CN104825551B - A kind of buckwheat shell chromocor extract and application - Google Patents
A kind of buckwheat shell chromocor extract and application Download PDFInfo
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Abstract
The present invention provides a kind of buckwheat shell chromocor extracts, and using ultrasonic wave assisted extraction method, flavone compound in buckwheat shell, solid-liquid ratio 1 are extracted by extractant of ethanol solution:30, extraction time 15min, concentration of alcohol 80%.Under this extraction conditions basis, buckwheat shell flavones yield and DPPH free radical scavenging activities are respectively 1.60% and 85.62%；Separation and purification is carried out to the buckwheat shell flavones that extraction obtains using hole resin again, is in technological parameter：Adsorption flow rate 2mL/min, sample solution mass concentration are 6.0mg/mL, 2~3BV of upper prop volume, washing volume 3BV, eluant, eluent concentration of alcohol 70%, elution flow rate 2mL/min, reusable 6 times of resin, the purity of buckwheat shell flavones can be increased to 76.17% by 30.82%, and purity improves 2.47 times.It is tested by type II diabetes rat model, buckwheat shell chromocor extract has a better role to type II diabetes rat, and hypoglycemic effect is better than Metformin hydrochloride.And the experiment proved that buckwheat shell chromocor extract is mixture, except also containing other unknown materials containing flavones and flavonoid beyond the region of objective existence.
Description
Technical field
The invention belongs to field of medicaments, and in particular to buckwheat shell extracting flavonoids method and the buckwheat shell flavones obtained with this method
The application of extract and buckwheat shell chromocor extract in terms of preparing auxiliary prevention and improving the food and drug of blood glucose.
Background technology
Buckwheat (Fagopyrum esculentum) belong to polygonaceae（Polygonaceae）Buckwheat platymiscium is draft Shuangzi
Leaf cereal crops have high nutrition, enjoy " kings of five cereals " good reputation.China's plantation buckwheat is only second to Russia, is buckwheat second
Big producing country[1], cultivation history is long, mainly there is sweet cherry roots（F.esculentumMoench）And duck wheat（F.tataricum
(L.) Gaertn）Two kinds.The nutritional ingredient contained in buckwheat has starch, protein, fat, vitamin and minerals
Deng[2-3], active ingredient has flavones, protein, aliphatic acid, phytosterol and mineral element etc.[4].Medical research shows, buckwheat
It is diabetes, coronary heart disease, hyperpietic with reducing blood lipid, hypoglycemic, blood pressure lowering, anti-cancer and cancer-preventing, protection angiocarpy and other effects
Optimum food, developed into plurality kinds of health care product and emerged.
At present, the primary raw material of buckwheat process is buckwheat seed, and buckwheat shell is the by-product in buckwheat process, can be used as resting the head on
Core filler material.But this is only the extremely least a portion of utilization of buckwheat shell, most of or be directly dropped, do not obtain effectively with
Sufficiently utilize.
The purpose of the present invention is to solve the problems, such as that buckwheat shell is discarded, and provide buckwheat shell extracting flavonoids method.
Buckwheat shell extracting flavonoids method, it includes：
1st, buckwheat shell carries out dry, crushing after removal of impurities processing, obtains buckwheat shell powder；
2nd, 20%-95% ethyl alcohol is mixed with buckwheat shell powder, solid-liquid ratio 1:20-1:50th, extraction time is 10-20 min, is obtained
Buckwheat shell chromocor extract；
The extraction is aided in using ultrasonic wave；
The concentration of alcohol is 80%；
The solid-liquid ratio 1:30；
Buckwheat shell chromocor extract obtained is through macroreticular resin Dynamic Adsorption-elution, evaporative removal solvent；
The macroreticular resin is D101 type macroreticular resins；
Dynamic Adsorption-the elution requirement is：Adsorption flow rate 2mL/min, sample solution mass concentration for 6.0mg/mL, on
2~3BV of column volume, washing volume 3BV, eluant, eluent concentration of alcohol 70%, elution flow rate 2mL/min, reusable 6 times of resin.
Another object of the present invention is to provide a kind of buckwheat shell chromocor extract and application.
A kind of buckwheat shell chromocor extract, it is extracted with above-mentioned buckwheat shell extracting flavonoids method.
