Decursin Production from Hairy Root Culture of Angelica gigas

Abstract

Angelica gigas Nakai, commonly known as Korean Angelica, is a perennial herb belonging to the Umbelliferae family and one of the most important medicinal plants in Korea. For thousands of years, the roots of A. gigas has been used as traditional Oriental herbal medicine to treat anemia, abdominal pain, injuries, migraine, arthritis, and female afflictions, and the material has also been prescribed for health-promoting effects [Chi and Kim, 1970; Choi et al., 2003; Sarker and Nahar, 2004]. Decursin (Fig. 1) and decursinol angelate (a decursin isomer), the major secondary metabolites of A. gigas, have significant neuroprotective, anticancer, and anti-androgen-receptor signaling activities [Kang et al., 2005; Yim et al., 2005; Guo et al., 2007]. Various species of bacteria can transfer genes to higher plants, among which Agrobacterium rhizogenes, a Gramnegative soil bacterium, is one of the most widely studied. A. rhizogenes infects the plant cell and leads to the formation of hairy roots [Signs and Flores, 1990]. In many plants, hairy root cultures have proven to be an efficient production system for secondary metabolites. Such cultures have genetic and biochemical stabilities, rapid growth rate, and the ability to synthesize natural compounds at levels comparable to those of intact plants [Giri and Narasu, 2000; Guillon et al., 2006]. To the best of our knowledge, this is the first report on decursin production by hairy root culture of A. gigas. Here, we describe the production of decursin by hairy root cultures of A. gigas transformed with A. rhizogenes R1000. A. rhizogenes strain R1000 cultures were grown to the log phase at 28oC on a gyratory shaker (180 rev/min) in liquid Luria-Bertani medium. The bacterial cells were collected by centrifugation for 10 min at 2000 rpm, and resuspended at a cell density of A600 = 0.5 in MS [Murashige and Skoog, 1962] liquid inoculation medium. Young leaf and stem of A. gigas were taken from plants grown in vitro and were cut at the ends into sections of 0.7×0.7 cm2 and 0.7 cm. Excised leaves and stems were dipped into A. rhizogenes culture in the liquid inoculation medium for 10 min, blotted dry on a sterile filter paper, and incubated in the dark at 25oC on the agar-solidified MS medium. After 2 days of co-cultivation, the explant tissues were transferred to a hormone-free solid MS medium containing 200 mg/L Timentin. Within 4 weeks numerous hairy roots were observed emerging from the wound sites. The hairy roots were separated from the explant tissue and subcultured in the dark at 25oC on the agar-solidified MS medium. After repeated transfers to fresh media, rapidly growing hairy root cultures were obtained. Wild type root cultures were established by inoculating MS liquid medium with excised roots from A. gigas seedlings grown in vitro. Isolated wild type and hairy roots (200 mg) were transferred to 30 mL MS liquid medium, containing 30 g/ L sucrose, in 100 mL flasks. Wild type and hairy root cultures were maintained at 25oC on a gyratory shaker (100 rev/min) in a growth chamber. After 24 days of culture, hairy roots were harvested. Dry weight and decursin contents were determined. Three flasks were used for each culture condition, and the experiments were performed in duplicates. Hairy roots were dried at −80oC in brown paper bags for at least 48 h using a freeze-dryer. Dried samples were ground into a fine powder using a mortar and pestle. Samples (0.5 g) were extracted with 30 mL of 70:30 (% v/v) ethanol-water at 50oC in a water bath for 1 h. After *Corresponding author Phone: +82-42-821-5730; Fax: +82-42-822-2631 E-mail: supark@cnu.ac.kr