CN104825551A - Buckwheat shell flavonoid extract and application thereof - Google Patents
Buckwheat shell flavonoid extract and application thereof Download PDFInfo
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Abstract
The invention provides a buckwheat shell flavonoid extract. A flavonoid compound in a buckwheat shell is extracted with an ethanol solution as an extracting agent by employing an ultrasonic assisted extraction method, wherein the material/liquid ratio is 1 to 30; the extraction time is 15 minutes; the ethanol concentration is 80%; on the basis of the extraction condition, the yield of buckwheat shell flavonoid and the scavenging rate of DPPH (diphenyl picryl hydrazinyl) free radical are respectively 1.60% and 85.62%; the extracted buckwheat shell flavonoid is separated and refined with porous resin; the technological parameters are as follows: the adsorption flow rate is 2mL/min; the mass concentration of sampling liquid is 6.0mg/mL; the column loading volume is 2-3BV; the washing volume is 3BV; the ethanol concentration of an eluent is 70%; the eluting flow rate is 2mL/min; the resin can be repeatedly used for six times; the purity of the buckwheat shell flavonoid can be improved to 76.17% from 30.82%; the purity is improved by 2.47 times; through a model test for rats with type 2 diabetes mellitus, the buckwheat shell flavonoid extract has a relatively good improvement effect on rats with the type 2 diabetes mellitus; and the hpoglycemic effect is superior to that of metformin hydrochloride. An experiment proves that the buckwheat shell flavonoid extract is a mixture and also contains other unknown substances besides flavonoids and flavonoid compounds.
Description
Technical field
The invention belongs to field of medicaments, be specifically related to Buckwheat shell extracting flavonoids method and the Buckwheat shell flavone extract that obtains by the method and Buckwheat shell flavone extract in the auxiliary prevention of preparation and the application that improves in the food of blood glucose and medicine.
Background technology
Semen Fagopyri Esculenti (
fagopyrum esculentum) belong to Polygonaceae (Polygonaceae) Fagopyrum plant, be the dicotyledonous cereal crops of draft, there is high nutrition, enjoy " kings of grain rice " good reputation.China's plantation Semen Fagopyri Esculenti is only second to Russia, is the second largest manufacturing country of Semen Fagopyri Esculenti
[1], cultivation history is long, mainly contain sweet cherry roots (
f.esculentummoench) and Radix Et Rhizoma Fagopyri Tatarici (
f.tataricum(L.) Gaertn) two kinds.The nutritional labeling contained in Semen Fagopyri Esculenti has starch, protein, fat, vitamin and mineral etc.
[2-3], active component has flavone, protein, fatty acid, plant sterol and mineral element etc.
[4].Medical research shows, and Semen Fagopyri Esculenti has the effects such as blood fat reducing, blood sugar lowering, blood pressure lowering, anti-cancer and cancer-preventing, protection cardiovascular, is the optimum food of diabetes, coronary heart disease, hyperpietic, has now been developed and has been developed into plurality kinds of health care product and emerges.
At present, the primary raw material of buckwheat process is Semen Fagopyri Esculenti seed, and Buckwheat shell is the by-product in buckwheat process, can be used as pillow core inserts.But the utilization of this few part that is only Buckwheat shell, great majority or be directly dropped, do not obtain effectively and fully utilizing.
The object of the invention is the problem gone out of use for solving Buckwheat shell, and Buckwheat shell extracting flavonoids method is provided.
Buckwheat shell extracting flavonoids method, it comprises:
1, Buckwheat shell carry out dry after remove impurity process, pulverize, obtain Buckwheat shell powder;
2,20%-95% ethanol mixes with Buckwheat shell powder, and solid-liquid ratio is 1:20-1:50, extraction time is 10-20 min, obtains Buckwheat shell flavone extract;
Described extraction adopts ultrasonic assistant;
Described concentration of alcohol is 80%;
Described solid-liquid ratio 1:30;
Obtained Buckwheat shell flavone extract through macroporous resin dynamic adsorption-eluting, evaporative removal solvent;
Described macroporous resin is D101 type macroporous resin;
Described dynamic adsorption-elution requirement is: adsorption flow rate 2mL/min, sample solution mass concentration are 6.0mg/mL, upper prop volume 2 ~ 3BV, washing volume 3BV, eluant 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 flavone extract and application.