A kind of application of buckwheat shell chromocor extract in terms of hypoglycemic drug is prepared.
The present invention is illustrated in detail below：
1. using ultrasonic wave assisted extraction method, flavone compound in buckwheat shell is extracted by extractant of ethanol solution, is adopted
With response phase method, using flavones yield and DPPH free radical scavenging activities as response, to concentration of alcohol, extraction time and feed liquid
Than three Extraction techniques are optimized, it is determined that optimum extraction condition is：Solid-liquid ratio 1:30, extraction time 15min, second
Determining alcohol 80%.Under this extraction conditions basis, buckwheat shell flavones yield and DPPH free radical scavenging activities are respectively 1.60% He
85.62%。
2. the research of separation and purification is carried out to the buckwheat shell flavones that extraction obtains using macroreticular resin, the results showed that：It chooses
The different macroreticular resin of tetra- kinds of polarity of D101, NKA-9, ADS-17, S-8 carries out Static Adsorption to buckwheat shell flavones, passes through absorption
The measure of rate, desorption efficiency and antioxidation activity, it is determined that D101 types resin is the adsorbent of most suitable research；Dynamic Adsorption-
Elution optimal purifying process parameter be：Adsorption flow rate 2mL/min, sample solution mass concentration for 6.0mg/mL, upper prop volume 2~
3BV, washing volume 3BV, eluant, eluent concentration of alcohol 70%, elution flow rate 2mL/min, reusable 6 times of resin.In this condition
Under, the purity of buckwheat shell flavones can be increased to 76.17% by 30.82%, and purity improves 2.47 times, and purification effect is good.
3. build type II diabetes rat model, animal pattern is randomly divided into six groups, be respectively normal group, model group,
Metformin hydrochloride positive controls（100mg/kg）, LBHE groups（50mg/kg）, MBHE groups（100mg/kg）, HBHE groups
（200mg/kg）, study through buckwheat shell extract（BHE）Improve type II diabetes rat generally life and physiological and biochemical index
Effect, the results showed that：Rat modeling success after disposable injection STZ；Internal animal test results are as it can be seen that BHE can mitigate glycosuria
The symptom that sick rat becomes thin improves liver index, renal index and spleen index；2 weeks high dose BHE of continuous gavage（200mg/kg）It can be aobvious
Writing reduces rat fasting blood-glucose level；Middle and high dosage group significantly improves glucose tolerance after being administered 28 days, while BHE
It is horizontal and improve the level of HDL-C in serum that TC, TG, LDL-C in rat blood serum can be significantly reduced, with high dose in each dosage group
It is the most notable that BHE groups improve blood lipid level effect.BHE has a better role to type II diabetes rat.
4.BHEs and BHEs-A also has AGEs in MGO-BSA and GO-BSA systems inhibition, and BHEs-A works as concentration
For 0.2mg/mL when, inhibit AGEs generation ability be respectively 58.97 % and 46.69 %, be significantly higher than same concentration in the same time
The inhibitory activity of lower AG（p<0.05）;SDS-PAGE the results shows also have a certain impact to the generation situation for the BSA that is saccharified；
5. when flavones content is identical, BHEs-A >=isoquercitrin>Rutin >=BHEs=rutin and isoquercitrin mix mark >=reed
Fourth, Quercetin and isoquercitrin mix mark>AG>Quercetin, and significant difference is not presented for BHEs-A and isoquercitrin（p<0.05）,
But BHEs-A is mixture, and rutin contained therein and isoquercitrin content be not high, illustrates that isoquercitrin has stronger inhibition
The activity of AGEs generations, and other active components are likely present in BHEs-A.
The present invention provides a kind of buckwheat shell chromocor extract, using ultrasonic wave assisted extraction method, using ethanol solution to carry
Take flavone compound in agent extraction buckwheat shell, solid-liquid ratio 1:30, extraction time 15min, concentration of alcohol 80%.Item is extracted herein
Under part basis, buckwheat shell flavones yield and DPPH free radical scavenging activities are respectively 1.60% and 85.62%；Again using hole resin to carrying
The buckwheat shell flavones obtained carries out separation and purification, is in technological parameter：Adsorption flow rate 2mL/min, sample solution mass concentration are
6.0mg/mL, 2~3BV of upper prop volume, washing volume 3BV, eluant, eluent concentration of alcohol 70%, elution flow rate 2mL/min, resin can
It reuses 6 times, the purity of buckwheat shell flavones can be increased to 76.17% by 30.82%, and purity improves 2.47 times.Pass through II type sugar
The sick rat model experiment of urine, buckwheat shell chromocor extract have a better role to type II diabetes rat, hypoglycemic effect
Better than Metformin hydrochloride.And the experiment proved that buckwheat shell chromocor extract is mixture, except containing flavones and flavone compound
Also contain other unknown materials outside.
