Biotransformation of Cinnamic Acid, p-coumaric Acid, Caffeic Acid, and Ferulic Acid by Plant Cell Cultures of Eucalyptus perriniana

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1 Biosci. Biotechnol. Biochem., 74 (9), , 2010 Biotransformation of Cinnamic Acid, p-coumaric Acid, Caffeic Acid, and Ferulic Acid by Plant Cell Cultures of Eucalyptus perriniana Hisashi KATSURAGI, 1 Kei SHIMDA, 2 Naoji KUBTA, 2 Nobuyoshi NAKAJIMA, 3;y Hatsuyuki HAMADA, 4 and Hiroki HAMADA 5;y 1 Sunny Health Co., Ltd., Nakajima Bilg., 8-8 Kabuto-cho, Nihonbashi, Chuo-ku, Tokyo , Japan 2 Department of Chemistry, Faculty of Medicine, ita University, 1-1 Hasama-machi, ita , Japan 3 Industry, Government, and Academic Promotional Center, Regional Cooperative Research rganization, kayama Prefectural University, Soja, kayama , Japan 4 National Institute of Fitness and Sports in Kanoya, 1 Shiromizu-cho, Kagoshima , Japan 5 Department of Life Science, Faculty of Science, kayama University of Science, 1-1 Ridai-cho, Kita-ku, kayama , Japan Received April 30, 2010; Accepted May 14, 2010; nline Publication, September 7, 2010 [doi: /bbb.335] Biotransformations of phenylpropanoids such as cinnamic acid, p-coumaric acid, caffeic acid, and ferulic acid were investigated with plant-cultured cells of Eucalyptus perriniana. The plant-cultured cells of E. perriniana converted cinnamic acid into cinnamic acid -D-glucopyranosyl ester, p-coumaric acid, and 4---D-glucopyranosylcoumaric acid. p-coumaric acid was converted into 4---D-glucopyranosylcoumaric acid, p-coumaric acid -D-glucopyranosyl ester, 4--- D-glucopyranosylcoumaric acid -D-glucopyranosyl ester, a new compound, caffeic acid, and 3---Dglucopyranosylcaffeic acid. n the other hand, incubation of caffeic acid with cultured E. perriniana cells gave 3---D-glucopyranosylcaffeic acid, 3--(6---Dglucopyranosyl)--D-glucopyranosylcaffeic acid, a new compound, 3---D-glucopyranosylcaffeic acid -D-glucopyranosyl ester, 4---D-glucopyranosylcaffeic acid, 4---D-glucopyranosylcaffeic acid -D-glucopyranosyl ester, ferulic acid, and 4---D-glucopyranosylferulic acid. 4---D-Glucopyranosylferulic acid, ferulic acid -D-glucopyranosyl ester, and 4---D-glucopyranosylferulic acid -D-glucopyranosyl ester were isolated from E. perriniana cells treated with ferulic acid. Key words: biotransformation; glycosylation; phenylpropanoid; plant-cultured cells; Eucalyptus perriniana Phenylpropanoids, such as cinnamic acid, p-coumaric acid, caffeic acid, and ferulic acid, are naturally occurring anti-oxidants that act as effective scavengers of free radicals. 1 3) It is well known that shikimic acid is metabolized in plant cells to cinnamic acid and p- coumaric acid, which are further converted into caffeic acid and ferulic acid. n the other hand, plant-cultured cells are ideal systems for propagating rare plants and for studying the biosynthesis of secondary metabolites. Furthermore, plant-cultured cells are considered to be useful agents for biotransformation reactions due to their biochemical potential to produce specific secondary metabolites. 4) The reactions involved in the biotransformation of organic compounds by plant-cultured cells include oxidation, reduction, hydroxylation, esterification, methylation, isomerization, hydrolysis, and glycosylation. Hydroxylation and glycosylation are characteristic biotransformation reactions in such cells because hydroxylases and glycosyltransferases are widespread in plants. 