A kind of Buckwheat shell flavone extract, it extracts by above-mentioned Buckwheat shell extracting flavonoids method.
A kind of Buckwheat shell flavone extract is preparing the application in hypoglycemic drug.
Elaborate the present invention below:
1. adopt ultrasonic assistant extraction method, be that extractant extracts flavone compound in Buckwheat shell with alcoholic solution, adopt response phase method, with flavone yield and DPPH free radical scavenging activity for response value, concentration of alcohol, extraction time and solid-liquid ratio three Extraction techniques are optimized, determining optimum extraction condition is: solid-liquid ratio 1:30, extraction time 15min, concentration of alcohol 80%.Under this extraction conditions basis, Buckwheat shell flavone yield and DPPH free radical scavenging activity are respectively 1.60% and 85.62%.
2. adopt macroporous resin to carry out the research of separation and purification to the Buckwheat shell flavone that extraction obtains, result shows: the macroporous resin choosing D101, NKA-9, ADS-17, S-8 tetra-kinds of polarity different carries out static adsorption to Buckwheat shell flavone, by the mensuration of adsorption rate, desorption efficiency and antioxidant activity, determine the adsorbent that D101 type resin is most suitable research; The best purifying process parameter of dynamic adsorption-eluting is: adsorption flow rate 2mL/min, sample solution mass concentration are 6.0mg/mL, upper prop volume 2 ~ 3BV, washing volume 3BV, eluant concentration of alcohol 70%, elution flow rate 2mL/min, reusable 6 times of resin.Under this condition, the purity of Buckwheat shell flavone can bring up to 76.17% by 30.82%, and purity improves 2.47 times, and purification effect is good.
3. build type Ⅱdiabetes mellitus rat model, animal pattern is divided into six groups at random, be respectively normal group, model group, metformin hydrochloride positive controls (100mg/kg), LBHE group (50mg/kg), MBHE group (100mg/kg), HBHE group (200mg/kg), study and improve type Ⅱdiabetes mellitus rat through Buckwheat shell extract (BHE) and generally live and the effect of physiological and biochemical index, result shows: rat modeling success after disposable injection STZ; In body, animal test results is visible, and BHE can alleviate the symptom that diabetes rat is become thin, and improves liver index, renal index and spleen index; Continuous gavage 2 weeks high dose BHE(200mg/kg) can significantly reduce rat fasting blood-glucose level; Administration after 28 days middle and high dosage group glucose tolerance is significantly improved, simultaneously BHE significantly can reduce TC, TG, LDL-C level in rat blood serum and improve the level of HDL-C in serum, improves blood lipid level effect the most remarkable in each dosage group with high dose BHE group.BHE has a better role to type Ⅱdiabetes mellitus rat.
4.BHEs and BHEs-A also has inhibition to AGEs in MGO-BSA and GO-BSA system; BHEs-A is when concentration is 0.2mg/mL; the ability suppressing AGEs to generate is respectively 58.97 % and 46.69 %, is significantly higher than the inhibit activities (p<0.05) with AG under concentration in the same time; The display of SDS-PAGE result also has a certain impact to the generation situation of saccharifying BSA;
5. when flavones content is identical, BHEs-A >=isoquercitrin > rutin >=BHEs=rutin and the mixed mark >=rutin of isoquercitrin, Quercetin and isoquercitrin mixed mark >AG> Quercetin, and BHEs-A and isoquercitrin do not present significant difference (p<0.05), but BHEs-A is mixture, wherein contained rutin and isoquercitrin content not high, illustrate that isoquercitrin has the activity of stronger suppression AGEs generation, and may also there is other active component in BHEs-A.
