CN117659219A - Henan dendrobium polysaccharide and preparation method and application thereof - Google Patents
Henan dendrobium polysaccharide and preparation method and application thereof Download PDFInfo
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- CN117659219A CN117659219A CN202311298690.9A CN202311298690A CN117659219A CN 117659219 A CN117659219 A CN 117659219A CN 202311298690 A CN202311298690 A CN 202311298690A CN 117659219 A CN117659219 A CN 117659219A
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- Prior art keywords
- polysaccharide
- dendrobium
- dendrobium nobile
- nobile polysaccharide
- preparation
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Classifications
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- A—HUMAN NECESSITIES
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Abstract
The invention provides a dendrobium nobile polysaccharide and a preparation method and application thereof, and relates to the field of plant polysaccharide extraction. The preparation method of the dendrobium nobile polysaccharide comprises the steps of degreasing treatment, water extraction and alcohol precipitation method extraction, starch removal, deproteinization, dialysis, DEAE-52 anion exchange chromatographic column primary purification and Sephadex-G gel column secondary purification. The invention provides a preparation method of dendrobium nobile polysaccharide for the first time, and the dendrobium nobile polysaccharide prepared by the method has remarkable blood fat reducing and antioxidation effects. The preparation method of the dendrobium nobile polysaccharide and the research on the medicinal value of the dendrobium nobile polysaccharide can further promote the development and utilization of dendrobium nobile medicinal materials, and are beneficial to promoting the sustainable development of local poverty-relieving industry. The preparation method of the dendrobium nobile polysaccharide provided by the invention has the advantages of stable process and simple and convenient operation, and can obtain high-purity and high-activity polysaccharide, thereby having important practical significance.
Description
Technical Field
The invention relates to the field of plant polysaccharide extraction and application, in particular to a dendrobium nobile polysaccharide, and a preparation method and application thereof.
Background
Dendrobe belongs to the genus dendrobe of the orchidaceae family, and the description of Shennong Bencao Jizhu is: dendrobium nobile, sweet and neutral in taste. Mainly hurting middle energizer, removing arthralgia, descending qi, tonifying five viscera, consumptive disease, emaciation, strengthening yin, taking thick intestines and stomach for a long time, and reducing weight and prolonging life. Henan dendrobium nobile (Dendrobium henanense J.L.Liu et L.X.Gao), stem clusters, cylinders, reverse-folded bends, she Ju round needle-like shape, near-revolutionary, sepals and petals white, capsules, inverted egg-like needle-like shape. The Henan dendrobium nobile has the effects of benefiting stomach, promoting fluid production, nourishing yin and clearing heat.
The plant polysaccharide is a natural high molecular polymer, has low toxic and side effects on human bodies, and has a high diversity of spatial configuration and connection modes compared with other biological macromolecules, and shows various biological activities. The research on chemical components of dendrobe is mainly focused on macromolecular substances such as polysaccharide, which suggests that the polysaccharide is a main active substance of dendrobe.
At present, the research on dendrobium polysaccharide at home and abroad mainly focuses on dendrobium species such as dendrobium huoshanense, dendrobium nobile, dendrobium candidum or dendrobium fimbriatum, and the like, including aspects of extraction and separation, structural characterization and activity research of dendrobium polysaccharide, but the research on the active ingredients of dendrobium huoshanense is very rare. This is because it is well known by those skilled in the art that the quality of Dendrobium huoshanense is the best among Dendrobium huoshanense medicinal material varieties, and Dendrobium huoshanense is the most common counterfeit product of Dendrobium huoshanense. The price difference of the two is very huge in the market, and the market price of dendrobium huoshanense is about 2000-4000 yuan/kg, and the price of dendrobium huoshanense is 50-100 yuan/kg at present. This has led to even further little research in the art on the active ingredients of dendrobium huoshanense. How to extract the dendrobium nobile polysaccharide with medicinal activity has specific medicinal value and is yet to be studied.
Disclosure of Invention
Therefore, the invention aims to provide the dendrobium nobile polysaccharide, and the preparation method and the application thereof, so as to promote the development and the utilization of dendrobium nobile medicinal materials.
In order to achieve the above purpose, the present invention provides the following technical solutions:
in a first aspect, the invention provides a method for preparing dendrobium huoshanense polysaccharide, which comprises the following steps:
(1) Degreasing raw medicinal material powder of dendrobium huoshanense, and drying to obtain degreased medicinal material powder;
(2) Extracting the defatted medicinal material powder by adopting a water extraction and alcohol precipitation method to obtain an alcohol precipitate;
(3) Taking the alcohol sediment for removing starch, deproteinizing and dialyzing to obtain coarse dendrobium nobile polysaccharide;
(4) And (3) performing primary purification on the dendrobium huoshanense crude polysaccharide by using a DEAE-52 anion exchange chromatographic column, performing secondary purification on the component polysaccharide eluted by ultrapure water by using a Sephadex-G gel column, collecting the corresponding eluted component, concentrating and freeze-drying to obtain the dendrobium huoshanense polysaccharide.
Further, in the step (1), degreasing treatment is carried out by adopting ethanol with the volume concentration of 50% -90%, and ethanol with the volume concentration of 80% is preferred; the mass volume ratio of the dendrobium huoshanense raw material powder to the ethanol is 1-1.5 in terms of g/mL: 2 to 6, preferably 1:4, a step of; adding ethanol into the raw medicinal powder of dendrobium huoshanense, and vibrating for 12-48 hours, preferably 24 hours at room temperature.
