CN106755266A - Hypoglycemic, pancreas islet protection activity test method of Moringa leaf flavone extract and application thereof - Google Patents
Hypoglycemic, pancreas islet protection activity test method of Moringa leaf flavone extract and application thereof Download PDFInfo
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Abstract
Hypoglycemic, pancreas islet protection activity test method the invention discloses Moringa leaf flavone extract and application thereof, it is comprised the steps of：The preparation of S1, chromocor extract；S2, glucose uptake experiment：According to glucose uptake vivacity K；S3, glycolipid metabolism activity test：Calculate the measure and insulin index of rat fat index content, rat blood serum SOD and MDA contents；S4, the experiment of pancreas islet protection activity：Calculate islet cell mass rate of change M and beta Cell of islet growth rate N.The present invention calculates glucose uptake vivacity K; determine the change of blood sugar rate of rat; rat fat index content; the measure and insulin index of rat blood serum SOD and MDA contents; and islet cell mass rate of change M and beta Cell of islet growth rate N; comprehensive descision goes out the hypoglycemic of Moringa leaf flavone extract, pancreas islet protection activity index, and the utilization and exploitation to leaf of Moringa provide a quantitative standard.
Description
Technical field
The present invention relates to biomedicine technical field, the especially hypoglycemic of Moringa leaf flavone extract, pancreas islet protection activity
Test method and application thereof.
Background technology
Diabetes are one group of metabolic diseases being characterized with hyperglycaemia.Hyperglycaemia be then due to defect of insulin secretion or
Its biological agent is damaged, or both have concurrently and cause.Long-standing hyperglycaemia during diabetes, cause various tissues, particularly eye,
Kidney, heart, blood vessel, chronic lesion, the dysfunction of nerve.The method that there is no radical cure diabetes at present, but by various treatments
Means can control diabetes.The hypoglycemic research of natural plants is more and more popular, and research display leaf of Moringa powder also has
Play the role of hypoglycemic.Moringa (morigaoleifera) category Moringaceae, Moringa, originate from the sub- Himalaya of north India
Mountain zone.India and African country are usually used in the diseases such as treatment diabetes, hypertension, cardiovascular disease, obesity.In recent years, it is peppery
Wood has introducing and planting in China Guangdong, Yunnan, Hainan and Sichuan etc..Leaf of Moringa is approved for new resource food within 2012, respectively
Plant Moringa product development burning hot, be widely used in the fields such as food, health products, purification of water quality, cosmetics.
The leaf of Moringa flavones (TFM) that the AB-8 macroporous resin purifications such as Chen Ruijiao are crossed is used for diabetic mice, carries out
Hypoglycemic activity is observed, and be finally found that, TFM has significant hypoglycemic activity, moreover it is possible to increase SOD in serum content, reduces MDA contents, and
On the blood sugar of non-diabetic mice without influence.Also there are some researches show can effectively reduce blood sugar in using moringa oleifera leaf extractive 3h
Level, and as increase effect of dosage is also improved, but its validity wants substandard hypoglycemic agent.Moringa adaptability
Extensively, resistance to extensive agriculture, leaf of Moringa nutritive value is abundant, healthcare function is various, and Moringa plantation and leaf of Moringa product are carried out in China
Market and commercial promise it is wide.Now to the exploitation of leaf of Moringa resource still, in the starting stage, but passed through substantial amounts of detection and
Experiment proves that leaf of Moringa is a kind of pollution-free food of safety and Health, and Moringa in 2012 has been approved as China newly by ministry of Health of China
Resource food.The composition that leaf of Moringa is reported is the change of most study in current leaf of Moringa exploitation based on flavone compound
Compound, its chief active shows as anti-oxidant, hypoglycemic, protection liver etc..Nowadays the shape that global diabetic's quantity surges
Under gesture, the natural protective agents demand to the disease is greatly increased, and leaf of Moringa flavones because of it while just have blood sugar decreasing effect,
Rich in nutrition content, Small side effects, the advantages such as diabetic complication that improve, and leaf of Moringa flavones raw material possesses collection convenience, money
Source is enriched, and the features such as extraction process is simple, leaf of Moringa flavones is provided with the potentiality as preventing and treating diabetes medicament, and this is probably
The further developing direction of leaf of Moringa.But hypoglycemic, pancreas islet protection activity the experiment tool there is presently no Moringa leaf flavone extract
The report of body index and Moringa leaf flavone extract related application, judges hypoglycemic, the pancreas islet protection of Moringa leaf flavone extract
Activity index.
The content of the invention
It is an object of the invention to overcome the deficiencies in the prior art, there is provided a kind of hypoglycemic, the pancreas of Moringa leaf flavone extract
Island protection activity test method and application thereof, can interpolate that hypoglycemic, the pancreas islet protection activity of Moringa leaf flavone extract.
The purpose of the present invention is achieved through the following technical solutions：The hypoglycemic of Moringa leaf flavone extract, pancreas islet are protected
Shield activity test method, it is comprised the steps of：
The preparation of S1, chromocor extract：Leaf of Moringa flavone powder 80-100mg is weighed, 1mL is settled to, mixed, warp
0.22um filters are filtered, and are distributed into 5 equivalent samples, stand-by；
S2, glucose uptake experiment：After experiment is digested with 0.25% trypsin solution of HepG2 cell solutions, inoculation
In culture plate, CO is then placed in together2Cultivated in incubator, wherein, the cell concentration of HepG2 cell solutions is 0.8-1.5
×105Individual/mL, after after cell attachment, discards original fluid, with initial glucose in glucose kit each hole nutrient solution of measure
Concentration, be calculated as G1, serum-free medium is changed, hungry 10-12h adds DMEM sugared nutrient solution 160-200 μ L high, then add respectively
Enter 20-50 μ L samples, determine the concentration of glucose in each hole nutrient solution after incubation 24-28h with glucose kit, be calculated as G2,
The consumption G of glucose is calculated using below equationC,Wherein, n is the hole count of experiment in culture plate, root
According to glucose uptake vivacity K, judgement sample glucose uptake is active,G0For the isoquercitrin of same volume is tested
Sample carries out the glucose utilization that glucose uptake experiment is obtained, and the content of isoquercitrin is in isoquercitrin test specimen
0.0156mg/mL, glucose uptake vivacity K are more than or equal to 1, then the glucose uptake activity for marking the sample is excellent, Portugal
Grape Sugar intake vivacity K is less than 1, then it is good to mark the glucose uptake activity of the sample, and K is small for glucose uptake vivacity
In 0.5, then it is not good to mark the glucose uptake activity of the sample；
S3, glycolipid metabolism activity test：Take artificial induction's diabetes B rat model sample sets and control group each 10
, wherein, the standard of artificial induction's diabetes B rat model is fasting blood-glucose big more than 7.8mmol/L or 2h-plasma glucose
In 11.1mmol/L, sample sets and control group are given the physiological saline of equivalent by the dosage of sample sets, in it is determined that rat Cheng Mo works as
Its unified beginning gastric infusion, continuous gavage 21 days, daily 8：00AM-9:00AM timings are administered once on an empty stomach, are given again after gavage
The feed and drinking-water of equivalent are given, the change of blood sugar rate for taking each group rat respectively, rat fat index content, rat serum are taken after 21 days
The calculating of the measure and insulin index of clear SOD and MDA contents；
The measure and insulin index of change of blood sugar rate, rat fat index content, rat blood serum SOD and MDA contents
Calculating need by the blood specimen collection of rat, after concrete operations are rat last day gavage, fasting can't help water overnight.With second
Ehterization animal, vena ophthalmica clump blood sampling, after placing 1h in 37 DEG C, 4 DEG C of refrigerator overnights are centrifuged 10min with 3000r/min, collect
Serum, -20 DEG C save backup.Blood sampling puts to death rat after finishing, and takes liver, pancreas and is fixed in Bo Enshi liquid, does histotomy
It is standby.Liver is taken in 4 DEG C of PBS of pH7.0, Apoptosis by Flow Cytometry is standby with the cycle.Liver, pancreas are taken in RNA
Protective agent, -80 DEG C of preservations, is cooked quantitative fluorescent PCR standby.Liver, pancreas are taken in glutaraldehyde, 4 DEG C of preservations, Ultrastructural Pathology is seen
Examine standby.
