Study On The Regularity Distribution Of D-chiro-inositol In Buckwheat And Its Antioxidative Effect In Cells

D-chiro-inositol(DCI)is an isomer of inositol is widely found in legumes,buckwheat and pumpkin.Epidemiological studies have shown that DCI plays an important role in a variety of human health effects.But it is easily lost in patients with metabolic abnormalities or chronic diseases and the timely supplement of DCI from food can relieve the symptoms.Buckwheat is the only plant that can be used as staple food in all the resources rich in DCI and is the main dietary source of DCI.At present,there is little information about the distribution of DCI in buckwheat and the current methods of DCI can not be widely used because of fussy and time-consuming pre-treatment,high-cost and harsh condition.It has been found that can play varieties of physiological functions,such as anti-hyperglycemic and anti-cancer,which are related to inhibiting oxidative stress.At present,it has been reported that DCI can play an antioxidant role in animal models and in vitro chemical reaction models.There are still rare reports on the inhibition of oxidative stress in cell models.Therefore,in this study we firstly established an economical and practical method for accurate quantification of DCI in buckwheat samples by ordinary equipment in view of the shortage of existing detection methods.Then,the new method was applied to analyze the content and the existing form of DCI in different varieties of buckwheat,different parts of the buckwheat plant during different growth period and different milling fractions of buckwheat seed.We comprehensively and systematically investigated the regularity of distribution of DCI in buckwheat.Finally,we researched the inhibitory effect of DCI on oxidative stress induced by palmitic acid in HepG2 cells.The main contents and major conclusions of this study were as follows:(1)The newly established method for the determination of the DCI in buckwheat is HPLC-UV with pre-column derivatization using the common C18 column and it is an economical and practical method.The detection wavelength was 229 nm and the mobile phase was acetonitrile and water with gradient elution.The best derivatization conditions were as follows: the dosage of the derivatization reagent was 20 μL;the concentration of NaOH was 7 mol/L;the derivation temperature was 25℃ and the time was 10 min.The linearity of standard curve within the range of 0.05～2 mg/mL.The limit of detection(LOD)was 5 μg/mL and The limit of quantitation(LOQ)was 10 μg/mL.The recovery of DCI from buckwheat was 99.65% ～ 112.59%.(2)There are some differences of the content and the existing form of DCI in different varieties of buckwheat.Zhaoku NO.2(7.00±0.04 mg/g)and Dingtian NO.1(6.84±0.10 mg/g)had the higher DCI contents(p < 0.05)and DCI in buckwheat are mostly in bound form.In addition,the results found that there is no significant difference of the total DCI content between tartary buckwheat and common buckwheat.But the tartary buckwheat easier to accumulate free DCI while the common buckwheat easier to bound DCI.(3)The results of the DCI content in different parts of the buckwheat plant during different growth period showed that the order of DCI content in the buckwheat plant from high to low in the growth process was: maturation period,grain period,flowering period,seedling-leaf period and the DCI content of leaves the highest in maturation period(9.35±0.81mg/g).Moreover,The results showed that DCI was mainly stored in the aerial part of buckwheat plants and DCI in the flower and seeds is mainly in bound forms while in roots and stems mainly exists in the free form.(4)There are some differences of the content of DCI in different milling fractions of buckwheat seed.Results found that the fraction of 80-100 mesh and the 60-80 mesh contained highest total DCI,followed by 40-60 mesh and the lower part of the 100 mesh sieve,and the upper part of the 40 mesh sieve was the lowest(p < 0.05).(5)The results of cell experiments showed that DCI can inhibit the oxidative stress of HepG2 cells induced by palmitic acid(PA).has a protective effect on oxidative stress induced by palmitic acid in HepG2 cells.On the one hand,DCI could improve the activity of antioxidant enzymes such as SOD and GR(p < 0.05).The other hand,DCI can could restore mitochondrial membrane potential(p < 0.05)and protect mitochondrial function,and then inhibit cell oxidative damage.