Further, in the step (2), in the water extraction process, the feed liquid ratio of the defatted medicinal material powder to water is 1 in terms of g/mL: 10 to 50, preferably 1:30; the extraction temperature is 60-90 ℃, preferably 80 ℃; the extraction time is 1-3 h, preferably 2h; in the alcohol precipitation process, the water extract is decompressed and concentrated to 1/4 to 1/8 of the original volume, preferably 1/5; ethanol is added to a final concentration of 60 to 80 percent, preferably 80 percent, and the mixture is kept stand for 12 to 48 hours, preferably 24 hours.
Further, in the step (3),
in the process of removing starch, taking the alcohol precipitate for redissolution, and then adding alpha amylase for enzymolysis, wherein the adding amount of the alpha amylase is 1-3 mu L/g, preferably 1 mu L/g, based on the mass of the raw medicinal material powder of dendrobium huoshanense; the enzymolysis temperature is 30-60 ℃, preferably 60 ℃; the enzymolysis time is 0.5-3 h, preferably 1h; concentrating the enzymolysis liquid to 1/2-1/6, preferably 1/3, of the original volume after enzymolysis, centrifuging to remove starch and other insoluble precipitates, and obtaining starch-removed sugar liquid;
in the deproteinization process, the volume ratio of the starch removing sugar solution to chloroform to n-butanol is 20-50: 2-10: shaking vigorously for 1-5 h after mixing 0.5-2, centrifuging to remove protein layers, repeating for 3-10 times, and removing residual organic solvent in the sugar solution under reduced pressure, wherein the preferable volume ratio is 30:5:1, violently shaking for 2 hours, and repeating for 7 times;
in the dialysis process, a dialysis bag with the molecular weight cut-off of more than 3500Da is adopted for running water dialysis for 2-3 days, preferably 2 days, deionized water is further used for dialysis for 12-48 hours, preferably 24 hours, and the concentrated solution is subjected to vacuum freeze drying, so that the dendrobium nobile polysaccharide is obtained.
Further, in the step (4),
in the primary purification process, the method sequentially uses ultrapure water and sodium chloride solution with the concentration from low to high to elute at the elution speed of 5-8 mL/min, preferably 5mL/min;
in the secondary purification process, a Sephadex-G100 gel column is adopted, and the elution is carried out by ultrapure water, wherein the elution speed is 2-3 mL/min, and preferably 2mL/min.
Further, in the step (4),
in the primary purification process, collecting the ultrapure water eluting component, concentrating, dialyzing and freeze-drying to obtain the ultrapure water eluting component polysaccharide;
in the second-stage purification process, the elution components of the left and right tubes with the highest absorbance value of the elution curve are collected.
In a second aspect, the invention provides the dendrobium nobile polysaccharide obtained by the preparation method.
Further, the molecular weight of the dendrobium nobile polysaccharide in Henan is 2 multiplied by 10 4 ～9×10 4 Da, preferably 8X 10 4 Da。
Further, the monosaccharide composition of the dendrobium huoshanense polysaccharide is fucose, galactose, glucose and mannose, wherein the molar ratio of the fucose, the galactose, the glucose and the mannose is 0.221:0.02:0.32:0.403.
in a third aspect, the invention provides the dendrobium nobile polysaccharide prepared by the method or the application of the dendrobium nobile polysaccharide in preparing hypolipidemic and/or antioxidant drugs.
In a fourth aspect, the invention provides an application of the dendrobium huoshanense polysaccharide in preparing medicines for treating nonalcoholic liver injury and/or treating oxidative stress.
The technical scheme of the invention has the following advantages:
the invention provides a preparation method of dendrobium nobile polysaccharide for the first time, which comprises the steps of degreasing treatment, water extraction and alcohol precipitation method extraction, starch removal, deproteinization, dialysis, DEAE-52 anion exchange chromatographic column primary purification and Sephadex-G gel column secondary purification. The dendrobe polysaccharide monosaccharide prepared by the method comprises fucose, galactose, glucose and mannose. The invention also unexpectedly discovers that the dendrobium nobile polysaccharide prepared by the method has remarkable blood fat reducing and antioxidation effects, and in particular, in vitro pharmacodynamics experiments prove that the dendrobium nobile polysaccharide prepared by the method has the functions of DPPH free radical, hydroxyl free radical (OH) and superoxide anion free radical (OOH) - ) And ABTS free radicals have a certain degree of scavenging effect; feeding high-sugar and high-fat feed to establish a non-alcoholic liver injury rat model, and in vivo pharmacodynamics experiments prove that the dendrobium nobile polysaccharide prepared by the invention has liver protection effect and can reduce the oxidative stress level; the in vivo pharmacodynamics experiment proves that the dendrobium nobile polysaccharide prepared by the invention can improve D-galactose-induced oxidative damage, has an anti-oxidative stress effect, and can reach the level basically same as that of dendrobium huoshanense polysaccharide by comparing with the dendrobium huoshanense polysaccharide.
The invention breaks through the knowledge and prejudice of the field on the dendrobium huoshanense, and has larger advantages in market price based on the dendrobium huoshanense.
