1 Indian Journal of Science and Technology http://www.indjst.org Vol.1 No 7 (Dec. 2008) Improved production of the indole alkaloid canthin-6-one from cell suspension culture of Brucea javanica (L.) Merr. ME.Wagih1, G.Alam2, S.Wiryowidagdo2 and K.Attia3 1Department of Biology, Faculty of Science, University of New Brunswick, Saint John, NB E2L 4L5, Canada. 2Department of Pharmacy, Faculty of Natural Sciences, Hasanuddin University, Indonesia. 3Institute of Genetic Engineering, Fudan University, Shanghai, 200433, China. [email protected] Abstract: In an attempt to increase the productivity homeostatic effects. In China, the fruit is known as of the pharmaceutical compound canthin-6-one “Ya-Tan-Tze” and are used to treat malaria and from cell suspension culture of Brucea javanica, a amoebic dysentery and as an insecticide (Liu et al., high cell density culture and improved culture 1990) and anti-cancer (Anderson et al., 1983). conditions have been investigated with focus on The isolation of the indole alkaloid canthin-6- culture conditions and stages of growth. The one from Brucea sp. has been reported by some highest yield of canthin-6-one, 26.72 mg g-l dry cell, workers (Harris et al., 1985; Liu et al., 1990). The was produced intracellular in 50 g L-1 cell mass. compound and its hydroxylated and methoxylated The established cell suspensions were harvested derivatives have demonstrated cytotoxic effect and at the age of 40 days and placed into MS medium marked activities against malaria, leukaemia, containing 1.0 mg L-1 2,4-D, 1.0 mg L-1 NAA, 0.1 carcinoma, keratinocytes of guinea-pig ear, and mg L-1 Kinetin and 10 mg L-1 tryptophan for 33-40 bacteria (Anderson et al., 1983). days, approximately when the cultures were at Conventionally, indole alkaloid canthin-6-one is their late stationary phase of growth. Addition of 20 obtained from field grown intact plants. However, mg L-1 tryptophan did not show a significant plants are seasonal and the quality of a metabolite difference in canthin-6-one production (p>0.05). may vary due to uncontrolled fluctuations in Higher than 10 mg L-1 of tryptophan showed growing conditions and geographical variations. adverse effect on the concentration of canthin-6- Growing intact plants is also confined to certain one alkaloid (p=0.01). Qualitative analysis of climate. In addition, because Brucea spp. are slow chloroform-extracts of dried cells from suspension growing trees and the fruits are not readily cultures revealed spots on TLC; and the identity of available, cell culture offers an alternative method canthin-6-one was established by comparison of its for the production of alkaloids. The expression of Rf value (Rf=0.37), its colour reaction with alkaloids in cell suspension is an interesting Dragendorff reagent and ammonium sulphate and phenomenon as the alkaloids are not detectable in its behaviour in UV with an authentic sample. vegetative plant parts (Cordell, 1981). Quantification of canthin-6-one alkaloid using TLC- The pattern of secondary metabolism in cell densitometry scanner in response to different cultures is a combination of many interacting concentration of tryptophan as a precursor showed factors, including the physiological status of the a concentration-dependent response. The culture, its origin (tissue type), age, and degree of production of canthin-6-one at 26.72 mg g-1 dried development. Manipulation of the medium cell weight in this study, establishes a methodology constituents and the physicals environments in of an improved procedure compared to those which cell cultures are grown should be useful in previously reported and offers an opportunity for improving the production of the characteristics production of canthin-6-one alkaloid at an industrial secondary metabolites. Therefore, cell suspension level. cultures have several advantages; rapid growth Keywords: Canthin-6-One, cell suspension, Brucea rate, easy to manipulation, uniform, natural, offers javanica, secondary metabolites. the possibility of quality control and availability Introduction independent of climatic environment, and have The tree plant Brucea javanica (L.) Merr is a limited number of cell types. member of the family Simaroubaceae, which is Canthin-6-one alkaloid has been expressed known for its medicinal value to some Asian and successfully produced in cell suspension countries. The fruits of this plant have been used in cultures of Brucea spp. The yield of this alkaloid the traditional medicine of Indonesia and China was found to be two fold greater in the cells (Hasbi, 1979; Alam et al. 1995). In Indonesia the compared to that in the medium (Liu et al., 1990). fruit is known as “Buah Makassar” and have While this suggests active excretion of alkaloids demonstrated anti-malaria, anti-pyretic, and into the medium, the cells remain the target source iSee category: Research article “Canthin-6-One from cell culture” by Wagih et al. Indian Society for Education and Environment Indian J.Sci.Technol. 