According to the requirement of kit specification, to the glucose (Glu) in rat blood serum, superoxide dismutase (SOD),
MDA (MDA), HDL-C (HDL-C), LDL-C (LDL-C), T-CHOL (T-
CHO), triglycerides (TG), insulin (INS), glucagon-like-peptide-1 (GLP-1) are measured.
According to the rat fasting blood-glucose value for measuring and FPI value, the following pancreas islet index of correlation is calculated.
HOMA-IR=FPG × FINS/22.5
HOMA-IS=1/ (FPG × FINS)
IAI=Ln [1/ (FPG × FINS)]
HOMA- β (%)=20 × FINS/ (FPG-3.5)
FPG：Fasting blood sugar, mmol/L；FINS：Fasting insulin level, mIU/L.
HOMA (Homeostasis model assessment), also known as HOMA steady-state models, extensively should turn at present
For clinical evaluation diabetes patient's insulin sensitivity, the common counter of insulin resistance level and islet beta cell function.
HOMA-IR (Homeostasis Model of Insulin Resistance) is the insulin resistance water for evaluating individuality
Flat index, the HOMA-IR indexes of normal individual are 1, and with the elevated rising of insulin resistance level, HOMA-IR indexes will
Higher than 1.HOMA-IS is the index for evaluating the insulin sensitivity of individuality, HOMA-IS (Homeostasis Model of
Insulin Secretion) index raises with the rising of insulin resistance.After the index takes natural logrithm, can
Clinically evaluated another index insulin action index IAI (Insulin Affect of insulin resistance
Index).HOMA- β are the indexs for evaluating the islet beta cell function of individuality.The HOMA- β indexes of normal individual are 100%.
In diabetic population, HOMA- β (Homeostasis Model of β-cell Function) index can be different because of disease process
And deviate normal value, and islet beta cell function reduction then its numerical value reduction, then its numerical value is raised for function enhancing.Zhao Wenjie is with Sang Bai
Skin is research object, around insulin resistance, using three-dimensional cell model and whole animal model, inquires into root bark of white mulberry reduction blood sugar
Effect and its mechanism.
Insulin index of the invention shows that leaf of Moringa flavones gavage can reduce HOMA- after 21 days compared with control group
IR, improves HOMA-IS, reduces IAI, improves HOMA- β, illustrates taking for leaf of Moringa flavones, it is possible to decrease Insulin Resistance of Rats water
Flat, enhancing body improves insulin action to insulin sensitivity, strengthens the effect of islet beta cell function, leaf of Moringa flavones mouthful
Take 30mg/k.
Wherein, the feed of artificial induction's diabetes B rat model is according to document《The foundation of diabetes B rat model
And its application in auxiliary hyperglycemic function evaluation》Described method, selection optimum formula prepares high-sugar-fat-diet, i.e.,
The sucrose of the lard of basal feed+10%+20%.Basal feed, sucrose are crushed, lard low-grade fever is mixed after melting, it is suitable to add water to
Humidity, then it is the cylindrical particle of diameter about 1.5cm, 5-8cm long to pelletize.After the completion of granulation, it is placed in 40 DEG C of constant temperature air blast and does
It is extremely dry in dry case.Prepare high-sugar-fat-diet sealing be stored in lucifuge, dry at.Each preparation amount feeds no more than rat
Measure within one week, prevent feed spoilage, fed again after the feed drying do not eaten up for a day.Experimental rat feeds in 550 × 400 ×
In the cage of 200mm, periodicity of illumination 12h：Ventilation, constant about 25 DEG C of keeping temperature in 12h, feeding floor.Periodic replacement bedding and padding, every time
Sterilization liquid disinfectant mouse cage during replacing, it is ensured that big mouse cage health.Sufficient drinking-water is given to be fed 30 days with high-sugar-fat-diet.31st
My god, water 24h is can't help in fasting.With 5 ‰ STZ solution, (0.1mM, pH4.5 citrate buffer of 4 DEG C of precoolings are fresh to match somebody with somebody within 32nd day
System, lucifuge dissolving), rat is injected by 40mg/kg body weight doses disposable celiac, whole rats have all been injected in 30min
Finish.Hereafter drinking-water is given with feed feeding, daily timing feeding, 9：00 and 17:00, two times a day, changed according to rat urine volume and padded
Material, keeps bedding and padding to dry.
S4, the experiment of pancreas islet protection activity：By sample sets in glycolipid metabolism activity test and the rat pancreas ripple of control group
Engler's liquid is fixed, and by after dehydration, transparent, waxdip, with FFPE, section, thickness is 4.5-5.5 μm, and 30-45 DEG C of baking oven adds
Heat fixation, HE dyeing and aldehyde fuchsine stain are carried out to pancreas section, are dehydrated, and transparent, mounting in basis of microscopic observation, and is taken pictures
Record, according to the quantity A of islet cells in the pancreas section that HE is dyeed2With islet cell mass A in control group1, calculate pancreas islet thin
Born of the same parents amount change M, whereinA2、A110 respectively in sample combination control group under the section of HE dyeing
The average value of rat pancreas cell quantity, the red area areal calculation pancreas islet β according to representated by aldehyde fuchsine stain beta Cell of islet
Cell growth rate N, wherein,B2、B1Lower 10 of the section of aldehyde fuchsine stain respectively in sample combination control group
The average value of the red area area representated by beta cells of isolated rat islets quantity.
Hematoxylin-eosin (Hematoxylin-EosinStaining, HE) decoration method, is histology, embryology, pathology
Teaching is learned with most basic, most popular technical method in scientific research.Mainly make endonuclear chromatin with intracytoplasmic core
Sugared body colours the composition red coloration of hyacinthine, cytoplasm and extracellular matrix, but pancreas islet is distinguished not substantially with pancreatic cell, and
Several cells of pancreas islet are difficult to differentiate between.The β cells of pancreas islet are basocytes, and aldehyde-fuchsin is to special protein and containing sulfate radicals
Mucopolysaccharide has very strong affinity, and elastomer is combined very tightly, in addition to basocyte, can be coloured very well.It is multiple using aldehyde
Red colouring is dyeed to pancreas, makes pancreatic islet alpha, β cells high-visible, is usually used in observing islet cells.
