CN104688800B - Application of the total saponins from Cornus officinalis in the drug of preparation prevention and treatment diabetic complication - Google Patents
Application of the total saponins from Cornus officinalis in the drug of preparation prevention and treatment diabetic complication Download PDFInfo
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Abstract
The invention discloses the applications of the total saponins from Cornus officinalis anti-diabetic in Fructus Corni, belong to biomedicine technical field.The main active total saponins from Cornus officinalis extracted in Fructus Corni can be used for preparing the drug for the treatment of diabetes, solves the problems such as Western medicine hypoglycemic approach is single, side effect is big, provides foundation for active ingredient of Chinese herbs hypoglycemic.Total saponins from Cornus officinalis of the invention is safer, it is more effective, mild persistently, have no toxic side effect, and mechanism of action manifold effect, multiple target point, multi-functional, curative effect is not only in that reduction blood glucose, while also playing a significant role in prevention and treatment diabetic complication.Therefore, total saponins from Cornus officinalis is worth in terms of improving diabetes with considerable development and application.
Description
Technical field
The invention belongs to biomedicine technical fields, and in particular to the application of total saponins from Cornus officinalis anti-diabetic.
Background technique
Caused by diabetes (Diabetes Mellitus, DM) are a kind of absolute or opposite hyposecretion by insulin
Incretion metabolism disease characterized by hyperglycemia, the disease incidence of DM is in increase trend year by year in the world, it has also become after
The third-largest uninfection after cardiovascular disease and malignant tumour, seriously affects human health and quality of life.Now I
Ratio of state's diabetes B in diabetic increasingly increases, up to 93.7% or more.The change for diabetes that there is now
It learns drug and there is the disadvantages of toxic side effect is big, single target spot works, and natural active components of plants toxic side effect is small, more
Site effect, it is cheap the advantages that have become exploitation antidiabetic medicine important sources and plant origin treatment sugar
Urinate an important channel of sick screening bioactive compounds.
Fructus Corni (Cornus officinalis Sieb.et Zucc.) is Cornaceae Macrocarpium plant, also known as is said
Pulp of dogwood fruit, fructus corni, medicine jujube etc. are rare one of the woody medicinal plants of China's tradition, and first recorded in Shennong's Herbal, main product is in China
The ground such as Zhejiang, Henan, Shaanxi, Anhui, Shanxi and Hubei, based on artificial cultivation.Its drying and ripening pulp can be used as medicine, sour,
Puckery, tepor returns liver and kidney channel, tool liver-kidney tonifying, arresting seminal emission prevent prolapse, Heat Diabetes and other effects, often appears in the hypoglycemic prescription of Chinese medicine
In.Studies have shown that saponin(e is one of important biomolecule active constituent of Fructus Corni, has and adjust immune, inhibition alpha-glucosidase
The effects of activity, liver protection, anti-arrhythmia, but specifically improve diabetes and hypoglycemic mechanism at present also about Fructus Corni saponin(e
It is still not clear, needs further to be inquired into.
Summary of the invention
The purpose of the present invention is to provide the application of total saponins from Cornus officinalis anti-diabetic, the main work extracted from Fructus Corni
Property ingredient total saponins from Cornus officinalis can be used for preparing the drugs for the treatment of diabetes, solve that Western medicine hypoglycemic approach is single, side effect is big etc.
Problem provides foundation for active ingredient of Chinese herbs hypoglycemic.
The present invention is to be achieved through the following technical solutions:
Application of the total saponins from Cornus officinalis in the drug and/or health care product for preparing anti-diabetic.
The drug and/or health care product is enhancing body sugar tolerance, increases insulin sensitivity index or reduce insulin
Resist the drug and/or health care product of index.
The drug and/or health care product is the drug and/or health care product for improving Abnormality of Glycolipid Metabolism.
The drug and/or health care product is the medicine for reducing the content of total cholesterol, triglycerides and low-density lipoprotein
Object and/or health care product.
The drug and/or health care product is the drug and/or health care product of increasing high density lipoprotein cholesterol level.
The drug and/or health care product is the drug and/or health care product for increasing liver oxidative stress.
The drug and/or health care product is the drug and/or health care product for reducing liver organization MDA, SOD content.
The drug and/or health care product is to reduce inflammatory factor IL-6, TNF-α, the drug of CRP level and/or health care
Product.
The drug and/or health care product is to improve insulin resistance by intervening skeletal muscle insulin signal transduction pathway
Drug and/or health care product.
The drug and/or health care product be raise GLUT-4, INSR, PI3K and AKT mRNA expression drug and/
Or health care product
Compared with prior art, the invention has the following beneficial technical effects:
The invention discloses the hypoglycemic application of total saponins from Cornus officinalis, the present invention handles each group with total saponins from Cornus officinalis respectively
Diabetic mice, as a result, it has been found that total saponins from Cornus officinalis is obviously improved the fasting blood-glucose and sugar tolerance of diabetic mice；It improves
Diabetic mice fasting insulin and insulin sensitivity index reduce diabetic mice insulin resistance index；It can reduce and rouge
It is metabolized the content of closely related total cholesterol, triglycerides and low-density lipoprotein, improves high-density lipoprotein cholesterol
Content；It increases diabetic mice and increases liver oxidative stress, can reduce liver organization MDA, SOD content；Sugar can be reduced
Urinate sick mouse relevant inflammatory factors: IL-6, TNF-α, CRP.From molecular level to diabetic mice skeletal muscle GLUT-4, INSR,
The measurement of PI3K and AKT gene mRNA expression, the results showed that GLUT-4, INSR, PI3K and AKT can be improved in total saponins from Cornus officinalis
The expression of gene mRNA.Therefore, total saponins from Cornus officinalis has apparent hypoglycemic effect.