The invention provides a kind of Buckwheat shell flavone extract, adopt ultrasonic assistant extraction method, is that extractant extracts flavone compound in Buckwheat shell, solid-liquid ratio 1:30, extraction time 15min, concentration of alcohol 80% with alcoholic solution.Under this extraction conditions basis, Buckwheat shell flavone yield and DPPH free radical scavenging activity are respectively 1.60% and 85.62%; Hole resin is adopted to carry out separation and purification to extracting the Buckwheat shell flavone obtained again, at technological parameter be: adsorption flow rate 2mL/min, sample solution mass concentration are 6.0mg/mL, upper prop volume 2 ~ 3BV, washing volume 3BV, eluant concentration of alcohol 70%, elution flow rate 2mL/min, reusable 6 times of resin, the purity of Buckwheat shell flavone can bring up to 76.17% by 30.82%, and purity improves 2.47 times.Tested by type Ⅱdiabetes mellitus rat model, having a better role to type Ⅱdiabetes mellitus rat of Buckwheat shell flavone extract, hypoglycemic effect is better than metformin hydrochloride.And the experiment proved that Buckwheat shell flavone extract is mixture, also contain other unknown materials except containing flavone and flavonoid beyond the region of objective existence.
Accompanying drawing explanation
The DPPH free radical scavenging activity of Fig. 1 tetra-kinds of macroporous resin stripping liquids;
Fig. 2 static adsorption dynamic experiment result;
The dynamic adsorption curve of total flavones under Fig. 3 different in flow rate;
The dynamic adsorption curve of total flavones under Fig. 4 variable concentrations;
Dynamic adsorption curve under the different upper prop volume of Fig. 5;
The determination of distilled water volume of Fig. 6 eluting impurity;
Fig. 7 different ethanol concentration is on the impact of elution property;
Fig. 8 elution flow rate is on the impact of elution property;
Fig. 9 resin access times are on the impact of the absorption property of Buckwheat shell flavone;
Figure 10 is the HPLC analysis result 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 standard substance, BHEs and BHEs-A compares the inhibitory action that AGEs is formed in Glucose-BSA system; Wherein: R: rutin; Q: Quercetin; I: isoquercitrin; R & I: rutin mixes with Quercetin; R & Q & I: rutin, Quercetin and isoquercitrin mixture;
Figure 12 is standard substance, BHEs and BHEs-A compares the inhibitory action that AGEs is formed in Fructose-BSA system; Wherein: R: rutin; Q: Quercetin; I: isoquercitrin; R & I: rutin mixes with Quercetin; R & Q & I: rutin, Quercetin and isoquercitrin mixture;
Figure 13 BHE is on the impact of diabetes rat liver index;
Figure 14 BHE is on the impact of diabetes rat spleen index;
Figure 15 BHE is on the impact of diabetes rat renal index;
Figure 16 is on the impact of diabetes rat T-CHOL;
Figure 17 BHE is on the impact of diabetes rat triglyceride;
Figure 18 BHE is on the impact of diabetes rat LDL-C;
Figure 19 BHE is on the impact of diabetes rat HDL-C.
Detailed description of the invention
the optimal extraction technology of flavone compound in embodiment 1 Buckwheat shell
Select natural Buckwheat shell to carry out dry (60 DEG C, forced air drying 4h) after remove impurity process, pulverize, cross 60 mesh sieves.Accurately take 1.0g Buckwheat shell powder, be placed in 50mL centrifuge tube, add certain density alcoholic solution mix homogeneously, sucking filtration after employing ultrasound assisted extraction method extraction certain hour, filtrate is settled to 100mL and saves backup.Adopt Three factors (concentration of alcohol, solid-liquid ratio, extraction time) three hydraulic test design, use response surface optimization method, Buckwheat shell extracting flavonoids condition is optimized.
Buckwheat shell flavonoid content measures and adopts " health food inspection and assessment technical specification " (2003 editions) NaNO
2-Al (NO
3)
3described in method.
The assay method of DPPH free radical scavenging activity: draw Semen Fagopyri Esculenti extracting solution 50 μ L respectively, adding 150 μ L 40mmol/L DPPH solution, is experimental group A
i; It is color comparator A that 50 μ L Semen Fagopyri Esculenti extracting solution add 150 μ L distilled water
j; Negative control A
obe that 50 μ L distilled water add 150 μ L DPPH solution.Add and terminate rear mix homogeneously, leave standstill 5min, use microplate reader to survey its absorbance at 517nm place.The computing formula of clearance rate is as follows:
Be optimized extraction conditions by Design-Expert8.0.5b software, solid-liquid ratio is 1:20-1:50, extraction time is 10-20 min, concentration of alcohol is 20%-95%.Preferred assembled scheme is solid-liquid ratio 1:30, extraction time 15min, concentration of alcohol 80%, and the predictive value of Buckwheat shell flavone yield and DPPH free radical scavenging activity is respectively 1.59% and 86.13% with this understanding.