5 9) Several studies of the extraction and purification of phenylpropanoid glycosides from plants have been reported ) Recently, it was reported that Haematococcus pluvialis biotransformed phenylpropanoids, viz., ferulic acid and p-coumaric acid, into vanillin, vanillic acid, vanillyl alcohol, and protocatechuic acid, 13) but little attention has been paid to the biotransformation, such as hydroxylation and glycosylation, and metabolic pathway of phenylpropanoids in plant-cultured cells. Here we report the biotransformation of cinnamic acid, p-coumaric acid, caffeic acid, and ferulic acid by plantcultured cells of Eucalyptus perriniana. Materials and Methods Substrates. Cinnamic acid, p-coumaric acid, caffeic acid, and ferulic acid, which were used as substrates, were purchased from Aldrich Chemical (St. Louis, M). Cell line and culture conditions. Cultured E. perriniana cells were subcultured at 4-week intervals on solid Murashige and Skoog (MS) medium ( ml in a 300-ml conical flask) containing 3% sucrose, 10 mmol/l 2,4-dichlorophenoxyacetic acid, and 1% agar (adjusted to ph 5.7) at 25 C in the dark. A suspension culture was started by transferring the cultured cells to ml of liquid medium in a 300-ml conical flask, and this was incubated on a rotary shaker (120 rpm) at 25 C in the dark. Prior to use in this study, part of the callus tissues (fr. wt, 40 g) was transplanted to freshly prepared MS medium ( ml in a 300-ml conical flask) and grown with continuous shaking for 2 weeks on a rotary shaker (120 rpm). Biotransformation and purification of products. To a 0-ml flask containing 200 ml of MS medium and suspension-cultured cells ( g) y To whom correspondence should be addressed. Nobuyoshi NAKAJIMA, Tel/Fax: ; nkmt-nakajima@fhw.oka-pu.ac.jp; Hiroki HAMADA, Tel: ; Fax: ; hamada@dls.ous.ac.jp Abbreviations: CSY, correlation spectroscopy; HMBC, heteronuclear multiple-bond correlation; HPLC, high performance liquid chromatography; HRFABMS, high resolution fast atom bombardment mass spectrometry; NMR, nuclear magnetic resonance; TMS, tetramethylsilane

2 of E. perriniana was added 15 mg of substrate. The cultures were incubated at 25 C for 96 h on a rotary shaker (120 rpm) in the dark. After the incubation period, the cells and medium were separated by filtration with suction. The extraction and purification procedures for the biotransformation products were performed according to previously reported methods. 14,15) The yield of products was determined on the basis of the peak area from HPLC, and was expressed as a percentage relative to the total amount of whole reaction products extracted. Biotransformation of Phenylpropanoids 1921 Analysis of the products. 1 H and 13 C NMR, H H CSY, C H CSY, and HMBC spectra were recorded using a Varian XL-400 spectrometer in pyridine-d 5 solution, and the chemical shifts were expressed in (ppm), referring to TMS. The HRFABMS spectra were measured using a JEL MStation JMS-700 spectrometer (JEL, Tokyo). The structures of the products were determined on the basis of analysis of their HRFABMS, 1 H and 13 C NMR, H-H CSY, C-H CSY, and HMBC spectra. The spectral data of new compounds were as follows: 4---D-Glucopyranosylcoumaric