Compared with existing oral hypoglycemic agents, total saponins from Cornus officinalis of the invention is safer, more effective, mild lasting, nontoxic
Side effect, and it is mechanism of action manifold effect, multiple target point, multi-functional, curative effect is not only in that reduction blood glucose, while in prevention and treatment diabetes
Also play a significant role in complication.Therefore, total saponins from Cornus officinalis has considerable development and application in terms of improving diabetes
Value.
Detailed description of the invention
Fig. 1 is influence result of the total saponins from Cornus officinalis to diabetic mice oral glucose tolerance (OGTT)；
Fig. 2 is influence result of the total saponins from Cornus officinalis to area (AUC) under diabetic mice glucose tolerance curve；
Fig. 3 is each group mice pancreatic tissue HE coloring pathological section figure of 400 × times light microscopic observation；
Wherein, A: normal group；B: model group；C: melbine group (150mgkg-1)；D: low dose group (50mgkg-
1)；E: middle dose group (100mgkg-1)；F: high dose group (200mg kg-1)；
Fig. 4 is each group mouse liver tissue HE coloring pathological section figure of 200 × times light microscopic observation；Wherein, A: normal
Group；B: model group；C: melbine group (150mgkg-1)；D: low dose group (50mgkg-1)；E: middle dose group
(100mg·kg-1)；F: high dose group (200mgkg-1)；
Fig. 5 is the influence that total saponins from Cornus officinalis expresses diabetic mice musculature GLUT-4mRNA；
Fig. 6 is the influence that total saponins from Cornus officinalis expresses diabetic mice musculature INSR mRNA；
Fig. 7 is the influence that total saponins from Cornus officinalis expresses diabetic mice musculature PI-3K mRNA；
Fig. 8 is influence of the total saponins from Cornus officinalis to diabetic mice musculature AKT mRNA relative expression quantity.
Specific embodiment
Below with reference to specific embodiment, the present invention is described in further detail, it is described be explanation of the invention and
It is not to limit.
One, experimental material
Fructus Corni is collected in Shaanxi Fuping in October, 2013, is accredited as Fructus Corni through Shaanxi Normal University professor Xiao Yaping
Section's plant Fructus Corni (Cornus officinalis Sieb.et Zucc.).
The Fructus Corni dried pulp being enucleated is taken, is crushed, it is spare after 40 meshes excessively.21 times of 70% ethanol solution is added
→ 5min → suction filtration is extracted under the conditions of microwave power 417W, ultrasonic power 369W, fling to ethyl alcohol, water-saturated n-butanol extraction 3
Secondary → evaporated under reduced pressure, distilled water redissolution → HPD-300 type macroporous resin purification (adsorption time 3h, sample concentration 3.5mgmL-
1,50% ethyl alcohol of eluant strength, elution volume 8BV) reduced pressure of → eluent, vacuum freeze drying is up to the total soap of Fructus Corni
Glycosides dried frozen aquatic products, through total saponins from Cornus officinalis in detection dried frozen aquatic products, purity 37.36%.
Experiment reagent: STZ (Sigma Co., USA)；Diabecron sustained-release tablet (the limited public affairs of Hebei mountain nurse scholar's medicine company
Department, authentication code: national drug standard H20123024)；Doctor's jade for asking rain blood glucose meter (Houmeide Biological Sci-Tech Co., Ltd.)；Glycerol
Three esters (TG) assay kit (Changchun Hui Li Bioisystech Co., Ltd, lot number: 20110411)；Total cholesterol (TC) measurement examination
Agent box (Changchun Hui Li Bioisystech Co., Ltd, lot number: 20130626)；High-density lipoprotein cholesterol (HDL-C) measurement examination
Agent box (Changchun Hui Li Bioisystech Co., Ltd, lot number: 20130626) low density lipoprotein cholesterol (LDL-C) measurement examination
Agent box (Changchun Hui Li Bioisystech Co., Ltd；Lot number: 20130626)；Mouse islets element enzyme-linked immunoassay kit (north
Jing Keyingmei Science and Technology Ltd.)；(Science and Technology Ltd., lot number: 20140723) are built up in Nanjing to malonaldehyde (MDA) testing cassete；
(Science and Technology Ltd., lot number: 20140723) are built up in Nanjing to total number born (T-SOD) testing cassete；BCA protein quantification
Kit (Xi'an He Te Biotechnology Co., Ltd)；Mouse interleukin 6 (IL-6) enzyme-linked immunoassay kit, mouse
Tumor necrosis factor α (TNF-α) enzyme-linked immunoassay kit, mouse c reactive protein enzyme-linked immunoassay kit (Beijing section
Ying Mei Science and Technology Ltd.)；TRNzol total RNA extraction reagent (TIANGEN Biotech (Beijing) Co., Ltd., article No.: DP405-
02)；PrimeScriptTMRT reagent Kit with gDNA Eraser (TaKaRa (precious biology), article No.: RR047B)； Premix Ex TaqTMII (Tli RNaseH Plus), ROX plus (TaKaRa (precious biology), article No.:
RR82LR)；DL2000DNA Marker (TaKaRa (precious biology), article No.: 3427Q)；(Xi'an Guoan biotechnology has cholesterol
Limit company)；Sucrose, Pig cholate, sodium chloride, citric acid, sodium citrate are that analysis is pure.
