100335 (332)

Biosci. Biotechnol. Biochem., 74 (9), 100335-1–5, 2010

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

y Hisashi KATSURAGI,1 Kei SHIMODA,2 Naoji KUBOTA,2 Nobuyoshi NAKAJIMA,3; y Hatsuyuki HAMADA,4 and Hiroki HAMADA5;

1Sunny Health Co., Ltd., Nakajima Bilg., 8-8 Kabuto-cho, Nihonbashi, Chuo-ku, Tokyo 103-0026, Japan 2Department of Chemistry, Faculty of Medicine, Oita University, 1-1 Hasama-machi, Oita 879-5593, Japan 3Industry, Government, and Academic Promotional Center, Regional Cooperative Research Organization, Okayama Prefectural University, Soja, Okayama 719-1197, Japan 4National Institute of Fitness and Sports in Kanoya, 1 Shiromizu-cho, Kagoshima 891-2390, Japan 5Department of Life Science, Faculty of Science, Okayama University of Science, 1-1 Ridai-cho, Kita-ku, Okayama 700-0005, Japan

Received April 30, 2010; Accepted May 14, 2010; Online Publication, September 7, 2010 [doi:10.1271/bbb.100335]

Biotransformations of such as biochemical potential to produce specific secondary cinnamic acid, p-coumaric acid, caffeic acid, and ferulic metabolites.4) The reactions involved in the biotransfor- acid were investigated with plant-cultured cells of mation of organic compounds by plant-cultured cells EucalyptusAdvance perriniana. The plant-cultured View cells of include oxidation, reduction, hydroxylation, esterifica- E. perriniana converted cinnamic acid into cinnamic tion, methylation, isomerization, hydrolysis, and glyco- acid -D-glucopyranosyl ester, p-coumaric acid, and sylation. Hydroxylation and glycosylation are character- 4-O- -D-glucopyranosylcoumaric acid. p-Coumaric acid istic biotransformation reactions in such cells because was converted into 4-O- -D-glucopyranosylcoumaric hydroxylases and glycosyltransferases are widespread in acid, p-coumaric acid -D-glucopyranosyl ester, 4-O- - plants.5–9) Several studies of the extraction and purifica- D-glucopyranosylcoumaric acid -D-glucopyranosyl tion of glycosides from plants have ester, a new compound, caffeic acid, and 3-O- -D- been reported.10–12) Recently, it was reported that glucopyranosylcaffeic acid. On the other hand, incuba- Haematococcus pluvialis biotransformed phenylpropa- tion of caffeic acid with cultured E. perriniana cells noids, viz., ferulic acid and p-coumaric acid, into , gave 3-O- -D-glucopyranosylcaffeic acid, 3-O-(6-O- -D- vanillic acid, vanillylProofs alcohol, and protocatechuic acid,13) glucopyranosyl)- -D-glucopyranosylcaffeic acid, a new but little attention has been paid to the biotransformation, compound, 3-O- -D-glucopyranosylcaffeic acid -D-glu- such as hydroxylation and glycosylation, and metabolic copyranosyl ester, 4-O- -D-glucopyranosylcaffeic acid, pathway of phenylpropanoids in plant-cultured cells. 4-O- -D-glucopyranosylcaffeic acid -D-glucopyranosyl Here we report the biotransformation of cinnamic acid, ester, ferulic acid, and 4-O- -D-glucopyranosylferulic p-coumaric acid, caffeic acid, and ferulic acid by plant- acid. 4-O- -D-Glucopyranosylferulic acid, ferulic acid cultured cells of Eucalyptus perriniana. -D-glucopyranosyl ester, and 4-O- -D-glucopyranosyl- ferulic acid -D-glucopyranosyl ester were isolated from Materials and Methods E. perriniana cells treated with ferulic acid. Substrates. Cinnamic acid, p-coumaric acid, caffeic acid, and Key words: biotransformation; glycosylation; phenyl- ferulic acid, which were used as substrates, were purchased from propanoid; plant-cultured cells; Eucalyptus Aldrich Chemical (St. Louis, MO). perriniana Cell line and culture conditions. Cultured E. perriniana cells were subcultured at 4-week intervals on solid Murashige and Skoog (MS) Phenylpropanoids, such as cinnamic acid, p-coumaric medium (100 ml in a 300-ml conical flask) containing 3% sucrose, acid, caffeic acid, and ferulic acid, are naturally occur- 10 mmol/l 2,4-dichlorophenoxyacetic acid, and 1% agar (adjusted to ring anti-oxidants that act as effective scavengers of free pH 5.7) at 25 C in the dark. A suspension culture was started by radicals.1–3) It is well known that transferring the cultured cells to 100 ml of liquid medium in a 300-ml is metabolized in plant cells to cinnamic acid and p- 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 coumaric acid, which are further converted into caffeic (fr. wt, 40 g) was transplanted to freshly prepared MS medium (100 ml acid and ferulic acid. On the other hand, plant-cultured in a 300-ml conical flask) and grown with continuous shaking for 2 cells are ideal systems for propagating rare plants and for weeks on a rotary shaker (120 rpm). studying the biosynthesis of secondary metabolites. Furthermore, plant-cultured cells are considered to be Biotransformation and purification of products. To a 500-ml flask useful agents for biotransformation reactions due to their containing 200 ml of MS medium and suspension-cultured cells (100 g)