When islet tissue HE is dyeed, it is found that the pancreas islet generation lesion of higher group of blood sugar is more serious, especially pancreas islet pars intermedia
Position, cell quantity is reduced, and is found by aldehyde fuchsine stain, and it is beta Cell of islet that the cell of reduction is most of, illustrates that rat occurs
Hyperglycaemia is probably, because β cells there occurs apoptosis, to reduce insulin secretion, and taking for isoquercitrin is generated and phosphoric acid
Xi Gelieting similar effect, reduces the generation of β Apoptosis, so as to protect beta Cell of islet, makes diabetes rat insulin
Secretory volume increases, so as to occur in that the phenomenon that blood sugar declines.
Research to moringa oleifera leaf extractive both at home and abroad at present, it was demonstrated that Moringa leaf flavonoid compound have reduction sugar tolerance,
Hypoglycemic, reducing blood lipid etc. is acted on.The leaf of Moringa flavones ethanol reflux extraction reported, time-consuming, less efficient, and only with
Optimized by Orthogonal Test extraction process.Microwave abstracting is extracted for flavone compound, extraction efficiency is high, time-consuming, save solvent,
It is environmental protection, cost-effective.Response phase method can study the reciprocation of several factors, more and more widely compared with Orthogonal Method
It is used in solution Multivariable.
Described leaf of Moringa flavone powder is obtained by following methods：Leaf of Moringa powder is taken with 50-60%v/v ethanol as solvent,
Solid-liquid ratio is 1:50-60g/ml, carries out microwave abstracting, collects extract solution, wherein, microwave power is 300-400W, extraction time
It is 6-10min, then extract solution filtration, Rotary Evaporators are concentrated under reduced pressure, reclaims ethanol to dry, 12 is stood after absolute ethyl alcohol redissolution
Hour filtration, removes water-solubility impurity, and filtrate rotated evaporation under reduced pressure concentration again removes ethanol, highly concentrated with distilled water diluting
Degree flavonoids solution is packed as 2mL/ pipes in 15mL screw socket centrifuge tube with cover, is placed in vacuum cryogenic temperature freezing drying instrument 48h, and flavones is complete
Complete freezing is powder, after taking-up, sealing, lucifuge, -80 DEG C of preservations.
The following is using microwave extraction method experiment of single factor, the main influence for considering concentration of alcohol, the influence of extraction time,
The influence of microwave power, the influence of liquid ratio.
5 parts of leaf of Moringa powder are weighed, every part of 1g is extracted in Microwave Extraction Apparatus.Be separately added into 50%, 60%, 70%,
80%th, 90% ethanol, microwave power 200w, extraction time 6min, liquid ratio 50:1, as a result see Fig. 1.By Fig. 1, ethanol volume integral
Number is at 60%, and flavones yield is maximum, after 60%, yield continuous decrease.Accordingly, as preferred, ethanol volume is selected
Fraction is 60%.
5 parts of leaf of Moringa powder are weighed, every part of 1g adds 60% ethanol, microwave power 200w, liquid ratio 50:1, respectively by carrying
Time 6min, 8min, 10min, 12min, 14min is taken to be extracted in Microwave Extraction Apparatus.Result is shown in Fig. 2.From Figure 2 it can be seen that at any time
Between extend, general flavone yield is raised, upon extracting between for 8min when, highest is reached, accordingly, as preferred, during selective extraction
Between be 8min.
5 parts of leaf of Moringa powder are weighed, every part of 1g adds 60% ethanol, liquid ratio 50:1, microwave power be respectively 100w,
200w, 300w, 400w, 500w, extract 8min.Result is shown in Fig. 3.As seen from Figure 3, as extraction power increases, general flavone yield
Increase, highest when power reaches 400w then declines during higher than 400w.Accordingly, as preferred, selective extraction power is 400w.
Weigh 5 parts of leaf of Moringa powder, every part of 1g, respectively according to 30:1、40:1、50:1、60:1、70:1 adds 60% ethanol,
Microwave power 400w, extracts 8min, is extracted in Microwave Extraction Apparatus.Result is shown in Fig. 4.From fig. 4, it can be seen that as liquid ratio increases,
Flavones yield is consequently increased, when liquid ratio is 60:When 1, general flavone yield highest, more than 60:1 occurs declining.Therefore, select
It is 60 to select liquid ratio:1.
The hole count of culture plate is 96 holes in described glucose uptake test procedure.
Described experiment with HepG2 cell solutions is obtained by following methods：HepG2 cells are inoculated in Tissue Culture Flask
In, RPMI-1640 is added, it is placed in 37 DEG C, cultivates in the 5%CO2 incubators of saturated humidity, 1 nutrient solution is changed every 2d,
Passed on after 3d, passage number 1:4, the cell in growth period of taking the logarithm is tested.
Glucose uptake vivacity K is calculated according to by glucose uptake tester, by glycolipid metabolism activity test
Determine the change of blood sugar rate of rat, rat fat index content, the measure and insulin of rat blood serum SOD and MDA contents
Index, and by pancreas islet protection activity experiment calculation islet cell mass rate of change M and beta Cell of islet growth rate N, comprehensively sentence
Break and hypoglycemic, the pancreas islet protection activity index of Moringa leaf flavone extract, the utilization and exploitation to leaf of Moringa provide one and determine
The standard of amount, wherein, contain more than 50%, rat fat index more than 1, change of blood sugar rate for glucose uptake vivacity K
Not exceeded, the rat blood serum SOD growth rates of amount be 10-20%, MDA slips more than 7%, HOMA-IR indexes more than 1, meter
Islet cell mass rate of change M is calculated more than 6, Moringa leaf flavone extracts of the beta Cell of islet growth rate N more than 5 is used to drop
The medicine and medical product of hypoglycemia, repairing pancreas β cells secrete insulin functions.
Yu little Rong etc. in the research that HPLC determines Xinjiang mulberry leaf Content of Chlorogenic Acid, rutin, isoquercitrin and Quercetin, respectively
It is chlorogenic acid, rutin, isoquercitrin, Quercetin to plant composition peak sequence.Guo Juliang etc. is green in HPLC determines rough branch Hypericum Chinense
In the research of ortho acid, rutin, Hyperoside, Quercetin and Kaempferol, various composition peak sequence is chlorogenic acid, rutin, different Mongolian oak
Skin glycosides, Quercetin.And isoquercitrin and quercetin content highest, respectively 41.42% in leaf of Moringa flavones sample in the present invention
With 21.97%, illustrate in leaf of Moringa flavones based on isoquercitrin and two kinds of compositions of Quercetin.Both compositions belong to flavones
Alcohol compound, such compound is the important branch of flavone compound one, is one of many medium-height grass the effective elements of the medicines, is had
The multiple pharmacological effects, the heat as chemical synthesis research such as prevention and cure of cardiovascular disease, removing free radical, anticancer, antibacterial, anti-inflammatory
Point.It is 71.80% that a upper chapter obtains ethyl acetate extract flavones content, and isoquercitrin accounts for the 41.42% of the position, that is, account for flavones
The 57.69% of composition, thus infers, isoquercitrin is the main component of leaf of Moringa flavones.
