CN107778374A - The preparation of sugarcane leaf polyose and its application of Prevention diabetes - Google Patents
The preparation of sugarcane leaf polyose and its application of Prevention diabetes Download PDFInfo
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- CN107778374A CN107778374A CN201710968485.7A CN201710968485A CN107778374A CN 107778374 A CN107778374 A CN 107778374A CN 201710968485 A CN201710968485 A CN 201710968485A CN 107778374 A CN107778374 A CN 107778374A
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- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
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Abstract
The invention discloses a kind of preparation method of sugarcane leaf polyose, and its application in terms of Prevention diabetes.Inventor extracts from sugarcane top and obtains sugarcane leaf polyose, is furtherd investigate using Non-obese diabetic mice, it is found that sugarcane leaf polyose has prevention effect to Non-obese diabetic mice diabetes.Experimental result is shown, 12 NOD mouse of model group, morbidity 8, the incidence of disease 66.7%, 12 NOD mouse of sugarcane leaf polyose group, fall ill 3, the incidence of disease 25%, the incidence of disease substantially reduces (P ＜ 0.05), and it is relevant that its mechanism may can improve in NOD mouse bodies oxidation resistance in inflammatory reaction and enhancing NOD Mice Bodies with sugarcane leaf polyose.Accordingly, sugarcane leaf polyose, especially for type 1 diabetes, has certain application prospect in terms of Prevention diabetes.
Description
Technical field
The invention belongs to preparation and its Prevention glycosuria in treating diabetes field, more particularly to a kind of sugarcane leaf polyose
The application of disease.
Background technology
The diabetes model of chemical induction is widely used in diabetes because of the advantages that its modeling success rate is high, and method is simple
Research, but the diabetes model of chemical induction can not the real pathogenetic mechanism of glycosuria in analog machine body.Spontaneous Diabetic
Animal model is that animal produces naturally under field conditions (factors), or occurs the dynamic of similar human diabetes performance due to gene mutation
Thing model, the model is closer to the natural onset of human diabetes and development.
Non-obese diabetic mice (Non-obese diabetic, NOD) is cultivated from ICR/Jcl mouse inbreeding,
Insulin-dependent diabetes mellitus, i.e. type 1 diabetes can spontaneously occur.Its pathogenesis and mankind's type 1 diabetes pathogenesis
It is similar, disease early stage (4~5 week old) occur inflammation of pancreatic islet, progressive destroy beta Cell of islet, when 12 week old occur it is bright
Aobvious diabetic symptom, 80% is can reach to female mice cumulative life-incidence during 30 week old, is the main of current research type 1 diabetes
One of animal model.
The content of the invention
The technical problem to be solved in the present invention is to provide the preparation of sugarcane leaf polyose and its application of Prevention diabetes.
In order to solve the above technical problems, the present invention uses following technical scheme：
The preparation method of sugarcane leaf polyose, by sugarcane top through water extract-alcohol precipitation, sediment drying sugarcane leaf polyose.
The preparation method of above-mentioned sugarcane leaf polyose, dry sugarcane top is cut into segment, adds boiling to boil, soaked 2h, add water
To powder was covered, extracted 3 times in 100 DEG C, each 2h；Filtering, merging filtrate, filtrate are concentrated into proportion 1.10-1.15, concentrate
3 times of 95% ethanol of amount are added, is sufficiently stirred, stands 12h；Lower sediment is collected, 55 DEG C of drying, crush and produce sugarcane in baking oven
Leaf polyose powder.
Application of the sugarcane leaf polyose in the medicine for preparing Prevention diabetes.
Diabetes are type 1 diabetes.
The medicine of Prevention diabetes can improve in body oxidation resistance in inflammatory reaction and reinforcement.
Sugarcane leaf polyose is prepared as described above method preparation.