y To whom correspondence should be addressed. Nobuyoshi NAKAJIMA, Tel/Fax: +81-866-94-2157; E-mail: [email protected]; Hiroki HAMADA, Tel: +81-86-256-9473; Fax: +81-86-256-8468; E-mail: [email protected] Abbreviations: COSY, correlation spectroscopy; HMBC, heteronuclear multiple-bond correlation; HPLC, high performance liquid chromatog- raphy; HRFABMS, high resolution fast atom bombardment mass spectrometry; NMR, nuclear magnetic resonance; TMS, tetramethylsilane 100335-2 H. KATSURAGI et al. of E. perriniana was added 15 mg of substrate. The cultures were 100 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 previ- ously reported methods.14,15) The yield of products was determined on (%) Yield the basis of the peak area from HPLC, and was expressed as a percentage relative to the total amount of whole reaction products 50 extracted.

Analysis of the products. 1H and 13C NMR, H–H COSY, C–H COSY, and HMBC spectra were recorded using a Varian XL-400 spectrometer in pyridine-d5 solution, and the chemical shifts were expressed in (ppm), referring to TMS. The HRFABMS spectra were measured using a JEOL MStation JMS-700 spectrometer (JEOL, Tokyo). The structures of the products were determined on the basis of 48 96 analysis of their HRFABMS, 1H and 13C NMR, H-H COSY, C-H Time (h) COSY, and HMBC spectra. The spectral data of new compounds were as follows: Fig. 1. Time-Course of the Biotransformation of Cinnamic Acid (1) by Cultured Cells of E. perriniana. The substrate, cinnamic acid (1, 15 mg), was incubated with 100 g 4-O--D-Glucopyranosylcoumaric acid -D-glucopyranosyl ester of E. perriniana suspension cell cultures at 25 C on a rotary shaker (6). HRFABMS m=z ðM þ NaÞþ: Calcd. for C H O Na: 511.1377, 21 28 13 (120 rpm) in the dark. Yields of 1 ( ), 2 ( ), 3 ( ), and 4 ( ) are Found: 511.1382; 1H NMR (400 MHz, pyridine-d ): 4.05–4.67 5 H plotted. (12H, m, H-20,200,30,300,40,400,50,500,60,600), 5.25 (1H, d, J ¼ 7:2 Hz, H-10), 6.11 (1H, d, J ¼ 7:6 Hz, H-100), 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 13 O O (2H, d, J ¼ 8:0 Hz, H-2, 6); C NMR (100 MHz, pyridine-d5): C 62.7 (C-60), 63.0 (C-600), 70.7 (C-40), 70.9 (C-400), 74.0 (C-200), 74.1 (C-20), OH OGlc Advance0 00 0 00 View00 77.6 (C-3 ), 77.9 (C-3 ), 78.0 (C-5 ), 78.2 (C-5 ), 99.8 (C-1 ), 102.5 0 (C-1 ), 109.9 (C-3, C-5), 115.0 (C-8), 124.6 (C-2, C-6), 127.5 (C-1), 1 2 (9%) 142.2 (C-7), 155.0 (C-4), 170.2 (C-9).

