CN108456259A - Application of the anoectochilus roxburghii polyose in acute alcohol-induced hepatic injury drug - Google Patents
Application of the anoectochilus roxburghii polyose in acute alcohol-induced hepatic injury drug Download PDFInfo
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Abstract
The invention discloses a kind of application of anoectochilus roxburghii polyose in acute alcohol-induced hepatic injury drug, high-speed countercurrent chromatography purifies anoectochilus roxburghii polyose（ARPS）, then dialysis desalting purifying, is freeze-dried to obtain roxburgh anoectochilus terminal bud holosaccharide（ARPS）.The ARPS molecular weight is 22090 Da, is mainly made of mannose, ribose, glucose, galactolipin and arabinose, and the molar ratio of each monosaccharide is:Mannose：Ribose：Glucose：Galactolipin：Arabinose=1.00：8.47：47.30：1.17：1.19.Glycosidic bond is α configurations, and triple helix conformation, purity 95.01% is not present.ARPS to the protective effect of acute Alcoholic Hepatic Injury the experimental results showed that：40 mg/kg and 80 mg/kg anoectochilus roxburghii polyose dosage protection groups can reduce ALT, and AST is horizontal, increase the content of GSH and SOD in liver, reduce MDA, NO, TG, H2O2With the content of TNF α, the activity of NOS is reduced, there is protective effect to alcohol-induced acute liver.
Description
Technical field
The present invention relates to a kind of application of anoectochilus roxburghii polyose drug in acute alcohol-induced hepatic injury protective effect.
Background technology
The livers property disease such as hepatitis, liver fibrosis and hepatic sclerosis is increasingly prominent, and never good drug can be thorough
Treatment has been one of emphasis, the hot spot in medical research field since the research unanimously of hepatic.In particular with people's life water
Flat raising, popularity of wine increase year by year, the incidence of alcoholic liver disease (Alcoholic liver disease, ALD)
It is in rising trend, thus the drug for studying prevention ALD is very necessary.
Alcoholic liver disease pathogenesis is complex, is not fully understood at present, but ethyl alcohol and its metabolite are to liver
Toxic effect, oxidative stress and TNF-α abnormal secretion be one of the main reasons.Ethyl alcohol is metabolized again in liver, most of
It is metabolized as acetaldehyde by alcohol dehydrogenase, then acetic acid is metabolized as by acetaldehyde dehydrogenase, is finally metabolized as carbon dioxide and water, second
Alcohol can also pass through Cytochrome P450（cytochrome-450）Approach is metabolized, and CYP450 enzyme systems are most important in hepatomicrosome
Mixed-function oxidase, CYP450 enzymes are there are many hypotype：CYP1A, CYP3A, CYP2E1 and CYP2C19 etc., two kinds of metabolism
Approach can generate a large amount of active oxygen species ROS, and the polyunsaturated fatty acid that ROS can attack membrane phospholipid forms ester matter
Peroxide such as malonaldehyde（MDA）, active oxygen is not only converted to activity chemistry agent by ester matter peroxide, but also anti-by chain type
The effect that active oxygen should be amplified causes mitochondrial membrane and cell membrane damage, membrane fluidity to decline to damage liver cell, 80 %
ALT and AST are present in mitochondria, and the activity in serum is relatively low under normal circumstances, when mitochondria and cell membrane damage, ALT
Cell is disengaged with AST and enters blood, and the activity of serum alt and AST is caused to increase.Due to the aggregation of active oxygen, cause liver thin
The antioxidant consumption of born of the same parents increases, and SOD and GSH is made to decline.A large amount of TG is accumulated in liver with low-density lipoprotein（Very
Low desity lipoprotein, VLDL）Form transport liver, into blood, the TG contents in blood is made accordingly to increase.
LPS is mainly bacterial secretory in enteron aisle, can increase the permeability of intestinal epithelial cell after heavy drinking, space between cells is caused to increase
Add, microvillus deformation and the morphological changes such as mitochondria swelling, therefore LPS largely enters blood, then by portalsystem into
Enter liver.Bacteria lipopolysaccharide（LPS）The free radical and MDA generated with metabolism of the ethyl alcohol in liver can activate Kuffer thin
Born of the same parents make the inflammatory factors such as its TNF secretion-α.The cytokine profiles such as LPS, TNF-α can induce the expression of NOS, generate a large amount of
The NO of NO, high concentration can inhibit synthesis and the damage dna structure of Hepatocyte matter, directly inhibit mitochondrial respiratory, keep liver thin
Intracellular energy metabolism impairment and cause hepatocellular apoptosis and necrosis.
