Plant Cell, Tissue and Organ Culture (2005) 80: 151155

Springer 2005

Rosmarinic acid production by Coleus forskohlii hairy root cultures

Wei Li1, Kazuo Koike1, Yoshihisa Asada2, Takafumi Yoshikawa2 & Tamotsu Nikaido1,*
Faculty of Pharmaceutical Sciences, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan; School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan (*requests for oprints: Fax: +81-47-472-1404; E-mail: nikaido@phar.toho-u.ac.jp)
2 1

Received 7 October 2003; accepted in revised form 4 May 2004

Key words: Coleus forskohlii, elicitor, hairy root, methyl jasmonate, rosmarinic acid, salicylic acid, yeast extract

Abstract Coleus forskohlii hairy root cultures were found to produce forskolin and rosmarinic acid (RA) as the main metabolites. The growth and RA production by C. forskohlii hairy root cultures in various liquid media were examined. The hairy root cultures showed good growth in hormone-free Murashige and Skoog medium containing 3% (w/v) sucrose (MS medium), and Gamborg B5 medium containing 2% (w/v) sucrose (B5 medium). RA yield reached 4.0 mg (MS medium) and 4.4 mg (B5 medium) after 5 weeks of culture in a 100 ml ask containing 20 ml of each medium. Hairy root growth and RA were also investigated after treatment with various concentrations of yeast extract (YE), salicylic acid (SA) and methyl jasmonic acid (MJA). RA production in a 100 ml ask containing 20 ml B5 medium reached 5.4 mg (1.9 times more than control) with treatment of 0.01 or 1% (w/v) YE, 5.5 mg (2.0 times more than control) with treatment of 0.1 mM SA, and the maximum RA content with 9.5 mg per ask (3.4 times more than control) was obtained in the hairy roots treated with 0.1 mM MJA. These results suggest that MJA is an eective elicitor for production of RA in C. forskohlii hairy root cultures. Abbreviations: B5 medium Gamborg B5 medium containing 2% (w/v) sucrose; MJA methyl jasmonate; MS medium Murashige and Skoog medium containing 3% (w/v) sucrose; NN medium Nitsch and Nitschs medium containing 2% (w/v) sucrose; RA rosmarinic acid; SA salicylic acid; WP medium Woody plant medium containing 2% (w/v) sucrose; YE yeast extract

Introduction Rosmarinic acid (RA), an ester of caeic acid and 3,4-dihydroxyphenyllactic acid, is a natural compound most commonly occurring in species of the Lamiaceae and Boraginaceae. RA has been reported to have a multitude of biological activities, including antibacterial, antioxidative, antiviral and antiinammatory eects, which makes it a valuable product for the pharmaceutical and cosmetic industries (Kim et al., 2001). The rst plant cell cultures accumulating RA were derived from

Coleus blumei (Razzaque and Ellis, 1977; Zenk et al., 1977). The production and biosynthetic regulation of RA have been studied in suspension cells of Lithospermum erythrorhizon (Mizukami et al., 1993) and root cultures of Salvia ocinalis (Hippolyte et al., 1992) and Ocimum basilicum (Tada et al., 1996) and Heliotropium peruvianum (Motoyama et al., 1996). Coleus forskohlii hariy root cultures induced by Agrobacterium rhizogenes MAFF 03-01724, were reported to produce forskolin, a diterpene which shows positive inotropic, positive chronotropic