Applicant it is multiple test result indicate that, when leaf of Moringa flavones concentration is 10 times of isoquercitrin concentration, to glucose
Consumption be higher than isoquercitrin, but difference is not notable.This is probably, because not only containing isoquercitrin in leaf of Moringa, also to contain Mongolian oak
Pi Su, chlorogenic acid and Kaempferol, this several composition are respectively provided with anti-oxidant, anti-inflammatory effect, can promote the growth of cell, improve Portugal
Grape sugar consumption amount, the synergy of these compositions improves consumption of the leaf of Moringa flavones to glucose.Leaf of Moringa flavones has body
Interior hypoglycemic activity, also shows glucose consumption activity higher in vitro, and its content highest composition isoquercitrin is in vitro
Also hypoglycemic activity is presented with, while recently also there are some researches show isoquercitrin has regulation blood sugar and blood fat and improving pancreas islet thin
The effect of born of the same parents' function.So as to infer, isoquercitrin is probably the main component of leaf of Moringa flavones hypoglycemic activity.
The beneficial effects of the invention are as follows：Glucose uptake vivacity K is calculated by glucose uptake tester, by sugar
Lipid metaboli activity test determines the change of blood sugar rate of rat, rat fat index content, rat blood serum SOD and MDA contents
Determine and insulin index, and by pancreas islet protection activity experiment calculation islet cell mass rate of change M and beta Cell of islet
Growth rate N, comprehensive descision goes out the hypoglycemic of Moringa leaf flavone extract, pancreas islet protection activity index, to leaf of Moringa utilization with open
Hair provides a quantitative standard.
Brief description of the drawings
Fig. 1 is influence curve figure of the concentration of alcohol of the present invention to flavones yield；
Fig. 2 is influence curve figure of the extraction time of the invention to flavones yield；
Fig. 3 is influence curve figure of the microwave power of the present invention to flavones yield；
Fig. 4 is influence curve figure of the liquid ratio of the present invention to flavones yield；
Fig. 5 is the micro- enlarged drawing of the section of control group HE dyeing of the present invention；
Fig. 6 is the micro- enlarged drawing of the section of inventive samples group HE dyeing；
Fig. 7 is the micro- enlarged drawing of the section of control group aldehyde fuchsine stain of the present invention；
Fig. 8 is the micro- enlarged drawing of the section of inventive samples group aldehyde fuchsine stain.
Specific embodiment
Technical scheme, but protection scope of the present invention are described in further detail with reference to the accompanying drawings and examples
It is not limited to as described below.
Embodiment 1
The hypoglycemic of Moringa leaf flavone extract, pancreas islet protection activity test method, it is comprised the steps of：
The preparation of S1, chromocor extract：Leaf of Moringa flavone powder 100mg is weighed, 1mL is settled to, mixed, filtered through 0.22um
Device is filtered, and is distributed into 5 equivalent samples, stand-by；
S2, glucose uptake experiment：After experiment is digested with 0.25% trypsin solution of HepG2 cell solutions, inoculation
In culture plate, CO is then placed in together2Cultivated in incubator, wherein, the cell concentration of HepG2 cell solutions is 1.0 × 105
Individual/mL, after after cell attachment, discards original fluid, and the dense of initial glucose in each hole nutrient solution is determined with glucose kit
Degree, is calculated as G1, serum-free medium is changed, hungry 10h adds the DMEM μ L of sugared nutrient solution 180 high, then is separately added into 30 μ L samples,
The concentration of glucose in each hole nutrient solution is determined after incubation 24h with glucose kit, G is calculated as2, Portugal is calculated using below equation
The consumption G of grape sugarC,Wherein, n is the hole count of experiment in culture plate, is according to glucose uptake activity
Number K, judgement sample glucose uptake activity, K=1.2, G0For the isoquercitrin test specimen of same volume carries out glucose uptake
The glucose utilization that experiment is obtained, the content of isoquercitrin is 0.0156mg/mL in isoquercitrin test specimen；
S3, glycolipid metabolism activity test：Take artificial induction's diabetes B rat model sample sets and control group each 10
, wherein, the standard of artificial induction's diabetes B rat model is that fasting blood-glucose is more than 7.8mmol/L, sample sets and control group
The physiological saline of equivalent is given by the dosage of sample sets, in it is determined that the rat Cheng Mo same day unified beginning gastric infusion, continuous gavage
21 days, daily 8：00AM-9:00AM timings are administered once on an empty stomach, give the feed and drinking-water of equivalent after gavage again, are taken after 21 days
Take the change of blood sugar rate of each group rat respectively, rat fat index content, the measure and pancreas of rat blood serum SOD and MDA contents
The calculating of island element index, wherein, control group blood sugar rises to 19.04 from 16.27, and sample sets drop to 10.12, blood from 16.34
Sugared rate of change is that 55%, rat fat index content is not exceeded, specially LDL-C2.36mmol/L, HDL-C0.34mmol/L,
T-CHO2.11mmol/L, TG1.21mmol/L, rat blood serum SOD contents are 160.92U/mL in control group, in sample sets
It is 185.81U/mL, SOD rates of change are 15%, and rat blood serum MDA contents are 30.68U/mL in control group, in sample sets
It is 28.45U/mL, MDA rates of change are that 7.8%, HOMA-IR indexes are 1.2；
S4, the experiment of pancreas islet protection activity：By sample sets in glycolipid metabolism activity test and the rat pancreas ripple of control group
Engler's liquid is fixed, and by after dehydration, transparent, waxdip, with FFPE, section, thickness is 4.5-5.5 μm, and 30-45 DEG C of baking oven adds
Heat fixation, HE dyeing and aldehyde fuchsine stain are carried out to pancreas section, are dehydrated, and transparent, mounting in basis of microscopic observation, and is taken pictures
Record, according to the quantity A of islet cells in the pancreas section that HE is dyeed2With islet cell mass A in control group1, calculate pancreas islet thin
Born of the same parents amount change M, as shown in Figure 5, Figure 6, whereinA2、A1HE dyeing respectively in sample combination control group
Section under 10 average values of rat pancreas cell quantity, A1In to have complete mouse Stem Cells quantity be close to 2, A2In
It is that islet cell mass rate of change M is 6 close to 15-20 or so to have complete mouse Stem Cells quantity；
Red area areal calculation beta Cell of islet growth rate N according to representated by aldehyde fuchsine stain beta Cell of islet, wherein,B2、B1The lower 10 beta cells of isolated rat islets quantity of the section of aldehyde fuchsine stain respectively in sample combination control group
The average value of representative red area area, B2Middle red area area (darker regions) is B1The 6-7 of middle red area area
Times, islet cell mass rate of change N is 5；.
Described leaf of Moringa flavone powder is obtained by following methods：Leaf of Moringa powder is taken with 60%v/v ethanol as solvent, feed liquid
Than being 1:60g/ml, carries out microwave abstracting, collects extract solution, wherein, microwave power is 400W, and extraction time is 8min, then
Extract solution is filtered, and Rotary Evaporators are concentrated under reduced pressure, and reclaims ethanol to dry, is stood 12 hours after absolute ethyl alcohol redissolution and is filtered, and is removed
Water-solubility impurity, filtrate rotated evaporation under reduced pressure concentration again, removes ethanol, with distilled water diluting, high concentration flavonoids solution point
It is 2mL/ pipes in 15mL screw socket centrifuge tube with cover to fill, and is placed in vacuum cryogenic temperature freezing drying instrument 48h, and it is powder that flavones is freezed completely
End, after taking-up, sealing, lucifuge, -80 DEG C of preservations.