Invention content
The purpose of the present invention is to provide a kind of anoectochilus roxburghii polyose drug and its in acute alcohol-induced hepatic injury protective effect
In application, anoectochilus roxburghii polyose to the protective effect of acute Alcoholic Hepatic Injury the experimental results showed that：40 mg/kg and 80
Mg/kg anoectochilus roxburghii polyose dosage protection groups can reduce ALT, and AST is horizontal and reduces the content of TG, increase in liver GSH and
The content of SOD reduces MDA, NO, H2O2With the content of TNF-α, the activity of NOS is reduced, alcohol-induced chmice acute liver is damaged
Wound has protective effect.
Anoectochilus roxburghii polyose of the present invention, is extracted using water extraction and alcohol precipitation method, extraction conditions：Roxburgh anoectochilus terminal bud herb dried and crushed
70 mesh sieve is crossed, several grams of roxburgh anoectochilus terminal bud herb powder is weighed, is 1 by the feed liquid mass ratio of roxburgh anoectochilus terminal bud herb powder and distilled water:10~
1:20 are added distilled water, in 50 DEG C ~ 60 DEG C ultrasonic extraction 30 min, 4200 r/min 15 min of centrifugation, take supernatant, repeat
It carries 2 times, merges extracting solution rotary evaporation three times and be concentrated into removing protein after small size, obtain small size concentrate, body is then added
Product is the absolute ethyl alcohol of 3 ~ 5 times of the small size volume of the concentrated liquid, alcohol precipitation, and sediment is collected by centrifugation in 4 DEG C of overnight precipitations of deepfreeze,
It is freeze-dried to constant weight.Establish high-speed countercurrent chromatography purifying anoectochilus roxburghii polyose（ARPS）Purifying process, then with film dialyse into
One step desalting and purifying.The condition of HSCCC is：Double-aqueous phase system is 12.5%PEG1000-20% potassium hydrogen phosphates（pH=6.8）, rotating speed
For 850 r/min, upper phase（Mobile phase）Flow velocity be 1.0 mL/min.5% phend-sulphuric acid tracing detection, merges identical group
Point, low pressure rotary evaporation is concentrated into small size, is purified with the further dialysis desalting of the film of M12000 molecular cut offs, using molybdic acid
Whether phosphate dialyses completely in ammonium detection dialyzate, merges the component in bag filter, and rotary evaporation concentration is freeze-dried, i.e.,
Obtain anoectochilus roxburghii polyose.Anoectochilus roxburghii polyose purity 95.01% is measured, molecular weight is 22090 Da, mainly by mannose, ribose, Portugal
Grape sugar, galactolipin and arabinose composition, and the molar ratio of each monosaccharide is:Mannose：Ribose：Glucose：Galactolipin：I
Uncle sugar=1.00：8.47 ：47.30 ：1.17 ：1.19.Glycosidic bond is α configurations, triple helix conformation is not present, and under AFM mirrors
Observation shows close agglomerating, the spheroidal of its molecule aggregation, and diameter is about 120 ~ 380 nm.The acquisition of anoectochilus roxburghii polyose is follow-up
Material base has been established in experiment.
Anoectochilus roxburghii polyose of the present invention is preparing the application in treating acute alcohol-induced hepatic injury drug.
A kind of two innovative points of application of anoectochilus roxburghii polyose drug of the present invention in acute alcohol-induced hepatic injury protective effect：
Innovative point one：Using lentinan as positive controls, make experiment more scientific and precise.
Innovative point two：High low dosage anoectochilus roxburghii polyose dosage protection group is devised, and in terms of Mechanism Study relatively comprehensively
Rationally, activity research generally is carried out to roxburgh anoectochilus terminal bud crude extract in document, and traditional Chinese medicine ingredients are more complicated, gold of the present invention
Line lotus purity of polysaccharide is up to 95.01%, and activity research is more convincing.
