Ultrasonic Extraction Process Of Pinostrobin From Pigeonpea Leaves And Its Effect On Osteoblasts MC3T3-E1

In this paper,the content of pinostrobin in pigeonpea leaves was detected by HPLC-UV method,and ultrasonic extraction conditions of pinostrobin were optimized thro?gh orthogonal design experiments.The proliferation,differentiation and mineralization effects of pinostrobin and pigeonpea leaves extract on osteoblast MC3T3-E1 were also studied.Results are as follows:1.The HPLC-UV method for the determination of pinostrobin in pigeonpea leaves was developed as follows:Chromatographic column HiQ sil C18W(4.6mm ? ×250mm,5?m);Mobile phase:(A)methanol-(B)water(v/v),0-15min A:75%-70%,15-25 min A:70%-85%,25-40min A:85%-75%;flow rate 1 mL/min;column temperature 25?;detection wavelength 287nm.The determination of pinostrobin HPLC-UV condition showed that the R.S.D.of the precision and repeatability with this method were all less than 3%.2.The ultrasonic extraction conditions of pinostrobin in pigeonpea leaves,were optimized by orthogonal design L9(34)and the optimal extraction conditions were obtained as follows:Methanol concentration 80%,extraction time 60min,extraction temperature 50? and solid-liquid ratio 1:40(g/mL).Under optimum conditions,the extraction yield of pinostrobin reached 1.832mg/g,and methanol extract exhibited good antioxidant activity with an IC50 value of 0.281 mg/ml,which was similar to ascorbic acid IC50 value of 0.202 mg/mL.Result shows that the extraction rate of optimization of pinostrobin in pigeonpea leaves is reliable.the methanol extract of pinostrobin has a good antioxidant activity with an IC50 value of 0.281 mg/ml.3.The effect of pinostrobin on proliferation of MC3T3-E1 cells was studied by MTT assay.Result shows that the proliferation effect of pinostrobin on MC 3T3-E1 cells is the most significant when compared with pigeonpea extract and estradiol,and the proliferation rate of MC 3T3-E1 cells treated by 40?g/mL pinostrobin for 24h is(26.27 ± 0.95)%.4.The content of NO in osteoblast MC 3T3-E1 treated by pinostrobin and pigeonpea extract was determined by nitrate reduction assay.The results showed that the content of NO in MC 3T3-E1 cells treated by 40?g/mL pinostrobin for 24h was(63.15 ± 2.15)?mol/L,which is signicant lower than that of control(82.63±3.48)?mol/L,lower NO content may be atributed to proliferation of MC3T3-E1 osteoblasts.5.Differentiation of osteoblast MC3T3-E1 treated by pinostrobin and pigeonpea leaf extract was studied using colorimetry.The result showed that pigeonpea leave extract and pinostrobin can promot the differentiation of osteoblast MC 3T3-E1.The differentiation effect of osteoblast MC 3T3-E1 treated by 40?g/mL pinostrobin for 4d,was the most obvious and the differentiation rate is(37.022±1.638)U/gprot.6.The content of COL I in osteoblast MC 3T3-E1 treated by pinostrobin was determined by enzyme-linked immunosorbent assay.Finding that the 10-40 ?g/mL pinostrobin have different effects on the content of COL.The content of COL I in MC 3T3-E1 cells treated by pinostrobin for 5d was(6.207±0.256)ng/106 ?mol/L,which is signicant more than that of control.7.The concentration of pinostrobin between 10 ?g/mL and 40 ?g/mL were applied to the osteoblast MC 3T3-E1 for 14d,21d,28d,MC 3T3-E1 was dyed by Von Kossa method,the result showed that the same concentration of pinostrobin treated the number of mineralized nodules increasing along with the extension of time in dosing,after 28d treatment osteoblast have the largest number of mineralized nodules,calcium deposition area increases,the osteoblast mineralization nodule number to 177.21 ± 25.86.The results showed pinostrobin has promoting effect on osteoblast mineralization effect,and there are time and dose-dependent.8.Osteocalcin(OC)gene expression in osteoblast MC 3T3-E1 treated by pinostrobin was determined by real-time fluorescence quantitative PCR(real-time qPCR).OC gene expression of osteoblast MC 3 T3-E1 treated by pinostrobin for 24h shows an upregulated trend with the increasing concentration of pinostrobin.the relative expression of OC mRNA of osteoblast MC 3T3-E1 treated by 40?g/mL pinostrobin was up to 8.3 times higher than that of control group,which shows pinostrobin can promote the expression of osteoblast OC gene.9.The reverse effect of pinostrobin on proliferation inhibition of osteoblast MC 3T3-E1 pro treated by dexamethasone was studied by MTT assay.Proliferation rate of osteoblast MC 3T3-E1 treated by pinostrobin and pigeonpea leaves extract were higher than that of hormone group and lower than control group.10.The reversed effect of pinostrobin on differentiation inhibition of osteoblast MC 3T3-E1 pro treated by dexamethasone was studied by colorimetry method.The result showed that the contents of ALP in osteoblast MC 3T3-E1 spiked by pinostrobin and pigeonpea leaves extract were higher than that of hormone group and lower than control group.When the concentration of pinostrobin is 40?g/mL,the osteoblast MC 3T3-E1 were treated for 4d.the content of ALP in pinostrobin group and pigeonpea leaves extract group was(10.235±0.283)U/gprot and(8.358± 0.893)U/gprot,respectively.The result showed that pinostrobin can weaken the inhibitory effect of dexamethasone on osteoblast MC 3T3-E1 differentiation.