The preparation method of the dendrobium nobile polysaccharide provided by the invention has the advantages of stable process and simple and convenient operation, and can obtain high-purity and high-activity polysaccharide, thereby having important practical significance.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is an elution curve of a DEAE-52 anion exchange chromatography column for primary purification of Dendrobium nobile crude polysaccharide in the examples of the present invention;
FIG. 2 is an elution curve of the second-stage purification of the Sephadex-G100 gel column of the ultrapure water-eluted fraction polysaccharide in the example of the present invention;
FIG. 3 is an appearance and morphology diagram of dendrobium huoshanense polysaccharide DNPW prepared by the embodiment of the invention;
FIG. 4 is a scanning electron microscope image of dendrobe polysaccharide DNPW prepared by the embodiment of the invention;
FIG. 5 is an ultraviolet spectrum of dendrobe polysaccharide DNPW prepared by the embodiment of the invention;
FIG. 6 is an infrared spectrum of dendrobe polysaccharide DNPW prepared by the embodiment of the invention;
FIG. 7 is a view of HE-stained sections of liver tissue from each group of rats in Experimental example 3 of the present invention;
FIG. 8 is a graph showing the effect of the dendrobium huoshanense polysaccharide and dendrobium huoshanense polysaccharide on SOD, MDA, GSH-PX and T-AOC in liver tissue in experimental example 4 of the present invention;
FIG. 9 is a graph showing the effect of the dendrobium huoshanense polysaccharide and dendrobium huoshanense polysaccharide on SOD, MDA, GSH-PX and T-AOC in brain tissue in experimental example 4 of the present invention;
FIG. 10 is a graph showing the effect of the dendrobium huoshanense polysaccharide and dendrobium huoshanense polysaccharide on SOD, MDA, GSH-PX and T-AOC in kidney tissue in experimental example 4 of the present invention;
FIG. 11 is the effect of the dendrobium huoshanense polysaccharide and dendrobium huoshanense polysaccharide on SOD, MDA, GSH-PX and T-AOC in serum in experimental example 4 of the present invention.
Detailed Description
The following examples are provided for a better understanding of the present invention and are not limited to the preferred embodiments described herein, but are not intended to limit the scope of the invention, any product which is the same or similar to the present invention, whether in light of the present teachings or in combination with other prior art features, falls within the scope of the present invention.
The specific experimental procedures or conditions are not noted in the examples and may be followed by the operations or conditions of conventional experimental procedures described in the literature in this field. The materials or instruments used are all conventional products commercially available, including but not limited to those used in the examples of the present application.
Examples
The embodiment provides a preparation method of dendrobium huoshanense polysaccharide, which comprises the following specific steps:
(1) Degreasing treatment for raw medicinal materials of dendrobium huoshanense
Accurately weighing 200g of dried stem powder of Dendrobium nobile, adding 800mL of 80% ethanol, shaking in a shaking table at room temperature for 24h, filtering, and oven drying to obtain defatted medicinal material powder;
(2) Henan dendrobe polysaccharide extraction
Taking the defatted medicinal material powder obtained in the step (1), adding 30 times of water, extracting for 2 hours in a water bath at 80 ℃, concentrating the obtained extracting solution under reduced pressure to 1/5 of the original volume, adding ethanol into the concentrated solution to a final concentration of 80%, standing for 24 hours, precipitating with ethanol, and filtering to obtain a precipitate to obtain an alcohol precipitate;
(3) Removing starch, removing protein, and dialyzing
Weighing 100g of alcohol sediment, re-dissolving, adding alpha amylase (the enzyme addition amount is 1 mu L/g based on the mass of dendrobium nobile stem powder), carrying out enzymolysis for 1h at 60 ℃, concentrating the enzymolysis liquid to 1/3 of the original volume, centrifuging (5000 rpm/10 min) to remove starch and other insoluble precipitates, and obtaining starch-removing sugar solution, wherein the starch-removing sugar solution is prepared by the following steps: chloroform: mixing n-butanol at a volume ratio of 30:5:1, shaking vigorously for 2h, centrifuging (5000 rpm/10 min), removing protein layer, repeating for 7 times, removing residual organic solvent in sugar solution under reduced pressure, dialyzing with dialysis bag with molecular weight cutoff of 3500Da for 48h, dialyzing with deionized water for 24h, and vacuum freeze-drying the concentrated solution to obtain 14g of herba Dendrobii crude polysaccharide;
(4) Separation and purification of dendrobium huoshanense polysaccharide
Purifying coarse herba Dendrobii polysaccharide with DEAE-52 anion exchange chromatographic column, sequentially eluting with ultrapure water and sodium chloride solution with concentration of 0.1, 0.2, 0.3, 0.4 and 0.5mol/L respectively, loading 200mg with sample rate of 5mL/min, collecting 20 tubes for each eluting component, measuring OD value of each tube by phenol sulfuric acid method, and drawing elution curve, as shown in figure 1, concentrating, dialyzing, and lyophilizing to obtain ultrapure water eluting component polysaccharide; performing secondary purification on the polysaccharide of the elution component of ultrapure water by using a Sephadex-G100 gel column, eluting with ultrapure water, wherein the loading amount is 50mg, the eluting speed is 2mL/min, collecting 80 tubes, measuring the OD value of each tube by using a phenol sulfuric acid method, drawing an elution curve, collecting the elution component of the highest absorbance value of the elution curve and the elution component of the left tube and the right tube, concentrating, and freeze-drying to obtain the dendrobium nobile polysaccharide, which is named DNPW.
Experimental example 1 determination of physicochemical Properties of DNPW
1. Topographical features
The appearance of DNPW was photographed with a camera, and DNPW was white flocculent powder as shown in FIG. 3.
Microscopic morphology of DNPW was observed by scanning electron microscopy, as shown in fig. 4, the DNPW was in the form of an irregular sheet with a smooth surface.
2. Ultraviolet spectrogram
DNPW is prepared into a dendrobium nobile polysaccharide solution with the concentration of 0.1mg/mL, and an ultraviolet spectrum is obtained by scanning the dendrobium nobile polysaccharide solution with the wavelength range of 190-800 nm by an ultraviolet-visible spectrophotometer. As shown in fig. 5, the absorption curve was smooth, indicating a low protein content in DNPW.