Description of the drawings
Fig. 1 is influence diagram of the anoectochilus roxburghii polyose to alcoholic liver injury mouse liver body index.Compared with blank control group,#
P <0.05；Compared with model control group,*P<0.05,n=8。
Fig. 2 is the level view of serum alt.Compared with blank control group,#P <0.05；Compared with model control group,*P<
0.05,n=8。
Fig. 2-1 is the level view of AST in serum.Compared with blank control group,#P <0.05；Compared with model control group,*
P<0.05,n=8。
Fig. 3 is TG level views in mice serum.Compared with blank control group,#P <0.05；Compared with model control group,*P
<0.05,n=8。
Fig. 4 is that the oxidative stress GSH of anoectochilus roxburghii polyose contains spirogram.Compared with blank control group,#P <0.05；With
Model control group compares,*P<0.05,n=8。
Fig. 4-1 is the oxidative stress SOD vigor figures of anoectochilus roxburghii polyose.Compared with blank control group,#P <0.05；
Compared with model control group,*P<0.05,n=8。
Fig. 5 is influence diagram of the anoectochilus roxburghii polyose to ester matter peroxidating.Compared with blank control group,#P <0.05；With model
Control group compares,*P<0.05,n=8。
Fig. 6 is anoectochilus roxburghii polyose to H2O2The influence diagram of concentration.Compared with blank control group,#P <0.05；With model pair
Compare according to group,*P<0.05,n=8。
Fig. 7 is influence diagram of the anoectochilus roxburghii polyose to TNF-α concentration.Compared with blank control group,#P <0.01；With model
Control group compares,*P<0.05,n=8。
Fig. 8 is influence diagram of the anoectochilus roxburghii polyose to total NOS vigor.Compared with blank control group,#P <0.05；With model
Control group compares,*P<0.05,n=8。
Fig. 8-1 is influence diagram of the anoectochilus roxburghii polyose to NO contents.Compared with blank control group,#P <0.05；With model pair
Compare according to group,*P<0.05,n=8。
Fig. 9 observes for hepatic tissue HE coloring pathological sections（×100）Scheme, a in figure：NC；b: ALI；c: 40 mg/
kgARPS-ALI；d: 80 mg/kgARPS-ALI；e: 100 mg/kgLentian-ALI.
Figure 10 is protective effect figure of the anoectochilus roxburghii polyose to alcoholic liver injury.
Specific implementation mode
With reference to embodiment, the present invention will be described in detail：
1.1 laboratory apparatus and experiment material
1.1.1 laboratory apparatus
TGL-16C high speed tabletop centrifuges（Anting Scientific Instrument Factory, Shanghai）；TDL-40B desk centrifuges（Town in Shanghai pavilion section
Learn instrument plant）；S648 electric heating constant-temperature water-bath tanks（Seven factory of Shanghai Medical instrument）；UV-250 ultraviolet-uisible spectrophotometers（Day
This Shimadzu）；90-2 time constant-temperature magnetic stirring apparatus（Industrial Co., Ltd. of upper Nereid section）；KQ-500DB type numerical control supersonics are clear
Wash device（Kunshan Ultrasonic Instruments Co., Ltd.）；OptimaTM, LE-80K ultracentrifuge（Beckman Coulter are public
Department）；Microfuge22R refrigerated centrifuges（Beckman companies）；The 1500 type MULISKAN SPECTRUM microplate reader U.S.
（Thermo scientfic companies）；CK40-F200 inverted microscopes（Olymbus optical CO.LTD,Japan）；
BX41TF microscopes（Olymbus optical CO. LTD, Japan）；DP70 digital vedio recording micro-image systems
（Olymbus optical CO. LTD, Japan）;
Experiment material
Roxburgh anoectochilus terminal bud holosaccharide（Purity 95.01%, self-control）；Lentinan（Purity 75%, U.S. logical sequence biology）；It is saturated picric acid, the third ammonia
Sour transaminase (ALT) kit, aspartic transaminase (AST) kit, BCA method protein quantifications kit, superoxides
Mutase (SOD) kit, malonaldehyde (MDA) kit, micro reduced glutathione (GSH) kit, nitric oxide close
Enzyme (NOS) assay kit, tumor necrosis factor-alpha (TNF-α) enzyme-linked immunologic detecting kit, TG assay kits（Enzyme is even
Join colorimetric method/single reagent type）, haematoxylin, eosin stain anticreep glass slide and coverslip（Bioengineering Research Institute is built up in Nanjing）；
58 DEG C ~ 60 DEG C of colleges and universities' paraffin wax fusing point（Shanghai Hua Shen rehabilitation materials Co., Ltd）；Dimethylbenzene, sodium chloride and formalin
（Analyze pure, Xilong Chemical Co., Ltd）；Absolute ethyl alcohol（Analyze pure, Sinopharm Chemical Reagent Co., Ltd.）；Kunming ♂
Mouse 18-22g and mouse high pressure feed（Wu Shi Experimental Animal Centers）.