Testing water used is Millipore ultrapure water, 0.9% physiological saline；Analyze ethyl alcohol (Tianjin day power chemistry examination
Agent Co., Ltd)；Paraformaldehyde (Beijing chemical reagents corporation)；RNA saves liquid (TIANGEN Biotech (Beijing) Co., Ltd.).
Two, experimental method
1, the foundation of diabetes model
The kunming mice (16 ± 2g of weight) 75 of healthy male, 5 week old of cleaning grade, buys in Xi'an Communications University's medicine
Institute's animal center.The formula composition of high-sugar-fat-diet is as follows: basal feed 66.98%, lard 10%, sucrose 20%, gallbladder are solid
Alcohol 3%, sodium taurocholate 0.02%.
Mouse adaptive feeding 3 days, two groups are randomly divided into, one group 10, gives basal feed；One group 65, give height
Rouge high sugar feed.After 4 weeks, after high-carbonhydrate diet group high in fat is deprived of food but not water 12h, it is injected intraperitoneally STZ (citrate buffer solution preparation)
60mg·kg-1, aforesaid operations are repeated after 72h, co-injection 4 times, establish diabetes model.(fasting is not after last time injection 72h
Prohibit water 12h) tail point venous blood sampling detection fasting blood-glucose, it chooses blood glucose value and is greater than 7.8mmolL-1Mouse as diabetes mould
Type mouse.
The successful mouse of modeling is randomly divided into 5 groups, specific grouping and dosage are as shown in table 1.Each group mouse is every
Day stomach-filling is primary, stomach-filling volume 0.2mL, and continuous gavage 4 weeks.Blank group gives common chow diet during administration, remaining is each
Group gives high-sugar-fat-diet.
Grouping and corresponding dosage of the table 1 at mould mouse
2, collection of specimens and processing
After gastric infusion 4 weeks, mouse is deprived of food but not water 12h, and eyeball takes blood, 3000rmin-1It is separated after centrifugation 10min
It is to be measured to be placed in -80 DEG C of preservations after packing for serum.Disconnected neck is put to death, and takes each group mouse liver, pancreas, muscle rapidly, wherein clip
0.5g liver organization is placed in physiological saline in case subsequent homogenate detects；Remaining liver, pancreas (normal saline flushing is clean) group
Knit be soaked in 4% paraformaldehyde solution fix it is to be checked；Musculature is soaked in RNA and saves in liquid, saves backup in -80 DEG C.
3, the measurement of index of correlation
1) morphological index is observed
The mental status, motion frequency, hair color, diet, drinking-water of each group mouse and urination etc. change during observation experiment, and
The changes of weight of record weekly.
2) fasting plasma glucose
The fasting blood-glucose (FBG) of each group mouse, is deprived of food but not water 12h before measurement, using blood glucose meter tail during detection administration
Sharp venous blood sampling detects fasting blood-glucose, and monitoring is primary every two weeks.
3) Diagnostic Value of Fasting Serum insulin level (FINS), insulin sensitivity index (ISI) and insulin resistance index (INI) are surveyed
It is fixed
After stomach-filling 4 weeks, each group mice serum is taken to detect insulin concentration, is tried using Reverse transcriptase method Enzyme-linked Immunosorbent Assay
It tests (ELISA) directly to be measured, operating process is carried out in strict accordance with kit specification.
According to the method that Guangwei LI proposes, insulin sensitivity index is falling for FBG and Diagnostic Value of Fasting Serum insulin concentration product
Number, and its natural logrithm is taken to be analyzed, i.e. ISI=-Ln [1/ (FBG × FINS)].
According to steady-state model HOMA-IR analytic approach, IRI=(FINS × FBG)/22.5 calculates insulin resistance index,
Reflect the insulin resistance degree of each group mouse with this.
4) oral glucose tolerance (OGTT) measures
OGTT experiment is carried out after being administered 4 weeks, the specific operation method is as follows: after the last administration, each group mouse is deprived of food but not water
12h, blood glucose value when measuring its fasting blood-glucose as 0min, immediately stomach-filling 2gkg-1Glucose solution (physiological saline configuration),
Blood glucose value after measurement fills sugared respectively when 30,60,120min.
According to approximate trapezoid area formula:
[(FBG+BG30min)×1/4+(BG30min+BG60min)×1/4+(BG60min+BG120min)×1/2+(BG120min+
BG180min) × 1/2], calculate area (AUC) under glucose tolerance curve.
5) blood lipids index measures
Isolated serum is used to detect total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-
C), low density lipoprotein cholesterol (LDL-C), is measured using semi-automatic biochemical analyzer, in strict accordance with corresponding reagent box
Specification is operated.