Description of the drawings
The DPPH free radical scavenging activities of tetra- kinds of macroreticular resin stripping liquids of Fig. 1;
Fig. 2 Static Adsorption dynamic experiment results；
The Dynamic Adsorption curve of Fig. 3 lower general flavones different in flow rate；
The Dynamic Adsorption curve of general flavone under Fig. 4 various concentrations；
Dynamic Adsorption curve under Fig. 5 difference upper prop volumes；
Fig. 6 elution impurity distilled water volumes determine；
Influence of Fig. 7 different ethanol concentrations to elution property；
Influence of Fig. 8 elution flow rates to elution property；
Influence of Fig. 9 resins access times to the absorption property of buckwheat shell flavones；
Figure 10 is the HPLC analysis results of buckwheat shell extract B HEs；Wherein (A) marks product testing result (1) rutin
(2) isoquercitrin (3) Quercetin；(B) buckwheat shell extract testing result；
Figure 11 is the inhibitory action ratio that standard items, BHEs and BHEs-A are formed AGEs in Glucose-BSA systems
Compared with；Wherein：R：Rutin; Q：Quercetin; I：Isoquercitrin; R&I：Rutin mixes with Quercetin; R&Q&I：Rutin, Quercetin
And isoquercitrin mixture；
Figure 12 is the inhibitory action ratio that standard items, BHEs and BHEs-A are formed AGEs in Fructose-BSA systems
Compared with；Wherein：R：Rutin; Q：Quercetin; I：Isoquercitrin; R&I：Rutin mixes with Quercetin; R&Q&I：Rutin, Quercetin
And isoquercitrin mixture；
Influences of Figure 13 BHE to diabetes rat liver index；
Influences of Figure 14 BHE to diabetes rat spleen index；
Influences of Figure 15 BHE to diabetes rat renal index；
Influences of the Figure 16 to diabetes rat T-CHOL；
Influences of Figure 17 BHE to diabetes rat triglycerides；
Influences of Figure 18 BHE to diabetes rat LDL-C；
Influences of Figure 19 BHE to diabetes rat HDL-C.
Specific embodiment
The optimal extraction technology of flavone compound in 1 buckwheat shell of embodiment
Natural buckwheat shell is selected to carry out drying after removal of impurities processing（60 DEG C, forced air drying 4h）, crush, cross 60 mesh sieves.Accurately
1.0g buckwheat shell powder is weighed, is placed in 50mL centrifuge tubes, certain density ethanol solution is added in and is uniformly mixed, using ultrasonic wave added
Extraction method extraction filters after a certain period of time, and filtrate is settled to 100mL and saves backup.Using three factors（Concentration of alcohol, solid-liquid ratio,
Extraction time）Three hydraulic tests design, and with response surface optimization method, buckwheat shell extracting flavonoids condition is optimized.
Buckwheat shell flavonoid content, which measures, to be used《Health food is examined and assessment technique specification》（2003 editions）
NaNO2-Al(NO3)3Described in method.
