CN106474145B - Application of the Polysaccharides from Leaves of Moringa oleifera in preparation prevention and treatment alcoholic liver injury drug and food - Google Patents
Application of the Polysaccharides from Leaves of Moringa oleifera in preparation prevention and treatment alcoholic liver injury drug and food Download PDFInfo
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- CN106474145B CN106474145B CN201611071660.4A CN201611071660A CN106474145B CN 106474145 B CN106474145 B CN 106474145B CN 201611071660 A CN201611071660 A CN 201611071660A CN 106474145 B CN106474145 B CN 106474145B
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- moringa oleifera
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
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- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation or decoction
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Abstract
The invention belongs to field of natural product chemistry, a kind of application of Polysaccharides from Leaves of Moringa oleifera in preparation prevention and treatment alcoholic liver injury drug and food is disclosed.The Polysaccharides from Leaves of Moringa oleifera is slightly mentioned using water extraction and alcohol precipitation method, is purified in conjunction with the process flows such as 14000rpm centrifugation, de- albumen, AB-8 type large pore resin absorption column, purer Polysaccharides from Leaves of Moringa oleifera can be obtained.Polysaccharides from Leaves of Moringa oleifera has significant protective effect to hepatic injury, is confirmed by animal experiment, Polysaccharides from Leaves of Moringa oleifera all has preventive and therapeutic effect to acute and subacute alcoholic liver injury.Polysaccharides from Leaves of Moringa oleifera can be applied well in exploitation prevention and treatment alcoholic liver injury drug and functional food.
Description
Technical field
The invention belongs to field of natural product chemistry, in particular to a kind of Polysaccharides from Leaves of Moringa oleifera is in preparation prevention and treatment alcoholic liver damage
Application in vulnerary object and food.
Background technique
Alcoholic liver disease (Alcoholic Liver Disease, ALD) is the damage of alcoholic liver caused by long-term alcohol
Wound, the acute and chronic hepatic injury based on liver metabolism disorder.Clinically it is mainly shown as three kinds of forms: alcoholic fatty liver,
Alcoholic hepatitis and alcoholic cirrhosis, these three forms can be independent or be mixed.According to the statistics of the World Health Organization: the whole world
There are about 1500~20,000,000 people excessive drinkings, wherein 10%~20% has different degrees of alcoholic liver disease.In American-European countries, Alcoholic
One of the main reason for hepatopathy is young and middle-aged dead；It shows in the epidemiological survey of North China, China from early 1980s
To the beginning of the nineties, ratio of the wine-head in general population rises to 14.3% from 0.21%；The beginning of this century, south and Midwest
Province epidemiological survey shows that the crowd of drinking increases to 30.9%~43.4%.Alcoholic liver disease accounts for same period hepatopathy inpatient
Ratio is constantly rising, and increases to 21.3% in 1996 from 4.2% in 1991.Especially in recent years, as China's economy increases
Long, people's social life mode development in pluralism, the total amount of China's pre-capita consumption alcohol and the total number of persons for consuming alcohol all exist
It rises year by year, alcohol abuse and alcohol dependence have become the whole world or even a Chinese public health problem to become increasingly conspicuous, secondary
Alcoholic liver injury also become a very important disease in China.Therefore, searching and research and development can effectively prevent alcohol
The drug and health food of property hepatopathy are very urgent.
Currently, alcoholic liver injury is temporarily without specific medicament and treatment method.In prevention and treatment, still take to reduce alcohol and take the photograph
Enter, the measure based on discovery early and early control state of an illness etc.；When there is obvious hepatic injury then with liver protection and supportive treatment phase
In conjunction with therapy, drug therapy is non-specific anti-inflammatory mostly from liver protection anti-fibrosis, anti-oxidant etc. to set about.Although currently having
The Chinese medicines such as research report pueraria lobata can be used for Antialcoholic liver-protecting, but most of Chinese medicine toxic side effects, only can be used as drug, cannot
Long-term consumption, as food；And reported most plants are all that liver-protecting efficacy ingredient is indefinite, with Plant crude extract
It uses, limits the range used.So find and using have in natural plant resource Antialcoholic liver-protecting effect it is single it is a kind of at
Point, and developed into the extensive concern of drug and health food by medicine and food service industry.A kind of liver-protecting efficacy in plant
The discovery of ingredient is safe and effective, quality controllable for developing, and can effectively prevent the health food of alcoholic liver injury, drug has
Great meaning.
Moringa (Moringa oleifera Lam.) is Moringa suborder Moringaceae (Moringaceae) Moringa
(Moringa) plant, is perennial tropical and subtropical zone deciduous tree, and Yin Qigen has acid, thus thus gain the name Moringa.Moringa
Root, leaf, okra fruit it is edible, contain several mineral materials, vitamin, nutriment is very more；In addition, its root, skin, leaf, fruit
It can make medical material, therefore Moringa has the good reputations such as " tree of miracle ", " diamond in plant ".Moringa has a variety of medicinal valences
Value, can treat the diseases such as ulcer, hypertension, hyperlipidemia, cancer, diabetes, inflammation, be the plant of great research and development potentiality
Object.
It is external at present about Moringa is anti-oxidant and the research of liver protection reports that the scholars such as Sharifudin are with to acetyl
Amino phenols (APAP) inducing mouse generate hepatic injury, then using N-acetylcystein as control drug, have studied leaf of Moringa and
Therapeutic effect of the flower extract to mouse liver injury, the results showed that leaf of Moringa and the flower extract Mouse Liver caused by APAP excess
Damage has potential therapeutic effect (Sharifudin SA, Fakurazi S, Hidayat MT, et al.Therapeutic
potential of Moringa oleifera extracts against acetaminophen-induced
Hepatotoxicity in rats [J] .Pharmaceutical Biology, 2013,51 (3): 279-288).Sharma etc.