6) liver organization index of correlation measures
Liver organization is homogenized to grinding, 4000rmin under condition of ice bath-1It is centrifuged 10min, separation supernatant is made
10% liver homogenate liquid.According to the needs of different index determinings, homogenate can be diluted to (normal saline dilution) to suitable dense
Degree.The index that detection is needed in liver organization includes MDA, SOD, IL-6, TNF-α and CRP, and the measuring method of each index is all in accordance with it
Kit specification is operated.
7) organ index measures
Mouse is broken after neck execution, its liver, kidney, spleen are taken, and normal saline flushing is clean and weighs weight in wet base, calculates each dirty
Device index.Calculation formula: organ index (%)=(organ weights/mouse weight) × 100%.
8) pathological study
1. taking out the mouse liver being soaked in fixer, pancreatic tissue, conventional gradients ethanol wash gradually sloughs tissue
In moisture, dimethylbenzene is transparent；
2. paraffin embedding: transparent tissue block is placed in the paraffin dissolved, wax-dissolving box heat preservation is put into, it is complete to paraffin
It is embedded after full immersion tissue block；
3. slice: by embedded wax stone with 6 μm of slicer interval serial section, investing the glass slide of fixer processing
On, 40 DEG C of constant temperature dryings；
4. HE is dyed: dimethylbenzene sloughs the paraffin in slice before dyeing, through drop graded ethanol dehydration, distilled water washing, Soviet Union
Another name for dyes 10min, and tap water rinses, and hydrochloride alcohol breaks up 5s, eosin stains 3min, rises graded ethanol dehydration, and dimethylbenzene is saturating
It is bright, resinene mounting；
5. light microscopic observation each group mouse liver, pancreatic tissue Pathologic changes, and shoot photo preservation.
4, the expression measurement of the important target gene mRNA of mice skeletal tissue skeletal muscle insulin signal path
1) extraction of sample total serum IgE
Sample Total RNAs extraction is carried out using TRNzol total RNA extraction reagent, experimental implementation is carried out by product description, tool
Gymnastics is made as follows:
Tissue is put into the mortar being pre-chilled and is ground, after tissue samples at after powdered:
1. Trizol is added, room temperature preservation 5 minutes；
2. chlorination imitates 0.2ml, with forced oscillation centrifuge tube, mix well, places -10 minutes 5 minutes at room temperature；
3. drawing upper strata aqueous phase (inhaling 70%) after 12000rpm high speed centrifugation 15 minutes into another new centrifuge tube pipe, pay attention to
The protein substance that be not drawn between two layers of water phase.New pipe is moved into, the pre- cold isopropanol of isometric -20 DEG C is added, it is sufficiently reverse
It mixes, is placed in 10 minutes on ice；
4. 12000rpm high speed from 15 minutes after carefully discard supernatant, by 1mlml-1The ratio of Trizol is added 75%
DEPC ethanol washing precipitating (4 DEG C of preservations), washing precipitate, oscillation mixes, 12000 rpm high speed centrifugation 5 minutes at 4 DEG C；
5. discarding ethanol liquid, 5 minutes are placed at room temperature sufficiently to dry precipitating, it is heavy that the processed water dissolution of DEPC is added
It forms sediment, freezes in -80 DEG C.
2) concentration and purity detecting of total serum IgE
Using the OD value (260nm, 280nm) of Nanodrop2000 ultraviolet specrophotometer measurement RNA, it is pure to calculate total serum IgE
Degree and concentration illustrate that RNA purity meets the requirements when OD260nm/OD280nm is within the scope of 1.8-2.0.
Above-mentioned 5 μ L of Total RNAs extraction liquid is taken, carries out 1% agarose gel electrophoresis to it, deposition condition 100V, 5min, gel
Imaging system observed result and preservation of taking a picture identify that mentioned RNA whether there is signature band according to result.
3) the external reverse transcription of RNA
Using PrimeScriptTMRT reagent Kit with gDNA Eraser carries out cDNA reverse transcription, experiment behaviour
Make to carry out by product description, concrete operations are as follows:
1. except genomic DNA reacts
By 2 ingredient of table in preparing reaction mixture on ice, in order to guarantee the accuracy of reaction solution preparation, every reaction is carried out
When, Master Mix first should be prepared by the amount of stoichiometric number+2, be then dispensed into each reaction tube again, be eventually adding RNA sample.
2 reverse transcription reaction system I of table
By above-mentioned sample blending, 42 DEG C are incubated for 2 minutes.
2. reverse transcription reaction
It, should be first by the amount preparation of stoichiometric number+2 when carrying out every reaction in order to guarantee the accuracy of reaction solution preparation
Then Master Mix dispenses 10 μ l into each reaction tube again, carry out reverse transcription reaction immediately after soft mixing.
3 reverse transcription reaction system II of table
37 DEG C of incubations 15 minutes are loaded by above-mentioned, 85 DEG C are incubated for 5 seconds, i.e. acquisition cDNA, -20 DEG C of refrigerators of storage are spare.