3-O-(6-O--D-Glucopyranosyl)--D-glucopyranosylcaffeic acid (9). þ HRFABMS: m=z ðM þ NaÞ : Calcd. for C21H28O14Na: 527.1428, þ 1 Found: 527.1425 ½M þ Na ; H NMR (400 MHz, pyridine-d5): H 0 00 0 00 0 00 0 00 0 00 4.07–4.57 (12H, m, H-2 ,2 ,3,3 ,4,4 ,5,5 ,6,6 ), 4.99 (1H, d, O O ¼ 00 ¼ 0 J 7:2 Hz, H-1 ), 5.10 (1H, d, J 7:6 Hz, H-1 ), 6.55 (1H, d, OH OH 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, HO GlcO 13 00 0 3 (5%) 4 (2%) H-7); C NMR (100 MHz, pyridine-d5): C 63.0 (C-6 ), 69.3 (C-6 ), Proofs 70.9 (C-40, C-400), 74.2 (C-200), 74.3 (C-20), 77.5, 77.7 (C-30, C-300), 78.1 (C-50, C-500), 99.5 (C-10), 100.0 (C-100), 114.1 (C-8), 114.9 (C-2), 115.5 Fig. 2. Biotransformation Pathway of Cinnamic Acid (1) by Plant- (C-5), 122.0 (C-6), 125.9 (C-1), 145.5 (C-7), 147.0 (C-3), 148.5 (C-4), Cultured Cells of E. perriniana. 165.0 (C-9). time course of the conversion of 1 was followed. As Time-course experiments. Suspension cells (100 g) of E. perriniana Fig. 1 indicates, cinnamic acid (1) was converted into 2 were partitioned to eight flasks containing 200 ml of MS medium. at an early stage of incubation, and the products 3 and 4 Substrate (15 mg) was administered to each of the flasks, and the were slightly produced. This indicates that glucosylation 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 at the carboxyl group of cinnamic acid occurred pre- medium were separated by filtration. The extraction and analysis dominantly, rather than hydroxylation at the 4-position procedures were as described above. of cinnamic acid to p-coumaric acid. The biotransforma- tion pathway of cinnamic acid is shown in Fig. 2. Results and Discussion Five compounds 4–8 were produced by incubation of cultured E. periniana cells with p-coumaric acid (3). After 96 h, incubation, the biotransformation products The structures of these products were identified as 4-O- were isolated from the cultured suspension cells of -D-glucopyranosylcoumaric acid (4, 24%), p-coumaric E. perriniana, which had been treated with cinnamic acid -D-glucopyranosyl ester (5, 10%),17) 4-O--D- acid (1). Three compounds 2–4 were obtained, and no glucopyranosylcoumaric acid -D-glucopyranosyl ester additional conversion products were observed under (6, 15%), caffeic acid (7, 7%), and 3-O--D-glucopyr- careful HPLC analysis. The structures of the products anosylcaffeic acid (8, 3%).17) were identified on the basis of their HRFABMS, 1H and The HRFABMS spectrum of product 6 showed a 13C NMR, H–H COSY, C–H COSY, and HMBC pseudomolecular ion ½M þ Naþ peak at m=z 511.1382, spectra as cinnamic acid -D-glucopyranosyl ester (2, consistently with a molecular formula of C21H28O13 16) 9%), p-coumaric acid (3, 5%), and 4-O--D-glucopyr- (calcd. 511.1377 for C21H28O13Na), suggesting that anosylcoumaric acid (4, 2%).17) Hydroxylation regiose- two new hexoses were introduced to the substrate. The lectively occurred at the 4-position of cinnamic acid (1) 1H NMR spectrum of 6 displayed two anomeric proton to give p-coumaric acid (3), followed by glucosylation signals, at 5.25 (1H, d, J ¼ 7:2 Hz) and 6.11 (1H, d, of 3 to 4. J ¼ 7:6 Hz). The sugar component in product 6 was In order to determine the ability of cultured E. determined to be -D-glucopyranose, according to the perriniana cells to biotransform cinnamic acid (1), the chemical shifts of the sugar carbon signals. The Biotransformation of Phenylpropanoids 100335-3