Above-mentioned roxburgh anoectochilus terminal bud holosaccharide（Purity 95.01%）Polysaccharide in roxburgh anoectochilus terminal bud is extracted using following water extraction and alcohol precipitation methods,
The specific steps are：Extraction conditions：Roxburgh anoectochilus terminal bud herb dried and crushed crosses 70 mesh sieve, weighs several grams of roxburgh anoectochilus terminal bud herb powder, down payment
The feed liquid mass ratio of line lotus herb powder and distilled water is 1:10~1:20 are added distilled water, in 50 DEG C ~ 60 DEG C ultrasonic extractions 30
Min, 4200 r/min centrifuge 15 min, and supernatant, repetition is taken to carry 2 times, merge extracting solution rotary evaporation three times and are concentrated into corpusculum
Removing protein after product obtains small size concentrate, and it is the absolute ethyl alcohol of 3 ~ 5 times of the small size volume of the concentrated liquid, alcohol that volume, which is then added,
Heavy, sediment, freeze-drying to constant weight is collected by centrifugation in 4 DEG C of overnight precipitations of deepfreeze.Establish high-speed countercurrent chromatography purifying
Anoectochilus roxburghii polyose（ARPS）Purifying process, then with the further desalting and purifying of film dialysis.The condition of HSCCC is：Double-aqueous phase system
For 12.5%PEG1000-20% potassium hydrogen phosphates（pH=6.8）, rotating speed is 850 r/min, upper phase（Mobile phase）Flow velocity be 1.0
mL/min.5% phend-sulphuric acid tracing detection merges same composition, and low pressure rotary evaporation is concentrated into small size, is cut with M12000
The further dialysis desalting purifying of the film of molecular weight is stayed, detects whether phosphate in dialyzate dialyses completely using ammonium molybdate, merges
Component in bag filter, rotary evaporation concentration, is freeze-dried to get anoectochilus roxburghii polyose.Anoectochilus roxburghii polyose purity 95.01% is measured,
Molecular weight is 22090 Da, is mainly made of mannose, ribose, glucose, galactolipin and arabinose, and each monosaccharide rubs
You are at ratio:Mannose：Ribose：Glucose：Galactolipin：Arabinose=1.00：8.47 ：47.30 ：1.17 ：1.19.Glucosides
Key is α configurations, triple helix conformation is not present, and AFM shows close agglomerating, the spheroidal of its molecule aggregation under the microscope, directly
Diameter is about 120 ~ 380 nm.The acquisition of anoectochilus roxburghii polyose is that subsequent experimental has established material base；
1.2 experimental method
1.2.1 weighing, dispensing
95.01% anoectochilus roxburghii polyose of above-mentioned purity is directly used in following administration applications；
1.2.2 grouping and administration
Kunming ♂ mouse 40, weight about 18 ~ 22g, it is more that adaptability is randomly divided into blank group, model group, roxburgh anoectochilus terminal bud after raising 3 days
It is sugared high（80 mg/kg）, it is low（40 mg/kg）Dosage protection group, lentinan（100 mg/kg）Positive controls, every group 8.