3. Infrared spectrogram
Weighing DNPW 2mg, adding 100mg KBr dry powder, mixing and grinding in an agate mortar, tabletting, and placing on a Fourier infrared spectrum scanner at 4000cm -1 ～400cm -1 Scanning analysis was performed in range. Absorption peak wave numbers are 3426cm respectively -1 、2938cm -1 、1742cm -1 、1644cm -1 、882cm -1 It can be seen from FIG. 6 that the molecular structure contains hydroxyl, methylene, carbonyl, and pyranose rings.
4. Molecular weight
The molecular weight of DNPW was measured using a Shimadzu 2010A high performance liquid chromatography system equipped with a Shimadzu RID-20A detector and a TSK-gel G4000 PWXL column (8.0 mm. Times. 300mm,Borui Saccharide,Biotech.Co.Ltd), the mobile phase was ultrapure water at a temperature of 40℃and a flow rate of 0.6mL/min. Calculating molecular weight by calibration curve prepared from serial dextran standard substances to obtain molecular weight of 8X10 4 Da。
5. Monosaccharide composition
Taking DNPW 5mg and 0.05M trifluoroacetic acid 10mL, placing into a closed ampoule bottle, hydrolyzing for 30min at 100 ℃, drying the reaction mixture with nitrogen, and evaporating with methanol to eliminate redundant acid; taking a proper amount of sample, dissolving the sample in water, and filtering the sample by a microporous filter membrane; dionexis-5000, a 100mM aqueous sodium hydroxide solution, was used as the mobile phase, and the flow rate was 0.5mL/min, through the Carbpac of Dionex corporation, U.S. Thermo Fisher Scientific, was used TM The monosaccharide composition of DNPW was analyzed on a PA20 column (3 mm. Times.150 mm) and the results are shown in Table 1.
TABLE 1DNPW monosaccharide composition results
| Monosaccharide composition | Fucose | Galactose | Glucose | Mannose |
| Molar ratio of | 0.221 | 0.02 | 0.32 | 0.403 |
Experimental example 2 in vitro pharmacodynamic experiments of DNPW
1. Polysaccharide solution preparation
Taking a proper amount of DNPW sample, and precisely weighing. Preparing polysaccharide solutions with the concentration of 0.1mg/mL, 0.2mg/mL, 0.4mg/mL, 0.6mg/mL, 0.8mg/mL, 1mg/mL, 2mg/mL, 3mg/mL, 4mg/mL and 5mg/mL respectively by distilled water for later use; ascorbic acid (Vc) was used as a positive control.
2. Radical scavenging rate determination
The DPPH radical, hydroxyl radical, superoxide anion radical or ABTS radical scavenging rate determination is calculated using equation (1):
DPPH radical: taking 1.0mL of polysaccharide solution with different concentrations, adding 2.0mL of DPPH (2, 2-diphenyl-1-pyridohydrazino) -ethanol solution with concentration of 0.2mM, reacting at room temperature in the dark for 30min, zeroing with deionized water, and measuring OD value (marked as A) at 517nm 1 ) The method comprises the steps of carrying out a first treatment on the surface of the The polysaccharide solution was replaced with deionized water and its OD was measured (designated A 0 ) The method comprises the steps of carrying out a first treatment on the surface of the The DPPH was replaced with deionized water and its OD was measured (designated A 2 ). The DPPH radical scavenging ability of ascorbic acid at the same concentration was measured as positive by the same methodSex control.
Hydroxyl radical: respectively taking 1.0mL of polysaccharide solutions with different concentrations, and adding 1.0 mLFASO 4 (6mmol/L)，1.0mL H 2 O 2 (6 mmol/L) mixing, incubating at 37deg.C for 10min, adding 1.0mL salicylic acid-ethanol solution (6 mmol/L), mixing, incubating at 37deg.C for 30min, zeroing with deionized water, and measuring OD value (denoted as A) at 510nm with spectrophotometer 1 ) The method comprises the steps of carrying out a first treatment on the surface of the The polysaccharide solution was replaced with deionized water and its OD was measured (designated A 0 ) The method comprises the steps of carrying out a first treatment on the surface of the Substitution of deionized water for H 2 O 2 The OD value was measured (denoted as A 2 ). The same method was used to determine the hydroxyl radical scavenging capacity of the same concentration of ascorbic acid as a positive control.
Superoxide anion radical: determination of the ability of DNPW to scavenge superoxide anion free radical Using NADH-NBT-PMS (reduced coenzyme I-phenazine methosulfate-azotetrazolium) System 1.0mL of polysaccharide solution was mixed with 1.0mL of 557. Mu. Mol/L NaDH (reduced coenzyme I disodium salt) -Na solution, 1.0mL of PMS (N-methylphenoxazine methosulfate) solution at 45. Mu. Mol/L, 1.0mL of NBT (nitrotetrazolium chloride) solution at 108. Mu. Mol/L, incubated at 25℃for 5min, zeroed with deionized water, and the OD (denoted A) was determined by spectrophotometry at 510nm 1 ) The method comprises the steps of carrying out a first treatment on the surface of the The polysaccharide solution was replaced with deionized water and its OD was measured (designated A 0 ) The method comprises the steps of carrying out a first treatment on the surface of the The NADH-NBT-PMS reaction solution was replaced with deionized water, and the OD value thereof was measured (designated A 2 ). The superoxide anion radical scavenging capacity of the same concentration of ascorbic acid was measured as a positive control by the same method.