The problem of lacking effective natural drug for treating diabetes, inventor extract that to obtain sugarcane top more from sugarcane top
Sugar, furtherd investigate using Non-obese diabetic mice (Non-obese diabetic, NOD), find sugarcane leaf polyose
There is prevention effect to Non-obese diabetic mice diabetes.Experimental result shows, 12 NOD mouse of model group, morbidity 8
Only, the incidence of disease 66.7%, 12 NOD mouse of sugarcane leaf polyose group, fall ill 3, the incidence of disease 25%, the incidence of disease substantially reduces (P ＜
0.05), its mechanism may can improve inflammatory reaction in NOD mouse bodies with sugarcane leaf polyose and strengthen antioxygen in NOD Mice Bodies
Change ability is relevant.Accordingly, sugarcane leaf polyose, especially for type 1 diabetes, necessarily should have in terms of Prevention diabetes
Use prospect.
Brief description of the drawings
Fig. 1 is the result figure that sugarcane leaf polyose influences on NOD mouse weights.
Fig. 2 is result figure of the sugarcane leaf polyose to NOD mouse islets tissue effects, in figure：(A) blank control group, (B) mould
Type control group, (C) FTY720 control groups, (D) sugarcane leaf polyose group.
Embodiment
Sugarcane leaf polyose is to NOD diabetes mices prevention effect and its Mechanism Study
1 laboratory apparatus and material
1.1 laboratory apparatus
High speed freezing centrifuge, model：X1R, Thermo Scientific companies of the U.S.；- 20 DEG C of refrigerators, model：BC-
45M, Guangdong Meidi Group Co. Ltd.；CO2Constant incubator, model：MCO-18AIC, Japanese SANYO companies；All-wave length enzyme
Mark instrument, model：Epoch, BioTek Co., Ltds of the U.S.；Electronic scale, model：JCS-300, Zhejiang Kaifeng Group Co., Ltd；
Roche brilliance type blood glucose meter and blood sugar test paper, German Roche Co., Ltds.
1.2 experiment reagent
FTY720, uncommon love (Shanghai) the chemical conversion industry Development Co., Ltd of ladder, lot number F1018；Mouse islets element Elisa reagents
Box, lot number 15019657, mouse IL-2Elisa kits, lot number 01019659, mouse TNF-alpha Elisa kits, batch
Number 03019658, mouse IL-17Elisa kits, lot number 03019660, the magnificent technology Co., Ltd in Wuhan；Total super oxygen
Compound mutase (SOD) WST-1 method kits, lot number 20141013, nitric oxide (Nitric Oxide) microplate method kit,
Lot number 20141020, MDA (Malondialdehyde) TBA method kits, lot number 20141021, glutathione peroxidase
Enzyme (Glutathione Peroxidase) colorimetric kit, lot number 20150419, Bioengineering Research Institute is built up in Nanjing.
Wherein, 2- (4- n-octyls ethyl) -2- amino-propanediol hydrochlorides (2-amino- { 2- (2- [4-
Octylphenyl] ethyl } -1,3-propanediol hydrochloride) abbreviation FTY720 is a kind of young from parasitic cicada
The aminopropane diol compound that the active component ISP-1 isolated in the ascus bacteria culture fluid of worm obtains through modifying for chemical structure,
Chemical structural formula is as follows.
FTY720 is a kind of new immunodepressant, and existing literature research proves that it prevents onset diabetes to nod mouse
Effectively, therefore this experiment selects FTY720 as positive control drug.
The preparation of 1.3 sugarcane leaf polyoses
Dry sugarcane top is cut into segment, suitable quantity of water is added and boils, soak 2h, add water to powder was covered, carried in 100 DEG C
Take 3 times (amount of water is defined to cover powder), each 2h；Filtering, merging filtrate, filtrate are concentrated into (80 DEG C of proportion 1.10-1.15
Survey), concentrate adds 3 times of 95% ethanol of amount, is sufficiently stirred, stands 12h；Ethanol is recovered under reduced pressure in supernatant, concentration, dry to do
Cream or powder preserve；Lower sediment is collected, 55 DEG C of drying, crush and produce sugarcane top polysaccharide powder in baking oven.