The protective effect for having studied the acetsminophen that Moringa induces 7,12- dimethylbiphenyl [a] anthracene (DMBA), finds its energy
Reduce the liver aspartate transaminase (AST), alanine aminotransferase (ALT) and alkaline phosphorus of the liver injury model mouse of DMBA induction
Sour enzyme (ALP) is horizontal, the pathological change of the significant liver organization for reversing DMBA induction.The result shows that Moringa induces mouse DMBA
Hepatocellular injury have good liver protection and antioxidation potential (Sharma V, Paliwal R, Janmeda P, et
Al.Chemopreventive efficacy of Moringa oleifera pods against 7,12-
dimethylbenz[a]anthracene induced hepatic carcinogenesis in mice[J].Asian
Pacific Journal of Cancer Prevention, 2012,13 (6): 2563-2569).Sinha etc. has studied Moringa
Protective effect of the leaf extract to lipid peroxidation injury caused by radiation.Mouse is exposed to 60 Co- after being administered 15 days
γ radiation, the variations such as liver lipid peroxidation, the reduced glutathione of observation irradiation model mice, the results showed that the leaf of Moringa
Extract can prevent hepatic lipid peroxidation damage (Sinha M, Das DK, Datta S, et al.Amelioration of
ionizing radiation induced lipid peroxidation in mouse liver by Moringa
Oleifera Lam leaf extract [J] .Indian Journal of Experimental Biology, 2012,50
(3): 209-215).But in the above research, leaf of Moringa or flower are for fighting DMBA and APAP compound and 60 Co- γ spokes
Hepatic injury caused by penetrating is to take crude extract, is not damaged about single a kind of chemical component such as Polysaccharides from Leaves of Moringa oleifera resisting alcoholic liver
The research and application of wound.
Summary of the invention
In order to overcome the shortcomings and deficiencies of the prior art described above, exist the primary purpose of the present invention is that providing Polysaccharides from Leaves of Moringa oleifera
Application in preparation prevention and treatment alcoholic liver injury drug and food.
The purpose of the present invention is realized by following proposal:
Application of the Polysaccharides from Leaves of Moringa oleifera in preparation prevention and treatment alcoholic liver injury drug and food.
The Polysaccharides from Leaves of Moringa oleifera is prepared by following methods:
(1) it stocks up: leaf of Moringa dry, pulverize, cross 80 meshes, it is spare to obtain leaf of Moringa powder；
(2) degreasing: taking leaf of Moringa powder obtained in step (1), and petroleum ether reflux degreasing is added, then filters and filters gained
Slag is dry, spare；
(3) it extracts: taking dry filter residue obtained in step (2), take water as a solvent, solid-liquid ratio is 1:15~1:25,60
DEG C~80 DEG C at extract 0.5~1.5h, extraction time be 3 times, merge Aqueous extracts, be centrifuged through 14000rpm, take supernatant, obtained peppery
The wooden leaf Thick many candies solution；
(4) alcohol precipitation: leaf of Moringa Thick many candies solution obtained in step (3) is concentrated into 1/2~1/3, with dehydrated alcohol tune
Alcohol content precipitates 12h, filtering takes precipitating, obtains leaf of Moringa Thick many candies to 75%~85%；
(5) by leaf of Moringa Thick many candies deionized water dissolving obtained in step (4), 1/5 body of polysaccharide liquid removing protein: is added
Long-pending Sevag reagent (chloroform and n-butanol volume ratio 4: 1), 30~35min of magnetic agitation after mixing, 4500rpm centrifugation
20min is handled 3 times repeatedly；
(6) it purifies: the product after removing protein in step (5) being added in adsorption resin column, is eluted, is washed with distilled water
Eluent is concentrated into the 1/5 of original volume, obtains concentrate by separation of flow speed 2mL/min, and concentrate is placed in 80 DEG C of water bath methods to phase
It is the medicinal extract of 0.16~0.25 (using water as reference material) to density, is dried in vacuo at 60 DEG C, obtains Polysaccharides from Leaves of Moringa oleifera powder.
The corresponding petroleum ether for using 3mL of the leaf of Moringa powder that the amount of petroleum ether used in step (2) is every 1g；Described returns
Stream degreasing refers to reflux twice, and each return time is 6h；The boiling range of petroleum ether used is 60 DEG C~90 DEG C.
Extraction conditions described in step (3) are preferred are as follows: solid-liquid ratio 1:20, in 80 DEG C of extraction 1h, extraction time 3
It is secondary.
Alcohol content described in step (4) is preferably 80%.
The dosage of Sevag reagent described in step (5) is more to obtain after leaf of Moringa Thick many candies deionized water dissolving
The 1/5 of liquid glucose volume；The Sevag reagent is the mixed liquor of chloroform and n-butanol that volume ratio is 4:1.
Adsorption resin column described in step (6) is AB-8 type large pore resin absorption column；The relative density is with water
For reference material, preferably 0.20.
The formulation ingredients of the prevention and treatment alcoholic liver injury drug and food include Polysaccharides from Leaves of Moringa oleifera and country's license
The auxiliary materials such as the acceptable adhesive of the food grade of addition, lubricant, flavoring agent.
The acceptable auxiliary material is preferably at least one in Icing Sugar, mannitol, microcrystalline cellulose, soluble starch etc.
Kind.
The prevention and treatment alcoholic liver injury drug can be various dosage forms, as granule, tablet, capsule, oral solution,
Powder etc..