ABTS radical: mixing 7 mmol/LABSS solution (2, 2' -dinitrogen-bis-3-ethylbenzothiazoline-6-sulfonic acid) and 2.45mmol/L ammonium persulfate solution, and oxidizing for 12h in dark to obtain ABTS mixed solution; the ABTS mixture was diluted with PBS solution at pH 7.4 to a absorbance of 0.80 at 734nm wavelength. Mixing 2mL of ABTS mixture with 1.0mL of polysaccharide solution, standing at room temperature for 6min, zeroing with deionized water, and measuring OD value (denoted as A) at 734nm 1 ) The method comprises the steps of carrying out a first treatment on the surface of the The polysaccharide solution was replaced with deionized water and its OD was measured (designated A 0 ) The method comprises the steps of carrying out a first treatment on the surface of the The ABTS mixture was replaced with deionized water and its OD was measured (designated A 2 ). Production of ascorbic acid at the same concentrationIts ABTS radical scavenging ability was determined as a positive control in the same manner.
3. Statistical analysis
Single-factor analysis of variance (one-way ANOVA) and IC using SPSS17.0 software 50 (half inhibitory concentration) and the results are expressed as mean.+ -. SD. The results of the radical scavenging rate measurements are shown in Table 2 and indicate that DNPW is effective on DPPH radicals, hydroxyl radicals (OH), superoxide anion radicals (OOH) - ) And ABTS radicals, and exhibit dose dependency.
TABLE 2 in vitro oxidative free radical scavenging results by DNPW
Experimental example 3 in vivo pharmacodynamic experiments of DNPW-nonalcoholic liver injury
1. Animal grouping, model building and dosing regimen
60 SPF-class SD male rats were fed with normal feed, were fed with free water and were foraged during one week. Following the adaptation period, the rats were examined for TG and TC levels in fasting serum and randomly aliquoted into a normal control group (C-CON), a normal polysaccharide-added high dose group (C-DNPW-H), a model group (C-HC), a positive control group (C-st+hc), a polysaccharide low dose group (C-DNPW-l+hc), and a polysaccharide high dose group (C-DNPW-h+hc), 10 each. The whole process of the groups C-CON and C-DNPW-H is used for giving normal feed, the other groups are replaced by high-sugar high-fat feed, the C-ST+HC groups are filled with 4mg/kg of simvastatin every day, and the C-DNPW-L+HC and C-DNPW-H groups are filled with 40mg/kg, 160mg/kg and 160mg/kg of dendrobium nobile polysaccharide DNPW every day. The canthus was bled the day before the end of the experiment. Before the last administration, the rats were fasted without water for 12h. Weighing, taking blood from abdominal aorta 30min after administration, dislocation, and killing. Heart, liver, epididymis and abdominal adipose tissues were taken and weighed.
2. Index measurement
Whole blood was collected in a clean 1.5mL centrifuge tube, left at room temperature for 2 hours, then placed in a refrigerator at 4 ℃ overnight, and after the blood coagulated blood clot, centrifuged at 3500×g for 10min, serum was isolated. Triglyceride (TG), total Cholesterol (TC), low density lipoprotein (LDL-C) and high density lipoprotein (HDL-C), AST (glutamic oxaloacetic transaminase), glutamic pyruvic transaminase (ALT) content in rat serum, malondialdehyde (MDA), superoxide dismutase (CAT), glutathione reductase (GSH-Px) and Catalase (CAT) content in serum are measured according to the specification of the kit.
The rat liver tissue was rinsed in pre-chilled physiological saline, the blood was removed, the surface water was blotted with filter paper, and weighed. Adding pre-cooled physiological saline with a mass concentration of 9 times of 0.9% into part of liver tissue, rapidly grinding in ice water bath in a glass homogenizer to obtain tissue homogenate, centrifuging at 12000 Xg for 15min at 4deg.C, and collecting supernatant. Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), and total antioxidant capacity (T-AOC) levels were measured according to kit instructions.
3. Liver histological examination
Liver tissue was obtained from the same portion of the right lobe of each rat liver at about 0.5 cm.times.1 cm, 10% formalin-fixed, dehydrated in ethanol, paraffin-embedded, sectioned (thickness 4-5 cm), hematoxylin and eosin stained (HE), and the degree of damage of the liver tissue was observed by using Nikon Eclipse E100 microscope manufactured by Nikon Corp.
4. Statistical method
One-way ANOVA (one-way ANOVA) was performed using SPSS7.0 software. Results are expressed as mean±sd. ## Represents p<0.01, with a significant difference compared to the normal control group; * represents p<0.05, with a significant difference compared to the model group, ** represents p<0.01, has a very significant difference compared with model group C-HC
5. Analysis of results
(1) Effect of DNPW on rat body weight and organ index
The results of analysis of the effect of DNPW on rat body weight and organ index are shown in Table 3. As can be seen from the data in table 3, the last body weight of the model group was significantly increased (p < 0.01) compared to the normal control group, and the last body weight of the rats was significantly decreased (p < 0.01) after treatment with different doses of dendrobium huoshanense polysaccharide DNPW; meanwhile, the weight of the rats in the C-DNPW-H group is obviously reduced compared with that of the rats in the model group, and the rats tend to be normal. However, the liver index of the rats in the model group is increased to a certain extent compared with that in the normal group, but the results are not obvious, the liver index of the rats is obviously increased (p < 0.05) compared with that in the model group after the treatment of the polysaccharide high-dose group, and the results show that the dendrobium nobile polysaccharide DNPW has a protective effect on the liver of the non-alcoholic liver injury rats.