The assay method of DPPH free radical scavenging activities：50 μ L of buckwheat extracting solution are drawn respectively, add in 150 μ L 40mmol/
L DPPH solution is experimental group Ai；50 μ L buckwheat extracting solutions add 150 μ L distilled water to be color comparator Aj；Negative control AoFor 50 μ L
Distilled water adds 150 μ L DPPH solution.It is uniformly mixed after addition, stands 5min, survey its suction at 517nm using microplate reader
Luminosity.The calculation formula of clearance rate is as follows：
Extraction conditions are optimized by Design-Expert8.0.5b softwares, solid-liquid ratio 1:20-1:50th, extract
Time is 10-20 min, concentration of alcohol 20%-95%.Preferred assembled scheme is solid-liquid ratio 1:30, extraction time 15min, second
Determining alcohol 80%, buckwheat shell flavones yield and the predicted value of DPPH free radical scavenging activities are respectively 1.59% He with this condition
86.13%。
2 buckwheat shell flavones process for refining of embodiment optimizes
（1）The pretreatment of macroreticular resin
95% ethyl alcohol of the macroreticular resin of tetra- kinds of models of D101, NKA-9, ADS-17, S-8 is impregnated for 24 hours, it is carried out just
Step removal of impurities.After macroreticular resin is fully swollen, ethyl alcohol is filtered off, repeats to wash for several times with distilled water, up to no alcohol taste.Then
It is first impregnated for 24 hours with 5% HCl solution, is then washed to pH=7.0 with distilled water；Then impregnated for 24 hours, finally with 5% NaOH solution
It spends distillation and is washed to pH=7.0.It is soaked in distilled water, saves backup.
（2）Macroreticular resin basic performance measures
The calculating of adsorbance, adsorption rate and desorption efficiency.The four kinds of each 2.0g of resin handled well are weighed, add in buckwheat shell flavones
（BHE）Solution 50mL（6.0mg/mL）, it is protected from light sealing, constant temperature water bath concussion（25 DEG C, 100r/min, for 24 hours）.After concussion,
Check experiment is done with unadsorbed BHE solution.After adsorption test, after filtering out adsorption liquid and rinsing absorption repeatedly with distilled water
Macroreticular resin washes away the solution colour of resin surface, adds in 70% ethanol solution 50mL, is put into the concussion of shaken cultivation case for 24 hours
(25 DEG C, 100rpm) after concussion, measure the concentration of flavones in stripping liquid, calculate resin desorption rate.Calculation formula is：
C0 in formula --- flavones concentration in sample liquid before absorption（mg/mL）
C1 --- flavones concentration in solution after absorption（mg/mL）
C2 --- flavones concentration in stripping liquid,
V --- liquor capacity（mL）,
W --- resin quality（g）.
The results are shown in Table 1, and D101 types resin has flavone compound very strong adsorption rate, and flavones adsorbance reaches
31.48mg/g, -8 type resin of polarity S also have stronger adsorption capacity to Flavonoid substances；But S-8 types desorption performance is not so good as D101
Type resin.Consider the adsorption rate and desorption efficiency of various resins, D101 type macroreticular resin better performances.Again due to D101 type trees
Fat belongs to low pole macroreticular resin, therefore illustrates that the Flavonoid substances in buckwheat shell are mainly low pole ingredient.
DPPH free radical scavenging activities measure.Method is the same as described in embodiment 1.The results are shown in Figure 1, the solution of D101 type resins
Imbibition concentration reaches more than 90% in 0.5mg/mL；Four kinds of macroreticular resins are descending to the clearance rate of DPPH to be followed successively by：D101
＞ NKA-9 ＞ ADS-17 ＞ S-8.The result shows that the Flavonoid substances antioxidation activity of D101 type resin adsorptions is best.
Static Adsorption dynamic test.BHE solution 50mL is taken in triangular flask, adds in 2.0g macroreticular resins, sealing is placed in
Concussion 7h in constant temperature oscillator (25 DEG C, 100r/min), every 1h samplings once.After sampling, NaNO is used2-Al(NO3)3Method
General flavone content is measured respectively, and adsorbance and adsorption rate are calculated according to flavones content, and dynamics song is drawn according to test data
Line.The results are shown in Figure 2, and D101 macroreticular resins basically reach adsorption equilibrium to the absorption of buckwheat shell Flavonoid substances in 8 h,
Meet the needs of industrialized production.