acid -D-glucopyranosyl ester (6). HRFABMS m=z ðm þ NaÞ þ : Calcd. for C 21 H Na: , Found: ; 1 H NMR (400 MHz, pyridine-d 5 ): H (12H, m, H-2 0,2 00,3 0,3 00,4 0,4 00,5 0,5 00,6 0,6 00 ), 5.25 (1H, d, J ¼ 7:2 Hz, H-1 0 ), 6.11 (1H, d, J ¼ 7:6 Hz, H-1 00 ), 6.55 (1H, d, J ¼ 16:0 Hz, H-8), 7.24 (2H, d, J ¼ 8:0 Hz, H-3, 5), 7.51 (1H, d, J ¼ 16:0 Hz, H-7), 7.60 (2H, d, J ¼ 8:0 Hz, H-2, 6); 13 C NMR ( MHz, pyridine-d 5 ): C 62.7 (C-6 0 ), 63.0 (C-6 00 ), 70.7 (C-4 0 ), 70.9 (C-4 00 ), 74.0 (C-2 00 ), 74.1 (C-2 0 ), 77.6 (C-3 0 ), 77.9 (C-3 00 ), 78.0 (C-5 0 ), 78.2 (C-5 00 ), 99.8 (C-1 00 ), (C-1 0 ), (C-3, C-5), (C-8), (C-2, C-6), (C-1), (C-7), (C-4), (C-9). 3--(6---D-Glucopyranosyl)--D-glucopyranosylcaffeic acid (9). HRFABMS: m=z ðm þ NaÞ þ : Calcd. for C 21 H Na: , Found: ½M þ NaŠ þ ; 1 H NMR (400 MHz, pyridine-d 5 ): H (12H, m, H-2 0,2 00,3 0,3 00,4 0,4 00,5 0,5 00,6 0,6 00 ), 4.99 (1H, d, J ¼ 7:2 Hz, H-1 00 ), 5.10 (1H, d, J ¼ 7:6 Hz, H-1 0 ), 6.55 (1H, d, J ¼ 15:6 Hz, H-8), 7.01 (1H, dd, J ¼ 8:0, 1.9 Hz, H-6), 7.08 (1H, d, J ¼ 8:0 Hz, H-5), 7.55 (1H, d, J ¼ 2:0, H-2), 7.70 (1H, d, J ¼ 15:6 Hz, H-7); 13 C NMR ( MHz, pyridine-d 5 ): C 63.0 (C-6 00 ), 69.3 (C-6 0 ), 70.9 (C-4 0, C-4 00 ), 74.2 (C-2 00 ), 74.3 (C-2 0 ), 77.5, 77.7 (C-3 0, C-3 00 ), 78.1 (C-5 0, C-5 00 ), 99.5 (C-1 0 ),.0 (C-1 00 ), (C-8), (C-2), (C-5), (C-6), (C-1), (C-7), (C-3), (C-4), (C-9). Time-course experiments. Suspension cells ( g) of E. perriniana were partitioned to eight flasks containing 200 ml of MS medium. Substrate (15 mg) was administered to each of the flasks, and the mixtures were incubated on a rotary shaker at 25 C. At 12-h intervals, one of the flasks was taken out from the rotary shaker, and the cells and medium were separated by filtration. The extraction and analysis procedures were as described above. Results and Discussion After 96 h, incubation, the biotransformation products were isolated from the cultured suspension cells of E. perriniana, which had been treated with cinnamic acid (1). Three compounds 2 4 were obtained, and no additional conversion products were observed under careful HPLC analysis. The structures of the products were identified on the basis of their HRFABMS, 1 H and 13 C NMR, H H CSY, C H CSY, and HMBC spectra as cinnamic acid -D-glucopyranosyl ester (2, 9%), 16) p-coumaric acid (3, 5%), and 4---D-glucopyranosylcoumaric acid (4, 2%). 17) Hydroxylation regioselectively occurred at the 4-position of cinnamic acid (1) to give p-coumaric acid (3), followed by glucosylation of 3 to 4. In order to determine the ability of cultured E. perriniana cells to biotransform cinnamic acid (1), the Fig. 1. Time-Course of the Biotransformation of Cinnamic Acid (1) by The substrate, cinnamic acid (1, 15 mg), was incubated with g of E. perriniana suspension cell cultures at 25 C on a rotary shaker (120 rpm) in the dark. Yields of 1 ( ), 2 ( ), 3 ( ), and 4 ( ) are plotted. H 1 3 (5%) H H Glc 2 (9%) Glc time course of the conversion of 1 was followed. As Fig. 1 indicates, cinnamic acid (1) was converted into 2 at an early stage of incubation, and the products 3 and 4 were slightly produced. This indicates that glucosylation at the carboxyl group of cinnamic acid occurred predominantly, rather than hydroxylation at the 4-position of cinnamic acid to p-coumaric acid. The biotransformation pathway of cinnamic acid is shown in Fig. 2. Five compounds 4 8 were produced by incubation of cultured E. periniana cells with p-coumaric acid (3). The structures of these products were identified as 4-- -D-glucopyranosylcoumaric acid (4, 24%), p-coumaric acid -D-glucopyranosyl ester (5, 10%), 17) 4---Dglucopyranosylcoumaric acid -D-glucopyranosyl ester (6, 15%), caffeic acid (7, 7%), and 3---D-glucopyranosylcaffeic acid (8, 3%). 