TABLE 3 influence of DNPW on organ index and body weight in non-alcoholic liver injury rats
| Group of | Liver index (%) | Initial body weight (g) | Last body weight (g) |
| C-CON | 2.856±0.21 | 249.58±5.09 | 329.64±21.9 |
| C-HC | 2.622±0.31 | 242.60±8.21 | 323.78±18.8 ## |
| C-ST+HC | 3.306±0.22 | 245.51±7.22 | 371.7±22.3** |
| C-DNPW-H | 3.383±0.4 | 239.23±9.05 | 327.2±38.4 |
| C-DNPW-L+HC | 3.315±0.3 | 241.14±9.63 | 289.2±20.6 ## |
| C-DNPW-H+HC | 2.748±0.53* | 244.26±8.24 | 342.7±52.63 ## |
(2) Effect of DNPW on rat serum biochemical index
To evaluate the effect of dendrobe polysaccharide DNPW on a non-alcoholic liver injury rat model, the levels of ALT and AST in rat serum were tested, and the results are shown in Table 4, and compared with the C-CON group, ALT (p < 0.05) and AST (p < 0.01) are significantly increased after the C-HC group rats are continuously given high-sugar high-fat feed, which indicates that the liver of the non-alcoholic liver injury rat model is damaged; compared with the C-HC group, the ALT and AST levels of rats dosed with the dendrobium huoshanense polysaccharide DNPW are significantly reduced (p <0.05 and p < 0.01), and the liver damage degree is improved; meanwhile, in order to examine lipid metabolism level of the non-alcoholic liver injury rat, TC, TG, LDL-C, HDL-C level in serum is measured, and compared with C-CON group, TC, TG, LDL-C level in serum of the rat in the model group is obviously increased (p < 0.05), and HDL-C level is extremely obviously reduced (p < 0.01). Compared with the C-HC group, the positive control group, the polysaccharide low-dose group and the polysaccharide high-dose group can obviously reduce TC, TG, LDL-C level (p < 0.05), and meanwhile, the polysaccharide high-dose group can obviously raise HDL-C level, so that the dendrobium nobile polysaccharide DNPW in Henan can protect liver injury in a dose-dependent manner.
TABLE 4 influence of DNPW on the biochemical index of rat serum
(3) Effect of DNPW on oxidative stress index in rat serum
Table 5 shows the results of analysis of the effect of DNPW on the serum oxidative stress index of rats. As can be seen from table 5, serum SOD and GSH-Px, T-AOC, CAT activity were significantly reduced (p < 0.01) and MDA content was significantly increased (p < 0.01) compared to the C-CON group, indicating that the rats in the model group were subjected to oxidative damage; the positive control group, the polysaccharide low dose group and the polysaccharide high dose group significantly increased T-AOC, SOD, GSH-Px and CAT levels (p <0.05, p < 0.01) and significantly decreased MDA levels (p < 0.01) compared to the C-HC group, and furthermore, the content of each oxidative index enzyme of the C-DNPW-H group was similar to that of the normal control group, suggesting that the long-term supplementation of the dendrobium nobile polysaccharide did not affect the levels of these enzymes.
TABLE 5 influence of DNPW on oxidative stress index in rat serum
(4) Effect of DNPW on oxidative stress index in liver tissue
Table 6 shows the results of analysis of the effect of DNPW on oxidative stress index in liver tissue. As can be seen from table 6, SOD and GSH-Px, T-AOC, CAT activity was significantly reduced (p <0.01, p < 0.05) and MDA content was significantly increased (p < 0.01) in liver tissue of rats of C-HC group compared to C-CON group; compared with the C-HC group, the positive control group, the polysaccharide low-dose group and the polysaccharide high-dose group have the advantages of remarkably increasing T-AOC, SOD, GSH-Px and CAT levels (p <0.05 and p < 0.01) and extremely remarkably reducing MDA levels (p < 0.01), which suggests that the dendrobium nobile polysaccharide DNPW can improve liver injury of non-alcoholic liver injury rats.
TABLE 6 influence of DNPW on oxidative stress index in rat liver tissue
(5) HE staining analysis of influence of Dendrobium huoshanense on liver injury of non-alcoholic fatty liver rats
As can be seen from FIG. 7, the liver of FIG. 7, a C-CON and FIG. 5C-DNPW-H has normal histological features, clear nuclear structure, and the liver cells are radially arranged around the central vein, are polygonal, have prominent cell nuclei and have uniform cytoplasm; FIG. 7b C-HC group hepatocytes with mild oedema, mild steatosis and small inflammatory infiltrates, large lipid droplet vacuoles in the cytoplasm, these typical pathological features confirm the success of the long-term high-lipid induced non-alcoholic liver injury model; FIG. 7c C-ST+HC group hepatocytes were substantially normal with no apparent edema and lipid drop cavitation; fig. 7e and 7f show that the liver treated by the dendrobium nobile polysaccharide with low and high doses (DNPW-L and DNPW-H) has clear structure, regular arrangement and different degrees of alleviation of edema and lipid drop cavitation, and the dendrobium nobile polysaccharide DNPW has a certain improvement effect on nonalcoholic liver injury.