embodiment 2 Buckwheat shell flavone process for refining is optimized
(1) pretreatment of macroporous resin
By the macroporous resin 95% soak with ethanol 24h of D101, NKA-9, ADS-17, S-8 tetra-kinds of models, preliminary remove impurity is carried out to it.After macroporous resin is fully swelling, by ethanol elimination, repeat to wash for several times, until without alcohol taste with distilled water.Then first use 5% HCl solution soaking 24h, be then washed to pH=7.0 with distilled water; Then soak 24h by the NaOH solution of 5%, finally spend distilled water and be washed till pH=7.0.Be soaked in distilled water, save backup.
(2) macroporous resin key property measures
The calculating of adsorbance, adsorption rate and desorption efficiency.Take the four kinds of each 2.0g of resin handled well, add Buckwheat shell flavone (BHE) solution 50mL(6.0mg/mL), lucifuge seals, constant temperature water bath concussion (25 DEG C, 100r/min, 24h).After concussion terminates, do controlled trial not adsorb BHE solution.After adsorption test terminates, leach adsorption liquid and repeatedly rinse the macroporous resin after absorption with distilled water, wash away the solution colour of resin surface, add the alcoholic solution 50mL of 70%, put into shaken cultivation case concussion 24h (25 DEG C, 100rpm), after concussion terminates, measure the concentration of flavone in stripping liquid, calculate resin desorption rate.Computing formula is:
C0-in formula---flavone concentration (mg/mL) in sample liquid before absorption
C1---flavone concentration (mg/mL) in solution after absorption
C2---flavone concentration in stripping liquid,
V---liquor capacity (mL),
W---resin quality (g).
Result is as shown in table 1, and D101 type resin has very strong adsorption rate to flavone compound, and flavone adsorbance reaches 31.48mg/g, and polarity S-8 type resin also has stronger absorbability to Flavonoid substances; But S-8 type desorption performance is not as D101 type resin.Consider adsorption rate and the desorption efficiency of various resin, D101 type macroporous resin better performances.Again because D101 type resin belongs to low pole macroporous resin, therefore illustrate that the Flavonoid substances in Buckwheat shell is mainly low pole composition.
DPPH free radical scavenging activity measures.Method is with described in embodiment 1.As shown in Figure 1, the stripping liquid concentration of D101 type resin reaches more than 90% when 0.5mg/mL to result; The clearance rate of four kinds of macroporous resins to DPPH is descending to be followed successively by: D101 > NKA-9 > ADS-17 > S-8.Result shows, the Flavonoid substances antioxidant activity of D101 type resin absorption is best.
Static adsorption dynamic test.Get BHE solution 50mL in triangular flask, add 2.0g macroporous resin, sealing, be placed in concussion 7h in constant temperature oscillator (25 DEG C, 100r/min), every 1h sampling once.After sampling terminates, use NaNO
2-Al (NO
3)
3method measures general flavone content respectively, calculates adsorbance and adsorption rate according to flavones content, and draws kinetic curve according to test data.As shown in Figure 2, D101 macroporous resin reaches adsorption equilibrium to being adsorbed in 8 h of Buckwheat shell Flavonoid substances to result substantially, meets the needs of suitability for industrialized production.
(3) for D101 type macroporous resin, the dynamic adsorption-elution processes of Buckwheat shell flavone is optimized
The determination of optimal adsorption flow velocity.Get D101 type resin 44.0g, wet method dress post (post specification is 15 × 400mm, resin bed volume 40ml, with aquametry).Under the condition that upper column liquid concentration (6.0mg/ml) is identical with upper prop volume (100mL), carry out dynamic adsorption respectively with the flow velocity of 1mL/min, 2mL/min, 3mL/min, every 10mL collects an effluent.Take effluent volume as abscissa, outflow concentration is vertical coordinate mapping.Adsorption flow rate is determined according to leakage point (outflow liquid mass concentration is that sample solution mass concentration 10% is leakage point).As shown in Figure 3, when flow velocity is 1.0mL/min, leakage point occurs the latest result, near 90mL; Leak time during flow velocity 2.0mL/min near 60mL; When flow velocity is 3.0mL/min, near 40mL, just reach leakage point.When flow velocity increases, effluent volume when reaching leakage point constantly reduces, and this is because flow velocity is too fast, and the Flavonoid substances in the not yet timely adsorbent solution of resin, it just flows out with solution, therefore selects more to be conducive at a slow speed absorption.But flow velocity is crossed and cycle period can be caused slowly long, and production efficiency is low, thus causes economy and waste of time.Therefore, 2.0mL/min flow velocity loading is selected.