Mechanism of the invention:
Polysaccharides from Leaves of Moringa oleifera, to malonaldehyde (MDA), triglycerides (TG) in Models of Acute Alcoholic Liver Injury mice serum
It is significantly reduced effect, and is had to superoxide dismutase (SOD) and glutathione peroxidase (GSH-px enzyme) vigor
Humidification is conducive to the mitigation, defence and reparation of alcoholic liver injury.Polysaccharides from Leaves of Moringa oleifera can reduce subacute alcoholic liver damage
Wound model mice serum low density lipoprotein cholesterol (LDL-C), glutamic-oxalacetic transaminease (GOT), alanine aminotransferase
(GPT), total cholesterol (T-CHO), total bilirubin (T-BIL) content make hepatocyte function damage mitigate and restore normal.Due to
Malonaldehyde (MDA) is one of most important product of membrane lipid peroxidatio, its generation can cause big point of the biology such as protein, nucleic acid
The cross-linked polymeric of son makes function damage or the forfeiture of film, generates cytotoxicity；It is ground according to alcoholic liver injury pathogenesis
Study carefully discovery, alcoholic liver injury can cause content of triglyceride in hepatic tissue to be built up, and cholesterol biosynthesis is reinforced, and causes accordingly to carry
The change of lipoprotein content, so as to cause the function damage of liver cell, and alcoholic liver steatosis and associated serum index
Change is the early stage performance of alcoholic liver injury.Glutathione peroxidase (GSH-px) is the one kind being widely present in body
The enzyme that important catalyzing hydrogen peroxide decomposes.Its special catalysis reduced glutathione (GSH) is anti-to the reduction of hydrogen peroxide
It answers, protection membrane structure and fully functional effect can be played.The activated centre of GSH-px is selenocystein, and selenium is
The required part of GSH-px, every gram molecule enzyme contain 4 gram atom selenium.The vigor of measurement GSH-px, which can be used as, measures body selenium level
A biochemical indicator.Glutathione peroxidase (GSH-px) can promote hydrogen peroxide (H2O2) and reduced form gluathione
Peptide (GSH) reaction generates H2O and oxidized form of glutathione (GSSG), the vigor of glutathione peroxidase can use enzymatic reaction
Speed indicate, measure the consumption of reduced glutathione in this enzymatic reaction, then can find out the vigor of enzyme.Serum T-BIL
(total bilirubin, T-BIL) be bilirubin direct (direct bilirubin, DBIL) and indirect bilirubin (miL) it
With, be heme catabolism product.Serum bilirubin is mainly derived from red blood cell aging cracking and the hemoglobin that releases, and liver
The dirty metabolite clearance effect undertaken to bilirubin.Therefore, the reasons such as liver cell is impaired or extrahepatic duct blocks will cause serum
T-BIL is increased, and is the sensitive indicator in Liver function grade.Transaminase is essential important object in liver cell metabolic process
Matter, when liver cell is caused the pathology damages such as inflammation, necrosis by various influences, transaminase releasably enters blood, leads to serum
Transaminase increases, and serum transaminase is the important indicator of clinical reflection hepatocellular injury.
The present invention compared with the existing technology, have the following advantages and the utility model has the advantages that
1, preparation method of the invention can make leaf of Moringa Thick many candies yield increase, and after refinement treatment, energy
Obtain purer Polysaccharides from Leaves of Moringa oleifera.
2, Polysaccharides from Leaves of Moringa oleifera prepared by the present invention shows acute and subacute alcoholic liver injury model experiment mouse
Significant protective effect.Therefore, Polysaccharides from Leaves of Moringa oleifera prepared by the present invention can be applicable to anti-liver injury field especially alcoholic liver
Damage.
Detailed description of the invention
Fig. 1 is blank control group mouse hepatic tissue section figure in embodiment 6.
Fig. 2 is model group murine liver tissue slice map in embodiment 6.
Fig. 3 is Polysaccharides from Leaves of Moringa oleifera high dose group murine liver tissue slice map in embodiment 6.
Fig. 4 is Polysaccharides from Leaves of Moringa oleifera middle dose group murine liver tissue slice map in embodiment 6.
Fig. 5 is Polysaccharides from Leaves of Moringa oleifera low dose group murine liver tissue slice map in embodiment 6.
Specific embodiment
Below with reference to embodiment and attached drawing, the present invention is described in further detail, but embodiments of the present invention are unlimited
In this.
Agents useful for same of the present invention can routinely be bought unless otherwise specified from market.
Embodiment 1: the preparation of Polysaccharides from Leaves of Moringa oleifera
(1) it stocks up: leaf of Moringa dry, pulverize, cross 80 meshes, it is spare to obtain leaf of Moringa powder；
(2) degreasing: taking leaf of Moringa powder obtained in step (1), and the petroleum of 3 times of volumes (g: mL) of leaf of Moringa silty amount is added
Ether flows back degreasing 2 times, each 6h, filtering, spare by filter residue and drying；
(3) it extracts: taking dry filter residue obtained in step (2) to take water as a solvent, solid-liquid ratio 1:15 is extracted at 60 DEG C
0.5h, extraction time are 3 times, merge Aqueous extracts, are centrifuged through 14000rpm, take supernatant, obtain leaf of Moringa Thick many candies solution；
(4) alcohol precipitation: leaf of Moringa Thick many candies solution obtained in step (3) is concentrated into 1/2-1/3, is contained with dehydrated alcohol tune
Alcohol amount precipitates 12h, filtering takes precipitating, obtains leaf of Moringa Thick many candies to 75%；
(5) by leaf of Moringa Thick many candies deionized water dissolving obtained in step (4), 1/5 body of polysaccharide liquid removing protein: is added
Long-pending Sevag reagent (chloroform and n-butanol volume ratio 4: 1), 30~35min of magnetic agitation after mixing, 4500rpm centrifugation
20min is handled 3 times repeatedly.
