Entada spiralis Ridl. known as Sintok or Akar Beluru is the woody climber plant belong to Leguminoceae family. E. spiralis is an excellent foaming agent due to the presence of saponin. Thus, E. spiralis stem bark is established used as natural body soap, shampoo and washing agent. The possession of antioxidant activity has been revealed by E. spiralis ability to treat syphilis, insect bites and superficial skin diseases. This research aims to screen the major compounds by phytochemical screening test, verify the antioxidant compounds from crude extracts and fractions via in-vitro antioxidant activity assays, isolate the antioxidant compounds through chromatographic methods and characterize the structure of antioxidant compounds by using various spectroscopic techniques. The crude extract of E. spiralis leaves was prepared by macerating the leaves with petroleum ether, chloroform, and methanol solvent sequentially. Determination and evaluation of antioxidant activity was done through in-vitro study by using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, and 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation. Folin-Ciocalteu’s reagent method was used to measure total phenolic content and flavonoid content was evaluated by calorimetric method. The crude extract of E. spiralis leaves was fractionate by vacuum liquid chromatography (VLC) and liquid-liquid extraction. Isolation of antioxidant compounds was further with column chromatography technique and characterized by spectroscopic analysis. Methanol, chloroform, and petroleum ether crude extracts were analyzed with 5.53 %, 2.05 % and 2.55 % yield. Saponin and flavonoid were the major components. Methanol extract exhibited the highest antioxidant activity with IC50 (DPPH); 40.23 ± 2.66 ug/mL and IC50 (ABTS); 5.09 ± 0.53 ug/mL. The highest possession of phenolic content and flavonoid content; 124.67 ± 6.63 mg GAE/g and 51.67 ± 2.17 mg QE/g were also displayed by the methanol extract. Isoscutellarein, kaempferol-3-C-rutinoside, kaempferol-3-O-glucoside, and kaempferol glycoside were isolated from liquid–liquid extraction fraction while stearic acid and ethyl oleate were isolated from the fraction of vacuum liquid chromatography. IC50 (DPPH) of isoscutellarein; 112.18 ± 2.21 ug/mL, kaempferol-3-C-rutinoside; 136.04 ± 0.52 ug/mL, kaempferol-3-O-glucoside; 301.01 ± 3.02 ug/mL, and stearic acid; 187.49 ± 1.37 ug/mL. Ethyl oleate and kaempferol glycoside were showed negative results towards DPPH. Whereas, IC50 (ABTS) of isoscutellarein; 17.50 ± 0.26 ug/mL, kaempferol-3-C-rutinoside; 34.10 ± 1.13 ug/mL, kaempferol-3-O-glucoside; 14.65 ± 0.11 ug/mL, stearic acid; 5.69 ± 0.06 ug/mL, ethyl oleate; 474.37 ± 2.91 ug/mL, and kaempferol glycoside; 124.82 ± 6.60 ug/mL. In conclusion, the active crude extract and fraction was verified by several in-vitro antioxidant assays. Six antioxidant compounds were successfully isolated from this research study.

The anti-oxidant properties of both M. koenigii leaves and N. sativa seeds extracts have been associated with many of their pharmacological activities including neuroprotective potentials in experimental animal models. The purpose of the current study was to analyze the anti-oxidant properties and assess neuroprotective effects of the extracts in zebrafish and rat models. The solubility and thin layer chromatographic (TLC) techniques have been used as classical methods for physicochemical characterization. Experimental neuro-excitotoxicity was induced by AlCl3 (20 μg/mL) and MSG (475 μg/mL) in zebrafish embryos and larvae models through immersion technique while neuroinflammation by two-vessel occlusion (2VO) in healthy male Sprague Dawley rats. It was confirmed that N. sativa oil (NSO) and water soluble extract (WSE) of N. sativa seeds have different physicochemical properties while WSE has exhibited similar Rf value of 0.95 to that of both Tualang and Kelulut honeys. The presence of thymoquinone (TQ) in NSO was confirmed at (Rf = 0.86) compared to the standard TQ. M. koenigii leaves extract (MKLE) has showed the most potent anti-oxidant property with (IC50=7.63 μg/mL) followed by WSE (IC50= 33.32 μg/mL), NSO alone (IC50= 73.67 μg/mL) and NSO + WSE (IC50= 78.22 μg/mL) respectively against 1, 1-diphenyl-2-hydrazyl (DPPH). Both NSO (0.125 μg/mL) and WSE (80 μg/μmL) have shown to protect the deformities of neurotoxicity significantly (P < 0.05) in AlCl3-induced neurotoxic zebrafish embryo model only after 48 hours of post-induction (hpi). In addition, WSE has also exhibited to protect the deformities of excitotoxicity in both of MSG-induced embryos (50 µg/mL) and larvae (80 µg/mL) models significantly (P < 0.05) compared to that of MSG (475 µg/mL) after 48 hpi. 24 healthy adult male Sprague Dawley rats were randomly divided into four groups (n=6); Healthy Control (HC); 2VO-untreated (2VO); 2VO+NSO treated (NSO) and 2VO+MKLE treated (MKLE). The NSO (100%, 1 mL/kg of b.w) and MKLE (50 mg/kg/day orally) groups were pre-treated for 10 days prior to 2VO surgery and continued until all animals were sacrificed at the end of 10th postoperative week. Total RNA was extracted, purified and relatively quantified as per relative normalized gene expression (∆∆Cq) of two-step RT-qPCR assay with pre-designed QuantiTect® primers. There were significant (P<0.01) folds of difference in GFAP mRNA expression of NSO and HC groups as compared to that of untreated 2VO while there was no significant (P > 0.05) of GFAP mRNA expressions for NSO vs. HC and MKLE vs. 2VO. Conversely, GFAP mRNA expression for MKLE was significantly (P < 0.05) different from NSO group. There was a significantly (P < 0.05) down-regulated MAP2 mRNA expression in both 2VO and NSO groups as compared to that of HC. Yet, the MAP2 mRNA expressions in both NSO and MKLE treated groups were not significantly different (P > 0.05) to that of 2VO untreated. The overall findings suggest that MKLE could have mild neuroprotective potential via glutamate receptors only while N.sativa seeds extract could have superior neuroprotective activity via both of glutamate and MI muscarinic acetylcholine receptors. It is proposed that zebrafish embryo model of 24 hpf developed in this study could be used as a reliable tool to investigate neuroprotective potentials of any other crude extract or leading anti-AD drug in neurobehavioral sciences.