The determination of upper prop liquid mass concentration.Prepare 3.0mg/mL, 6.0mg/mL, 9.0mg/mL BHE solution loading respectively, and with the flow velocity dynamic adsorption of 1mL/min.Every 10mL collects an effluent.Take effluent volume as abscissa, outflow concentration is vertical coordinate mapping.Suitable loading mass concentration is determined according to leakage point (outflow liquid mass concentration is that sample solution mass concentration 10% is leakage point).As shown in Figure 4, as upper prop liquid mass concentration 3.0mg/mL, leakage point occurs the latest result, is about 140mL; When upper prop liquid mass concentration is 6.0mg/mL, leakage point appears at 90mL place; When upper prop liquid mass concentration is 9.0mg/mL, leakage point occurs too early, just reaching leakage point when 1BV and 40mL.When arriving leakage point, adsorbance is respectively 420mg, 540mg and 360mg, and excessive concentration is unfavorable for absorption as can be seen here.Therefore, 6.0mg/mL loading mass concentration is selected to carry out dynamic adsorption.
The determination of optimum upper column quantity.Adopt the loading mass concentration and loading speed optimized, carry out dynamic adsorption test by resin column.Every 10mL collects an effluent, and measures every pipe flow fluid flavone concentration respectively.Take effluent volume as abscissa, flavone concentration is that vertical coordinate draws adsorption curve, occurs that speed determines optimum upper column quantity according to leakage point.As shown in Figure 5, when the flavone concentration of effluent reaches 10% of load solution flavone concentration, be namely considered as saturated adsorption, now loading volume is about 90-100mL to result.Therefore optimum loading volume is selected to be 2-3BV.
The determination of distilled water volume of eluting impurity.With distilled water with the flow velocity eluting of 2mL/min, the eluent of every 10mL is collected once, and measures general flavone content respectively.As shown in Figure 6, distilled water carries out eluting with the flow velocity of 2mL/min, and when distilled water volume is 1BV, total flavones concentration reaches the highest, illustrates and is washed away by most of Buckwheat shell total flavones.When washing volume and reaching 150mL, detect that total flavones concentration is 0.01mg/mL, therefore distilled water 150mL.Because washing volume too much not only causes damage to adsorbing Flavonoid substances, extend manufacture cycle simultaneously.Therefore, washing volume 3BV is adopted to be best.
The determination of elution concentration.Get the upper prop liquid of suitable concentration, Optimum, dynamic adsorption under certain adsorption flow rate, under identical desorbing flow velocity, carry out desorbing as strippant with the ethanol of different volumes mark, every 1BV stripping liquid carries out collecting and measuring flavones content respectively, draws elution curve.Result is as shown in table 2.
70% ethanol elution rate reaches the highest; When concentration of alcohol reaches 90%, eluting rate does not have significant change, illustrate 70% ethanol and 90% ethanol all better to the elute effect of flavone.But compare the purity of eluate, 70% ethanol is higher than 90% ethanol.As shown in Figure 7, the ethanol elution speed of 30% and 50% is slow and have conditions of streaking, and the elution speed of 70% and 90% ethanol to adsorption liquid is very fast; Flavonoid substances all can all elute at 5BV by the ethanol of four kinds of concentration substantially.Therefore, 70% ethanol washes that elution speed is fast, eluting rate is high, eluate purity is high, and peak shape symmetry is good, is the volume fraction of best eluant.
The determination of elution flow rate.Get the upper prop liquid of suitable concentration, Optimum, dynamic adsorption under certain adsorption flow rate, under 1mL/min, 2mL/min, 3mL/min desorbing flow velocity, desorbing is carried out respectively as strippant with the ethanol of same volume mark, every 1BV stripping liquid carries out collecting and measuring flavones content respectively, draws elution curve.Result is as shown in table 3.
