METHODS: The antidiarrheal investigation was performed by using in vivo castor oil-induced diarrheal method whereas in vitro antimicrobial and antioxidant investigation have been implemented by disc diffusion and DPPH scavenging method respectively. Moreover, in silico studies were followed by molecular docking analysis of several secondary metabolites that were appraised with Schrödinger-Maestro v11.1 and Biovia Discovery Studio.

METHODS: In this work, the biochemical potential of M. buxifolia (Falc.) A. DC was explored and linked with its biological activities. Methanol and chloroform extracts from leaves and stems were investigated for total phenolic and flavonoid contents. Ultrahigh-performance liquid chromatography coupled with mass spectrometry (UHPLC-MS) was used to determine secondary-metabolite composition, while high-performance liquid chromatography coupled with photodiode array detection (HPLC-PDA) was used for polyphenolic quantification. In addition, we carried out in vitro assays to determine antioxidant potential and the enzyme-inhibitory response of M. buxifolia extracts.

RESULTS: Phenolics (91 mg gallic-acid equivalent (GAE)/g) and flavonoids (48.86 mg quercetin equivalent (QE)/g) exhibited their highest concentration in the methanol extract of stems and the chloroform extract of leaves, respectively. UHPLC-MS analysis identified a number of important phytochemicals, belonging to the flavonoid, phenolic, alkaloid, and terpenoid classes of secondary metabolites. The methanol extract of leaves contained a diosgenin derivative and polygalacin D, while kaempferol and robinin were most abundant in the chloroform extract. The methanol extract of stems contained a greater peak area for diosgenin and kaempferol, whereas this was true for lucidumol A and 3-O-cis-coumaroyl maslinic acid in the chloroform extract. Rutin, epicatechin, and catechin were the main phenolics identified by HPLC-PDA analysis. The methanol extract of stems exhibited significant 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical-scavenging activities (145.18 and 279.04 mmol Trolox equivalent (TE)/g, respectively). The maximum cupric reducing antioxidant capacity (CUPRAC) (361.4 mg TE/g), ferric-reducing antioxidant power (FRAP) (247.19 mg TE/g), and total antioxidant potential (2.75 mmol TE/g) were depicted by the methanol extract of stems. The methanol extract of leaves exhibited stronger inhibition against acetylcholinesterase (AChE) and glucosidase, while the chloroform extract of stems was most active against butyrylcholinesterase (BChE) (4.27 mg galantamine equivalent (GALAE)/g). Similarly, the highest tyrosinase (140 mg kojic-acid equivalent (KAE)/g) and amylase (0.67 mmol acarbose equivalent (ACAE)/g) inhibition was observed for the methanol extract of stems.

METHODS: Genes and proteins of phase II detoxifying antioxidant enzymes were analyzed by QuantiGenePlex 2.0 Assay and Western blot analysis.

RESULTS: PB significantly induced genes and proteins of phase II and antioxidant enzymes, NAD(P)H quinone oxidoreductase 1, and catalase in aging mice (p < 0.05). The expression of these enzymes were stimulated via translocation of Nrf2 into the nucleus, indicating the involvement of ARE, a cis-acting motif located in the promoter region of nearly all phase II genes.

METHOD: One hundred and twenty male C57BL/6 inbred mice were divided into three age groups: young (6 months old), middle-aged (12 months old), and old (18 months old). Each age group consisted of two control groups (distilled water and olive oil) and three treatment groups: Piper betle (50 mg/kg body weight), tocotrienol-rich fraction (30 mg/kg), and Chlorella vulgaris (50 mg/kg). The duration of treatment for all three age groups was two months. Blood was withdrawn from the orbital sinus to determine the antioxidant enzyme activity and the malondialdehyde level.

RESULTS: Piper betle increased the activities of catalase, glutathione peroxidase, and superoxide dismutase in the young, middle, and old age groups, respectively, when compared to control. The tocotrienol-rich fraction decreased the superoxide dismutase activity in the middle and the old age groups but had no effect on catalase or glutathione peroxidase activity for all age groups. Chlorella vulgaris had no effect on superoxide dismutase activity for all age groups but increased glutathione peroxidase and decreased catalase activity in the middle and the young age groups, respectively. Chlorella vulgaris reduced lipid peroxidation (malondialdehyde levels) in all age groups, but no significant changes were observed with the tocotrienol-rich fraction and the Piper betle treatments.