2 Indian Journal of Science and Technology http://www.indjst.org Vol.1 No 7 (Dec. 2008) of alkaloid production. So far the yield of alkaloids darkness at 25 ± 2 C° for one week prior to use as from the cell suspension culture is considered low inocula in various treatments. and, therefore, there is a need to further improve Five replicates of 1.0 g of cells inoculated into the procedure. Liu et al. (1990), have been 50ml of medium, supplemented with various successful in yielding 2 mg total alkaloids per 1 concentrations of L-tryptophan, were incubated on gram of cell dry weight. Nevertheless, they an orbital shaker at 120 rpm in darkness at 25 ± 2 suggested further investigation of parameters C° for a maximum of 40 days, and the cultures necessary for maximizing yields of these were assessed in eight time points; 0, 4, 12, 16, compounds. 21, 28, 33 and 40 day for the following During the course of study an attempt has characteristics (Arias-Castro et al., 1993; Torres, been made to maximise the yield of indole canthin- 1989): 6-one using cell suspension culture and tryptophan Height of cell sediments (hcs) or settled cell precursor. So far, the use of tryptophan in volume was monitored during the incubation period stimulating alkaloid production from cell using modified Erlenmeyer flasks with a branched suspension culture of B. javanica has not been measuring cylinder. Packed cell weight (pcw) was reported. However, Dicosmo and Towers (1984) determined by centrifuging 25 ml of cell have successfully used tryptophan to enhance suspension cultures in a pre-weighed indole alkaloid production in cell cultures of centrifugation tubes for one min at 1000 rpm, Catharanius roseus. removing supernatant and weighing the packed Materials and methods cells. PCW is expressed as g L-1. The height of cell Plant Material and Callus Culture sedimentation was monitored during the incubation Fruits of Brucea javanica from Muntilan, Centre period. Dry cell weight (dcw) was obtained by of Java province, were washed thoroughly and rinsing the pellets in 25 ml of distilled water and surface sterilised in 30 % BayclinTM (containing filtering the suspension on pre-weighed dry 5.5% sodium hypochlorite) for 15 minutes and then Whatman No.1 filter paper (Whatman Paper Ltd.) 0 washed three times in distilled sterile water. Fruits with suction, and drying cells at 60 C for 48 h were sliced and placed onto Murashige and Skoog followed by weighing and expressed as mg ml-1. (MS) medium (Murashige & Skoog, 1962) Analyses of cell culture with TLC for canthine-6- containing 3% sucrose and 1.0 mg L-1 2,4-D alone one alkaloids identification or with 1.0 mg L-1 NAA and 0.1 mg L-1 Kinetin at Aliquots of 500 mg dried cell pellets of pH 5.8 for callus initiation and growth. Calli were centrifuged (1000 rpm for 10 min) cell suspension routinely subcultured every four weeks. The latest cultures of Brucea javanica were ground in 25 ml of passage of friable granulated and globular calli methanol and the extract was filtered and was used to initiate cell suspension culture for evaporated to dryness. The residue was extracted alkaloid production. with 25 ml chloroform: water (1:1) mixture. The Establishment of Cell Suspension Culture chloroform layer was separated and analyzed for Cell suspension cultures were initiated by canthin-6-one using TLC on silica gel (GF-254) placing 5.0 g callus into 50 ml of liquid MS medium eluted with toluene: acetyl acetate (6:4). containing 1.0 mg L-1 2,4-D, 1.0 mg L-1 NAA and The aqueous extracts contained water soluble 0.1 mg L-1 Kinetin at pH 5.8 in 300 ml Erlenmeyer materials from samples of dried cells and authentic flask. The cultures were maintained at 25±2ºC samples of canthine-6-one alkaloid, as a standard under illumination (photon flux density of (generously donated by Professor Masayoshi approximately 150 µmol m-2 s-1) on an orbital Okano, University of Hiroshima, Japan), was shaker with 2 cm stroke and subcultured every 10 brought to 85.5% with absolute ethanol. The days for four times. Treatments were applied by suspension was vortexed for about 5 min and transferring 25 ml of cell biomass into liquid MS centrifuged at 3000g for 10 min. The supernatant medium containing 1 mg L-1 NAA and 0.1 mg L-1 fraction containing ethanol extractable materials Kinetin and tryptophan precursor at 0, 10, 20, 40, samples along with the authentic samples of and 80 mg L-1. canthine-6-one alkaloid were directly used for TLC. Growth parameters of cell suspension When the solvent has reached the top of the Cell suspensions used in the growth study were plate, the plate were removed from the solvent preconditioned, by inoculating 1:10 (w/v, 1.0 g cell tray, air-dried, and the separated components of pellet from the inoculum stock in 50 ml of liquid MS the mixture were visualized under UV lamp, medium containing 1.0 mg L-1 2,4-D, 1.0 mg L-1 fluoresced everywhere except where the organic NAA and 0.1 mg L-1 Kinetin.