152 and hypotensive activity, inhibits thrombocyte aggregation, and decreases intraocular pressure (Sasaki et al., 1998). In a further phytochemical investigation of the hairy root cultures, we found that the hairy root cultures also produce RA as a main metabolite. The present report describes the optimal culture conditions for RA production and the enhancement of RA production by treatment with yeast extract (YE), salicylic acid (SA) and methyl jasmonate (MJA). HPLC analysis of RA Hairy roots cultured in a 100 ml ask containing 20 ml each medium were harvested using a lter paper to remove the medium. The hairy roots were then extracted with 20 ml methanol at 25 C for 1 h under ultrasonic treatment. A part of the methanolic solution (1 ml) was ltered through a 0.45 lm membrane lter. The ltrate (10 ll) was subjected to HPLC using Senshu Pak pegasil ODS (4.6 mm internal diameter 150 mm) at 40 C and eluted with 35% acetonitrile containing 0.01% phosphate at a ow rate of 1.0 ml min)1. The column euent was monitored at 280 nm with SPD-M10A diode array detector (Shimazu). Rt, 15.0 min. Time course of growth of C. forskohlii hairy root cultures in B5, WP, MS and NN media Hairy roots (ca. 0.06 g) were inoculated into hormone-free B5, WP (Lloyd and McCown, 1980), MS (Murashige and Skoog, 1962) and NN (Nitsch and Nitsch, 1969) liquid media (20 ml per 100 ml ask) and cultured in the dark at 25 C (50 rpm). The hairy roots were harvested periodically (once a week, 15 weeks) and the growth (fresh weight) and RA contents were determined. Five asks were used for each experiment. Eect of YA, SA and MJA on RA production Concentrated solutions of YE, SA or MJA in hormone-free B5 medium were autoclaved, then appropriate volumes were added to 3 week-old hairy roots cultured in hormone-free B5 medium to give nal concentrations of 0.01, 0.1, 1% (w/v) for YE, 0.01, 0.1, 1 mM for SA, and 0.1, 0.5, 1 mM for MJA. The hairy roots were harvested at day 1 and 7, respectively. The growth (fresh weight) and RA contents were determined. Controls were hairy root cultures to which the same volume of medium without YE, SA or MJA had been added. All experimental results were the mean of six replicates.

Material and methods Isolation and structure determination of RA C. forskohlii hairy roots were induced by infection of Agrobacterium rhizogenes MAFF 03-01724 (Sasaki et al., 1998). Mass-culture was carried out in B5 medium (Gamborg et al., 1968) at 25 C in the dark, at 50 rpm on a rotary shaker, using 500 ml Erlenmeyer ask containing 200 ml medium. After 4 weeks of culture, the hairy roots were harvested. The freeze-dried hairy roots (52.2 g) were extracted three times with methanol (each 1000 ml). Evaporation of methanol under reduced pressure from the combined extract gave the methanol extract (21.41 g). The extract was then partitioned between ethyl acetate and water. Removal of the solvent from the ethyl acetate phase under reduced pressure below 40 C yielded the ethyl acetate extract (2.60 g). The ethyl acetate extract was subjected to silica gel chromatography and eluted with chloroform/methanol (1:1) to give crude RA (200 mg). Further purication of crude RA (20 mg) by RPHPLC using Senshu Pak pegasil ODS-II (20 mm internal diameter 150 mm) with 27% acetonitrile containing 1% formic acid at a ow rate of 3.0 ml min)1 (Rt: 28 min), gave puried RA (18 mg) used for MS and NMR determination. RA: White amorphous powder. a23 D + 73.9 (c 1.15, methanol). Positive FAB-MS: 383 [M + Na]+, 361 [M + H]+. 1H-NMR (C5D5N) d: 3.50 (IH, dd, J 14.5, 8.5 Hz, 70 -Ha), 3.59 (IH, dd, J 14.5, 4.5 Hz, 70 -Hb), 5.94 (IH, dd, J 8.5, 4.5 Hz, 80 -H), 6.69 (IH, d, J 16.0 Hz, 8-H), 7.06 (IH, dd, J 8.0, 2.0 Hz, 6-H), 7.08 (IH, dd, J 8.0, 2.0 Hz, 60 -H), 7.18 (IH, d, J 8.0 Hz, 5H), 7.23 (IH, d, J 8.0 Hz, 5-H), 7.51 (IH, d, J 2.0 Hz, 2-H), 7.53 (IH, d, J 2.0 Hz, 20 -H), 8.04 (IH, d, J 16.0 Hz, 7-H).