Experimental example 4 in vivo pharmacodynamic experiments of DNPW-D-galactose-induced oxidative damage
1. Animal grouping, model building and dosing regimen
70 male KM mice were fed with normal feed, drinking water and foraging during one week. After the adaptation period, the three groups were randomly divided into a normal Control group (Control), a D-galactose Model group (Model), a VC Positive Control group (Positive), a Henan dendrobium polysaccharide low dose group (NL), a Henan dendrobium polysaccharide high dose group (NH), a Henan dendrobium polysaccharide low dose group (HL) and a Henan dendrobium polysaccharide high dose group (HH), each group comprising 10 groups. Except for the normal control group, each group was intraperitoneally injected with 0.2mL of D-galactose (150 mg/kg) per day, and 100mg/kg of aqueous polysaccharide solution, 400mg/kg of aqueous polysaccharide solution, 100mg/kg of VC aqueous solution, and equal volumes of physiological saline were respectively administered to the Henan dendrobium polysaccharide low-dose and high-dose groups and the Henan dendrobium huoshanense low-dose and high-dose groups on the same day after the intraperitoneal injection of D-galactose. The experiment lasted 30 days and on day 31, all mice were weighed after fasting and water deprivation for 12h. Blood is taken from the canthus; dissecting after neck removal and sacrifice, taking brain, liver and kidney, and preserving at-80deg.C.
The dendrobium nobile polysaccharide adopted in the experiment is DNPW prepared in the example.
The preparation method of dendrobium huoshanense polysaccharide DHPW adopted in the experiment is as follows:
weighing 200g of dry dendrobium huoshanense stem raw material powder, oscillating and degreasing for 24 hours at room temperature by adopting ethanol with the mass concentration of 80%, filtering after finishing, retaining sediment, and volatilizing ethanol to obtain defatted material powder for later use; distilled water is added into defatted medicinal material powder according to a feed liquid ratio of 1:30 (g/mL), extraction is carried out for 2h at 80 ℃, the obtained sugar solution is concentrated to 600mL, alpha-amylase (the enzyme addition amount is 1 mu L/g based on the mass of dendrobium huoshanense stem raw medicinal material powder) is added, enzymolysis is carried out for 1h at 60 ℃ to remove starch, the sugar solution after starch removal is concentrated to 1/3 of the original volume, and the sugar solution is prepared according to the following steps: chloroform: n-butanol=30: 5: adding chloroform and n-butanol according to the volume ratio of 1 to remove protein, removing residual organic reagent, dialyzing with distilled water for 48h (dialysis bag cutoff molecular weight is 3500 Da), concentrating to 20mL, and freeze drying to obtain herba Dendrobii crude polysaccharide.
200mg of dendrobium huoshanense crude polysaccharide is weighed and is loaded on a DEAE-52 anion exchange column, ultrapure water and sodium chloride solution with the concentration of 0.1, 0.2, 0.3, 0.4 and 0.5mol/L are sequentially used for eluting, the eluting speed is 5mL/min, 20 tubes are collected for each eluting component, the OD value of each tube is measured by a phenol sulfuric acid method, a standard curve is drawn, the ultrapure water eluting component is taken for concentration, dialysis and freeze drying, and the ultrapure water eluting component polysaccharide is named DHPW.
The molecular weight of DHPW was determined to be 2.4X10 4 Is a heteropolysaccharide mainly comprising glucose and mannose, and the specific monosaccharide composition molar ratio is fucose (Fuc): rhamnose (Rha): arabinose (Ara): galactose (Gal): glucose (Glc): xylopyranose (Xyl): mannose (Man): galacturonic acid (Gla-UA): glucuronic acid (Glc-UA) =1: 19.5:25.0:37.0:280.0:6.0:107.5:21.0:3.5.
2. index measurement
Whole blood was collected in a clean 1.5mL centrifuge tube, left at room temperature for 2 hours, centrifuged at 3500×g for 10min, and serum was separated. Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) levels, malondialdehyde (MDA) levels and total antioxidant capacity (T-AOC) in mouse serum were determined according to kit instructions.
Mouse liver, brain and kidney tissues were rinsed in pre-chilled saline, blood was removed, surface water was blotted with filter paper, and weighed. Adding 9 times of 0.9% pre-cooled physiological saline into part of liver, brain and kidney tissue, rapidly grinding in ice water bath in a glass homogenizer to obtain tissue homogenate, centrifuging at 12000 Xg for 15min at 4deg.C, and collecting supernatant. SOD, GSH-Px, MDA and T-AOC levels were determined according to the kit instructions.
3. Statistical method
One-way ANOVA (one-way ANOVA) was performed using SPSS17.0 software. Results are expressed as mean±sd; p values less than 0.05 are considered statistically significant. # p<0.05， ## p<0.01 represents a significant difference and a very significant difference, respectively, compared with the normal control group; * P is p<0.05，**p<0.01 represents a significant difference and a very significant difference, respectively, from the model group; & p<0.05， && p<0.01 represents a significant difference and a very significant difference, respectively, compared to the high dose group of dendrobium huoshanense.
4. Analysis of results
FIGS. 8-11 show the effect of two dendrobe polysaccharides on SOD, MDA, GSH-PX and T-AOC in liver tissue, brain tissue, kidney tissue and serum. As can be seen from the figure, the MDA levels in the liver tissue, kidney tissue, brain tissue and serum of the mice in the model group were significantly increased (p <0.05, p < 0.01) compared to the normal group, and the antioxidant enzyme SOD, GSH-PX activity and total antioxidant capacity T-AOC were significantly decreased (p <0.05, p < 0.01) compared to the normal group. Compared with a model group, the two polysaccharide groups can obviously reduce the MDA level, improve the activities of antioxidant enzymes SOD and GSH-PX and the total antioxidant capacity, which indicates that the Henan dendrobium polysaccharide and the Dendrobium huoshanense polysaccharide can improve D-Gal induced oxidative damage, wherein most index results of the Henan dendrobium polysaccharide and the Dendrobium huoshanense polysaccharide in different organs/serum have no obvious difference, which indicates that the Henan dendrobium polysaccharide also has the potential effect of improving D-Gal induced oxidative damage, has the effect of antioxidant stress, and has obvious price advantage so that the Henan dendrobium polysaccharide has wider application prospect.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.
