Isolation And Identification Of Effective Constituents From Moringa Oleifera Lam.leaves And Their Biological Activities

Moringa oleifera Lam., belonging to the member of Moringaceae family, is widely distributed in tropic and subtropical regions of India and Africa. In recent years, these plants are widely cultivated in Yunnan, Guangdong, Fujian, Taiwan and other provinces in China. M.oleifera is rich in nutrients and active substances, and has been reported to have various physiological activities. In addition, these plants have been widely used and developed in pharmaceutical and health care industries. The current research on M. oleifera confined to a single variety, while comparisons between different varieties are rarely reported. Therefore,the present work aims to explore the differences of chemical constituents and biological activities among varieties, which can provide certain theory basis for further full use of these plants.Response surface methodology(RSM) was used to optimize ultrasonic-assisted extraction(UAE) of total flavonoids from M. stenopetala leaves. The optimal conditions were enthanol concentration 70%(v/v), solid-liquid ratio 1:27(m/v), ultrasonic time 46 min, and temperature 50 °C. Under the condition, the yield and ORAC value were 48.93 ± 0.44 mg RE/g and 2747.17 ± 301.51 μmol TE/g. The crude extract was then purified by polyamide resin, the flavonoids content and ORAC value were 3.01 and 2.16 times, respectively. The crude and purified extracts showed strong DPPH and ABTS radical scavenging activities.The total flavonoids and polysaccharides were obtained by the UAE and heating reflux extraction(HRE). Their yields of total flavonoids were 21.30 g/100 g dry weight(DW), 22.29g/100 g DW, and 20.78 g/100 g DW, and flavonoid contents were 21.91%, 35.95%, and16.40%, respectively. Their yields of total polysaccharides were 6.18 g/100 g DW, 7.71 g/100 g DW, and 6.71 g/100 g DW, and polysaccharide contents were 34.80%, 43.30%, and 35.32%,respectively.The antioxidant, hyperglycemic and anticomplement activities of total flavonoids and polysaccharides from three species of M. oleifera were comparatively investigated. Results showed that flavonoids exhibited stronger antioxidant and hyperglycemic activities, while polysaccharides had a strong inhibitory effect on the complementary system. Flavonoids from pkm1 leaves had significant inhibitory activities of α-amylase and α-glucosidase. However,the flavonoids from M. stenopetala and pkm1 improved variety had weaker inhibition effect on α-amylase, and had no inhibitory effect on α-glucosidase. Polysaccharides from pkm1 showed weaker inhibitory effect on α-amylase and α-glucosidase. In the tested concentration range, polysaccharides from M. stenopetala and pkm1 improved variety exhibited no inhibition. The flavonoids from pkm1 showed some hemolysis inhibition activity, followed by M. stenopetala and pkm1 improved variety, which were lower than that of the positive control.Polysaccharides from M. stenopetala exhibited better hemolysis inhibition activity at low concentrations(0.25 ~ 0.5 mg/mL), and polysaccharides from pkm1 showed the strongest hemolysis inhibition at high concentrations(3 ~ 5 mg/mL), which were higher than the positive control.The crude flavonoids from pkm1 were partitioned with different solvents, which yielded petroleum ether fraction, ethyl acetate fraction, n-butanol fraction, and water fraction,respectively. Results showed that n-butanol fraction was found to have the strongest antioxidant activity, followed by the other three fractions. In addition, the n-butanol fraction and water fraction showed significant inhibitory activities against α-amylase andα-glucosidase, which were equal to the positive control. Among four fractions, the ethyl acetate fraction and n-butanol fraction were responsible for the anticomplement activities, and their EC50 were 2.32 mg/mL and 2.96 mg/mL, respectively.The n-butanol fraction was separated and purified by the polyamide resin column,Sephadex LH-20 gel column and preparative HPLC. One main compound was obtained and identified as Quercetin-3-O-β-glucoside by MS, 1H-NMR, and 13C-NMR analysis. The compound had significant antioxidant, α-glucosidase inhibitory and anticomplement activities.Its ORAC value was 12377.74 ± 361.54 μmol TE/g, which was 1.2 times higher than ascorbic acid, α-glucosidase inhibitory activity(0.039 mg/mL of EC50 value) was little lower than acarbose, and hemolysis inhibition(1.75 mg/mL of EC50 value) was higher than heparin at high concentrations(?4 mg/mL).