1,2, carry out dynamic desorption under 3mL/min flow velocity, the eluting rate of D101 type macroporous resin to Buckwheat shell flavone is respectively 93.12%, 92.65%, 81.97%, and known 1mL/min and 2mL/min is all better to the elute effect of Buckwheat shell flavone; 2mL/min eluate purity is higher.As shown in Figure 8, with 1mL/min flow velocity eluting, the high but curve peak shape of eluting rate is not concentrated, and hangover is serious and elution time is long; When flow velocity is 2mL/min and 3mL/min, peak shape is concentrated, and symmetry is good, does not have conditions of streaking, but the eluting rate of 3mL/min flow velocity is lower, fast and eluting rate is the highest with 2mL/min elution speed.Therefore, determine that elution flow rate is 2mL/min.
(4) research of number of times reused by resin
Carry out dynamic adsorption-elution test according to the condition optimized, repeated trials 15 times, calculates the adsorbance of total flavones respectively, draws adsorption curve.As shown in Figure 9, D101 type resin recycles in 1 ~ 6 process result, maintains 0.93 ~ 0.96mg/g to the adsorbance of total flavones, and from the 7th time, absorbability is obviously on a declining curve; Until between 11 ~ 15 times, adsorbance is only the low absorption horizontal extent of 0.36 ~ 0.38mg/g, can not continue to recycle if do not regenerated, therefore the reusing number of times and should be advisable within 6 times of D101 type macroporous resin.
By Buckwheat shell alcohol extracts (BHEs), macroporous resin leaches, and remaining liq is absorption.Absorption and the liquid do not adsorbed rotate evaporative removal solvent respectively under 60 DEG C of conditions, dry until solid is separated out 100 DEG C of Water Under baths with thermostat water bath, last vacuum drying treatment obtains dry adsorbate (BHEs-A) and non-adsorbate (BHEs-NA).
Adopt the NaNO announced
2-Al (NO
3)
3colorimetry and conventional H PLC method are carried out detection to the general flavone content in BHEs, BHEs-A, BHEs-NA and Flavonoid substances respectively and are analyzed.Result shows, the flavones content of three kinds of samples presents significant difference, and wherein BHEs is (390.28 ± 26.61) mg/g, accounts for 39.03% of Buckwheat shell crude extract; BHEs-A is (643.18 ± 43.95) mg/g, accounts for general flavone content residual in 64.32%, BHEs-NA of Buckwheat shell crude extract less, is only (106.88 ± 3.47) mg/g.As shown in Figure 10, whether occur in the time series analysis Buckwheat shell extract at peak containing this material according to Figure 10 (A) Plays product rutin (1), isoquercitrin (2), Quercetin (3), from Figure 10 (B), main containing rutin and isoquercitrin in Buckwheat shell extract, Quercetin does not detect in BHEs.
embodiment 7 flavonoid standards compares nonenzymatic glycosylation is inhibiting with Buckwheat shell extract
Determine whether main active component is flavone compound further, compare the inhibition between standard substance and BHEs and BHEs-A, with the rutin contained in known Buckwheat shell, isoquercitrin, and do not detect Quercetin and hybrid standard product as inhibitor, join in reactant liquor, detect its inhibitory action to AGEs.
Standard concentration is 0.08mM and 0.32mM.For Glucose-BSA (as Suo Shi Figure 11 (A)), when reaction carries out 1 month, except Quercetin, each sample all shows certain inhibition, and in dose-dependence, inhibition is followed successively by from high to low: BHEs-A >=isoquercitrin > rutin >BHEs=rutin and the mixed mark=rutin of isoquercitrin, Quercetin and isoquercitrin mixed mark >AG> Quercetin; For Fructose-BSA system (as Suo Shi Figure 12 (A)), when reaction carries out 1 month, inhibition is followed successively by from high to low: BHEs-A=isoquercitrin > rutin=BHEs=rutin and isoquercitrin mixed mark > rutin, Quercetin and isoquercitrin mixed mark >AG> Quercetin.When two individual system react 3 months (as Suo Shi Figure 11 (B), Figure 12 (B)), the inhibition of all samples all generally raises, and BHEs-A, BHEs, isoquercitrin and rutin all show very high inhibition.Therefore, when flavones content is identical, 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 will be the AG of 0.68mmol/L higher than concentration far away.But BHEs-A is mixture, wherein contained rutin and isoquercitrin content not high, illustrates that isoquercitrin has the activity of stronger suppression AGEs generation, and may also there is other active component in BHEs-A.