（3）By taking D101 type macroreticular resins as an example, optimize Dynamic Adsorption-elution processes of buckwheat shell flavones
Optimal adsorption flow velocity determines.Take D101 type resin 44.0g, wet method dress post（Column specification be 15 × 400mm, resin
Bed volume 40ml, uses aquametry）.In upper column liquid concentration（6.0mg/ml）With upper prop volume（100mL）Under the same conditions, respectively
Dynamic Adsorption is carried out with the flow velocity of 1mL/min, 2mL/min, 3mL/min, an efflux is collected per 10mL.With effluent volume
For abscissa, outflow concentration is mapped for ordinate.According to leakage point（Outflow liquid mass concentration is sample solution mass concentration 10%
As leakage point）Determine adsorption flow rate.The results are shown in Figure 3, when flow velocity be 1.0mL/min when, leakage point occur the latest,
Near 90mL；It is leaked when during flow velocity 2.0mL/min near 60mL；When flow velocity is 3.0mL/min, just reach near 40mL
Leakage point.When flow velocity increases, effluent volume when reaching leakage point constantly reduces, this is because flow velocity is too fast, resin
Flavonoid substances in not yet timely adsorbent solution, just flow out with solution, therefore selection is more advantageous to adsorbing at a slow speed.But it flows
Speed can cause cycle period long slowly excessively, low production efficiency, so as to cause economy and waste of time.Therefore, 2.0mL/min is selected
Flow velocity loading.
Upper prop liquid mass concentration determines.3.0mg/mL, 6.0mg/mL, 9.0mg/mL BHE solution loading are prepared respectively,
And with the flow velocity Dynamic Adsorption of 1mL/min.An efflux is collected per 10mL.Using effluent volume as abscissa, efflux is dense
It spends and maps for ordinate.According to leakage point（Outflow liquid mass concentration is leakage point for sample solution mass concentration 10%）It determines to close
Suitable loading mass concentration.The results are shown in Figure 4, and as upper prop liquid mass concentration 3.0mg/mL, leakage point occurs the latest, about
140mL；Leakage point is at 90mL when upper prop liquid mass concentration is 6.0mg/mL；When upper prop liquid mass concentration is 9.0mg/mL
It is too early that leakage point occurs, and just reaches leakage point in 1BV, that is, 40mL.When reaching leakage point adsorbance be respectively 420mg,
540mg and 360mg, it can be seen that excessive concentration is unfavorable for adsorbing.Therefore, 6.0mg/mL loadings mass concentration is selected into Mobile state
Absorption.
Optimum upper column quantity determines.Using the loading mass concentration and loading speed optimized, carried out by resin column
Dynamic adsorption test.An efflux is collected per 10mL, and measures often pipe efflux flavones concentration respectively.Using effluent volume as
Abscissa, flavones concentration draw adsorption curve for ordinate, speed occur according to leakage point and determine optimum upper column quantity.As a result
As shown in figure 5, when the flavones concentration of efflux reaches the 10% of load solution flavones concentration, that is, be considered as saturation absorption, at this time on
Sample volume is about 90-100mL.Therefore optimum loading volume is selected as 2-3BV.
Elute determining for impurity distilled water volume.It is eluted with distilled water with the flow velocity of 2mL/min, the eluent per 10mL
It collects once, and measures general flavone content respectively.As shown in Figure 6, distilled water is eluted with the flow velocity of 2mL/min, works as distillation
General flavone concentration reaches highest when water volume is 1BV, illustrates to wash away most of buckwheat shell general flavone.When washing volume reaches
During 150mL, general flavone concentration is detected as 0.01mg/mL, therefore distilled water 150mL.Because wash volume excessively not only
It causes damages to having adsorbed Flavonoid substances, extends manufacture cycle simultaneously.Therefore, it is optimal to use washing volume 3BV.
Elution concentration determines.The upper prop liquid of suitable concentration, Optimum is taken, the dynamic under certain adsorption flow rate
Absorption is desorbed under identical desorption flow velocity by the use of the ethyl alcohol of different volumes fraction as strippant, received per 1BV stripping liquids
Collect and measure flavones content respectively, draw elution curve.The results are shown in Table 2.
70% ethanol elution rate reaches highest；When concentration of alcohol reaches 90%, eluting rate does not have significant change, illustrates 70%
Ethyl alcohol and 90% ethyl alcohol are all preferable to the elution effect of flavones.But compare the purity of eluate, 70% ethyl alcohol is higher than 90% ethyl alcohol.Such as
Shown in Fig. 7,30% and 50% ethanol elution speed is slow and has trailing phenomenon, and 70% and 90% ethyl alcohol is to the elution speed of adsorption liquid
Comparatively fast；The ethyl alcohol of four kinds of concentration substantially can all elute Flavonoid substances in 5BV.Therefore, 70% ethyl alcohol elution speed
Degree is fast, eluting rate is high, eluate purity is high, and peak shape symmetry is good, is the volume fraction of optimal eluant, eluent.