After applicability is raised 3 days, gastric infusion, once a day, the administration capacity of 0.2 mL/10g, blank group and model group are given and wait bodies
Long-pending distilled water, model group, roxburgh anoectochilus terminal bud holosaccharide are high after 3 h（80 mg/kg）, it is low（40 mg/kg）Dosage protection group, mushroom are more
Sugar（100 mg/kg）50% ethyl alcohol of positive controls gavage（12 mL/kg）, continuous 9 days, after last gavage, it is deprived of food but not water, 12
It weighs after h, cuts mouse beard, eyeball takes blood, 4000 r/min to centrifuge 15 min and take supernatant, surveys ALT, AST and TG.Mouse
It is rapid to dissect after disconnected neck is put to death, ligamentum falciforme hepatis is cut, mouse is taken out and takes liver, check the shape and color and luster of liver, whether there is or not fill
Blood, bleeding, extravasated blood and fat lesion etc., weigh, and separately take 0.3g hepatic tissues, are cleaned with physiological saline, and 0.9% physiological saline is made
10% liver homogenate surveys liver protein content, GSH, SOD, MDA, NOS, NO and TNF-α etc..Cut each one piece 3 of intermediate leaf, left outside leaf
Mm is thick, and 10% neutral formalin fixes 24 h or so, makees paraffin section, and HE dyeing makes pathology of hepar sections observation, remains
- 70 DEG C of remaining liver organization saves backup.
Biochemical Indices In Serum detects
Serum alanine aminotransferase (ALT), aspartate amino transferase (AST) are surveyed by kit method (reitman-frankel method)
Content, by kit method (GPO-PAP enzyme process) survey serum triglyceride (TG) content.
Hepatic tissue biochemical indicator detects
10% liver tissue homogenate surveys hepatic tissue protein content, GSH, SOD, MDA, NOS, NO, H by kit method2O2And TNF-
α etc..Malonaldehyde (MDA) uses thiobarbituricacidα- (TBA) method, superoxide dismutase (SOD) to use xanthine oxidizing process,
H2O2Using molybdic acid development process, TNF-α is using biotin Double antibody sandwich-ELISA (ELISA) method.
Pathology of hepar is observed
Materials with it is fixed to cut each one piece of 3 mm of leaf, left outside leaf among mouse liver thick, put at once physiological saline rinse with
Drift removes peripheral blood, removes adipose tissue etc. extra around tissue, and 20 times of 10% neutral formalins of tissue volume fix the left sides 24 h
The right side, distilled water rinse twice, it are carefully pressed from both sides out, marks and is fitted into embedded box.
Murine liver tissue dehydration is transparent and waxdip detailed process is shown in Table 1.
1 mouse liver of table is dehydrated transparent waxdip flow
Table1 The process of dehydration,transparentation and soaked wax
After paraffin embedding, slice, exhibition piece and baking piece paraffin wax embedding embedding, cooling slice, slice thickness is 4 μm；40
DEG C ~ 43 DEG C of water-baths exhibition pieces, the processed anticreep glass slide fishing piece of poly-D-lysine, 60 DEG C of 4 h or 37 DEG C of roasting pieces are overnight.
Dewaxing water suction, HE dyeing detailed process are shown in Table 2.
2 mouse liver of table dewaxing water suction HE dyeing flows
Table2 The process of dewaxing,soaked water and HE staining
Mounting and microscopy
1.3 result
Roxburgh anoectochilus terminal bud holosaccharide is to the protective effect of alcoholic liver injury as a result, following blank group is indicated with NC；Model group ALI tables
Show；Roxburgh anoectochilus terminal bud holosaccharide low dosage（40 mg/kg）Protection group and high dose（80 mg/kg）Protection group group is respectively with 40 mg/kg
ARPS-ALI and 80 mg/kg ARPS-ALI are indicated；Lentinan（100 mg/kg）100 mg/kg mushrooms of positive controls
Polysaccharide-ALI is indicated；
1.3.1 influence of the anoectochilus roxburghii polyose to alcoholic liver injury mouse liver index
Independent samples t test is carried out with SPSS20.0 softwares, is test stone with α=0.05, the results are shown in Table 3 and Fig. 1, with blank
Group is compared, and model group liver body index significantly increases（P ﹤ 0.01）Illustrate modeling success；Compared with model group roxburgh anoectochilus terminal bud holosaccharide it is high,
Low dose group can reduce liver body index（P ﹤ 0.01）.
3 anoectochilus roxburghii polyose of table to alcoholic liver injury mouse liver body index influence ()
Table3 Effect of thee ARPS on hepatosomatic index in alcoholic
liverinjury mice ()
Note：Compared with blank control group,*P<0.05,**P<0.01；Compared with model control group,△P<0.05,△△P<0.0l,n=
8
1.3.2 influence of the anoectochilus roxburghii polyose to alcoholic liver injury mice serum ALT and AST level
Independent samples t test is carried out with SPSS20.0 softwares, is test stone with α=0.05, the results are shown in Table 4 and Fig. 2, Fig. 2-1,
Compared with blank group, AST, ALT in model group serum are significantly increased（P ﹤ 0.05）, illustrate modeling success；It is compared with model group
Roxburgh anoectochilus terminal bud holosaccharide height and low dose group can reduce ALT and AST（P ﹤ 0.05）.