13C NMR spectrum of 6 showed two anomeric carbon and 4-O--D-glucopyranosylferulic acid (14, 1%).17) resonances, at 99.8 and 102.5. Thus the structure of Cultured E. perriniana cells catalyzed the regioselective compound 6 was determined to be 4-O--D-glucopyr- methylation of caffeic acid (7) to ferulic acid (13). No anosylcoumaric acid -D-glucopyranosyl ester. The formation of caffeic acid -D-glucopyranosyl ester was disaccharide product 6 had not previously been found. identified. The HRFABMS spectrum of 9 included a pseudomo- These results demonstrated that E. perriniana cells lecular ion ½M þ Naþ peak at m=z 527.1425, which is regioselectively hydroxylated at the 3-position of p- consistent with a molecular formula of C21H28O14 1 coumaric acid (3) to produce caffeic acid (7). The time (calcd. 527.1428 for C21H28O14Na). The HNMR course in the conversion of 3 indicates that glucosylation spectrum of 9 included proton signals at 4.99 (1H, d, at the phenolic hydroxyl group and the carboxyl group J ¼ 7:2 Hz) and 5.10 (1H, d, J ¼ 7:6 Hz), indicating the of p-coumaric acid occurred first, and that disaccharide presence of two -anomers in the sugar moiety. The 1H product 6 was formed subsequently (Fig. 3). The and13C NMR spectra of 9 indicate that it was a - biotransformation pathway of p-coumaric acid is shown gentiobiosyl analog.15) Furthermore, the HMBC spec- in Fig. 4. trum included correlations between the anomeric proton Next, the conversion of caffeic acid (7) by cultured signal at 5.10 (H-10) and the carbon signal at 147.0 E. perriniana cells was investigated. Seven products (C-3), and between the anomeric proton signal at 4.99 were isolated, and were identified as 3-O--D-gluco- (H-100) and the carbon signal at 69.3 (C-60). These pyranosylcaffeic acid (8, 25%), 3-O-(6-O--D-gluco- findings confirm that the inner -D-glucopyranosyl pyranosyl)--D-glucopyranosylcaffeic acid (9, 6%), 3- residue was attached to the phenolic hydroxyl group O--D-glucopyranosylcaffeic acid -D-glucopyranosyl at the 3-position of caffeic acid (7), and that the pair ester (10, 9%),18) 4-O--D-glucopyranosylcaffeic acid of -D-glucopyranosyl residues were 1,6-linked. Thus (11, 7%),17) 4-O--D-glucopyranosylcaffeic acid -D- product 9 was identified as 3-O-(6-O--D-glucopyrano- glucopyranosyl ester (12, 3%),19) ferulic acid (13, 5%), syl)--D-glucopyranosylcaffeic acid. The gentiobioside Advance View9 product was a new compound. The time-course experiment indicated that no glyco- 100 sylation at the carboxyl group of 13 occurred, probably due to the low yield of 13 (Fig. 5). The biotransforma- tion pathway of caffeic acid is shown in Fig. 6. On the other hand, cultured E. periniana cells Yield (%) Yield glucosylated ferulic acid (13)to4-O--D-glucopyrano- sylferulic acid (14, 22%), ferulic acid -D-glucopyrano- 50 syl ester (15, 5%),17) and 4-O--D-glucopyranosylferulic acid -D-glucopyranosyl ester (16, 14%).20) The time- course of the conversionProofs 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.