4) real-time real-time quantitative PCR
1. design of primers: real-time PCR testing goal gene primer is synthesized by Beijing Invitrogen company.Four
The primer sequence of kind target gene is shown in Table 4:
4 primer sequence of table
2. reaction system: using Premix Ex TaqTMII (Tli RNaseH Plus), ROX plus into
Row amplification, experimental implementation are carried out by product description.Amplification program: 95 ° of 30sec, (95 DEG C of 5sec, 60 DEG C of 40sec) × 45
Circulation.Real-time reaction system is shown in Table 5:
5 real-time reaction system of table
Above-mentioned amplification procedure is carried out using 7500 type fluorescence quantitative PCR instrument of ABI, it is relatively fixed using GAPDH as internal reference
Amount 2-△△CTMethod carries out data analysis, its calculation formula is:
Three, data processing
Data statistics is analyzed using SPSS16.0 statistical software, and measured data are indicated with mean ± standard deviation (± s),
Mean compares with variance analysis between multiple groups, compare two-by-two variance it is neat when examined with LSD, when heterogeneity of variance, uses Dunnett ' s T3
It examines, P < 0.05 is variant statistical significance.
Four, experimental result
1, influence of the total saponins from Cornus officinalis to diabetic mice glycometabolism and Pancreas Disease Neo-Confucianism
1) ordinary circumstance
High-carbonhydrate diet joint injection low dosage STZ modeling diabetic mice high in fat, tail point take blood examination to survey fasting blood-glucose, blood glucose
Value >=7.8mmolL-1As modeling success.In 65 (2 because of death of the fighting) mouse for participating in modeling, 50 mouse
FBG≥7.8mmol·L-1, at mould rate up to 79%.
During administration, the normal mouse state of mind of organizing is good, and activity is normal, is quick on the draw, freely, hair color has light for activity
Pool, urine volume is normal, and weight gain is obvious；There is apparent " more than three " symptom (more drinks, more foods, diuresis), spirit in model group mouse
Dispirited, happiness, which flocks together to curl up, sleeps, and activity is reduced, slow movement, the withered tarnish of the in disorder color of chaeta；After being administered 3 weeks, middle and high dosage group and
, there is different degrees of improvement in the case where melbine group mouse, and wherein high dose group, melbine group improve more apparent, essence
Refreshing state improves, and activity increases, and hair color is smooth, gloss.
2) total saposins ring diabetic mice ghost image
Original body mass no significant difference before each group mouse is administered, after stomach-filling 4 weeks, normal group mouse weight, which is stablized, to be increased, mould
Type group mouse weight more normally organizes significant decrease (P < 0.05).Treatment group (melbine group, low, middle and high dose groups) Mice Body
More normal group of weight dramatically increases (P < 0.05) compared with model group, the results are shown in Table 6 without significant difference.
6 total saponins from Cornus officinalis of table to diabetic mice weight influence ()
Note: compared with blank group,*P < 0.05,**P<0.01；Compared with model group,△P < 0.05,△△P<0.01
3) total saposins influence diabetic mice fasting blood-glucose (FBG)
It is administered during 4 weeks, measures a fasting blood-glucose every 2 weeks.Compared with normal group, diabetic mice (model group,
Melbine group, low, middle and high dose groups) FBG significantly increases (P < 0.05).After being administered 2 weeks, each administration group is compared with model group FBG
There is different degrees of decreasing trend, wherein low, middle and high dose groups reduce significant (P < 0.05), middle and high dosage group mouse FBG
Reach extremely significant decline (P < 0.01), melbine group has decreasing trend, but no difference of science of statistics；After being administered 4 weeks, with model group
It compares, each administration group mouse FBG occurs extremely significant decline (P < 0.01), and becoming of reducing of melbine group and high dose group
Gesture becomes apparent from, and the results are shown in Table 7:
7 total saponins from Cornus officinalis of table the fasting blood-glucose of diabetic mice is influenced ()
Note: compared with blank group,*P < 0.05,**P<0.01；Compared with model group,△P < 0.05,△△P<0.01
4) influence of the total saposins to diabetic mice FINS, ISI and IRI
After being administered 4 weeks, each group mouse Diagnostic Value of Fasting Serum insulin content is detected, and calculate its corresponding insulin sensitivity index
With resistance index.Compared with normal group, the extremely significant reduction (P < 0.01) of model group mouse FINS and ISI, the extremely significant liter of IRI
High (P < 0.01).After administration is intervened, compared with model group, melbine group, middle and high dosage group FINS, ISI are in extremely significant rising
(P < 0.01), IRI are in extremely significant decline (P < 0.01), show that total saponins from Cornus officinalis is increasing insulin secretion and sensibility, delaying
Play the role of good in terms of solution insulin resistance, the results are shown in Table 8:
8 total saponins from Cornus officinalis of table is to diabetic mice Fasting insulin level (FINS), insulin sensitivity index (ISI)
And insulin resistance index (IRI) influence ()
Note: compared with blank group,*P < 0.05,**P<0.01；Compared with model group,△P < 0.05,△△P<0.01
5) influence of the total saposins to diabetic mice oral glucose tolerance (OGTT)
By measuring to each group mouse oral sugar tolerance, 9 and Fig. 1,2 the results are shown in Table.By graph results it is found that each group is small
Mouse is after the glucose solution of stomach-filling 2g/kg, and in 0.5h, blood glucose value reaches peak；Normal group mouse blood glucose value base after 1h
Originally postprandial normal level is returned to；Model group mouse is constantly in hyperglycemia state after reaching blood glucose value peak, and with normal group
There is extremely significant difference (P < 0.01)；There is downward trend in blood glucose value after 1h for melbine group, middle and high dosage group, in 3h
Fall is obvious, is in compared with model group extremely significant difference (P < 0.01).Each group glucose tolerance in mice area under the curve compares knot
For fruit it is found that compared with normal group, diabetic mice (model group, melbine group, low, middle and high dose groups) AUC is extremely significant
It increases (P < 0.01)；Compared with model group, melbine group, low, middle and high dose groups AUC have it is extremely significant decline (P <
0.01)。
9 total saponins from Cornus officinalis of table to diabetic mice oral glucose tolerance (OGTT) influence ()
Note: compared with blank group,*P < 0.05,**P<0.01；Compared with model group,△P < 0.05,△△P<0.01
6) influence of the total saposins to diabetic mice organ index
Each group mice organs index results are shown in Table 10 after measured.As shown in Table 10, compared with normal group, model group Mouse Liver
Dirty, kidney and index and spleen index significantly increase (P < 0.01)；Compared with model group, it is each be administered intervention group mouse liver, kidney and
Index and spleen index has decreasing trend, and wherein melbine group significantly reduces (P < 0.05), and total saposins administration group decreasing trend is in agent
Dependence is measured, and middle and high dosage group lowers trend significantly (P < 0.01).
