Bioactive constituents and anti-inflammatory effect of agarwood leaf extract and its nano-encapsulated as halal therapeutic alternative

Abstract

The serious adverse effects associated with the prolonged use of synthetic anti-inflammatory medications have raised concerns in the pharmaceutical field. In response, the pharmaceutical industry has begun to embrace the halal built-in approach to meet the desired unified attributes of safety and high quality. This work is focused on Aquilaria malaccensis (Family Thymelaeaceae ), an agarwood-producing tree geographically distributed in Indomalesian rainforests. Different parts of the tree have been traditionally used to treat several medical disorders including inflammatory-related conditions. However, the scientific evaluation of the anti-inflammatory related traditional claims of this plant has been very scarce. In this study, A. malaccensis ethanolic leaf extract (ALEX-M) is subjected to characterization via phytochemical screening, FTIR, GC-MS and LC-MS. The extract was also subjected to isolation of bioactive compounds with anti-inflammatory potential using column chromatography. The structures of the isolated compounds were elucidated using HREIMS, IR, UV, ID and 2D NMR spectroscopy. Polymeric nanocapsules were fabricated using water-in-oil-in-water (w/o/w) emulsion method uing Tragacanth gum as a natural and Halal polymer to encapsulate the extract. The toxicity and anti-inflammatory activity of the free extract (ALEX-M) and nanoencapsulated extract (ALEX-M-PNCs) were assessed in-vitro on RA W264.7 macrophages and in Zebrafish embryos. The findings revealed that a total of 58 compounds were tentatively identified in the extract using LC-Q-TOFMS, some of which have been previously reported to exhibit anti-inflammatory activity. The dichloromethane and ethyl acetate fractions yielded eight compounds including one terpenoid, four flavonoids and three benzophenones. DCM-C2, DCM-C4, EA-CS and EA-C7 demonstrated the highest inhibition of NO production in LPS-stimulated RAW 264.7 macrophages with ECso=27.75, 35.25, 16.65 and 27.78 μg/mL, respectively. The extract-loaded nanocapsules (ALEX-M-PNCs) were spherical in shape with desirable size (167.13±1.24 nm) and polydispersity index (0.29±0.01) and demonstrated relatively high encapsulation efficiency (87.36±1.81 %). ALEX-M demonstrated high viability on RAW 264.7 cells and towards zebrafish embryos at high concentrations in contrast to nanocapsules that showed relatively higher cytotoxicity. ALEX-M and ALEX-M-PNCs demonstrated ability to reduce NO production in LPS-stimulated macrophages in a dose-dependent manner with ECso values of 164. 790±6.318 μg/mL and 5.50 ± 0.31 μg/mL, respectively. A significant reduction in the amount of NO was also observed with ALEX-M and ALEX-M-PNCs in LPS-treated zebrafish embryos with EC50 values of 43 .09± 7 .67 μg/mL and 11.92±2.61 μg/mL, respectively. The results confirmed the traditional use of A. malaccensis in the management of inflammation and supports its use as a safe and Halal therapeutic alternative. In addition, nanoencapsulation of the extract potentiated its anti-inflammatory effect. Further in-depth research can be conducted to elucidate the mechanisms underlying the anti-inflammatory effect of the extract.