PURPOSE: This study aimed to investigate the anti-aging potential of CC extracts and fractions, particularly their inhibition of collagenase, MMP-1 and MMP-3 activities in human dermal fibroblasts CCD-966SK, followed by isolation, identification and analysis of their bioactive constituents.

STUDY DESIGN AND METHODS: DPPH assay was firstly used to evaluate the antioxidant activity throughout the bioactivity-guided fractionation. Cell viability was determined using MTS assay. Collagenase activity was examined, while MMP-1 and MMP-3 expression were measured using qRT-PCR and western blotting. Then, chemical identification of pure compounds isolated from CC fractions was done by using ESIMS, 1H and 13C NMR spectroscopies. HPLC analyses were carried out for bioactive fractions to quantify the major components.

RESULTS: Throughout the antioxidant activity-guided fractionation, fractions CC-E2 and CC-E3 with antioxidant activity and no toxicity towards CCD-966SK cells were obtained from CC 75% ethanol partitioned layer (CC-E). Both fractions inhibited collagenase activity, MMP-1 and MMP-3 mRNA and protein expression, as well as NF-κB activation induced by TNF-α in CCD-966SK cells. 14 compounds, which mainly consists of flavonoids and their glycosides, were isolated. Quercitrin (14.79% w/w) and quercetin (11.20% w/w) were major compounds in CC-E2 and CC-E3, respectively, as quantified by HPLC. Interestingly, both fractions also inhibited the MMP-3 protein expression synergistically, compared with treatment alone.

METHODOLOGY: This randomised, blinded end-point, placebo-controlled clinical trial with a parallel design involved 36 healthy male subjects who took either an oral placebo or TRE at doses of 80, 160 or 320 mg daily for 2 mo. Baseline and end-of-treatment measurements of vitamin E concentration, arterial compliance [assessed by aortic femoral pulse wave velocity (PWV) and augmentation index (AI)], ASBP, plasma TAS, serum TC and LDL-C were taken.

RESULTS: Baseline tocotrienol isomer concentrations were low and not detectable in some subjects. Upon supplementation, all TRE-treated groups showed significant difference from placebo for their change in alpha, gamma and delta tocotrienol concentrations from baseline to end of treatment. There was a linear dose and blood level relationship for all the isomers. There was no significant difference between groups for their change in PWV, AI, plasma TAS, ASBP, TC or LDL-C from baseline to end of treatment. Groups 160 mg (p = 0.024) and 320 mg (p = 0.049) showed significant reductions in their ASBP. Group 320 mg showed a significant 9.2% improvement in TAS.

RESULTS: Higher calcium concentrations (1.5 and 2% w/v) increased calcium concentration in the peel and pulp tissues, maintained firmness, and reduced anthracnose incidence and severity. While leakage of calcium-treated fruit was lower for 1.5 and 2% calcium treatments compared to the control, microscopic results confirmed that pulp cell wall thickness was higher after 6 days in storage, for the 2% calcium treatment compared to the control. Calcium-treated fruit also had higher total antioxidant activity and total phenolic compounds during storage.

METHODS: Diabetes was induced using streptozotocin (60 mg/kg, i.v.) followed by nicotinamide (210 mg/kg, intraperitoneal (i.p.)). MAD (50 mg/kg) was administered orally for 4 weeks, commencing 15 days after induction of diabetes; resveratrol (10 mg/kg) was used as a positive control. Fasting blood glucose, plasma insulin, HbA1c, liver and lipid parameters were measured, along with antioxidant enzymes and malondialdehyde as an index of lipid peroxidation; histological and immunohistochemical studies were also undertaken.

KEY FINDINGS: MAD normalized the elevated fasting blood glucose levels. This was associated with increased plasma insulin concentrations. MAD alleviated oxidative stress by improving enzymatic antioxidants and reducing lipid peroxidation. Histopathological examination showed significant recovery of islet structural degeneration and an increased area of islets. Immunohistochemical staining showed increased insulin content in islets of MAD-treated rats.