10 total saponins from Cornus officinalis of table to the influence situation of each organ index of diabetic mice ()
Note: compared with blank group,*P < 0.05,**P<0.01；Compared with model group,△P < 0.05,△△P<0.01
7) influence of the total saposins to diabetic mice Pancreas Disease Neo-Confucianism
The metamorphosis of light microscopic observation each group mice pancreatic.Normal group pancreas islet boundary is clear, and number is more, islet cells
Large number of and marshalling, cytoplasm is abundant, and core circle is (see Fig. 3 A) placed in the middle；More normal group is compared, model group mouse islets number
It largely reduces, islet cells is in irregular shape, disorganized, volume-diminished, and most cell cytosol vacuolar degenerations illustrate diabetes
Mouse model injury of pancreas is significant (see Fig. 3 B)；Melbine group mouse islets number is slightly reduced, and cell volume slightly reduces,
Part is in empty balloon-shaped (see Fig. 3 C)；Low dose group mouse islets number moderate is reduced, and islet cells volume slightly increases, cell
Quantity is reduced, still visible vacuolar degeneration (see Fig. 3 D)；Middle dose group mouse islets number is slightly reduced, and islet cells volume increases
Greatly, cell quantity increases, accidental vacuolar degeneration, endochylema distribution uniform (see Fig. 3 E).High dose group mouse islets number is slight
It reduces, islet cells volume increases, and cell quantity increased significantly, accidental vacuolar degeneration, and born of the same parents will uniformly (see Fig. 3 F).
2, influence of the total saponins from Cornus officinalis to diabetic mice glycolipid metabolism and liver pathology
1) influence of the total saposins to diabetic mice blood lipid
After drug therapy 4 weeks, mice serum is taken to detect each blood lipids index.Compared with normal group, model group mouse TC, TG,
The horizontal extremely significant raising (P < 0.01) of LDL-C, HDL-C level significantly reduce (P < 0.05).Compared with model group, melbine group
Mouse TG is reduced, HDL-C is increased, and have significant change (P < 0.01), and TC, LDL-C level also have decreasing trend, no statistics
Difference；Each dosage group mouse TC, TG, LDL-C level is in decreasing trend, compared with model group there were significant differences (P < 0.01), HDL-
C level significantly increases (P < 0.01) compared with model group, and wherein TG, HDL-C, LDL-C variation tendency are in dosage according to lazyness, as a result sees
Table 11:
11 total saponins from Cornus officinalis of table to diabetic mice blood lipid level influence ()
Note: compared with blank group,*P < 0.05,**P<0.01；Compared with model group,△P < 0.05,△△P<0.01
2) influence of the total saposins to diabetic mice liver pathology
Normal group murine liver tissue color is ruddy, and lobuli hepatis structure is normal under light microscopic, and liver cell is centered on central vein
It is arranged at strand, cell boundaries are clear, core is round and it is central (see Fig. 4 A) to be located at cell；Model group murine liver tissue whiting matter is crisp,
Liver rope disorder, liver cell volume increases, soft edge, nucleus shrinkage or cracking, visible fat vacuolar degeneration in endochylema, liver
Obvious lesion (see Fig. 4 B)；Melbine group lobuli hepatis structure is complete, and liver cell profile is clear compared with model group, it is seen that fat vacuole
Denaturation reduces (see Fig. 4 C)；The visible moderate steatosis of low dose group liver cell, vacuolar degeneration mitigate compared with model group (see figure
4D)；Middle dose group lobuli hepatis structure is substantially complete, it is seen that slight steatosis and vacuolar degeneration (see Fig. 4 E)；High dose group
Lobuli hepatis structure is complete, and disorder that hepatic cell cords is slight, caryoplasm is more visible, and steatosis and vacuolar degeneration height are alleviated (see figure
4F)。
3, influence of the total saponins from Cornus officinalis to diabetic mice liver inflammation and oxidative stress
1) influence of the total saposins to the diabetic mice liver inflammation factor
After being administered 4 weeks, detection each group mouse liver tissue IL-6, TNF-α and the variation of CRP inflammatory factor.With normal group phase
Than model group hepatic tissue IL-6, TNF-α and CRP level significantly increase (P < 0.01)；Compared with model group, melbine group
Hepatic tissue IL-6, TNF-α and CRP level are in decreasing trend, wherein TNF-α level be in significant changes (P < 0.01), IL-6 and
CRP variation is not statistically significant；Each dosage group murine liver tissue IL-6, TNF-α and CRP level all significantly reduce, compared with model group
There were significant differences (P < 0.01), and variation tendency is in dosage according to lazyness, the results are shown in Table 12:
