharide composition analysis was performed by HPLC, and the results are shown in FIG. 5. It shows that (1) the S-DOP1 polysaccharide mainly comprises mannose, glucose and arabinose, and simultaneously contains a small amount of galacturonic acid and glucuronic acid, and the molar ratio of each monosaccharide of mannose, glucose, arabinose, galacturonic acid and glucuronic acid is 5.63.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (9)
1. A method for preparing anti-hypoxia active dendrobium officinale polysaccharide by steam explosion is characterized by comprising the following steps:
1) Performing steam explosion treatment on the dendrobium officinale to obtain pretreated dendrobium officinale; the conditions of the steam explosion treatment were as follows: the charging coefficient of the blasting cavity is 0.7-0.9, the blasting steam pressure is 0.8-1.5 MPa, and the pressure maintaining time is 60-180 s;
2) Refining the pretreated dendrobium officinale in the step 1), then carrying out reflux extraction by using water, precipitating the obtained leaching solution by using an ethanol solution, and collecting precipitates to obtain crude polysaccharide;
3) Dissolving the crude polysaccharide obtained in the step 2) in water, removing protein, dialyzing by using a dialysis bag with molecular cut-off of 2000Da, and concentrating the polysaccharide solution in the dialysis bag;
4) Redissolving the polysaccharide solution concentrated in the step 3), loading the redissolved polysaccharide solution into a DEAE-Cellulose anion exchange column, continuously and linearly eluting by using 0-2 mol/L NaCl solution, detecting the polysaccharide content in each tube of eluent, drawing an elution curve, merging fractions according to the peak condition in the elution curve, and collecting polysaccharide with the weight-average molecular weight of 6.2-8.4 kDa as the anti-hypoxia active dendrobium officinale polysaccharide;
after the combined parts are combined in the step 4), purifying the combined parts; the purification method adopts SephadexG-100 sephadex column chromatography to separate each flow component, and uses deionized water to elute, so as to obtain the refined polysaccharide.
2. The method according to claim 1, wherein the conditions of the steam explosion treatment are as follows: the charging coefficient of the blasting cavity is 0.78, the pressure of the blasting steam is 1.0-1.3 MPa, and the pressure maintaining time is 80-160 s.
3. The method as claimed in claim 1, wherein the temperature of the reflux extraction in the step 2) is 70-100 ℃, the time of the reflux extraction is 2-3 h, and the number of reflux extractions is 2-3; the mass percentage of the ethanol solution is 70-100%.
4. The method of claim 1, wherein the protein is removed in step 3) by vortexing the crude polysaccharide solution with Sevage reagent to remove protein precipitate; the Sevage reagent is prepared by mixing chloroform and n-butanol according to the weight ratio of (3.5-4.5): 1 in a volume ratio; the volume ratio of the obtained crude polysaccharide solution to Sevage reagent is 1 (0.5-2), and the vortex mixing time is 5-10 min.
5. The method according to claim 1, wherein the flow rate of elution in step 4) is 0.5 to 0.8mL/min.
6. The anti-hypoxia active dendrobium officinale polysaccharide prepared by the method of any one of claims 1-5, which is characterized by comprising mannose, glucose, arabinose, galacturonic acid and glucuronic acid, wherein the content of total glucuronic acid is more than 3wt%; the purity of the polysaccharide is 90wt% -95 wt%, and the molar ratio of mannose, glucose, arabinose, galacturonic acid and glucuronic acid is (5.5-5.7), (2.1-2.15), (1.8-1.9), (0.26-0.28) and (0.08-0.1).
7. The use of the anti-hypoxic active dendrobium officinale polysaccharide of claim 6 for the preparation of a medicament for resisting acute hypoxic injury.
8. The use of claim 7, wherein the acute hypoxic-injury disease comprises acute altitude disease; the acute altitude disease comprises one or more of the following: acute mild altitude disease, high altitude pulmonary edema and high altitude cerebral edema.
9. The use of the anti-hypoxic active dendrobium officinale polysaccharide of claim 6 for preparing a functional food for alleviating acute hypoxic injury.
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| CN110437344A (en) * | 2019-09-17 | 2019-11-12 | 广西民族大学 | A kind of dendrobium candidum refined polysaccharide and preparation method thereof |
| CN110818812A (en) * | 2019-10-30 | 2020-02-21 | 韶关市丹霞铁皮石斛研究院 | Dendrobium officinale refined polysaccharide extract and application thereof |
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| CN104642702A (en) * | 2015-03-19 | 2015-05-27 | 郝燕 | Preparation method of noble dendrobium stem preserved fruits |
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| CN109651532A (en) * | 2018-11-21 | 2019-04-19 | 浙江农林大学 | A kind of dendrobium candidum Glucomannan |
| CN110437344A (en) * | 2019-09-17 | 2019-11-12 | 广西民族大学 | A kind of dendrobium candidum refined polysaccharide and preparation method thereof |
| CN110818812A (en) * | 2019-10-30 | 2020-02-21 | 韶关市丹霞铁皮石斛研究院 | Dendrobium officinale refined polysaccharide extract and application thereof |
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