the preparation of embodiment 3 type Ⅱdiabetes mellitus rat model
Raised at standardization Animal House by rat, conform one week, do not limit eclipse limit water, under 12h dark cycle in daytime condition, room ventilation condition is good, relative humidity 60-70%.Drink distilled water, keep the cleaning of environment, normal feedstuff of throwing something and feeding, change water every day, change bedding and padding respectively once.Laboratory is regularly sterilized.10 are randomly drawed for Normal group after one week.All the other 80 rats are fed high-sugar-fat-diet (wherein containing 10% sucrose, 10% Adeps Sus domestica, 5% cholesterol) 4 weeks.After 4 weeks, fasting 12h, lumbar injection streptozotocin (STZ) 30mg/kg, injects 1 time after 1 week again.Matched group does placebo to 1% buffer 30mg/kg, fasting blood sugar > 11.0 mmol/L's for becoming mould standard after 72h.2 pm every day injects, bolus during injection.
the grouping of embodiment 4 laboratory animal and process
Successful for modeling diabetes rat is divided into 5 groups at random, adds Normal group totally 6 groups.Experiment advance row fasting plasma glucose.The grouping of each group of rat and gavage disposition as follows:
Normal group: distilled water gavage 10mL/kg/d;
Model group: distilled water gavage 10mL/kg/d;
BHE high dose group (HBHE): diabetes rat is to BHE reagent 200mg/kg/d;
Dosage group (MBHE) in BHE: diabetes rat is to BHE reagent 100mg/kg/d;
BHE low dose group (LBHE): diabetes rat is to BHE reagent 50mg/kg/d;
Positive controls: metformin hydrochloride is to rat oral gavage 100mg/kg/d
Administration every day 1 time, successive administration 28d, all experimental rats all drink distilled water except testing requirement, do not limit and adopt hydromining food.
embodiment 5 Buckwheat shell extract is on the impact of type Ⅱdiabetes mellitus rat conventional live index and body weight
(1) ordinary circumstance
In process of the test, the Normal group mental status is good, agile, and fur is white and glossy; After rat disposable vein injection STZ, food-intake, amount of drinking water, urine volume all increase, and health starts to become thin, and lethargy, do not like motion, and rob water phenomenon once in a while, feces has acid smell, and hair color jaundice is matt; After administration, each dosage group and positive controls also have analogue, but comparatively model group mild degree.
(2) amount of drinking water
Polydipsia is one of feature of diabetes, and therefore the number of amount of drinking water can weigh the effect that treatment group improves the state of an illness in a way.After modeling success, administration 28 days, adds up weekly total amount of once drinking water.As shown in table 4.
Compared with model group, LBHE group rat amount of drinking water slightly declines, and obviously, HBHE group amount of drinking water declines the most obvious for MBHE group and the downward trend of HBHE group rat amount of drinking water.
(3) body weight
As shown in Table 5, model group rats body weight reduces before comparing modeling gradually; After the modeling of HBHE group, body weight first reduces, and administration 3 weeks rear body weight have obvious growth; MBHE group body weight first reduces, and beginning in the 4th week slightly increases, but body weight change is not obvious; The body weight of LBHE group reduces gradually.After positive controls modeling, body weight first reduces, and after administration 3 weeks, body weight starts to increase but trend is slow.The above results shows, rat model loses weight may caused by diabetes, and each treatment group has positive-effect to recovery rat body weight.
embodiment 6 Buckwheat shell extract is on the impact of the organ indexs such as type Ⅱdiabetes mellitus rat liver,spleen,kidney
Adopt conventional organ index assessment method, detect the impact of each dosage group on organ indexs such as type Ⅱdiabetes mellitus rat liver,spleen,kidneys.As shown in figs. 10-12, BHE can significantly improve the organ indexs such as liver liver,spleen,kidney to result.
embodiment 7 Buckwheat shell extract is on the impact of type Ⅱdiabetes mellitus rat fasting blood-glucose
Adopt blood glucose meter, to specifications time-and-motion study experimental rat blood glucose.Result is as shown in table 6.