(6) it purifies: Polysaccharides from Leaves of Moringa oleifera extracting solution obtained in step (5) is added in AB-8 type large pore resin absorption column,
It is eluted with distilled water, elution flow rate 2mL/min, eluent is concentrated into the 1/5 of original volume, obtains concentrate, by concentrate
It is placed in the medicinal extract that 80 DEG C of water bath methods to relative density is 0.16 (using water as reference material), is dried in vacuo, obtains peppery at 60 DEG C
The wooden leaf polyose powder.
Embodiment 2: the preparation of Polysaccharides from Leaves of Moringa oleifera
(1) it stocks up: leaf of Moringa dry, pulverize, cross 80 meshes, it is spare to obtain leaf of Moringa powder；
(2) degreasing: taking the leaf of Moringa powder in step (1), and the petroleum ether that 3 times of volumes (g: mL) of leaf of Moringa silty amount are added returns
Stream degreasing 2 times, each 6h, filtering are spare by filter residue and drying；
(3) it extracts: taking dry filter residue obtained in step (2) to take water as a solvent, solid-liquid ratio 1: 20 extracts at 80 DEG C
1h, extraction time are 3 times, merge Aqueous extracts, are centrifuged through 14000rpm, take supernatant, obtain leaf of Moringa Thick many candies solution；
(4) alcohol precipitation: leaf of Moringa Thick many candies solution obtained in step (3) is concentrated into 1/2-1/3, is contained with dehydrated alcohol tune
Alcohol amount precipitates 12h, filtering takes precipitating, obtains leaf of Moringa Thick many candies to 80%；
(5) by leaf of Moringa Thick many candies deionized water dissolving obtained in step (4), 1/5 body of polysaccharide liquid removing protein: is added
Long-pending Sevag reagent (chloroform and n-butanol volume ratio 4: 1), magnetic agitation 30-35min after mixing, 4500rpm are centrifuged 20min,
It handles 3 times repeatedly；
(6) it purifies: Polysaccharides from Leaves of Moringa oleifera extracting solution obtained in step (5) is added in AB-8 type large pore resin absorption column,
It is eluted with distilled water, elution flow rate 2mL/min, eluent is concentrated into the 1/5 of original volume, obtains concentrate, by concentrate
It is placed in the medicinal extract that 80 DEG C of water bath methods to relative density is 0.20 (using water as reference material), is dried in vacuo, obtains peppery at 60 DEG C
The wooden leaf polyose powder.
Embodiment 3: the preparation of Polysaccharides from Leaves of Moringa oleifera
(1) it stocks up: leaf of Moringa dry, pulverize, cross 80 meshes, it is spare to obtain leaf of Moringa powder；
(2) degreasing: taking the leaf of Moringa powder in step (1), and the petroleum ether that 3 times of volumes (g: mL) of leaf of Moringa silty amount are added returns
Stream degreasing 2 times, each 6h, filtering are spare by filter residue and drying；
(3) it extracts: taking dry filter residue obtained in step (2) to take water as a solvent, solid-liquid ratio 1: 25 extracts at 70 DEG C
1.5h, extraction time are 3 times, merge Aqueous extracts, are centrifuged through 14000rpm, take supernatant, obtain leaf of Moringa Thick many candies solution；
(4) alcohol precipitation: leaf of Moringa Thick many candies solution obtained in step (3) is concentrated into 1/2-1/3, is contained with dehydrated alcohol tune
Alcohol amount precipitates 12h, filtration takes and precipitates up to leaf of Moringa Thick many candies to 85%；
(5) by leaf of Moringa Thick many candies deionized water dissolving obtained in step (4), 1/5 body of polysaccharide liquid removing protein: is added
Long-pending Sevag reagent (chloroform and n-butanol volume ratio 4:1), magnetic agitation 30-35min after mixing, 4500rpm are centrifuged 20min,
It handles 3 times repeatedly；
(6) it purifies: Polysaccharides from Leaves of Moringa oleifera extracting solution obtained in step (5) is added in AB-8 type large pore resin absorption column,
It is eluted with distilled water, elution flow rate 2mL/min, eluent is concentrated into the 1/5 of original volume, obtains concentrate, by concentrate
It is placed in medicinal extract of 80 DEG C of water bath methods to relative density for 0.25 (with the reference material of water), is dried in vacuo, obtains peppery at 60 DEG C
The wooden leaf polyose powder.
Embodiment 4: the measurement test of Polysaccharides from Leaves of Moringa oleifera content
1. the preparation of reference substance solution: precision weighs the dry glucose 5mg to constant weight, is settled to 50mL's with distilled water
It in volumetric flask, shakes up, concentration 0.1mg/mL, then accurate absorption 1,2,3,4,5mL is set in 10mL volumetric flask respectively, with distillation
Water dilution constant volume shakes up, and obtains 0.01mg/mL, 0.02mg/mL, 0.03mg/mL, the control series of 0.04mg/mL, 0.05mg/mL
Product solution.
2. the drafting of standard curve: precision draws above-mentioned control series product solution 1mL, is placed in 10mL tool plug test tube, ice
Water-bath 5min is added 0.2% Anthrone Sulphuric acid test solution 4mL and shakes up, and is immediately placed on boiling water bath heating 10min, and taking-up is cooling with cold water
To room temperature, it is placed at room temperature for 10min, trap is measured at 620nm, another accurate absorption distilled water 1mL is placed in 10mL tool plug test tube
In, ice-water bath 5min is added 0.2% sulfuric acid anthrone test solution 4mL and shakes up, and is immediately placed on boiling water bath heating 10min, and cold water is used in taking-up
It is cooled to room temperature, is placed at room temperature for 10min or so, as blank control.With absorbance (A) for ordinate, glucose control product are dense
Spending (C) is abscissa, obtains glucose standard curve.