The hole count of culture plate is 96 holes in described glucose uptake test procedure.
Described experiment with HepG2 cell solutions is obtained by following methods：HepG2 cells are inoculated in Tissue Culture Flask
In, RPMI-1640 is added, it is placed in 37 DEG C, cultivates in the 5%CO2 incubators of saturated humidity, 1 nutrient solution is changed every 2d,
Passed on after 3d, passage number 1:4, the cell in growth period of taking the logarithm is tested.
Described Moringa leaf flavone extract is used to reduce blood sugar, the medicine of repairing pancreas β cells secrete insulin functions
And medical product.
Embodiment 2
The hypoglycemic of Moringa leaf flavone extract, pancreas islet protection activity test method, it is comprised the steps of：
The preparation of S1, chromocor extract：Leaf of Moringa flavone powder 80mg is weighed, 1mL is settled to, mixed, filtered through 0.22um
Device is filtered, and is distributed into 5 equivalent samples, stand-by；
S2, glucose uptake experiment：After experiment is digested with 0.25% trypsin solution of HepG2 cell solutions, inoculation
In culture plate, CO is then placed in together2Cultivated in incubator, wherein, the cell concentration of HepG2 cell solutions is 0.8 × 105
Individual/mL, after after cell attachment, discards original fluid, and the dense of initial glucose in each hole nutrient solution is determined with glucose kit
Degree, is calculated as G1, serum-free medium is changed, hungry 12h adds the DMEM μ L of sugared nutrient solution 160 high, then is separately added into 20 μ L samples,
The concentration of glucose in each hole nutrient solution is determined after incubation 28h with glucose kit, G is calculated as2, Portugal is calculated using below equation
The consumption G of grape sugarC,Wherein, n is the hole count of experiment in culture plate, is according to glucose uptake activity
Number K, judgement sample glucose uptake activity, K=1.05, G0For the isoquercitrin test specimen of same volume carries out glucose uptake
The glucose utilization that experiment is obtained, the content of isoquercitrin is 0.0156mg/mL in isoquercitrin test specimen；
S3, glycolipid metabolism activity test：Take artificial induction's diabetes B rat model sample sets and control group each 10
, wherein, the standard of artificial induction's diabetes B rat model is that fasting blood-glucose is more than 7.8mmol/L, sample sets and control group
The physiological saline of equivalent is given by the dosage of sample sets, in it is determined that the rat Cheng Mo same day unified beginning gastric infusion, continuous gavage
21 days, daily 8:00AM-9:00AM timings are administered once on an empty stomach, give the feed and drinking-water of equivalent after gavage again, are taken after 21 days
Take the change of blood sugar rate of each group rat respectively, rat fat index content, the measure and pancreas of rat blood serum SOD and MDA contents
The calculating of island element index, wherein, change of blood sugar rate is that 52%, rat fat index content is not exceeded, rat blood serum SOD rates of change
It is 13%, rat blood serum MDA rates of change are that 8%, HOMA-IR indexes are 1.1；
S4, the experiment of pancreas islet protection activity：By sample sets in glycolipid metabolism activity test and the rat pancreas ripple of control group
Engler's liquid is fixed, and by after dehydration, transparent, waxdip, with FFPE, section, thickness is 5 μm, and 30-45 DEG C of baking oven heating is solid
It is fixed, HE dyeing and aldehyde fuchsine stain are carried out to pancreas section, it is dehydrated, transparent, mounting, in basis of microscopic observation, and Taking Pictures recording,
According to the quantity A of islet cells in the pancreas section that HE is dyeed2With islet cell mass A in control group1, calculate islet cells number
Quantitative change rate M, as shown in Figure 5, Figure 6, whereinA2、A1HE dyeing cuts respectively in sample combination control group
10 average values of rat pancreas cell quantity under piece, islet cell mass rate of change M is 7；
Red area areal calculation beta Cell of islet growth rate N according to representated by aldehyde fuchsine stain beta Cell of islet, wherein,B2、B1The lower 10 beta cells of isolated rat islets quantity of the section of aldehyde fuchsine stain respectively in sample combination control group
The average value of representative red area area, islet cell mass rate of change N is 6；.
Described leaf of Moringa flavone powder is obtained by following methods：Leaf of Moringa powder is taken with 60%v/v ethanol as solvent, feed liquid
Than being 1:55g/ml, carries out microwave abstracting, collects extract solution, wherein, microwave power is 400W, and extraction time is 8min, then
Extract solution is filtered, and Rotary Evaporators are concentrated under reduced pressure, and reclaims ethanol to dry, is stood 12 hours after absolute ethyl alcohol redissolution and is filtered, and is removed
Water-solubility impurity, filtrate rotated evaporation under reduced pressure concentration again, removes ethanol, with distilled water diluting, high concentration flavonoids solution point
It is 2mL/ pipes in 15mL screw socket centrifuge tube with cover to fill, and is placed in vacuum cryogenic temperature freezing drying instrument 48h, and it is powder that flavones is freezed completely
End, after taking-up, sealing, lucifuge, -80 DEG C of preservations.
Described experiment with HepG2 cell solutions is obtained by following methods：HepG2 cells are inoculated in Tissue Culture Flask
In, RPMI-1640 is added, it is placed in 37 DEG C, cultivates in the 5%CO2 incubators of saturated humidity, 1 nutrient solution is changed every 2d,
Passed on after 3d, passage number 1:4, the cell in growth period of taking the logarithm is tested.
Described Moringa leaf flavone extract is used to reduce blood sugar, the medicine of repairing pancreas β cells secrete insulin functions
And medical product.