1.4 experimental animal
SPF levels 4 week old NOD/LtJ mouse 36 of female, SPF levels 4 week old ICR mouse 12 of female, purchased from Beijing Hua Fu
Health biotech inc, animal quality certification number：SCXK (capital) 2009-0004.Raise and tested in Guangxi Medical University
Animal center SPF level Animal Houses, room temperature (22 ± 2) degree, humidity (70 ± 5) %, ad lib drinking-water, lighting hours is in company with natural
Day-night change.
2 experimental methods
This experiment gives the treatment of sugarcane leaf polyose for continuous 16 weeks, research sugarcane leaf polyose is to NOD using NOD mouse as model
Whether mouse has prevention diabetes effect.Specific design and operation are as follows：
2.1 animal packets and administration
36 NOD mouse are divided into sugarcane leaf polyose group, FTY720 positive drug control groups and model by body weight stratified random
Control group, every group each 12；12 ICR mouse are classified as one group, are blank control group.Successive administration 16 weeks, dosing schedule is such as
Under：
(1) sugarcane leaf polyose group, the daily gavage 7g/kg sugarcane leaf polyoses of every mouse；
(2) FTY720 positive drug control groups, the daily gavage 1mg/kg FTY720 of every mouse；
(3) model control group, the physiological saline of the daily gavage equivalent of every mouse；
(4) blank control group, the physiological saline of the daily gavage equivalent of every mouse.
2.2 NOD mouse weights and blood sugar detection
A NOD mouse weight is weighed weekly；A NOD mouse fasting blood-glucose is determined weekly, determines evening before that day, is prohibited
Food can't help water 12 hours, puncture the mouse tail vein sterilized with cotton ball soaked in alcohol during measure with blood collecting pen, light firmly to squeeze blood, take tail
Venous blood one drips and is measured at once with blood glucose meter.The blood glucose value of every mouse weekly is recorded, with continuous two weeks fasting blood-glucoses
More than 11.1mmol/L, diagnose this NOD mouse and diabetes occur.
The collection and processing of 2.3 samples
It is diagnosed as having occurred the NOD mouse of diabetes, after falling ill 2 weeks, previous late fasting can't help water 12 hours, pluck eyeball
Take blood and dissect mouse and take pancreatic tissue；Remaining each group NOD mouse not fallen ill and blank control group ICR mouse, in administration 16
Zhou Hou, previous late fasting can't help water 12 hours, pluck eyeball and take blood and dissect mouse and take pancreatic tissue.Blood sample room temperature places 30 points
Clock, 3000rmin-14 DEG C of centrifugation 10min, separate serum, serum keeping is standby in -20 DEG C of refrigerators；Pancreatic tissue, which is put into, to be filled
The 5ml centrifuge tubes of paraformaldehyde, it is sent to the attached auspicious health hospital pathology department of Guangxi University of Chinese Medicine and does pathological section.
2.4 cell factor IL-2, TNF-α, IL-17 and Antioxidant Indexes NO, MDA, SOD, GSH-PX measure
NOD mice serums are taken, according to IL-2, TNF-α, IL-17ELISA kit specifications and NO, MDA, SOD, GSH-
The method of PX kit specifications, carry out the experimental implementation measure of kit.
2.5 statistical method
Experimental data is handled using the statistical softwares of SPSS 19.0, compares between multigroup mean and is examined using One-Way ANOVA
Test, be that difference is statistically significant with P ＜ 0.05.
3 experimental results
Influence of the 3.1 sugarcane leaf polyoses to NOD mouse general status
Model group NOD mouse increase with week old, and mouse chaeta is irregular, is faint in color, One's spirits are drooping, stool and urine excretion
Showed increased；Sugarcane leaf polyose group and FTY720 positive controls mouse increase with week old, in addition to morbidity mouse individually, remaining
Mouse chaeta is neat, and color is glossy, full of energy, and stool and urine excretion is normal.