12 total saponins from Cornus officinalis of table on diabetic mice liver organization IL-6, TNF-α, CRP influence ()
Note: compared with blank group, P < 0.01 * P < 0.05, * *；Compared with model group, P < 0.01 △ P < 0.05, △ △
2) influence of the total saposins to diabetic mice liver oxidative stress
The severity and scavenging activated oxygen energy that MDA, SOD horizontal reverse film projector body are attacked by free radical in liver organization
Power, and then reflect the extent of damage of liver function.Compared with normal group, the horizontal significant raising of MDA, SOD in model group hepatic tissue
(P<0.01)；Compared with model group, each intervention group MDA, SOD level that is administered all is in decreasing trend, wherein melbine group MDA
Level significantly reduces (P < 0.01), and SOD level is without significant difference；The significant decrease of total saposins administration group MDA, SOD level (P <
0.01), and it is in dose dependent, is shown in Table 13:
13 total saponins from Cornus officinalis of table on diabetic mice liver organization MDA, SOD influence ()
Note: compared with blank group,*P < 0.05,**P<0.01；Compared with model group,△P < 0.05,△△P<0.01
4, total saponins from Cornus officinalis expresses the important target gene of diabetic mice bone flesh skeletal muscle insulin signal path
It influences
1) influence of the total saposins to diabetic mice bone flesh GLUT-4 gene expression
Glucose Transporter 4 (GLUT-4) is the important link of insulin signal transduction approach, in the absorption of glucose sugar
It is acted on crucial speed limit is played in transhipment, is one of the important target gene for treating diabetes.Total saponins from Cornus officinalis is small to diabetes
The influence of murine skeletal muscle GLUT-4 gene expression, is shown in Fig. 5.As seen from the figure, compared with normal group, model group GLUT-4 relative expression
Amount is significant to lower (P < 0.01)；Compared with model group, melbine group, high dose group GLUT-4 relative expression quantity significantly raise (P
<0.01).It can be seen that the expression quantity of diabetic mice skeletal muscle GLUT-4mRNA can be improved in total saponins from Cornus officinalis.
2) influence of the total saposins to diabetic mice bone flesh INSR gene expression
Insulin must be with insulin receptor (INSR) on cell membrane in conjunction with adjusting blood glucose balance.Once body cell membrane
Lack this receptor, the effect of insulin will be affected, to slow down intake of the cell to glucose, increase blood glucose level.
Influence of the total saponins from Cornus officinalis to diabetic mice skeletal muscle INSR gene expression, is shown in Fig. 6.As seen from the figure, compared with normal group,
Model group INSR relative expression quantity significantly lowers (P < 0.01)；Compared with model group, melbine group, high dose group INSR phase
(P < 0.01) is significantly raised to expression quantity.It can be seen that total saponins from Cornus officinalis can raise diabetic mice skeletal muscle INSR mRNA's
Expression quantity.
3) influence of the total saposins to diabetic mice bone flesh PI-3K gene expression
PI-3K approach is the main path that insulin adjusts glycolipid metabolism, and intracellular GLUT-4 is finally promoted to be transferred to carefully
After birth surface, and then utilization and the Glycogen synthesis etc. for promoting grape cell sugar, adjust body blood glucose balance.Total saponins from Cornus officinalis pair
The influence of diabetic mice skeletal muscle PI-3K gene expression, is shown in Fig. 7.As seen from the figure, compared with normal group, model group PI-3K phase
(P < 0.01) is significantly lowered to expression quantity；Compared with model group, melbine group, high dose group PI-3K relative expression quantity are significant
It raises (P < 0.01).It can be seen that total saponins from Cornus officinalis can raise the expression quantity of diabetic mice skeletal muscle PI-3K mRNA.
4) influence of the total saposins to diabetic mice bone flesh AKT gene expression
AKT is the downstream molecules of insulin signaling pathway, and one of the important target spot for the treatment of diabetes, mediate insulin
The glucose metabolism of stimulation.Influence of the total saponins from Cornus officinalis to diabetic mice skeletal muscle AKT gene expression, is shown in Fig. 8.It can by figure
Know, compared with normal group, model group AKT relative expression quantity significantly lowers (P < 0.01)；Compared with model group, melbine group,
High dose group AKT relative expression quantity significantly raises (P < 0.01).It can be seen that total saponins from Cornus officinalis can raise diabetic mice skeletal muscle
The expression quantity of AKT mRNA.