4 serum alt of table and AST it is horizontal ()
Table4 Serum levels of ALT and AST ()
Note：Compared with blank control group,*P<0.05,**P<0.01；Compared with model control group,△P<0.05,△△P<0.0l,n=
8
1.3.3 serum triglyceride（TG）Horizontal influence
Independent samples t test is carried out with SPSS20.0 softwares, is test stone with α=0.05, the results are shown in Table 5 and Fig. 3, with blank
Group is compared, and model group TG contents significantly increase（P ﹤ 0.05）；It is compared with model group, roxburgh anoectochilus terminal bud holosaccharide height and low dose group
Significantly reduce TG contents in liver organization（P ﹤ 0.05, P ﹤ 0.01）.
5 anoectochilus roxburghii polyose of table on alcoholic liver injury mice serum TG levels influence ()
Table5 Effect of the ARPS on serum level of TG in alcoholic liver
injurymice ()
Note：Compared with blank control group,*P<0.05,**P<0.01；Compared with model control group,△P<0.05,△△P<0.0l,n=
8
1.3.4 influence of the anoectochilus roxburghii polyose to alcoholic liver injury mouse liver GSH and SOD content
Independent samples t test is carried out with SPSS20.0 softwares, is test stone with α=0.05, the results are shown in Table 6 and Fig. 4, Fig. 4-1,
Compared with blank group, GSH, SOD in model group hepatic tissue are significantly reduced（P ﹤ 0.05）；High low dose group is compared with model group can
It is horizontal to increase GSH and SOD（P ﹤ 0.05）.
6 anoectochilus roxburghii polyose of table to GSH, SOD value of alcoholic liver injury protective effect ()
Table6 GSH and SOD of the mode of the mice alcoholic liver damage()
| Group | GSH(µmol/gprot) | SOD(U/mgprot) |
| NC | 2.233±0.334 | 159.148±28.395 |
| ALI | 1.674±0.320* | 120.989±36.810* |
| 40 mg/kgARPS-ALI | 2.884±0.453△ | 159.095±46.432△ |
| 80 mg/kgARPS-ALI | 2.989±0.741△ | 175.330±54.243△ |
| 100 mg/kg lentinans-ALI | 2.234±0.695△ | 178.441±42.394△ |
Note：Compared with blank control group,*P<0.05,**P<0.01；Compared with model control group,△P<0.05,△△P<0.0l,n=
8
1.3.5 influence of the anoectochilus roxburghii polyose to alcoholic liver injury mouse liver MDA levels
Independent samples t test is carried out with SPSS20.0 softwares, is test stone with α=0.05, the results are shown in Table 7 and Fig. 5, with blank
Group is compared, and model group MDA contents significantly increase（P ﹤ 0.05）；Roxburgh anoectochilus terminal bud holosaccharide height and low dose group are compared with model group
Reduce MDA contents in liver（P ﹤ 0.05）.
7 anoectochilus roxburghii polyose of table to alcoholic liver injury mouse MDA levels influence ()
Table7 Effect of the ARPS on MDA in alcoholic liver injury mice ()
| Group | MDA contents（nmol/mgprot） |
| NC | 2.578±0.536 |
| ALI | 4.694±0.800* |
| 40 mg/kgARPS-ALI | 2.493±0.507△ |
| 80 mg/kgARPS-ALI | 2.595±0.474△ |
| 100 mg/kg lentinans-ALI | 2.687±0.242△ |
Note：Compared with blank control group,*P<0.05,**P<0.01；Compared with model control group,△P<0.05,△△P<0.0l,n=
8
1.3.6 anoectochilus roxburghii polyose is to alcoholic liver injury mouse liver H2O2Horizontal influence
Independent samples t test is carried out with SPSS20.0 softwares, is test stone with α=0.05, the results are shown in Table 8 and Fig. 6, with blank
Group is compared, model group H2O2Content significantly increases（P ﹤ 0.05）；Roxburgh anoectochilus terminal bud holosaccharide height and low dose group are compared with model group
Reduce H in liver2O2Content（P ﹤ 0.05）.