Results and discussion Phytochemical isolation of the methanol extract of C. forskohlii hairy root cultures gave a main product which was determined to be RA (Figure l)

153 It has been reported that RA biosynthesis was aected by various elicitors in a number of plant species. Namely, RA production was induced by MJA in Coleus blumei (Szabo et al., 1999) and Lithospermum erythrorhizon (Mizukami et al., 1993) cell suspension cultures, by acetylated SA in Origanum vulgare tissue cultures (Andarwulan and Shetty, 1999), by YE in Agastache rugosa cell suspension cultures (Kim et al., 2001), Salvia miltiorrhiza hairy root cultures (Chen et al., 2001), Lithospermum erythrorhizon (Mizukami et al., 1992) and Orthosiphon aristatus (Sumaryono et al., 1991) cell suspension cultures, but was reduced by YE in Salvia miltiorrhiza cell suspension cultures (Chen and Chen, 2000a, b). The growth of hairy roots and RA contents were determined at 1 and 7 days after addition of various elicitors, respectively. The growth of C. forskohlii hairy roots and RA accumulation were not increased in the cultures treated with various elicitors, in comparison to those in the control culture 1 day (24 h) after elicitation (data not shown). A similar observation was also made from experiments on treatment in suspension cultures of C. blumei (Szabo et al., 1999) and L. erythrorhizon (Mizukami et al., 1993) with YE and MJA. Seven days after addition of elicitors, RA accumulation was higher than in the control culture in almost all of the treatments, except for SA at 1 mM (Figure 4). The highest RA contents (9.49.5 mg per ask) were approximately 3.4 times greater than the control level, and were obtained in the hairy roots treated with 0.1

Figure 1. Structure of rosmarinic acid (RA).

by its positive FAB-MS (m/z 383 [M + Na]+) and 1 H-NMR data. To select an optimum medium, the growth and RA production in hormone-free B5, WP, MS and NN media was determined periodically (Figures 2, and 3). Hairy roots showed the largest growth (4.5 g per 100 ml ask) in the MS medium (Figure 2), and yielded highest amount of RA in the B5 medium (4.4 mg per 100 ml ask) at week 5 (Figure 3). The hairy roots gradually turned brown after 5 weeks in MS and B5 media. The rapid growth of the hairy roots in the MS medium continued at week 4. Hairy roots in the B5 medium showed almost the same growth rate with those in the MS medium until week 3, and the content of RA increased as the hairy roots grew and reached the maximum level during the stationary phase at week 5. Rapid RA production was observed after 4 weeks in the MS medium, and reached to 4.0 mg per 100 ml ask at week 5, which is almost the same amount as that in the B5 medium.

Figure 2. Time course of growth of C. forskohlii hairy roots cultured in hormone-free Gamborg B5 medium containing 2% (w/v) sucrose (B5), Murashige and Skoog medium containing 3% (w/v) sucrose (MS), Nitsch and Nitschs medium containing 2% (w/v) sucrose (NN) and Woody Plant medium containing 2% (w/v) sucrose (WP) for 5 weeks in the dark at 25 C. Data are mean SD of ve replicates.

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Figure 3. Time course of rosmarinic acid (RA) production in C. forskohlii hairy roots cultured in hormone-free Gamborg B5 medium containing 2% (w/v) sucrose (B5), Murashige and Skoog medium containing 3% (w/v) sucrose (MS), Nitsch and Nitschs medium containing 2% (w/v) sucrose (NN) and Woody Plant medium containing 2% (w/v) sucrose (WP) for 5 weeks in the dark at 25 C. Data are mean SD of ve replicates.

Figure 4. Growth and rosmarinic acid (RA) production of non-elicited (control) and elicitor-treated C. forskohlii hairy roots cultured 1 week after induction of various concentrations of yeast extract (YE), salicylic acid (SA) and methyl jasmonic acid (MJA) to 3-weekold cultures in hormone-free Gamborg B5 medium containing 2% (w/v) sucrose. Data are mean SD of six replicates.

and 0.5 mM MJA. However, additions of YE, SA or a higher concentration of MJA did not achieve a similar increase. Treatments with 0.01, 1% (w/v) YE or 0.1 mM SA only aorded RA levels with about 5.4 mg per ask, 2.0 times more than the control level. Furthermore, weaker elicitation effects were observed from the cultures treated with 0.1% (w/v) YE, 0.01 mM SA and 1.0 mM MJA. These results suggested that MJA was an eective elicitor with the concentration of MJA at 0.1 0.5 mM for the production of RA in C. forskohlii hairy roots.

Acknowledgements The authors wish to thank Dr Koichiro Shimomura (Tsukuba Medicinal Plant Research Station, National Institute of Health Sciences) for providing C. forskohlii hairy root culture.

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