3. measurement: weighing Polysaccharides from Leaves of Moringa oleifera prepared by experimental example 1, experimental example 2, experimental example 3 respectively, prepared with distilled water
1mg/mL sample solution takes 1mL to be placed in 10mL tool plug test tube, ice-water bath 5min, and 0.2% sulfuric acid anthrone test solution 4mL is added and shakes
It is even, it is immediately placed on boiling water bath heating 10min, taking-up is cooled to room temperature with cold water, is placed at room temperature for 10min, is measured and inhale at 620nm
Receipts degree, by concentration of glucose (C) in regression equation calculation test liquid.The results are shown in Table 1.
The measurement test data of 1 Polysaccharides from Leaves of Moringa oleifera content of table
From table 1 it follows that Polysaccharides from Leaves of Moringa oleifera content highest prepared by the method for embodiment 2, compared with Example 3,
Solution usage is fewer, and extraction time is also shorter, and content is higher, is more suitably applied in production technology.
Embodiment 5: influence test of the Polysaccharides from Leaves of Moringa oleifera to Models of Acute Alcoholic Liver Injury mouse
1. material:
(1) experimental animal:
SPF grades of Kunming (KM) kinds male mice 77,18~22 grams of weight, experimental animal credit number: SCXK (Guangdong)
2013-0034 is provided by Traditional Chinese Medicine University Of Guangzhou's Experimental Animal Center.Use credit number: SYXK (Guangdong) 2014-0136.
(2) reagent:
Bifendate (Biphenyldicarboxylate, BP) dripping pill, the limited public affairs of a Community in Baiyunshan, Guangzhou group of stars (medicine company) share
Department's production；The Polysaccharides from Leaves of Moringa oleifera being prepared in the embodiment of the present invention 2.MDA, TG, SOD, GSH-px enzyme detection kit are by south
Bioengineering Research Institute's offer is built up in capital.
2. experimental animal is grouped: 77 male Kunming strain mices are randomly divided into 6 groups, respectively blank control group, model group,
Bifendate (15mg/mL) group, the high, medium and low dosage of Polysaccharides from Leaves of Moringa oleifera of the present invention (concentration be respectively 200mg/mL, 100mg/mL,
50mg/mL) group, wherein blank control group 12, remaining each group each 13.
3. test method:
Adaptive feeding 5 days before testing, free diet observes and records the food ration and amount of drinking water of each group mouse daily.It is real
It tests first 16 days, each group intragastric administration on mice gives the tested material of various dose, except blank control group and model group intragastric administration on mice are given
It measures outside physiological saline, 2 tested materials are given once daily in each dosage Polysaccharides from Leaves of Moringa oleifera group mouse, fill by weight 0.12mL/10g dosage
Stomach, bifendate group mouse is by the dosage medication after people's daily quantity conversion, i.e. weight 0.12mL/10g dosage stomach-filling,
One time a day.Each group mouse weight is weighed weekly twice, to adjust corresponding tested material dosage.It tests the 17th day, starts modeling, remove
Outside blank control group, remaining each group mouse gavages 50% food grade alcohol 1 time by weight 0.1mL/10g dosage daily, continuously
Give 9 days alcohol, tested material give dosage and method is the same.Each group mouse gives corresponding tested material 3~5 again after modeling
It.Before experiment terminates, fasting 16h after last time stomach-filling weighs weight, then plucks eyeball and take blood, by the whole blood of acquisition 20
After placing 1h at DEG C, it is centrifuged 10min through 3 000rpm of revolving speed, serum is drawn in sterilizing 1.5mL centrifuge tube, is placed in -20 DEG C of ice
Case saves backup.Cervical dislocation puts to death mouse, weighs mice organs, liver is placed on ice platform, quickly cut with knife blade
Liver lobus dexter pays attention to taking right lobe of liver same area hepatic tissue to be divided into 2~3 parts and be distributed into clean cryopreservation tube and is placed in -80 DEG C of ice
It is saved backup in case.
4. Testing index and detection method
The measurement of 4.1 Triglycerides in Serum (TG), malonaldehyde (MDA) content.It is operated in strict accordance with kit specification
Step carries out.
Malonaldehyde (MDA) content, superoxide dismutase (SOD) activity, glutathione object peroxidase in 4.2 hepatic tissues
(GSH-px) active measurement.A liver organization for taking above-mentioned preservation, 10% liver homogenate is configured to cold saline, is pressed
The operation of kit specification operating procedure detects MDA content in liver, SOD activity, GSH-px activity.
4.3 statistical analysis
Experimental data is all made of 15.0 software package of SPSS and carries out statistical disposition, is as a result indicated with x ± s, and comparison among groups use
One-way analysis of variance, P < 0.05 indicate statistical difference.
5. test result: taking blood, separate serum, measure malonaldehyde (MDA), triglycerides (TG) content in mice serum；
Take liver organization detection MDA, superoxide dismutase (SOD enzyme) vigor and glutathione peroxidase (GSH-px enzyme) living
Power.As a result as shown in table 2~3.
Influence of 2 Polysaccharides from Leaves of Moringa oleifera of table to acute alcohol-induced hepatic injury mice serum TG and MDA content
Note: compared with model group, * indicates that P < 0.05, * * indicate P < 0.01.