Five, conclusion
1, influence of the total saponins from Cornus officinalis to diabetic mice glycometabolism and Pancreas pathology
The display of this experimental result, diabetic mice is during entire administration, and " three-many-one-little " symptom is significant, and weight subtracts
Gently, FBG is constantly in higher level (21.57mmol/L), and FINS, ISI are decreased obviously, and IRI then significantly rises, and pancreas islet occurs
Element is resisted, and OGTT is abnormal, and each organ index organizes significant difference with normal, and islet function is also badly damaged.
Upon administration, each group mouse situation makes moderate progress for total saponins from Cornus officinalis administration group and melbine group.It is wherein each
Group weight is on the rise；Melbine group and middle and high dosage group FBG downward trend are significant (P < 0.01), promote insulin
Secretion, ISI, OGTT and each organ index be obviously improved, and IRI significantly reduces (P < 0.01), and islet function obtains centainly
Protection, Insulin Resistance alleviated.In conclusion total saponins from Cornus officinalis, which has, reduces blood glucose, improve sugar tolerance, pancreas
Apparent dose dependent is presented in the performance of insulin resistance and the effect for protecting islet function, effect.
2, influence of the total saponins from Cornus officinalis to diabetic mice lipid metaboli and Pancreas pathology
In this experiment, model group mouse TC, TG, LDL-C are significantly increased, and HDL-C is then reduced, to a certain extent can be with
The blood fat disorder of analogy diabetic；There is obvious fat deposition and vacuolar degeneration in liver organization simultaneously, liver function seriously by
Damage.Melbine group and total saponins from Cornus officinalis administration group reduce TC, TG, LDL-C level to varying degrees, increase HDL-C,
Middle total saponins from Cornus officinalis administration group TG, HDL-C, LDL-C variation tendency is in dosage according to lazyness；Middle and high dosage group liver under light microscopic
The fat accumulation of tissue has certain alleviation, it is seen that total saponins from Cornus officinalis is improving diabetic mice disorders of lipid metabolism, liver function
Certain positive effect can be played in terms of protection.
3, influence of the total saponins from Cornus officinalis to diabetic mice liver inflammation and oxidative stress
The display of this experimental result, model group mouse liver tissue IL-6, TNF-α, CRP, MDA dramatically increase (P < 0.01),
SOD activity is then remarkably decreased (P < 0.01), shows that obvious inflammatory reaction occurs in liver, and MDA increase can reflect liver by freedom
Base attack damage is serious, and the reduction of SOD activity then shows that body Scavenging ability weakens, and oxidative stress increases.Mountain Zhu
Cornel total saposins administration group IL-6, TNF-α, CRP, MDA significantly reduce (P < 0.01), and SOD activity is in rising trend, and are in agent
Measure dependence.Show that total saponins from Cornus officinalis can alleviate liver inflammation, reduce MDA content, increases SOD activity, to increase liver
Oxidation resistance reduces liver oxidative stress, reduces the insulin resistance of liver organization.
4, influence of the total saponins from Cornus officinalis to the important target gene of diabetic mice skeletal muscle insulin signal path
This experimental selection GLUT-4, INSR, PI-3K and AKT tetra- crucial target genes are treated to study total saponins from Cornus officinalis
The molecular mechanism of diabetes.Experimental result shows, model group mice skeletal GLUT-4, GLUT-4, INSR, PI-3K and AKT
Expression significantly reduces, and shows that obstacle occurs in skeletal muscle insulin signal path, glucose metabolism is obstructed and insulin resistance occurs；
Melbine group and the significant liter of total saponins from Cornus officinalis high dose group mice skeletal GLUT-4, INSR, PI-3K and AKT expression
High (P < 0.01), shows that total saponins from Cornus officinalis can raise GLUT-4, INSR, PI-3K and AKT gene in insulin signaling pathway
Expression, to promote utilization of the skeletal muscle to glucose, thus it is speculated that its be total saponins from Cornus officinalis improve diabetic mice skeletal muscle
One of molecular mechanism of insulin resistance.
Claims (1)
1. application of the total saponins from Cornus officinalis in the drug of preparation prevention and treatment diabetic complication, it is characterised in that:
The drug is the content for reducing total cholesterol, triglycerides and low-density lipoprotein, increasing high density lipoprotein gallbladder
Sterol content reduces liver organization MDA, SOD content, and reduces the drug of inflammatory factor IL-6, TNF-α, CRP level；
Wherein, the total saponins from Cornus officinalis is prepared as follows:
The Fructus Corni dried pulp being enucleated is taken, is crushed, it is spare after 40 meshes excessively；Smashed Fructus Corni pulp powder 21 is added
70% ethanol solution again extracts 5min then under the conditions of microwave power is 417W, ultrasonic power 369W；Then filter,
Ethyl alcohol is flung to, is saturated with water extracting n-butyl alcohol 3 times, then evaporated under reduced pressure, distilled water are pure with HPD-300 type macroreticular resin after redissolving
Change processing, eluent are concentrated under reduced pressure, and vacuum freeze drying is up to total saponins from Cornus officinalis dried frozen aquatic products, the total saponins from Cornus officinalis dried frozen aquatic products
Moderate purity is 37.36%；
Wherein, the adsorption time of above-mentioned purification process is 3h, sample concentration 3.5mgmL-1, eluant, eluent be that concentration is 50%
Ethyl alcohol, elution volume 8BV.
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