Influence of the 3.2 sugarcane leaf polyoses to NOD mouse weights
From table 1, Fig. 1 results, model group NOD mouse are after administration the 8th week, and body weight increase is slow, body weight after 12 weeks
Start to be decreased obviously, body weight is significantly lower than other three groups (P ＜ 0.05) at 16 weeks；Sugarcane leaf polyose group body weight increase is normal, does not have
There is Body weight loss phenomenon, but body weight is slightly below FTY720 positive controls.
The sugarcane leaf polyose of table 1 influences on NOD mouse weights
Contrasted with model group, * P ＜ 0.05.
Influence of the 3.3 sugarcane leaf polyoses to NOD mouse invasion rates
The NOD mouse of 4 week old give sugarcane leaf polyose gavage and taken daily, and after 16 weeks, experimental result is shown successive administration,
2 are shown in Table, 12 NOD mouse of model control group, is diagnosed as diabetes 8, diabetes morbidity 66.7%, sugarcane leaf polyose
12 NOD mouse of group, are diagnosed as diabetes 3, diabetes morbidity 25%, and 12 NOD of FTY720 positive controls are small
Mouse, it is diagnosed as diabetes 1, diabetes morbidity 8.3%.As shown in table 2,8~12 weeks this periods upon administration, mould
Compared with sugarcane leaf polyose group and FTY720 positive controls, the model control group incidence of disease significantly raises the type control group incidence of disease.
After being administered 16 weeks, the total incidence of disease of sugarcane leaf polyose group diabetes is significantly lower than model control group (P ＜ 0.05), and experimental result shows
Show, sugarcane leaf polyose can prevent the generation of nod diabetes mices, but prevention effect is weaker than FTY720 positive controls.
The sugarcane leaf polyose of table 2 influences on NOD mouse invasions rate
Contrasted with model group, * P ＜ 0.05.
Influence of the 3.4 sugarcane leaf polyoses to NOD mouse islets element
As shown in table 3, after administration prevents, sugarcane leaf polyose group and FTY720 positive controls insulin contain NOD mouse
Measure (P ＜ 0.05) higher than model control group, but insulin content is not up to blank group control group level, sugarcane leaf polyose group pancreas
Island cellulose content is less than FTY720 positive controls.The results show, after prophylactic treatment, insulin secretion connects NOD mouse
Nearly normal level.
The sugarcane leaf polyose of table 3 to NOD mouse islets element influence (N=12)
Contrasted with model group, * P ＜ 0.05.
3.5 sugarcane leaf polyoses are to NOD mouse islets tissue pathology checking
As can be known from Fig. 2, naive mice pancreatic cell structural integrity is clear, and inflammatory cell leaching does not occur in islet cells
Profit, beta Cell of islet are intact.Model group mice pancreatic eucaryotic cell structure is irregular, a large amount of inflammatory cell infiltrations occurs, and pancreas islet is damaged,
Beta Cell of islet significantly reduces.FTY720 control groups and sugarcane leaf polyose group, though there is a small amount of inflammatory cell infiltrations, pancreas islet
Morphosis is preferable, and pancreas islet is not impaired serious, beta Cell of islet no abnormality seen.
3.6 sugarcane leaf polyoses divide cell factor IL-2, TNF-α and IL-17 in NOD mice serums sugarcane leaf polyose
Bleeding is flat to be influenceed
As shown in table 4, compared with model control group, sugarcane leaf polyose can reduce Th1 cytokines in NOD mice serums
IL-2, TNF-α and Th17 cytokines IL-17 expression, action effect is suitable with FTY720 positive controls, shows
Sugarcane leaf polyose can adjust CD4 unbalance in NOD Mice Bodies+T cell subgroup, suppress internal inflammatory reaction, prevent islet cells
It is damaged, prevents the generation of diabetes.