It is obtained by table 2, compared with blank control group mouse, malonaldehyde (MDA) and triglycerides in model group mice serum
(TG) content is significantly raised, has significant statistical difference (P < 0.05, P < 0.01), illustrates that alcohol induction acute liver damage is small
Mouse model is successfully established.Compared with model group, the high, medium and low dosage group mice serum triglycerides of Polysaccharides from Leaves of Moringa oleifera of the invention
(TG) it is decreased obviously (P < 0.05, P < 0.01), the most significant, positive control drug bifendate group mouse blood is declined with high dose group
Clear TG is also lower (P < 0.05) than model group mice serum TG level；In terms of each group mice serum MDA detection level, model group mouse
Serum is apparently higher than blank control group (P < 0.05)；Compared with model group, Polysaccharides from Leaves of Moringa oleifera middle dose group mice serum MDA content
(8.139 ± 3.764nmol/ml) is lower than model group mouse MDA (11.511 ± 2.015nmol/ml) content (P < 0.05), Moringa
Leaf polyose high and low dose group has the tendency that reduction.Therefore, Polysaccharides from Leaves of Moringa oleifera of the invention tests acute alcohol-induced hepatic injury small
Mouse model shows apparent hepatoprotective effect.
3 Polysaccharides from Leaves of Moringa oleifera of table influences acute alcohol-induced hepatic injury murine liver tissue MDA, SOD and GSH-px
Note: compared with model group, * indicates that P < 0.05, * * indicate P < 0.01.
It is obtained by table 3, compared with blank control group mouse, malonaldehyde (MDA) content is obvious in model group murine liver tissue
Increase, have significant statistical difference (P < 0.01), and SOD enzyme and GSH-px enzymatic activity significantly lower than blank control group (P <
0.05).After the high, medium and low dosage processing alcoholic liver injury model mice of Polysaccharides from Leaves of Moringa oleifera, it has been found that Polysaccharides from Leaves of Moringa oleifera
It is small lower than blank control group that each dosage group murine liver tissue MDA level is extremely remarkably decreased (P < 0.01) or even high dose group
Mouse MDA is horizontal, and the MDA level of bifendate group is also decreased obviously (P < 0.05).From hepatic tissue SOD enzyme and GSH-px enzymatic activity
It sees, the high, medium and low dosage group Mouse Liver GSH-px enzymatic activity of Polysaccharides from Leaves of Moringa oleifera obviously rises (P < 0.05), and Polysaccharides from Leaves of Moringa oleifera
The performance of low dose group Mouse Liver SOD enzyme activity significantly increases (P < 0.01).Therefore, compared with model group, leaf of Moringa of the invention
Polysaccharide is significantly reduced effect (P < 0.01) liver MDA, and to SOD and GSH-px enzyme activity have humidification (P < 0.05, P <
0.01), be conducive to improve the hepatic injury of model mice, Polysaccharides from Leaves of Moringa oleifera has defencive function effect to acute alcohol-induced hepatic injury.
Embodiment 6: influence test of the Polysaccharides from Leaves of Moringa oleifera to subacute alcoholic liver injury model mice
1. material:
(1) experimental animal:
SPF grades of Kunming (KM) kinds male mice 80,18~22 grams of weight, experimental animal credit number: SCXK (Guangdong)
2013-0034 is provided by Traditional Chinese Medicine University Of Guangzhou's Experimental Animal Center.Use credit number: SYXK (Guangdong) 2014-0136.
(2) reagent:
The Polysaccharides from Leaves of Moringa oleifera being prepared in the embodiment of the present invention 2；Sunflower liver-protecting tablet, is had by Heilongjiang Kuihua Yaoye Co.Ltd
The production of limit company, lot number: Z20003336；MDA, GOT, GPT, T-CHO, LDL-C and T-BIL detection kit are built up by Nanjing
Bioengineering Research Institute provides.
2. experimental animal is grouped: 80 male Kunming strain mices are randomly divided into 6 groups, respectively blank control group, model group,
Sunflower liver-protecting tablet (86mg/mL) group, (concentration is respectively 200mg/mL, 100mg/ to the high, medium and low dosage of Polysaccharides from Leaves of Moringa oleifera of the present invention
ML, 50mg/mL) group, wherein blank control group 10, remaining each group each 14.
3. test method:
Adaptive feeding 5 days, free diet before testing.Different tested materials is given in each group mouse oral stomach-filling, removes blank
Control group and model group intragastric administration on mice are given outside same amount of normal saline, each Polysaccharides from Leaves of Moringa oleifera group mouse be given once daily 2 times it is corresponding dense
The Polysaccharides from Leaves of Moringa oleifera of degree, by weight 0.12mL/10g dosage stomach-filling, sunflower liver-protecting tablet group mouse is by the daily quantity folding of people
Dosage medication after calculation, i.e. weight 0.12mL/10g dosage stomach-filling are continuously given 45 days one time a day.Mouse weighs body weekly
Weight twice, and adjusts given the test agent dosage by weight.In addition to blank control group, each group terminates first 14 days in experiment, presses daily
30% food grade alcohol is given in the oral stomach-filling of 10mL/kgbw.The stomach-filling time interval 4h of tested material and alcohol.Before experiment terminates
Fasting 12h, weighs weight, then plucks eyeball blood sampling.After the whole blood of acquisition is placed 1h at 20 DEG C, through 3 000rpmin of revolving speed
It is centrifuged 10min, serum is drawn in sterilizing 1.5mL centrifuge tube, is placed in -20 DEG C of refrigerators and saves backup.Mouse cervical dislocation is put to death,
Mice organs are weighed, liver is placed on ice platform, liver lobus dexter is quickly cut with knife blade, pay attention to taking right lobe of liver same area
Hepatic tissue is used for pathological examination, is put into 10% neutral formalin solution and fixes, remaining hepatic tissue is divided into 2~3 parts points
It is fitted into clean cryopreservation tube and is placed in -80 DEG C of refrigerators and save backup.