Elution flow rate determines.Take the upper prop liquid of suitable concentration, Optimum, the Dynamic Adsorption under certain adsorption flow rate,
It is solved respectively in the case where 1mL/min, 2mL/min, 3mL/min desorb flow velocity by the use of the ethyl alcohol of same volume fraction as strippant
It inhales, is collected per 1BV stripping liquids and measures flavones content respectively, draw elution curve.The results are shown in Table 3.
Dynamic desorption is carried out under 1,2,3mL/min flow velocitys, D101 types macroreticular resin is to the eluting rate point of buckwheat shell flavones
It Wei 93.12%, 92.65%, 81.97%, it is known that 1mL/min and 2mL/min is preferable to the elution effect of buckwheat shell flavones；
2mL/min eluate purity highers.As shown in figure 8, being eluted with 1mL/min flow velocitys, eluting rate is high but curve peak shape is not concentrated, and drags
Tail is serious and elution time is long；Peak shape is concentrated when flow velocity is 2mL/min and 3mL/min, and symmetry is good, without trailing phenomenon, but
The eluting rate of 3mL/min flow velocitys is relatively low, with 2mL/min elution speeds are fast and eluting rate highest.Accordingly, it is determined that elution flow rate is
2mL/min。
（4）Resin reuses the research of number
Dynamic Adsorption-elution test is carried out according to the condition optimized, repeats experiment 15 times, calculates the suction of general flavone respectively
Attached amount draws adsorption curve.The results are shown in Figure 9, during D101 types resin recycles 1~6 time, the absorption to general flavone
Amount maintains 0.93~0.96mg/g, and adsorption capacity is substantially on a declining curve since the 7th time；Until between 11~15 times, inhale
Attached amount is only the low absorption horizontal extent of 0.36~0.38mg/g, cannot continue cycling through use if do not regenerated, therefore D101 types are big
The reuse number of hole resin should be advisable within 6 times.
By buckwheat shell alcohol extracts（BHEs）, macroreticular resin filters out, and remaining liq is to adsorb.It adsorbs and unadsorbed
Rotary evaporation removes solvent to liquid under the conditions of 60 DEG C respectively, with thermostat water bath under the conditions of 100 DEG C drying with water bath until solid
Body is precipitated, and last vacuum drying treatment obtains dry adsorbate（BHEs-A）With unadsorbed object（BHEs-NA）.
Using the NaNO announced2-Al(NO3)3Colorimetric method and conventional H PLC methods are respectively to BHEs, BHEs-A, BHEs-
General flavone content and Flavonoid substances in NA are detected analysis.The result shows that the flavones content of three kinds of samples is presented significantly
Sex differernce, wherein BHEs are（390.28±26.61）Mg/g accounts for the 39.03% of buckwheat shell crude extract；BHEs-A is
（643.18±43.95）Mg/g, it is less to account for remaining general flavone content in 64.32%, BHEs-NA of buckwheat shell crude extract, only
For（106.88±3.47）mg/g.As shown in Figure 10, according to Figure 10（A）Plays product rutin（1）, isoquercitrin（2）, Quercetin
（3）Occur whether containing the substance in the time analysis buckwheat shell extract at peak, by Figure 10（B）It understands, in buckwheat shell extract
Rutin and isoquercitrin are mainly contained, Quercetin does not detect in BHEs.
7 flavonoid standards of embodiment and comparison of the buckwheat shell extract to non-glycosylation inhibitory action
Further determine that whether main active ingredient is flavone compound, compares standard items and BHEs and BHEs-A
Between inhibition, with the rutin contained in known buckwheat shell, isoquercitrin and do not detect Quercetin and hybrid standard product and make
It for inhibitor, is added in reaction solution, detects its inhibitory action to AGEs.
