Purification and Characterization of Active Protein/peptides and Studies of Biological Activities of Different Extracts from Nelumbo Nucifera Gaertin Seeds

Abstract

Acid phosphatase-I (Apase-I) from seeds of N. nucifera Gaertn was purified to electrophoretic homogeneity by combination of ammonium sulfate precipitation, size exclusion and ion exchange chromatography. SDS-PAGE of purified Apase-I gave a single band with molecular mass of 80 kDa under reducing and non-reducing conditions, indicating that the enzyme was a monomer. The purified enzyme showed maximum activity at 50 ºC and at pH 5. The Km, Vmax and Kcat for p-nitrophenyl phosphate were 132 mM, 10 mmol/min/mg and 6.7/s respectively. Apase-I activity was strongly inhibited by Zn,++ W,3+ weakly inhibited by Cu,++ Mo++ and Cr6+ and moderately activated by Mg2+. The enzyme was shown to be thermo-labile as it lost 50 % of its activity after incubation for 1 hour. The amino acid analysis of enzyme revealed high proportion of acidic amino acids, which is very similar to that of tomato Apase-I and lower than potato Apase. The in vitro antimicrobial, antioxidant, antileishmanial effects as well as cholinesterase and urease inhibitory activities of various fractions of N. nucifera were also determined followed by investigation the anti-acetylcholinesterase, antihyerglycemic, antihyperlipidemic activities and antioxidant enzymes effects of N. nucifera extracts in alloxan induced diabetic rats in vivo. Shade dried seed powder of N. nucifera was extracted with methanol (crude), suspended in distilled water and further fractionated to yield n-hexane (N1), chloroform (N2) , ethyl acetate (N3) and butanol (N4) soluble fractions while the remaining portion was used as aqueous fraction (N5). Nelumbo nucifera seeds volatile oil (essential oil, EO) as well as non-volatile oil (fixed oil, NnFO) were also obtained through standard procedures and their gas chromatography–mass spectrometry (GC-MS) analysis was carried out. The crude extract, subsequent fractions and EO were evaluated for their DPPH, ABTS and superoxide anion free radical scavenging and cholinesterase inhibitory activities. The N3 fraction and EO showed outstanding antioxidant activities with IC50 values of 191, 450 μg/mL (DPPH), 123, 221 μg/mL (ABTS) and 69, 370 μg/mL (superoxide anion) respectively. The N3 fraction and EO also caused significant inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with IC50 values of 70 ± 0.6, 64 ± 0.8 and 75 ± 0.3, 58 ± 0.2 μg/mL in dose-dependent manner. The presence of saponins, alkaloids and glycosides might attribute to the promising bioactivity of crude and fractions (detected in phytochemical tests) while EO constituted 19 phytochemicals (mainly oxygenated sesquiterpenes) responsible for its promising bioactivity. Crude methanolic extract, subsequent fractions and fixed oil (NnFO) were subjected to different biological activities and enzyme inhibition assays to investigate beneficial potential of this medicinal XVI plant. Promising antibacterial effects were shown by the N3 (69%) and N4 (65%) against Escherichia coli and Bacillus subtilis respectively, while maximum antifungal effect was exhibited by N4 against Candida glaberata (75%). All the extracts of N. nucifera showed potent cytotoxicity in Brine shrimp lethality assay. Potent antileishmanial activity was observed for N. nucifera N3 fraction (IC50 = 6.88 ±1.27 μg/mL) and NnFO (IC50 = 7.34 ±2.72 μg/mL). The N3 and N4 fractions also showed strong inhibition against both Jack Bean urease and Bacillus Pastry urease in dose dependent manner with IC50 values of 21.45±0.6, 35.76 ±0.4 and 28.65±.0.3, 44.87±0.2 μg/mL respectively. GC-MS analysis of the fix oil NnFO revealed the presence of 39 compounds mainly comprised of 20.8 % keto fatty acids with high content of palmitic acid (13 %) and nonanoic acid (11.89 %). The in vivo investigation of antidiabetic, antiacetylcholinesterase (AChE) and antioxidant enzymes effects of methanlic crude and isolated compounds, nuciferin and norcoclaurine from the seeds of N. nucifera in alloxan induced diabetic rats were performed. The alloxan (100 mg/kg b.w) induced diabetic rats (200-250 g) were divided into seven groups (n = 6). Group I; normal control, Group II; diabetic control, Group III; standard, Group lV-VII were methanolic crude extracts (100, 200 mg/kg), nuciferin and norcoclaurine (10 mg/kg b.w.), received for 15 days in dose dependent manner. The study included: examination of oral glucose, fasting blood glucose, serum lipid profile and checking of body weight changes. In oral glucose examination, within 60 and 80 minutes of sample induction, nuciferin and norcoclaurine significantly reduced blood glucose (P<0.05) and restored body weight in diabetic rats. Alloxan induced diabetic rats reported 30-50% reduction of blood glucose level (P<0.05) and improved 5-20% body weight at day 15 after ingestion of 100-200 mg/kg crude extracts and nuciferin and norcoclaurine (10 mg/kg b.w.). It also improved elevated biochemical parameters such as triglycerides (TG), low density lipoprotein (LDL), high density lipoprotein (HDL), total cholesterol (TC), serum urea and creatinine significantly. Antioxidant enzyme assays i.e. superoxide dismutase (SOD), catalase test (CAT), lipid peroxidation assay (TBARS), glutathione assay (GSH) and acetylcholinesterase (AChE) assays were performed. Nuciferin and norcoclaurine significantly reduced blood glucose (p<0.05) and restored body weight in diabetic rats. Diabetes caused decrease in the antioxidant enzymes level (SOD, CAT, GPx and GSH). However, nuciferin and norcoclaurine (10 mg/kg) significantly increased the antioxidant enzymes in diabetic groups. While, significant increase in TBARS level was observed in diabetic group. Nuciferin and norcoclaurine (10 mg/kg) prevented this increase of diabetic animals (p<0.05) as compared to glibenclmide. AChE levels were significantly decreased both in blood and brain of diabetic group XVII (p<0.05). Our results demonstrated that Nuciferin and norcoclaurine improved memory and interfered with the cholinergic signalling. Our findings have proven that various fractions of N.nucifera seeds possess cytotoxic, antimicrobial, antidiabetic, antiacetylcholinesterase and antioxidant enzymes effects in diabetic rats (P<0.05), which might be due to the presence of bioactive phytochemicals such as flavonoids, tannins, saponins and alkaloids. Further studies are required to isolate the major bioactive constituents and to verify the potent effects in both in vitro and in vivo results.