17) The HRFABMS spectrum of product 6 showed a pseudomolecular ion ½M þ NaŠ þ peak at m=z , consistently with a molecular formula of C 21 H (calcd for C 21 H Na), suggesting that two new hexoses were introduced to the substrate. The 1 H NMR spectrum of 6 displayed two anomeric proton signals, at 5.25 (1H, d, J ¼ 7:2 Hz) and 6.11 (1H, d, J ¼ 7:6 Hz). The sugar component in product 6 was determined to be -D-glucopyranose, according to the chemical shifts of the sugar carbon signals. The 4 (2%) Fig. 2. Biotransformation Pathway of Cinnamic Acid (1) by Plant- H

3 1922 H. KATSURAGI et al. 13 C NMR spectrum of 6 showed two anomeric carbon resonances, at 99.8 and Thus the structure of compound 6 was determined to be 4---D-glucopyranosylcoumaric acid -D-glucopyranosyl ester. The disaccharide product 6 had not previously been identified. These results demonstrated that E. perriniana cells regioselectively hydroxylated at the 3-position of p- coumaric acid (3) to produce caffeic acid (7). The time course in the conversion of 3 indicates that glucosylation at the phenolic hydroxyl group and the carboxyl group of p-coumaric acid occurred first, and that disaccharide product 6 was formed subsequently (Fig. 3). The biotransformation pathway of p-coumaric acid is shown in Fig. 4. Next, the conversion of caffeic acid (7) by cultured E. perriniana cells was investigated. Seven products were isolated, and were identified as 3---D-glucopyranosylcaffeic acid (8, 25%), 3--(6---D-glucopyranosyl)--D-glucopyranosylcaffeic acid (9, 6%), 3- --D-glucopyranosylcaffeic acid -D-glucopyranosyl ester (10, 9%), 18) 4---D-glucopyranosylcaffeic acid (11, 7%), 17) 4---D-glucopyranosylcaffeic acid -Dglucopyranosyl ester (12, 3%), 19) ferulic acid (13, 5%), Fig. 3. Time-Course of the Biotransformation of p-coumaric Acid (3) by The substrate, p-coumaric acid (3, 15 mg), was incubated with g of E. perriniana suspension cell cultures at 25 C on a rotary shaker (120 rpm) in the dark. Yields of 3 ( ), 4 ( ), 5 ( ), 6 ( ), 7 ( ), and 8 () are plotted. and 4---D-glucopyranosylferulic acid (14, 1%). 17) Cultured E. perriniana cells catalyzed the regioselective methylation of caffeic acid (7) to ferulic acid (13). No formation of caffeic acid -D-glucopyranosyl ester was found. The HRFABMS spectrum of 9 included a pseudomolecular ion ½M þ NaŠ þ peak at m=z , which is consistent with a molecular formula of C 21 H (calcd for C 21 H Na). The 1 HNMR spectrum of 9 included proton signals at 4.99 (1H, d, J ¼ 7:2 Hz) and 5.10 (1H, d, J ¼ 7:6 Hz), indicating the presence of two -anomers in the sugar moiety. The 1 H and 13 C NMR spectra of 9 indicate that it was a - gentiobiosyl analog. 