Standard concentration is 0.08mM and 0.32mM.For Glucose-BSA（Such as Figure 11（A）It is shown）, react into
During row 1 month, in addition to Quercetin, each sample all shows certain inhibition, and in dose-dependence, inhibition by height to
It is low to be followed successively by：BHEs-A >=isoquercitrin>Rutin>BHEs=rutin and isoquercitrin mix mark=rutin, Quercetin and isoquercitrin
Mixed mark>AG>Quercetin；For Fructose-BSA systems（Such as Figure 12（A）It is shown）, reaction carry out 1 month when, inhibition by
It is high to Low to be followed successively by：BHEs-A=isoquercitrin>Rutin=BHEs=rutin and isoquercitrin mix mark>Rutin, Quercetin and isoquercitrin
Glycosides mixes mark>AG>Quercetin.When two systems are reacted 3 months（Such as Figure 11（B）, Figure 12（B shown in)）, the inhibition of all samples
It generally raises, BHEs-A, BHEs, isoquercitrin and rutin all show very high inhibition.Therefore, when flavones content is identical
When, the inhibition of BHEs-A and isoquercitrin does not have significant difference, and concentration is the inhibition of the Flavonoid substances of 0.08mmol/L
Property to be significantly larger than concentration be 0.68mmol/L AG.But BHEs-A is mixture, and rutin contained therein and isoquercitrin contain
Amount is not high, illustrates that isoquercitrin has the activity of stronger inhibition AGEs generations, and other activity are likely present in BHEs-A
Ingredient.
The preparation of 3 type II diabetes rat model of embodiment
By rat in standardization animal house raising, adaptation environment one week, unlimited eclipse limit water, 12h daytime dark cycle conditions
Under, room ventilation condition is good, relative humidity 60-70%.Distilled water is drunk, keeps the cleaning of environment, feeds basal feed, often
It changes water, changes bedding and padding respectively once.Laboratory periodically sterilizes.10 are randomly selected after a week as Normal group.It is remaining 80 big
Mouse feeds high-sugar-fat-diet（Wherein containing 10% sucrose, 10% lard, 5% cholesterol）4 weeks.After 4 weeks, fasting 12h, intraperitoneal injection
Streptozotocin（STZ）30mg/kg is injected 1 time again after 1 week.Control group does placebo to 1% buffer solution 30mg/kg, after 72h
11.0 mmol/L of fasting blood sugar ＞ are into mould standard.Daily 2 pm injection, bolus during injection.
4 experimental animal of embodiment is grouped and processing
The successful diabetes rat of modeling is randomly divided into 5 groups, in addition totally 6 groups of Normal group.It is carried out on an empty stomach before experiment
Blood sugar detection.Each group rat is grouped and gavage disposition is as follows:
Normal group：Distilled water gavage 10mL/kg/d;
Model group：Distilled water gavage 10mL/kg/d;
BHE high dose groups（HBHE）：Diabetes rat gives BHE reagents 200mg/kg/d;
BHE middle dose groups（MBHE）：Diabetes rat gives BHE reagents 100mg/kg/d;
BHE low dose groups（LBHE）：Diabetes rat gives BHE reagents 50mg/kg/d;
Positive controls：Metformin hydrochloride is to rat oral gavage 100mg/kg/d
It is administered once daily, successive administration 28d, all experimental rats drink distilled water in addition to testing requirements, do not limit and adopt
Hydromining is eaten.
Influence of the 5 buckwheat shell extract of embodiment to type II diabetes rat routine life index and weight
（1）Ordinary circumstance
During experiment, the Normal group state of mind is good, agile, and fur is white and glossy；Rat is disposably quiet
After arteries and veins injection STZ, food-intake, amount of drinking water, urine volume increase, and body starts to become thin, apathetic, do not like movement, rob once in a while
Water phenomenon, excrement have acid smell, and hair color jaundice is matt；Each dosage group and positive controls also have analogue after administration, but
Compared with model group mild degree.
（2）Amount of drinking water
One of the characteristics of more drinks are diabetes, thus in a way amount of drinking water number can weigh treatment group and improve the state of an illness
Effect.It is administered 28 days after modeling success, counts total amount of once drinking water weekly.As shown in table 4.
Compared with model group, LBHE group rat amounts of drinking water are declined slightly, and MBHE groups and HBHE group rats amount of drinking water decline
Gesture is apparent, and HBHE groups amount of drinking water declines most apparent.