Embodiment 3
The hypoglycemic of Moringa leaf flavone extract, pancreas islet protection activity test method, it is comprised the steps of：
The preparation of S1, chromocor extract：Leaf of Moringa flavone powder 90mg is weighed, 1mL is settled to, mixed, filtered through 0.22um
Device is filtered, and is distributed into 5 equivalent samples, stand-by；
S2, glucose uptake experiment：After experiment is digested with 0.25% trypsin solution of HepG2 cell solutions, inoculation
In culture plate, CO is then placed in together2Cultivated in incubator, wherein, the cell concentration of HepG2 cell solutions is 1.5 × 105
Individual/mL, after after cell attachment, discards original fluid, and the dense of initial glucose in each hole nutrient solution is determined with glucose kit
Degree, is calculated as G1, serum-free medium is changed, hungry 12h adds the DMEM μ L of sugared nutrient solution 200 high, then is separately added into 50 μ L samples,
The concentration of glucose in each hole nutrient solution is determined after incubation 26h with glucose kit, G is calculated as2, Portugal is calculated using below equation
The consumption G of grape sugarC,Wherein, n is the hole count of experiment in culture plate, is according to glucose uptake activity
Number K, judgement sample glucose uptake activity, K=1.3, G0For the isoquercitrin test specimen of same volume carries out glucose uptake
The glucose utilization that experiment is obtained, the content of isoquercitrin is 0.0156mg/mL in isoquercitrin test specimen；
S3, glycolipid metabolism activity test：Take artificial induction's diabetes B rat model sample sets and control group each 10
, wherein, the standard of artificial induction's diabetes B rat model is that fasting blood-glucose is more than 7.8mmol/L, sample sets and control group
The physiological saline of equivalent is given by the dosage of sample sets, in it is determined that the rat Cheng Mo same day unified beginning gastric infusion, continuous gavage
21 days, daily 8：00AM-9:00AM timings are administered once on an empty stomach, give the feed and drinking-water of equivalent after gavage again, are taken after 21 days
Take the change of blood sugar rate of each group rat respectively, rat fat index content, the measure and pancreas of rat blood serum SOD and MDA contents
The calculating of island element index, wherein, change of blood sugar rate is that 62%, rat fat index content is not exceeded, rat blood serum SOD rates of change
It is 17%, rat blood serum MDA rates of change are that 9%, HOMA-IR indexes are 1.3；
S4, the experiment of pancreas islet protection activity：By sample sets in glycolipid metabolism activity test and the rat pancreas ripple of control group
Engler's liquid is fixed, and by after dehydration, transparent, waxdip, with FFPE, section, thickness is 5 μm, and 30-45 DEG C of baking oven heating is solid
It is fixed, HE dyeing and aldehyde fuchsine stain are carried out to pancreas section, it is dehydrated, transparent, mounting, in basis of microscopic observation, and Taking Pictures recording,
According to the quantity A of islet cells in the pancreas section that HE is dyeed2With islet cell mass A in control group1, calculate islet cells number
Quantitative change rate M, as shown in Figure 5, Figure 6, whereinA2、A1HE dyeing cuts respectively in sample combination control group
10 average values of rat pancreas cell quantity under piece, islet cell mass rate of change M is 8；
Red area areal calculation beta Cell of islet growth rate N according to representated by aldehyde fuchsine stain beta Cell of islet, wherein,B2、B1The lower 10 beta cells of isolated rat islets quantity of the section of aldehyde fuchsine stain respectively in sample combination control group
The average value of representative red area area, islet cell mass rate of change N is 6.5；.
Described leaf of Moringa flavone powder is obtained by following methods：Leaf of Moringa powder is taken with 60%v/v ethanol as solvent, feed liquid
Than being 1:60g/ml, carries out microwave abstracting, collects extract solution, wherein, microwave power is 300W, and extraction time is 8min, then
Extract solution is filtered, and Rotary Evaporators are concentrated under reduced pressure, and reclaims ethanol to dry, is stood 12 hours after absolute ethyl alcohol redissolution and is filtered, and is removed
Water-solubility impurity, filtrate rotated evaporation under reduced pressure concentration again, removes ethanol, with distilled water diluting, high concentration flavonoids solution point
It is 2mL/ pipes in 15mL screw socket centrifuge tube with cover to fill, and is placed in vacuum cryogenic temperature freezing drying instrument 48h, and it is powder that flavones is freezed completely
End, after taking-up, sealing, lucifuge, -80 DEG C of preservations.
Described experiment with HepG2 cell solutions is obtained by following methods：HepG2 cells are inoculated in Tissue Culture Flask
In, RPMI-1640 is added, it is placed in 37 DEG C, cultivates in the 5%CO2 incubators of saturated humidity, 1 nutrient solution is changed every 2d,
Passed on after 3d, passage number 1:4, the cell in growth period of taking the logarithm is tested.
Described Moringa leaf flavone extract is used to reduce blood sugar, the medicine of repairing pancreas β cells secrete insulin functions
And medical product.
Embodiment 4
The hypoglycemic of Moringa leaf flavone extract, pancreas islet protection activity test method, it is comprised the steps of：
The preparation of S1, chromocor extract：Leaf of Moringa flavone powder 100mg is weighed, 1mL is settled to, mixed, filtered through 0.22um
Device is filtered, and is distributed into 5 equivalent samples, stand-by；
S2, glucose uptake experiment：After experiment is digested with 0.25% trypsin solution of HepG2 cell solutions, inoculation
In culture plate, CO is then placed in together2Cultivated in incubator, wherein, the cell concentration of HepG2 cell solutions is 1.2 × 105
Individual/mL, after after cell attachment, discards original fluid, and the dense of initial glucose in each hole nutrient solution is determined with glucose kit
Degree, is calculated as G1, serum-free medium is changed, hungry 10h adds the DMEM μ L of sugared nutrient solution 200 high, then is separately added into 40 μ L samples,
The concentration of glucose in each hole nutrient solution is determined after incubation 24h with glucose kit, G is calculated as2, Portugal is calculated using below equation
The consumption G of grape sugarC,Wherein, n is the hole count of experiment in culture plate, is according to glucose uptake activity
Number K, judgement sample glucose uptake activity, K=1.12, G0For the isoquercitrin test specimen of same volume carries out glucose uptake
The glucose utilization that experiment is obtained, the content of isoquercitrin is 0.0156mg/mL in isoquercitrin test specimen；
S3, glycolipid metabolism activity test：Take artificial induction's diabetes B rat model sample sets and control group each 10
, wherein, the standard of artificial induction's diabetes B rat model is that fasting blood-glucose is more than 7.8mmol/L, sample sets and control group
The physiological saline of equivalent is given by the dosage of sample sets, in it is determined that the rat Cheng Mo same day unified beginning gastric infusion, continuous gavage
21 days, daily 8：00AM-9:00AM timings are administered once on an empty stomach, give the feed and drinking-water of equivalent after gavage again, are taken after 21 days
Take the change of blood sugar rate of each group rat respectively, rat fat index content, the measure and pancreas of rat blood serum SOD and MDA contents
The calculating of island element index, wherein, change of blood sugar rate is that 55%, rat fat index content is not exceeded, rat blood serum SOD rates of change
It is 12%, rat blood serum MDA rates of change are 7.9%, HOMA-IR indexes are 1.12；
S4, the experiment of pancreas islet protection activity：By sample sets in glycolipid metabolism activity test and the rat pancreas ripple of control group
Engler's liquid is fixed, and by after dehydration, transparent, waxdip, with FFPE, section, thickness is 5 μm, and 30-45 DEG C of baking oven heating is solid
It is fixed, HE dyeing and aldehyde fuchsine stain are carried out to pancreas section, it is dehydrated, transparent, mounting, in basis of microscopic observation, and Taking Pictures recording,
According to the quantity A of islet cells in the pancreas section that HE is dyeed2With islet cell mass A in control group1, calculate islet cells number
Quantitative change rate M, as shown in Figure 5, Figure 6, whereinA2、A1HE dyeing cuts respectively in sample combination control group
10 average values of rat pancreas cell quantity under piece, islet cell mass rate of change M is 8；
Red area areal calculation beta Cell of islet growth rate N according to representated by aldehyde fuchsine stain beta Cell of islet, wherein,B2、B1The lower 10 beta cells of isolated rat islets quantity of the section of aldehyde fuchsine stain respectively in sample combination control group
The average value of representative red area area, islet cell mass rate of change N is 5；.
