GC/MS Analysis and Secondary Metabolites Determination with Antioxidant and Anticancer Evaluation of Sequoia sempervirens (D. Don) Endl. through Micropropagated Cultures

Micro-cuttings of Sequoia sempervirens tree were carried out on Murashige and Skoog medium enriched with various concentrations of sucrose and/or growth regulators to induce callus or shootlet cultures. Gas chromatography/mass spectrometry (GC/MS) analysis of n-hexane extracts of the leaves of S. sempervirens mother plants, shootlets, and calli were investigated to identify nonpolar compounds. Likewise, their aqueous ethanol extracts were assessed quantitatively for polar secondary metabolites (phenolics, flavonoids, and triterpenoids) and then investigated to explore their antioxidant and cytotoxic activities. Maximum survival shootlets (100%) were recorded when the medium was provided by either (25 g/l sucrose + 2g/l activated charcoal) or (50 g/l sucrose + 0.5 mg/l kinetin) with an increase in means of shootlet length, fresh and dry weights. Moreover, GC/MS analysis of n-hexane extracts of the leaves of mother plants, shootlets, and calli revealed the identification of 43 (93.56 %), 42 (96.15 %), and 17 (95.05%) compounds, respectively. Aliphatic hydrocarbons were the major components in n-hexane extract of callus treated with 1.6 mg/l 2, 4-D + 0.8 mg/l NAA (62.53%), with high levels eicosane, 2-methyl (19.55%), and heptacosane (17.10 %) whereas a high percentage of monoterpene hydrocarbons compounds were found in n-hexane extract of shootlet treated with 50 g/l sucrose + 0.5 mg/l kinetin. The major compounds in n-hexane extract of shootlets and the leaves of mother plants were monoterpene hydrocarbons (79.35 and 69.23%), alpha-Phellandrene (36.02 and 35.93%) and alpha-pinene (31.41 and 10.57%), respectively. Furthermore, quantitative estimation of the total flavonoids, phenolics, and triterpenoids of the aqueous ethanol extracts showed that shootlet cultures treated with 50 g/l sucrose + 0.5 mg/l kinetin had the highest contents (2.514, 0.98, 172.640 mu g mg-1, d.w.) compared with those of leaves (2.061, 0.684, 172.672 mu g mg-1, d.w.), respectively. Moreover, the same shootlet cultures gave the most potent antioxidant activity and showed inhibition effects on the human liver (HepG-2) and breast (MCF-7) tumor cell lines compared to the other aqueous ethanolic extracts of the tested samples. Consequently, shootlet culture enriched with 50 g/l sucrose + 0.5 mg/l kinetin was the most biologically active medium and could be considered as an alternative source of bioactive metabolites.