（3）Weight
As shown in Table 5, model group rats weight is gradually reduced before comparing modeling；Weight first reduces after HBHE group modelings, gives
Medicine after 3 weeks weight have apparent growth；MBHE group weight first reduces, and the 4th week starts slightly to increase, but changes of weight unobvious；
The weight of LBHE groups is gradually reduced.Weight first reduces after positive controls modeling, and weight starts to increase after being administered 3 weeks but trend is delayed
Slowly.The above results show that rat model weight loss may be caused by diabetes, and each treatment group has just to recovering rat body weight
Effect.
Influence of the 6 buckwheat shell extract of embodiment to organ indexs such as type II diabetes rat liver,spleen,kidneys
Using conventional organ index evaluation assessment, each dosage group is detected to organ indexs such as type II diabetes rat liver,spleen,kidneys
Influence.As a result as shown in figs. 10-12, BHE can significantly improve the organ indexs such as liver liver,spleen,kidney.
Influence of the 7 buckwheat shell extract of embodiment to type II diabetes rat fasting blood-glucose
Using blood glucose meter, time-and-motion study experimental rat blood glucose to specifications.The results are shown in Table 6.
After medicine 28 days, HBHE groups, MBHE groups result and model group relatively have pole marked difference, and HBHE group blood glucose values are minimum,
Better than other groups, fasting blood glucose level decreases.As a result the buckwheat shell extract of doses is prompted to impaired fasting glucose
There is protective effect, certain blood sugar reducing function can be played to rat.
Influence of the 8 buckwheat shell extract of embodiment to type II diabetes Serum Lipids in Experimental HypercholesterolemicRats
Using automatic clinical chemistry analyzer, T-CHOL is measured according to routine operation（TC）, triglycerides（TG）, low-density
Lipoprotein（LDL-C）, high-density lipoprotein（HDL-C）It is horizontal.As a result as shown in figures 13-16, after being administered 28 days, BHE can be significantly
TC, TG, LDL-C are horizontal in reduction rat blood serum and improve the level of HDL-C in serum, improve blood in each dosage group with HBHE groups
Lipid level effect is the most notable.
Influence of the 9 buckwheat shell extract of embodiment to type II diabetes rat sugar tolerance and serum insulin level
Using conventional sugar tolerance and insulin level detection method, detection buckwheat shell extract is to rat sugar tolerance and serum
The influence of insulin level.As a result as shown in table 7,8, after being administered 28 days, middle and high dosage group significantly carries glucose tolerance
It is high；The BHE of each dosage is respectively provided with improvement result to the insulin secretion of type II diabetes rat, is extracted with the buckwheat shell of high dose
Object is the most notable to the protective effect of beta Cell of islet, and the secretion of insulin is promoted to reach normal level.It can be seen that BHE is to II
Patients with type Ⅰ DM rat has a better role.
Claims (2)
1. a kind of buckwheat shell chromocor extract, it is prepared by following methods：
1）Buckwheat shell carries out dry, crushing after removal of impurities processing, obtains buckwheat shell powder；
2）80% ethyl alcohol is mixed with buckwheat shell powder, solid-liquid ratio 1:30th, extraction time is 10-20 min；
Extraction is aided in using ultrasonic wave；It is extract obtained again through macroreticular resin Dynamic Adsorption-elution；
The macroreticular resin is D101 type macroreticular resins；
Dynamic Adsorption-the elution requirement is：Adsorption flow rate 2mL/min, sample solution mass concentration are 6.0mg/mL, column body
2~3BV of product, washing volume 3BV, eluant, eluent concentration of alcohol 70%, elution flow rate 2mL/min, resin are reused 6 times, obtain buckwheat
Shell chromocor extract.
2. a kind of application of the buckwheat shell chromocor extract described in claim 1 in terms for the treatment of type II diabetes drug is prepared.
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| CN105859701B (en) * | 2016-05-13 | 2019-04-19 | 南京财经大学 | A method of extracting separating flavone monomeric substance from sweet tea buckwheat shell |
| CN110656083A (en) * | 2019-08-30 | 2020-01-07 | 吉林农业大学 | Pre-adipocyte brown induction kit |
| CN110628710B (en) * | 2019-08-30 | 2022-11-22 | 吉林农业大学 | Application of buckwheat hull flavone in inducing preadipocyte differentiation |
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| CN112401122A (en) * | 2020-11-11 | 2021-02-26 | 贵州师范大学 | Comprehensive utilization and processing method of black rice buckwheat seeds |
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