Described leaf of Moringa flavone powder is obtained by following methods：Leaf of Moringa powder is taken with 60%v/v ethanol as solvent, feed liquid
Than being 1:60g/ml, carries out microwave abstracting, collects extract solution, wherein, microwave power is 400W, and extraction time is 6min, then
Extract solution is filtered, and Rotary Evaporators are concentrated under reduced pressure, and reclaims ethanol to dry, is stood 12 hours after absolute ethyl alcohol redissolution and is filtered, and is removed
Water-solubility impurity, filtrate rotated evaporation under reduced pressure concentration again, removes ethanol, with distilled water diluting, high concentration flavonoids solution point
It is 2mL/ pipes in 15mL screw socket centrifuge tube with cover to fill, and is placed in vacuum cryogenic temperature freezing drying instrument 48h, and it is powder that flavones is freezed completely
End, after taking-up, sealing, lucifuge, -80 DEG C of preservations.
Described experiment with HepG2 cell solutions is obtained by following methods：HepG2 cells are inoculated in Tissue Culture Flask
In, RPMI-1640 is added, it is placed in 37 DEG C, cultivates in the 5%CO2 incubators of saturated humidity, 1 nutrient solution is changed every 2d,
Passed on after 3d, passage number 1:4, the cell in growth period of taking the logarithm is tested.
Described Moringa leaf flavone extract is used to reduce blood sugar, the medicine of repairing pancreas β cells secrete insulin functions
And medical product.
Embodiment 5
The hypoglycemic of Moringa leaf flavone extract, pancreas islet protection activity test method, it is comprised the steps of：
The preparation of S1, chromocor extract：Leaf of Moringa flavone powder 80mg is weighed, 1mL is settled to, mixed, filtered through 0.22um
Device is filtered, and is distributed into 5 equivalent samples, stand-by；
S2, glucose uptake experiment：After experiment is digested with 0.25% trypsin solution of HepG2 cell solutions, inoculation
In culture plate, CO is then placed in together2Cultivated in incubator, wherein, the cell concentration of HepG2 cell solutions is 1.0 × 105
Individual/mL, after after cell attachment, discards original fluid, and the dense of initial glucose in each hole nutrient solution is determined with glucose kit
Degree, is calculated as G1, serum-free medium is changed, hungry 12h adds the DMEM μ L of sugared nutrient solution 180 high, then is separately added into 25 μ L samples,
The concentration of glucose in each hole nutrient solution is determined after incubation 28h with glucose kit, G is calculated as2, Portugal is calculated using below equation
The consumption G of grape sugarC,Wherein, n is the hole count of experiment in culture plate, is according to glucose uptake activity
Number K, judgement sample glucose uptake activity, K=1.4, G0For the isoquercitrin test specimen of same volume carries out glucose uptake
The glucose utilization that experiment is obtained, the content of isoquercitrin is 0.0156mg/mL in isoquercitrin test specimen；
S3, glycolipid metabolism activity test：Take artificial induction's diabetes B rat model sample sets and control group each 10
, wherein, the standard of artificial induction's diabetes B rat model is that fasting blood-glucose is more than 7.8mmol/L, sample sets and control group
The physiological saline of equivalent is given by the dosage of sample sets, in it is determined that the rat Cheng Mo same day unified beginning gastric infusion, continuous gavage
21 days, daily 8:00AM-9:00AM timings are administered once on an empty stomach, give the feed and drinking-water of equivalent after gavage again, are taken after 21 days
Take the change of blood sugar rate of each group rat respectively, rat fat index content, the measure and pancreas of rat blood serum SOD and MDA contents
The calculating of island element index, wherein, change of blood sugar rate is that 54%, rat fat index content is not exceeded, rat blood serum SOD rates of change
It is 11.5%, rat blood serum MDA rates of change are 9%, HOMA-IR indexes are 1.4；
S4, the experiment of pancreas islet protection activity：By sample sets in glycolipid metabolism activity test and the rat pancreas ripple of control group
Engler's liquid is fixed, and by after dehydration, transparent, waxdip, with FFPE, section, thickness is 5 μm, and 30-45 DEG C of baking oven heating is solid
It is fixed, HE dyeing and aldehyde fuchsine stain are carried out to pancreas section, it is dehydrated, transparent, mounting, in basis of microscopic observation, and Taking Pictures recording,
According to the quantity A of islet cells in the pancreas section that HE is dyeed2With islet cell mass A in control group1, calculate islet cells number
Quantitative change rate M, as shown in Figure 5, Figure 6, whereinA2, A1 are respectively cutting for HE dyeing in sample combination control group
10 average values of rat pancreas cell quantity under piece, islet cell mass rate of change M is 7；
Red area areal calculation beta Cell of islet growth rate N according to representated by aldehyde fuchsine stain beta Cell of islet, wherein,B2, B1 are respectively the lower 10 beta cells of isolated rat islets quantity of section of aldehyde fuchsine stain in sample combination control group
The average value of representative red area area, islet cell mass rate of change N is 7；.
Described leaf of Moringa flavone powder is obtained by following methods：Leaf of Moringa powder is taken with 50%v/v ethanol as solvent, feed liquid
Than being 1:60g/ml, carries out microwave abstracting, collects extract solution, wherein, microwave power is 400W, and extraction time is 10min, then
Extract solution is filtered, and Rotary Evaporators are concentrated under reduced pressure, and reclaims ethanol to dry, is stood 12 hours after absolute ethyl alcohol redissolution and is filtered, and is removed
Water-solubility impurity, filtrate rotated evaporation under reduced pressure concentration again, removes ethanol, with distilled water diluting, high concentration flavonoids solution point
It is 2mL/ pipes in 15mL screw socket centrifuge tube with cover to fill, and is placed in vacuum cryogenic temperature freezing drying instrument 48h, and it is powder that flavones is freezed completely
End, after taking-up, sealing, lucifuge, -80 DEG C of preservations.
Described experiment with HepG2 cell solutions is obtained by following methods：HepG2 cells are inoculated in Tissue Culture Flask
In, RPMI-1640 is added, it is placed in 37 DEG C, cultivates in the 5%CO2 incubators of saturated humidity, 1 nutrient solution is changed every 2d,
Passed on after 3d, passage number 1:4, the cell in growth period of taking the logarithm is tested.
Described Moringa leaf flavone extract is used to reduce blood sugar, the medicine of repairing pancreas β cells secrete insulin functions
And medical product.