Medicine is after 28 days, and HBHE group, MBHE group result compares with model group pole marked difference, and HBHE group blood glucose value is minimum, and be better than other groups, fasting blood glucose level decreases.The Buckwheat shell extract of result prompting doses has protective effect to impaired fasting glucose (IFG), can play certain blood sugar reducing function to rat.
embodiment 8 Buckwheat shell extract is on the impact of type Ⅱdiabetes mellitus Serum Lipids in Experimental HypercholesterolemicRats
Adopt automatic clinical chemistry analyzer, conveniently time-and-motion study T-CHOL (TC), triglyceride (TG), low density lipoprotein, LDL (LDL-C), high density lipoprotein (HDL-C) level.As shown in figures 13-16, administration is after 28 days for result, and BHE significantly can reduce TC, TG, LDL-C level in rat blood serum and improve the level of HDL-C in serum, improves blood lipid level effect the most remarkable in each dosage group with HBHE group.
embodiment 9 Buckwheat shell extract is on the impact of type Ⅱdiabetes mellitus rat carbohydrate tolerance and serum insulin levels
Adopt conventional carbohydrate tolerance and insulin level detection method, detect the impact on rat carbohydrate tolerance and serum insulin levels of Buckwheat shell extract.Result is as shown in table 7,8, and administration is after 28 days, and middle and high dosage group significantly improves glucose tolerance; The insulin secretion of BHE to type Ⅱdiabetes mellitus rat of each dosage all has improvement result, the most remarkable to the protective effect of beta Cell of islet with the Buckwheat shell extract of high dose, promotes that the secretion of insulin reaches normal level.As can be seen here, BHE has a better role to type Ⅱdiabetes mellitus rat.
Claims (10)
1. Buckwheat shell extracting flavonoids method, it comprises:
1) Buckwheat shell carry out dry after remove impurity process, pulverize, obtain Buckwheat shell powder;
2) 20%-95% ethanol mixes with Buckwheat shell powder, and solid-liquid ratio is 1:20-1:50, extraction time is 10-20 min, obtains Buckwheat shell flavone extract.
2. Buckwheat shell extracting flavonoids method according to claim 1, is characterized in that: described extraction adopts ultrasonic assistant.
3. Buckwheat shell extracting flavonoids method according to claim 2, is characterized in that: described concentration of alcohol is 80%.
4. Buckwheat shell extracting flavonoids method according to claim 3, is characterized in that: described solid-liquid ratio 1:30.
5. the Buckwheat shell extracting flavonoids method according to claim 1,2,3 or 4, is characterized in that: obtained Buckwheat shell flavone extract (BHE) is again through macroporous resin dynamic adsorption-eluting.
6. Buckwheat shell extracting flavonoids method according to claim 5, is characterized in that: described macroporous resin is D101 type macroporous resin.
7. Buckwheat shell extracting flavonoids method according to claim 6, is characterized in that: described dynamic adsorption-elution requirement is: adsorption flow rate 2mL/min, sample solution mass concentration are 6.0mg/mL, upper prop volume 2 ~ 3BV, washing volume 3BV, eluant concentration of alcohol 70%, elution flow rate 2mL/min, resin reuse 6 times.
8. a kind of Buckwheat shell flavone extract according to claim 1, it extracts by above-mentioned Buckwheat shell extracting flavonoids method.
9. a kind of Buckwheat shell flavone extract according to claim 1 is preparing the application in the food and medicine preventing and improve blood glucose.
10. the application of a kind of Buckwheat shell flavone extract according to claim 1 in the medicine of preparation protection beta Cell of islet, maintenance insulin normal level.
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| 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 |
| CN112022906A (en) * | 2020-08-28 | 2020-12-04 | 吉林农业大学 | Preparation method of buckwheat husk non-flavone substance |
| CN112401122A (en) * | 2020-11-11 | 2021-02-26 | 贵州师范大学 | Comprehensive utilization and processing method of black rice buckwheat seeds |
| CN117025200A (en) * | 2023-10-08 | 2023-11-10 | 吉林农业大学 | Biosensor for detecting flavonoid compounds and preparation method |
| CN117025200B (en) * | 2023-10-08 | 2023-12-22 | 吉林农业大学 | Biosensor for detecting flavonoid compounds and preparation method |
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