Elicitation of Anthraquinones with its Associated Enzymic and Non-Enzymic Antioxidant Responses in Morinda Elliptica Cell Culture

Morinda elliptica (Rubiaceae) cell suspension culture was used as a model system to understand the effects of elicitation and enzymic and non-enzymic antioxidant responses. The kinetic profiles of anthraquinone (AQ) compounds, hydrogen peroxide (H2O2) level, lipid peroxidation and antioxidant vitamins were determined for cultures grown in maintenance (M), intermediary (G) and production (P) medium strategies, and compared with the profiles from callus and leaf. Screening experiments were carried out to select the most suitable elicitors and effective elicitation factors that could enhance AQs without affecting cell growth in G medium. The selected elicitor was then used to study the effects on enzymic and non-enzymic antioxidant responses. High performance liquid chromatographic (HPLC) method was developed to identify and quantify AQ constituents from the two methods of elicitation – medium strategies and jasmonic acid (JA). P medium strategy produced the highest registered cell growth at 49 g l-1, intracellular AQ content at 42 mg g-1 (dry cell weight) DW and H2O2 level at 9 mol g-1 (fresh weight) FW, compared to other media. The extent of lipid peroxidation at 40.4 nmol g-1 FW and total carotenoids at 13.3 mg g-1 FW for cultures in P medium were comparable to the leaf. Vitamin C content in all culture systems was almost half the leaf content. On the other hand, vitamin E was around 400 to 500 µg g-1 FW in 7-day-old cultures from all medium strategies which was 2-fold to the leaf. In P(21) cultures, nordamnacanthal at 5.6 mg g-1 DW and 1-hydroxy-2-methylanthraquinone at 17.2 mg g-1 DW were detected 4-fold and 12-fold, respectively, higher than the roots. The mechanism to counter reactive oxygen species (ROS) in the whole plant and plant cell cultures is dynamic depending on the availability and level of different types of antioxidants. Different elicitors depending on the concentration and day of treatment exerted different effects on cell growth and AQ production. Fifty µM JA treated on day 12 with cultures harvested on day 15 enhanced AQ content to 39.6 mg g-1 DW, which was 2.1-fold to control. JA elicitor treatment during early and exponential growth phase showed significant AQ induction than stationary growth phase. With JA treatment on day 12 and cultures harvested on day 17, nordamnacanthal, 1-hydroxy-2-methylanthraquinone and lucidin-ω-methyl ether were registered at 3.3, 3 and 1.8 mg g-1 DW, which were 2, 3 and 4-fold, respectively, to control. Early exponential growth phase elicitation can induce extracellular AQ such as 1 g l-1 Aspergillus flavus (AF) elicitation which significantly promoted extracellular AQ at 5 mg l-1. In the early stage of elicitation, the increase in antioxidant activities as compared to control show that antioxidative mechanisms were functioning to counter the increase in H2O2 and lipid peroxidation. JA elicitation could enhance total carotenoids and vitamin E to the level comparable to P medium strategy and the leaf. The reduction of both catalase (CAT) and ascorbate peroxidase (APO) activities in elicited cultures after 6 days could suggest a greater antioxidative role being played by AQ, tocopherols and carotenoids. On the other hand, the higher vitamin C in elicited culture than control corresponds well with low APO, but high glutathione reductase (GR) activities suggest the need to maintain glutathione (GSH) level. The induction or reduction of antioxidant activities provides evidence for occurrence of oxidative burst in elicited cell cultures and the versatility of plant secondary compounds. The better understanding of stress responses in plant cell culture could pave the way for rational design and distribution of plant cell-based products.

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