The diabetes B rat model of zygotic induction 21 days.After being measured to the indices of rat, finally found that：
Leaf of Moringa flavones has the symptom for improving diabetes rat " three-many-one-little ", reduces blood sugar, improves oral glucose tolerance amount, subtracts
Light oxidative stress, suppression inflammatory reaction, reduce insulin resistance level, increase insulin sensitivity, strengthen beta Cell of islet work(
Can, and it reduces the effect of diabetes and cardiovascular disease risk.Simultaneously by HE dyeing, aldehyde fuchsine stain to different medication group rats
Pancreas islet observed.Finally found that, the effect to pancreas islet protection of leaf of Moringa flavones is preferable.Shown as under mirror, pancreas islet knot
Structure is more complete, and islet tissue is visible with acinus distinct, and islet cells is more, and preferably, marshalling, endochylema enriches form,
Karyon is most clear, only a small amount of cell degeneration or necrosis, reduces the lesion of diabetes rat beta Cell of islet.Therefore, isoquercitrin
Glycosides plays the role of protection to diabetes rat beta Cell of islet.Based on the above, test data applicant draws, for glucose uptake
Vivacity K is not exceeded more than 50%, rat fat index content more than 1, change of blood sugar rate, rat blood serum SOD growth rates are
10-20%, MDA slip more than 7%, HOMA-IR indexes more than 1, calculate islet cell mass rate of change M 6 with
On, Moringa leaf flavone extracts of the beta Cell of islet growth rate N more than 5 is used to reduce blood sugar, repairing pancreas β cells secretion pancreas
The medicine and medical product of island element function.
The above is only the preferred embodiment of the present invention, it should be understood that the present invention is not limited to described herein
Form, is not to be taken as the exclusion to other embodiment, and can be used for various other combinations, modification and environment, and can be at this
In the text contemplated scope, it is modified by the technology or knowledge of above-mentioned teaching or association area.And those skilled in the art are entered
Capable change and change does not depart from the spirit and scope of the present invention, then all should be in the protection domain of appended claims of the present invention
It is interior.
Claims (5)
1. the hypoglycemic of Moringa leaf flavone extract, pancreas islet protection activity test method, it is characterised in that it is comprised the steps of：
The preparation of S1, chromocor extract：Leaf of Moringa flavone powder 80-100mg is weighed, 1mL is settled to, mixed, filtered through 0.22um
Device is filtered, and is distributed into 5 equivalent samples, stand-by；
S2, glucose uptake experiment：After experiment is digested with 0.25% trypsin solution of HepG2 cell solutions, training is inoculated in
Support in plate, CO is then placed in together2Cultivated in incubator, wherein, the cell concentration of HepG2 cell solutions is 0.8-1.5 × 105
Individual/mL, after after cell attachment, discards original fluid, and the dense of initial glucose in each hole nutrient solution is determined with glucose kit
Degree, is calculated as G1, serum-free medium is changed, hungry 10-12h adds DMEM sugared nutrient solution 160-200 μ L high, then be separately added into 20-
50 μ L samples, determine the concentration of glucose in each hole nutrient solution with glucose kit after incubation 24-28h, be calculated as G2, use with
Lower formula calculates the consumption G of glucoseC,Wherein, n is the hole count of experiment in culture plate, according to grape
Sugar intake vivacity K, judgement sample glucose uptake activity,G0For the isoquercitrin test specimen of same volume enters
The glucose utilization that the experiment of row glucose uptake is obtained, the content of isoquercitrin is in isoquercitrin test specimen
0.0156mg/mL；
S3, glycolipid metabolism activity test：Artificial induction's diabetes B rat model sample sets and each 10 of control group are taken, its
In, the standard of artificial induction's diabetes B rat model is fasting blood-glucose more than 7.8mmol/L or 2h-plasma glucose is more than
11.1mmol/L, sample sets and control group are given the physiological saline of equivalent by the dosage of sample sets, in it is determined that the rat Cheng Mo same day
It is unified to start gastric infusion, continuous gavage 21 days, daily 8：00AM-9:00AM timings are administered once on an empty stomach, are given again after gavage
The feed of equivalent and drinking-water, take the change of blood sugar rate for taking each group rat respectively, rat fat index content, rat blood serum after 21 days
The calculating of the measure and insulin index of SOD and MDA contents；
S4, the experiment of pancreas islet protection activity：By sample sets in glycolipid metabolism activity test and the rat pancreas Bo Enshi of control group
Liquid is fixed, and by after dehydration, transparent, waxdip, with FFPE, section, thickness is 4.5-5.5 μm, and 30-45 DEG C of baking oven heating is solid
It is fixed, HE dyeing and aldehyde fuchsine stain are carried out to pancreas section, it is dehydrated, transparent, mounting, in basis of microscopic observation, and Taking Pictures recording,
According to the quantity A of islet cells in the pancreas section that HE is dyeed2With islet cell mass A in control group1, calculate islet cells number
Quantitative change rate M, whereinA2、A110 rats respectively in sample combination control group under the section of HE dyeing
The average value of Stem Cells quantity, the red area areal calculation beta Cell of islet according to representated by aldehyde fuchsine stain beta Cell of islet
Growth rate N, wherein,B2、B1Lower 10 rats of the section of aldehyde fuchsine stain respectively in sample combination control group
The average value of the red area area representated by beta Cell of islet quantity.
2. the hypoglycemic of Moringa leaf flavone extract as claimed in claim 1, pancreas islet protection activity test method, its feature exist
In：Described leaf of Moringa flavone powder is obtained by following methods：Leaf of Moringa powder is taken with 50-60%v/v ethanol as solvent, solid-liquid ratio
It is 1:50-60g/ml, carries out microwave abstracting, collects extract solution, wherein, microwave power is 300-400W, and extraction time is 6-
10min, then extract solution filtration, Rotary Evaporators are concentrated under reduced pressure, and reclaim ethanol to dry, and 12 hours are stood after absolute ethyl alcohol redissolution
Filtration, removes water-solubility impurity, and filtrate rotated evaporation under reduced pressure concentration again removes ethanol, and with distilled water diluting, high concentration is yellow
Ketone solution is packed as 2mL/ pipes in 15mL screw socket centrifuge tube with cover, is placed in vacuum cryogenic temperature freezing drying instrument 48h, and flavones freezes completely
It is powder to do, after taking-up, sealing, lucifuge, -80 DEG C of preservations.
3. the hypoglycemic of Moringa leaf flavone extract according to claim 1, pancreas islet protection activity test method, its feature exist
In:The hole count of culture plate is 96 holes in described glucose uptake test procedure.
4. the hypoglycemic of Moringa leaf flavone extract according to claim 1, pancreas islet protection activity test method, its feature exist
In:Described experiment with HepG2 cell solutions is obtained by following methods：HepG2 cells are inoculated in Tissue Culture Flask, plus
Enter RPMI-1640, be placed in 37 DEG C, cultivate in the 5%CO2 incubators of saturated humidity, 1 nutrient solution is changed every 2d, passed after 3d
Generation, passage number 1:4, the cell in growth period of taking the logarithm is tested.
5. the Moringa leaf flavone extract as described in claim 1-4 is any, it is characterised in that：Described leaf of Moringa extracting flavonoids
Thing is used to reduce blood sugar, the medicine and medical product of repairing pancreas β cells secrete insulin functions.
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| CN104173393A (en) * | 2014-07-29 | 2014-12-03 | 四川农业大学 | Method for extracting flavones from horseradish tree leaves |
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| CN104173393A (en) * | 2014-07-29 | 2014-12-03 | 四川农业大学 | Method for extracting flavones from horseradish tree leaves |
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| Title |
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| 吉莉莉: "辣木叶黄酮提取分离纯化及其主要成分异槲皮苷降血糖活性与机理研究", 《中国博士学位论文全文数据库医药卫生科技辑》 * |
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