4. Testing index and detection method
Low density lipoprotein cholesterol (LDL-C), glutamic-oxalacetic transaminease (GOT), alanine aminotransferase in 4.1 serum
(GPT), the measurement of total cholesterol (T-CHO), total bilirubin (T-BIL) content.In strict accordance with kit specification operating procedure
It carries out.
The measurement of malonaldehyde (MDA) content in 4.2 hepatic tissues.A liver organization for taking above-mentioned preservation, with cold physiology salt
Water is configured to 10% liver homogenate, operates by kit specification operating procedure, detects MDA content in liver.
4.3 liver histopathology inspections: being dyed using HE, and with optical microscopy, amplification factor is 400 × inspection Mouse Liver
The pathological section of tissue, observation degeneration of liver cells (steatosis, hydropic degeneration, endochylema cohesion, balloon sample become), necrosis of liver cells
Situations such as changing with inflammation, and report is made to each group murine liver tissue pathological examination results.
4.4 statistical analysis
Data statistic analysis is carried out using 20.0 software of SPSS.Between mice serum biochemical indicator, liver index multiple groups group
Mean compares using one-way analysis of variance (ANOVA), and when homogeneity of variance is examined with LSDL, S-N-K (S), when heterogeneity of variance
It is examined using Tamhane ' s T2 (M) and Dunnett ' s T3, the significance of difference of more each class mean, P < 0.05 is indicated
Statistical difference.
5. test result:
5.1 biochemistry detection results: taking blood, separates serum, measures LDL-C, GOT, GPT, T-CHO, T-BIL in mice serum
Content；Take liver organization detection MDA horizontal.According in SFDA " health care test evaluation technical specification " " to chemical damage
Have assistant protection function evaluation method (revised draft) " " correlation criterion, experimental result is determined.As a result such as table 4~5
It is shown.
Influence of 4 Polysaccharides from Leaves of Moringa oleifera of table to biochemical indicator content each in mice serum
Note: compared with model group, * indicates that P < 0.05, * * indicate P < 0.01.
It is obtained by table 4, subacute alcoholic hepatic injury model group mice serum LDL-C, GOT, GPT, T-CHO and T-BIL contain
Amount has significant statistical difference (P < 0.05, P < 0.01) obviously higher than blank control group, shows subacute alcoholic liver
Damage Establishment of mouse model success.
In addition to serum T-CHO is horizontal, each dosage group mice serum LDL-C, GOT, GPT, T-CHO, T-BIL of Polysaccharides from Leaves of Moringa oleifera
Content has different degrees of reduction (P < 0.05) compared with model group mouse.Polysaccharides from Leaves of Moringa oleifera damages subacute alcoholic liver
Wound has defencive function effect.
Influence of 5 Polysaccharides from Leaves of Moringa oleifera of table to murine liver tissue malonaldehyde (MDA) content
Note: * * indicates the P < 0.01 compared with model group.
It is obtained by table 5, subacute alcoholic hepatic injury model group mouse MDA content (0.422 ± 0.49 μm of ol/g) is significantly high
In blank control group (0.268 ± 0.29 μm of ol/g), difference has statistical significance (P < 0.01).Polysaccharides from Leaves of Moringa oleifera is high, in,
Low dose group murine liver tissue MDA level is substantially less than model group mouse (0.422 ± 0.49 μm of ol/g), has statistics poor
Different (P < 0.01) is prompted after giving Polysaccharides from Leaves of Moringa oleifera and intervening, and the biochemical indicator of model mice dysfunction of liver is restored,
Hepatocyte function damage mitigates.
The influence that 5.2 Polysaccharides from Leaves of Moringa oleifera change subacute alcoholic hepatic injury model mice hepatic pathology
To above-mentioned blank control group, model group, Polysaccharides from Leaves of Moringa oleifera high dose group, Polysaccharides from Leaves of Moringa oleifera middle dose group, leaf of Moringa
The murine liver tissue of polysaccharide low dose group carries out sections observation, as a result respectively such as Fig. 1, Fig. 2, Fig. 3, Fig. 4, shown in Fig. 5.
Wherein Fig. 1 is blank control group mouse hepatic tissue section figure, from figure 1 it appears that the arrangement of liver cell rope is whole
Together, cell is in polygon, is arranged radially and is demarcated clearly centered on central vein, liver cell nuclear is round, it is seen that double-core,
Phenomena such as liver organization structural integrity, no oedema and steatosis.
Fig. 2 is model group murine liver tissue slice map, from figure 2 it can be seen that lobuli hepatis structure is abnormal, liver cell arrangement
Disorder, the universal swelling and degeneration of liver cell and necrosis.The liver cell of necrosis is distributed in disperse shape, and centrilobular vein area liver cell is bad
Dead degree is most heavy, and liver interstitial has severe extravasated blood.
Fig. 3 is Polysaccharides from Leaves of Moringa oleifera high dose group murine liver tissue slice map, from figure 3, it can be seen that hepatic tissue structure is different
Chang Chengdu is substantially change, and lobuli hepatis structure is gradually recovered normally, and liver cell arrangement is in streak, the liver cell number of denaturation and necrosis
Amount is reduced, and liver cell double-core increases, and illustrates that hepatocyte function enhances, and liver cell division increases, hepatic sinusoid, central veins of liver and
Liver interstitial extravasated blood degree significantly mitigates.
