Micropropagation of Zingiber montanum Koenig and evaluation of its essential oils for antioxidative and antimicrobial activities

Zingiber montanum or locally known as bonglai is a rhizomatous perennial plant belonging to Zingiberaceae. This species is highly valued for its medicinal properties as it has been used in folk medicine for centuries. Pharmacological studies indicated that Z. montanum extracts possess antioxidant and antimicrobial agent. But in Malaysia, there is little study confirming these activities for Z. montanum. Moreover, this plant has been considered as underutilized especially in Malaysia. Conventional propagation means was hampered by slow propagation rate and the risk of disease transmittance through division by sectioning of the rhizomes. The fact that more planting materials are needed for further pharmacological importance discovery and for commercial planting, alternative propagation method by means of tissue culture is required. This study was conducted to provide scientific evidence for its antioxidative and antimicrobial potential and technique for its mass propagation to overcome the conventional propagation constraints of Z. montanum. Shoot of Z. montanum was induced in Murashige and Skoog (MS) basal media enriched with 4.44μM BAP + 4.65μM KIN and produced 6.3 shoots. The shoots were multiplied in 4.44μM BAP + 4.65μM KIN + 2.69μM NAA supplemented MS media produced 5.8 shoots per explant with shoot length of 7.0 cm. Shoots in the MS medium incorporated with 0.56 μM NAA showed good rooting response producing 20.4 roots per explant with root length of 6.3 cm. While, the essential oil analysis of the Z. montanum mother plant showed that the highest percentage of oil based on their fresh weight was obtained in rhizome (1.75% v/w), followed by the leaves (0.05% v/w) and pseudo-stem (0.02% v/w). GC-MS analysis of the rhizome oil revealed that 28 compounds were detected where β-phellandrene (31.94%), terpinen-4-ol (24.27%), 1-4-bis(methoxy)triquinacene (24.03%) were identified as major compounds while in the leaf oil, 84 compounds were detected with the dominant compounds being 1,4,-bis(methoxy)triquinacene (17.00%), followed by β-phellendrene (13.79%) and β-sesquiphellandrene (12.86%). Total phenolic assay indicated that Z. montanum had high phenolic content as high as 343.42 mg Gallic acid equivalent GAE/100g. The antioxidant activity of the oils was higher than that of BHT as revealed by DPPH free radicals scavenging and β–carotene bleaching assay proving that Z. montanum is a good source of antioxidant agents. On the other hand, antimicrobial assay of the oils revealed different susceptibility with Bacillus subtilis observed as the most susceptible microbe with Minimum Inhibitory Concentration (MIC) value of 0.025mg/μl. Fungi showed considerable resistance over the oils. The micropropagation protocol of Z. montanum in this present study could be potentially applied for its mass propagation to exploit its medicine properties. The present study revealed that even though Z. montanum oils exhibited weak to moderate antimicrobial activity but it exhibited relatively good antioxidant activity, confirming the potential of Z. montanum as a source of natural antioxidant to be used as natural preservatives in food, cosmetic and pharmacological products