Claims (10)
1. The preparation method of the dendrobium nobile polysaccharide is characterized by comprising the following steps:
(1) Degreasing raw medicinal material powder of dendrobium huoshanense, and drying to obtain degreased medicinal material powder;
(2) Extracting the defatted medicinal material powder by adopting a water extraction and alcohol precipitation method to obtain an alcohol precipitate;
(3) Taking the alcohol sediment for removing starch, deproteinizing and dialyzing to obtain coarse dendrobium nobile polysaccharide;
(4) And (3) performing primary purification on the dendrobium huoshanense crude polysaccharide by using a DEAE-52 anion exchange chromatographic column, performing secondary purification on the component polysaccharide eluted by ultrapure water by using a Sephadex-G gel column, collecting the corresponding eluted component, concentrating and freeze-drying to obtain the dendrobium huoshanense polysaccharide.
2. The method for preparing dendrobium nobile polysaccharide according to claim 1, wherein in the step (1), degreasing treatment is performed by adopting ethanol with the volume concentration of 50% -90%, preferably ethanol with the volume concentration of 80%; the mass volume ratio of the dendrobium huoshanense raw material powder to the ethanol is 1-1.5 in terms of g/mL: 2 to 6, preferably 1:4, a step of; adding ethanol into the raw medicinal powder of dendrobium huoshanense, and vibrating for 12-48 hours, preferably 24 hours at room temperature.
3. The method for preparing dendrobium nobile polysaccharide according to claim 1, wherein in the step (2), in the water extraction process, the feed liquid ratio of defatted medicinal material powder to water is 1 in terms of g/mL: 10 to 50, preferably 1:30; the extraction temperature is 60-90 ℃, preferably 80 ℃; the extraction time is 1-3 h, preferably 2h; in the alcohol precipitation process, the water extract is decompressed and concentrated to 1/4 to 1/8 of the original volume, preferably 1/5; ethanol is added to a final concentration of 60 to 80 percent, preferably 80 percent, and the mixture is kept stand for 12 to 48 hours, preferably 24 hours.
4. The method for preparing dendrobium nobile polysaccharide according to claim 1, wherein in the step (3),
in the process of removing starch, taking the alcohol precipitate for redissolution, and then adding alpha amylase for enzymolysis, wherein the adding amount of the alpha amylase is 1-3 mu L/g, preferably 1 mu L/g, based on the mass of the raw medicinal material powder of dendrobium huoshanense; the enzymolysis temperature is 30-60 ℃, preferably 60 ℃; the enzymolysis time is 0.5-3 h, preferably 1h; concentrating the enzymolysis liquid to 1/2-1/6, preferably 1/3, of the original volume after enzymolysis, centrifuging to remove starch and other insoluble precipitates, and obtaining starch-removed sugar liquid;
in the deproteinization process, the volume ratio of the starch removing sugar solution to chloroform to n-butanol is 20-50: 2-10: shaking vigorously for 1-5 h after mixing 0.5-2, centrifuging to remove protein layers, repeating for 3-10 times, and removing residual organic solvent in the sugar solution under reduced pressure, wherein the preferable volume ratio is 30:5:1, violently shaking for 2 hours, and repeating for 7 times;
in the dialysis process, a dialysis bag with the molecular weight cut-off of more than 3500Da is adopted for running water dialysis for 2-3 days, preferably 2 days, deionized water is further used for dialysis for 12-48 hours, preferably 24 hours, and the concentrated solution is subjected to vacuum freeze drying, so that the dendrobium nobile polysaccharide is obtained.
5. The method for preparing dendrobium nobile polysaccharide according to claim 1, wherein in the step (4),
in the primary purification process, the method sequentially uses ultrapure water and sodium chloride solution with the concentration from low to high to elute at the elution speed of 5-8 mL/min, preferably 5mL/min;
in the secondary purification process, a Sephadex-G100 gel column is adopted, and the elution is carried out by ultrapure water, wherein the elution speed is 2-3 mL/min, and preferably 2mL/min.
6. The method for preparing dendrobium nobile polysaccharide according to claim 1, wherein in the step (4),
in the primary purification process, collecting the ultrapure water eluting component, concentrating, dialyzing and freeze-drying to obtain the ultrapure water eluting component polysaccharide;
in the second-stage purification process, the elution components of the left and right tubes with the highest absorbance value of the elution curve are collected.
7. A dendrobe polysaccharide obtained by the method of any one of claims 1 to 6.
8. The dendrobium nobile polysaccharide of claim 7, wherein,
the molecular weight of the dendrobium huoshanense polysaccharide is 2 multiplied by 10 4 ～9×10 4 Da, preferably 8X 10 4 Da；
The monosaccharide composition of the dendrobium huoshanense polysaccharide comprises fucose, galactose, glucose and mannose, wherein the molar ratio of the fucose, the galactose, the glucose and the mannose is 0.221:0.02:0.32:0.403.
9. the application of the dendrobium nobile polysaccharide prepared by the method of any one of claims 1 to 6 or the dendrobium nobile polysaccharide of claim 7 or 8 in preparing hypolipidemic and/or antioxidant drugs.
10. Use of a dendrobium nobile polysaccharide prepared by the method of any one of claims 1 to 6 or a dendrobium nobile polysaccharide of claim 7 or 8 in the preparation of a medicament for treating non-alcoholic liver injury and/or treating oxidative stress.
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