Fig. 4 is Polysaccharides from Leaves of Moringa oleifera middle dose group murine liver tissue slice map, figure 4, it is seen that hepatic tissue structure changes
Become, degeneration of liver cells and necrosis are distributed in stove shape, and degree relatively low-dose group is light, and the liver cell in central veins of liver area restores just first
Often, the extravasated blood degree of the secondary liver cell for lobuli hepatis limiting plate area, hepatic sinusoid and liver interstitial substantially reduces, a large amount of cell infiltrations.
Fig. 5 is Polysaccharides from Leaves of Moringa oleifera low dose group murine liver tissue slice map, from figure 5 it can be seen that hepatic tissue structure has
Pathologic variation, necrosis of liver cells are in stove shape, and downright bad liver cell quantity is more compared with middle dose group, and lesion degree is also compared with middle dose group
Weight is sliced the inflammatory trifle that visible inflammatory cell hyperplasia is formed.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (9)
1. application of the Polysaccharides from Leaves of Moringa oleifera as single active ingredient in preparation prevention and treatment alcoholic liver injury drug, it is characterised in that
The effective dose of Polysaccharides from Leaves of Moringa oleifera every 10g weight daily is 6 ~ 24mg in the described prevention and treatment alcoholic liver injury drug；
The Polysaccharides from Leaves of Moringa oleifera is prepared by following methods:
(1) it stocks up: leaf of Moringa dry, pulverize, cross 80 meshes, it is spare to obtain leaf of Moringa powder；
(2) degreasing: taking leaf of Moringa powder obtained in step (1), and petroleum ether reflux degreasing is added, then filters and does gained filter residue
It is dry, it is spare；
(3) it extracts: taking dry filter residue obtained in step (2), take water as a solvent, solid-liquid ratio is 1:20 ~ 1:25, at 70 ~ 80 DEG C
0.5 ~ 1.5h of lower extraction, extraction time are 3 times, merge Aqueous extracts, are centrifuged through 14000rpm, take supernatant, it is slightly more to obtain leaf of Moringa
Sugar juice；
(4) alcohol precipitation: being concentrated into 1/2 ~ 1/3 for leaf of Moringa Thick many candies solution obtained in step (3), with dehydrated alcohol tune alcohol content
To 75% ~ 85%, 12h is precipitated, filtering takes precipitating, obtains leaf of Moringa Thick many candies；
(5) by leaf of Moringa Thick many candies deionized water dissolving obtained in step (4), Sevag reagent, mixing removing protein: is added
30 ~ 35min of magnetic agitation afterwards, 4500rpm are centrifuged 20min, handle 3 times repeatedly；
(6) it purifies: the product after removing protein in step (5) being added in adsorption resin column, is eluted with distilled water, elution stream
Eluent is concentrated into the 1/5 of original volume, obtains concentrate by fast 2mL/min, and concentrate is placed in 80 DEG C of water bath methods to relatively close
The medicinal extract that degree is 0.16 ~ 0.25, is dried in vacuo at 60 DEG C, obtains Polysaccharides from Leaves of Moringa oleifera powder.
2. Polysaccharides from Leaves of Moringa oleifera according to claim 1 prevents and treats alcoholic liver injury drug in preparation as single active ingredient
In application, it is characterised in that:
The corresponding petroleum ether for using 3mL of the leaf of Moringa powder that the amount of petroleum ether used in step (2) is every 1g；The reflux is de-
Rouge refers to reflux twice, and each return time is 6h；The boiling range of petroleum ether used is 60 DEG C ~ 90 DEG C.
3. Polysaccharides from Leaves of Moringa oleifera according to claim 1 prevents and treats alcoholic liver injury drug in preparation as single active ingredient
In application, it is characterised in that:
Sevag reagent described in step (5) is the mixed liquor of chloroform and n-butanol that volume ratio is 4:1；The Sevag examination
The dosage of agent is the 1/5 of the polysaccharide liquid volume obtained after leaf of Moringa Thick many candies deionized water dissolving.
4. Polysaccharides from Leaves of Moringa oleifera according to claim 1 prevents and treats alcoholic liver injury drug in preparation as single active ingredient
In application, it is characterised in that:
Adsorption resin column described in step (6) is AB-8 type large pore resin absorption column.
5. Polysaccharides from Leaves of Moringa oleifera according to claim 1 prevents and treats alcoholic liver injury drug in preparation as single active ingredient
In application, it is characterised in that:
Relative density described in step (6) is using water as reference material.
6. Polysaccharides from Leaves of Moringa oleifera according to claim 1 prevents and treats alcoholic liver injury drug in preparation as single active ingredient
In application, it is characterised in that:
Extraction conditions described in step (3) are as follows: solid-liquid ratio 1: 20, in 80 DEG C of extraction 1h, extraction time is 3 times；
Alcohol content described in step (4) is 80%；
Relative density described in step (6) is 0.20.
7. Polysaccharides from Leaves of Moringa oleifera according to claim 1 prevents and treats alcoholic liver injury drug in preparation as single active ingredient
In application, it is characterised in that:
The formulation ingredients of the prevention and treatment alcoholic liver injury drug include that Polysaccharides from Leaves of Moringa oleifera and country are permitted addible drug
The acceptable auxiliary material of grade.
8. Polysaccharides from Leaves of Moringa oleifera according to claim 7 prevents and treats alcoholic liver injury drug in preparation as single active ingredient
In application, it is characterised in that:
The acceptable auxiliary material is at least one of Icing Sugar, mannitol, microcrystalline cellulose, soluble starch.
9. Polysaccharides from Leaves of Moringa oleifera according to claim 7 prevents and treats alcoholic liver injury drug in preparation as single active ingredient
In application, it is characterised in that:
The prevention and treatment alcoholic liver injury drug is granule, tablet, capsule, oral solution or powder.
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