8 anoectochilus roxburghii polyose of table is to alcoholic liver injury mouse H2O2Horizontal influence ()
Table8 Effect of the ARPS on H2O2 in alcoholic liverinjury mice ()
Note：Compared with blank control group,*P<0.05,**P<0.01；Compared with model control group,△P<0.05,△△P<0.0l, n=
8
1.3.7 influence of the anoectochilus roxburghii polyose to alcoholic liver injury mouse liver TNF-α level
Independent samples t test is carried out with SPSS20.0 softwares, is test stone with α=0.05, the results are shown in Table 9 and Fig. 7, with blank
Group is compared, and model group TNF-α content significantly increases（P ﹤ 0.01）；It is compared with model group, the high low dose group of roxburgh anoectochilus terminal bud holosaccharide is equal
TNF-α content in liver can be reduced（P ﹤ 0.05）.
9 anoectochilus roxburghii polyose of table to alcoholic liver injury mouse TNF-α level influence ()
Table9 Effect of the ARPS on TNF-α inalcoholic liver injury mice ()
| Group | TNF-α content（ng/L） |
| NC | 55.451±6.314 |
| ALI | 74.561±4.693** |
| 40 mg/kgARPS-ALI | 58.760±7.811△△ |
| 80 mg/kgARPS-ALI | 60.412±3.793△ |
| 100 mg/kg lentinans-ALI | 54.642±7.451△△ |
Note：Compared with blank control group,*P<0.05,**P<0.01；Compared with model control group,△P<0.05,△△P<0.0l,n=
8
1.3.8 the influence of anoectochilus roxburghii polyose NOS total to alcoholic liver injury mouse liver and NO levels
Independent samples t test is carried out with SPSS20.0 softwares, is test stone with α=0.05, the results are shown in Table 10 and Fig. 8, Fig. 8-1,
Compared with blank group, the total NOS vigor of model group and NO contents significantly increase（P ﹤ 0.05）；Roxburgh anoectochilus terminal bud holosaccharide is compared with model group
High low dose group can reduce NOS vigor and NO contents in liver（P ﹤ 0.05）.
10 anoectochilus roxburghii polyose of table NOS levels total to alcoholic liver injury mouse influence ()
Table10 Effect of the ARPS on total NOS in alcoholic liver injury mice ()
| Group | NO contents（µmol/gprot） | Total NOS vigor（U/mgprot） |
| NC | 0.745±0.120 | 0.287±0.118 |
| ALI | 1.061±0.261* | 0.916±0.123* |
| 40 mg/kgARPS-ALI | 0.809±0.131△ | 0.308±0.133△ |
| 80 mg/kgARPS-ALI | 0.824±0.149△ | 0.376±0.192△ |
| 100 mg/kg lentinans-ALI | 0.929±0.137△ | 0.249±0.086△ |
Note：Compared with blank control group,*P<0.05,**P<0.01；Compared with model control group,△P<0.05,△△P<0.0l,n=
8
1.3.9 pathology of hepar is observed
The hepatic tissue cell arrangement of blank control group mouse is more neat as shown in Figure 9, and the arrangement of remaining each group liver cell is relatively dredged
Pine, occurs cavitation phenomena, and each group is showed no inflammatory cell and invades profit.Roxburgh anoectochilus terminal bud holosaccharide high and low dose group is compared with model group
There is different degrees of improvement result.
Anoectochilus roxburghii polyose concludes Figure 10 to the Protective effects of alcoholic liver injury.Anoectochilus roxburghii polyose damages alcoholic liver
The Protective effects of wound：The expression that may be by inhibiting CYP2E1 enzymes, reduces the generation of active oxygen species, reduces mitochondria
Film and cell membrane damage, to have the function that protect liver organization.