15) Furthermore, the HMBC spectrum included correlations between the anomeric proton signal at 5.10 (H-1 0 ) and the carbon signal at (C-3), and between the anomeric proton signal at 4.99 (H-1 00 ) and the carbon signal at 69.3 (C-6 0 ). These findings confirm that the inner -D-glucopyranosyl residue was attached to the phenolic hydroxyl group at the 3-position of caffeic acid (7), and that the pair of -D-glucopyranosyl residues were 1,6-linked. Thus product 9 was identified as 3--(6---D-glucopyranosyl)--D-glucopyranosylcaffeic acid. The gentiobioside product 9 was a new compound. The time-course experiment indicated that no glycosylation at the carboxyl group of 13 occurred, probably due to the low yield of 13 (Fig. 5). The biotransformation pathway of caffeic acid is shown in Fig. 6. n the other hand, cultured E. periniana cells glucosylated ferulic acid (13) to 4---D-glucopyranosylferulic acid (14, 22%), ferulic acid -D-glucopyranosyl ester (15, 5%), 17) and 4---D-glucopyranosylferulic acid -D-glucopyranosyl ester (16, 14%). 20) The timecourse of the conversion of 13 indicated that 16 predominantly accumulated in conformity with the formation of 14 (Fig. 7). The biotransformation pathway of ferulic acid is shown in Fig. 8. The results of this experiment indicate that the plantcultured cells of E. perriniana metabolized phenylpropanoids, including cinnamic acid, p-coumaric acid, caffeic acid, and ferulic acid, catalyzing the hydroxylation of cinnamic acid to p-coumaric acid, the hydroxylation of p-coumaric acid to caffeic acid, and the methylation of caffeic acid to ferulic acid. Additionally, both the phenolic hydroxyl group and the carboxyl H H Glc 4 (24%) Glc H 3 Glc Glc 6 (15%) H 5 (10%) H H Glc H H H 7 (7%) 8 (3%) Fig. 4. Biotransformation Pathway of p-coumaric Acid (3) by Plant-

4 Biotransformation of Phenylpropanoids 1923 Fig. 5. Time-Course of the Biotransformation of Caffeic Acid (7) by The substrate, caffeic acid (7, 15 mg), was incubated with g of E. perriniana suspension cell cultures at 25 C on a rotary shaker (120 rpm) in the dark. Yields of 7 ( ), 8 ( ), 9 ( ), 10 ( ), 11 ( ), 12 (), 13 ( ), and 14 ( ) are plotted. Fig. 7. Time-Course of the Biotransformation of Ferulic Acid (13)by The substrate, ferulic acid (13, 15 mg), was incubated with g of E. perriniana suspension cell cultures at 25 C on a rotary shaker (120 rpm) in the dark. Yields of 13 ( ), 14 ( ), 15 ( ), and 16 ( ) are plotted. Glc H GlcGlc H H 9 (6%) H H 7 H H H 8 (25%) H Glc H H Glc 10 (9%) Glc Glc 11 (7%) Glc 12 (3%) H 3 C H H 3 C H H 13 (5%) Glc 14 (1%) Fig. 6. Biotransformation Pathway of Caffeic Acid (7) by Plant- H 3 C H H 3 C H Glc 14 (22%) H Glc H 13 H 3 C Glc Glc 16 (14%) H 15 (5%) Fig. 8. Biotransformation Pathway of Ferulic Acid (13) by Plant- group of these compounds were glycosylated to give the corresponding mono- and disaccharides, including two new compounds, 4---D-glucopyranosylcoumaric acid -D-glucopyranosyl ester and 3--(6---D-glucopyranosyl)--D-glucopyranosylcaffeic acid. The time-course experiments on the biotransformation of p-coumaric acid, caffeic acid, and ferulic acid indicated that glycosylation at the phenolic hydroxyl group occurred preferentially to that at the carboxyl group. Recently, it was reported that Hematococcus pluvialis, a green unicellular alga, converted phenylpropanoids, ferulic acid and p-coumaric acid, into vanillin, vanillic acid, vanillyl alcohol, and protocatechuic acid. 13) No formation of phenylpropanoid glycosides in

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