The sugarcane leaf polyose of table 4 to IL-2, TNF-α and IL-17 in NOD mice serums influence (N=12)
Contrasted with model control group, * P ＜ 0.05；Contrasted with blank control group,&P ＜ 0.05.
3.7 sugarcane leaf polyoses are to influence of the sugarcane leaf polyose to Antioxidative Factors SOD, MDA, NO in NOD mice serums
As shown in table 5, compared with model control group, sugarcane leaf polyose can reduce NOD mice serums MDA, NO, GSH-PX and contain
Amount, increase SOD vigor in serum, show that sugarcane leaf polyose can improve body antioxidant levels, oxyradical is removed, so as to protect
Shield beta Cell of islet is no longer damaged by immunity.
The sugarcane leaf polyose of table 5 to SOD, NO, MDA and GSH-PX in NOD mice serums influence (N=12)
Contrasted with model control group, * P ＜ 0.05；Contrasted with blank control group,&P ＜ 0.05.
4 discuss
Type 1 diabetes are a kind of autoimmune diseases, relevant with autoimmunity Imbalance, by T cell mediateds,
Body immune system produces wadding disorderly, characterized by immunity inflammation of pancreatic islet and selective beta Cell of islet are damaged, causes beta Cell of islet
Destroyed, insulin secretion is definitely insufficient, causes type 1 diabetes.When type 1 diabetes are fallen ill, beta Cell of islet is destroyed mostly,
It is often intact less than less than 10% beta Cell of islet, the secretion of insulin much can not meet the demand of body, and patient is necessary
Long term injections insulin therapy.Therefore, the generation of type 1 diabetes can be prevented if any medicine, type 1 diabetes are carried out immune dry
In advance, related mechanism is studied, then with great clinical meaning and value.
One of main pathogenesis of type 1 diabetes is that Th1/Th2 cell subsets is unbalance in body.When Th1/Th2 cells
When subgroup mutually restricts maintenance balance, body is by immunoprotection, and beta Cell of islet is so as to being protected；When Th1/Th2 cell subsets
When unbalance, body can be then damaged by immune-mediated caused autoimmune destruction, beta Cell of islet, induced Diabetic.NOD
The generation of diabetes mice is similar with the mechanism that mankind's type 1 diabetes occur, and female NOD mouse were since birth 4 weeks, Th1
The concentration of cell subsets constantly increases, and reaches highest in 12~16 weeks Th1 cell subsets concentration, and now exactly onset diabetes are high
The peak phase.
This result of study is shown, takes the NOD mouse of sugarcane leaf polyose treatment in advance, and the incidence of disease of its diabetes is
25.0%, reduced compared to the model control group incidence of disease (66.7%)；After being treated with sugarcane leaf polyose, the NOD mouse of first morbidity,
Disease time also delays 2 weeks than model control group, it was demonstrated that sugarcane leaf polyose can prevent and delay the generation of NOD diabetes mices.
Pancreas pathology section display, is contrasted with model group, and sugarcane leaf polyose group mice pancreatic only has a small amount of inflammatory cells,
Pancreas Islet Structure is relatively intact, illustrates that sugarcane leaf polyose can adjust inflammatory reaction in body, and can reduce inflammation damage of the cell to pancreas islet
Wound, beta Cell of islet is protected, prevents and delay the generation of diabetes.
In NOD mouse model group serum, TH1 cytokines IL-2, TNF-α content and blank control group more rise
Height, body Immune system imbalance is prompted, is inflamed in machine；And the NOD mouse of sugarcane leaf polyose prevention are taken in advance, in serum
IL-2, TNF-α content reduce than model group, prompt sugarcane leaf polyose to improve inflammatory reaction in body, and it is thin to recover Th1/Th2
The balance of born of the same parents' subgroup, prevents pancreas islet from being damaged by LADA, prevents the generation of diabetes.