It discusses
Experiments have shown that 40 mg/kg and 80 mg/kg roxburgh anoectochilus terminal bud holosaccharide dosage groups can reduce ALT, AST is horizontal and reduces TG
Content, increase liver in GSH and SOD content, reduce MDA, NO, H2O2With the content of TNF-α, the activity of NOS, liver are reduced
Dirty pathological study shows that the hepatic tissue cell arrangement of blank control group mouse is more neat, and remaining each group liver cell arranges
Than more loose, cavitation phenomena is occurred, each group, which is showed no inflammatory cell, invades profit and compare each dosage of roxburgh anoectochilus terminal bud holosaccharide with model group
Group has different degrees of improvement result.The high low dosage protection group of anoectochilus roxburghii polyose has alcohol-induced acute liver
There is protective effect.Its Protective effects to alcoholic liver injury：The expression that may be by inhibiting CYP2E1 enzymes, reduces and lives
Property oxygen molecule generation, mitochondrial membrane and cell membrane damage are reduced, to have the function that protect liver organization.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
Any modification made by within refreshing and principle, equivalent replacement and improvement etc., should all be included in the protection scope of the present invention.
Claims (2)
1. a kind of anoectochilus roxburghii polyose, it is characterised in that：It is prepared by following methods：1）Roxburgh anoectochilus terminal bud herb dried and crushed crosses 70 mesh
Sieve, then weighs that roxburgh anoectochilus terminal bud herb powder is appropriate, is 1 by the feed liquid mass ratio of roxburgh anoectochilus terminal bud herb powder and distilled water:10~1:20
Distilled water is added, in 50 DEG C ~ 60 DEG C ultrasonic extractions, supernatant, repetition is taken to carry after centrifugation 2 times, merges extracting solution rotation three times and steams
Hair is concentrated into removing protein after small size, obtains small size concentrate, and it is 3 ~ 5 times of the small size volume of the concentrated liquid that volume, which is then added,
Sediment, freeze-drying to constant weight is collected by centrifugation in absolute ethyl alcohol, alcohol precipitation, 4 DEG C of overnight precipitations of deepfreeze；2）It is inverse to establish high speed
Flow chromatography purifies anoectochilus roxburghii polyose（ARPS）The condition of purifying process, HSCCC is：Double-aqueous phase system is pH=6.8
12.5wt%PEG1000-20wt% potassium hydrogen phosphates, rotating speed are 850 r/min, and the flow velocity of mobile phase is 1.0 mL/min, 5wt% benzene
Phenol-sulfuric acid method tracing detection merges same composition, and low pressure rotary evaporation is concentrated into small size, with M12000 molecular cut offs
The further dialysis desalting purifying of film detects whether phosphate in dialyzate dialyses completely using ammonium molybdate, merges in bag filter
Component, rotary evaporation concentration, freeze-drying is to get anoectochilus roxburghii polyose, and it is 22090 Da to measure anoectochilus roxburghii polyose molecular weight, mainly
It is made of mannose, ribose, glucose, galactolipin and arabinose, and the molar ratio of each monosaccharide is:Mannose：Ribose：Portugal
Grape sugar：Galactolipin：Arabinose=1.00：8.47 ：47.30 ：1.17 ：1.19, glycosidic bond is α configurations, and three strands of spiral shells are not present
Conformation is revolved, and AFM shows close agglomerating, the spheroidal of its molecule aggregation under the microscope, diameter is about 120 ~ 380 nm.
2. anoectochilus roxburghii polyose described in claim 1 is preparing the application in treating acute alcohol-induced hepatic injury drug.
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| CN111603538A (en) * | 2020-06-30 | 2020-09-01 | 华侨大学 | Application and preparation method of anoectochilus formosanus extract |
| CN115894731A (en) * | 2022-11-02 | 2023-04-04 | 福建中医药大学 | Anoectochilus roxburghii homogeneous polysaccharide and preparation method and application thereof |
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Cited By (5)
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| CN110507666A (en) * | 2019-10-14 | 2019-11-29 | 武汉华联科生物技术有限公司 | A kind of new application of selenium lentinan |
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| CN111603538B (en) * | 2020-06-30 | 2021-04-27 | 华侨大学 | Application of anoectochilus formosanus extract |
| CN115894731A (en) * | 2022-11-02 | 2023-04-04 | 福建中医药大学 | Anoectochilus roxburghii homogeneous polysaccharide and preparation method and application thereof |
| CN115894731B (en) * | 2022-11-02 | 2024-04-09 | 福建中医药大学 | Anoectochilus formosanus uniform polysaccharide and preparation method and application thereof |
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