It has now been found that the IL-17 of Thl7 cells secretion is relevant with the generation of pancreatitis and diabetes, it is 1 type glycosuria
The new target spot of disease, IL-17 is a kind of scorching prefactor, there is promotion inflammatory reaction, and in immunoreaction process, with other cells because
Son, act synergistically beta Cell of islet, causes islet beta-cell apoptosis.Experimental result shows, IL-17 contents in model control group serum
Rise, IL-17 contents in the NOD mice serums of sugarcane leaf polyose treatment are taken in advance and are declined to a great extent compared with model group, sugarcane top is more
Sugar can suppress the secretion of IL-17 inflammation prefactors, prevent the generation of diabetes, specific mechanism also need to deeper into research.
At present research clear and definite oxidative stress and diabetes relation it is very close, oxidative stress not only influence islet function with
Insulin resistance, and be the common upstream mechanism of 4 classical pathways of chronic complicating diseases of diabetes, therefore, oxidative stress is anti-
Control diabetes and its chronic complicating diseases provide new target spot and visual angle.Superoxide dismutase (SOD) vigor directly reflects machine
Internal antioxidant levels；The contents level of MDA (MDA) and nitric oxide (NO) can react the degree of lipid peroxidation,
The degree of cell damage can be judged indirectly；Glutathione peroxidase (GSH-PX) can directly remove harmful peroxidating
Thing, internal reducing agent is allowed to keep reducing condition.The morbidity of sugarcane leaf polyose prevention NOD mouse is given in experiment, as a result shows, sweet
MDA, NO and GSH-PX content reduce than model control group in sugarcane leaf polyose group serum, and SOD vigor increases than model control group,
Illustrate that sugarcane leaf polyose can strengthen oxidation resistance in NOD Mice Bodies, promote body to remove oxyradical, it is thin to improve pancreas islet β
The oxidative stress situation of born of the same parents, beta Cell of islet is protected, so as to prevent the generation of diabetes, this may be also the prevention of sugarcane leaf polyose
One of mechanism that NOD diabetes mices occur.
Claims (6)
- A kind of 1. preparation method of sugarcane leaf polyose, it is characterised in that：Sugarcane top is sweet through water extract-alcohol precipitation, sediment drying Sugarcane leaf polyose.
- 2. the preparation method of sugarcane leaf polyose according to claim 1, it is characterised in that：Dry sugarcane top is cut into small Section, adds boiling to boil, and soaks 2h, adds water to powder was covered, is extracted 3 times in 100 DEG C, each 2h；Filtering, merging filtrate, filtrate are dense Proportion 1.10-1.15 is reduced to, concentrate adds 3 times of 95% ethanol of amount, is sufficiently stirred, stands 12h；Lower sediment is collected, in baking 55 DEG C of drying, crush and produce sugarcane top polysaccharide powder in case.
- 3. application of the sugarcane leaf polyose in the medicine for preparing Prevention diabetes.
- 4. application according to claim 3, it is characterised in that：The diabetes are type 1 diabetes.
- 5. application according to claim 3, it is characterised in that：The medicine of the Prevention diabetes can improve body Oxidation resistance in interior inflammatory reaction and reinforcement.
- 6. application according to claim 3, it is characterised in that the sugarcane leaf polyose presses the preparation side of claim 1 or 2 It is prepared by method.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020063772A1 (en) * | 2018-09-30 | 2020-04-02 | 广西叶茂食品有限责任公司 | Use of sugar-containing sugarcane cell water beverage in treatment of diabetes |
-
2017
- 2017-10-16 CN CN201710968485.7A patent/CN107778374A/en active Pending
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| 何耀涛: "甘蔗叶多糖成分分析及其对1型糖尿病降糖作用研究", 《中国学位论文全文数据库》 * |
| 侯小涛 等: "甘蔗叶不同提取物对3种糖尿病模型的降血糖作用", 《华西药学杂志》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2020063772A1 (en) * | 2018-09-30 | 2020-04-02 | 广西叶茂食品有限责任公司 | Use of sugar-containing sugarcane cell water beverage in treatment of diabetes |
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