48 96 The results of this experiment indicate that the plant- Time (h) cultured cells of E. perriniana metabolized phenylpro- panoids, including cinnamic acid, p-coumaric acid, Fig. 3. Time-Course of the Biotransformation of p-Coumaric Acid caffeic acid, and ferulic acid, catalyzing the hydroxyla- (3) by Cultured Cells of E. perriniana. The substrate, p-coumaric acid (3, 15 mg), was incubated with tion of cinnamic acid to p-coumaric acid, the hydro- 100 g of E. perriniana suspension cell cultures at 25 C on a rotary xylation of p-coumaric acid to caffeic acid, and the shaker (120 rpm) in the dark. Yields of 3 ( ), 4 ( ), 5 ( ), 6 ( ), methylation of caffeic acid to ferulic acid. Additionally, 7 ( ), and 8 () are plotted. both the phenolic hydroxyl group and the carboxyl

O

OH

GlcO O O 4 (24%) OH OGlc

HO O GlcO

3 OGlc 6 (15%)

HO 5 (10%)

O O HO GlcO OH OH

HO HO 7 (7%) 8 (3%)

Fig. 4. Biotransformation Pathway of p-Coumaric Acid (3) by Plant-Cultured Cells of E. perriniana. 100335-4 H. KATSURAGI et al.

100 100 Yield (%) Yield Yield (%) Yield

50 50

48 96 Time (h)

48 96 Fig. 7. Time-Course of the Biotransformation of Ferulic Acid (13)by Time (h) Cultured Cells of E. perriniana. The substrate, ferulic acid (13, 15 mg), was incubated with 100 g Fig. 5. Time-Course of the Biotransformation of Caffeic Acid (7)by of E. perriniana suspension cell cultures at 25 C on a rotary shaker Cultured Cells of E. perriniana. (120 rpm) in the dark. Yields of 13 ( ), 14 ( ), 15 ( ), and 16 ( ) The substrate, caffeic acid (7, 15 mg), was incubated with 100 g of are plotted. E. perriniana suspension cell cultures at 25 C on a rotary shaker (120 rpm) in the dark. Yields of 7 ( ), 8 ( ), 9 ( ), 10 ( ), 11 ( ), Advance12 (), 13 ( ), and 14 ( ) are plotted. View O GlcGlcO OH O HO GlcO OH 9 (6%)

HO O 8 (25%) GlcO OGlc O HO HO OH 10 (9%) HO 7 O O HOProofs HO OH OGlc

GlcO GlcO 11 (7%) 12 (3%)

O O H CO H CO 3 OH 3 OH

HO GlcO 13 (5%) 14 (1%)

Fig. 6. Biotransformation Pathway of Caffeic Acid (7) by Plant-Cultured Cells of E. perriniana.

O H CO 3 OH

GlcO O O H3CO 14 (22%) HO OH OGlc

HO O GlcO H CO 13 3 OGlc 16 (14%)

HO 15 (5%)

Fig. 8. Biotransformation Pathway of Ferulic Acid (13) by Plant-Cultured Cells of E. perriniana. group of these compounds were glycosylated to give the glycosylation at the phenolic hydroxyl group occurred corresponding mono- and disaccharides, including two preferentially to that at the carboxyl group. new compounds, 4-O--D-glucopyranosylcoumaric acid Recently, it was reported that Hematococcus pluvia- -D-glucopyranosyl ester and 3-O-(6-O--D-glucopyra- lis, a green unicellular alga, converted phenylpropa- nosyl)--D-glucopyranosylcaffeic acid. The time-course noids, ferulic acid and p-coumaric acid, into vanillin, experiments on the biotransformation of p-coumaric vanillic acid, vanillyl alcohol, and protocatechuic acid, caffeic acid, and ferulic acid indicated that acid.13) No formation of phenylpropanoid glycosides in Biotransformation of Phenylpropanoids 100335-5 algae cells treated with these phenylpropanoids occur- 7) Melek FR, Miyase T, Ghaly NS, and Nabil M, Phytochemistry, red, suggesting that the biotransformation pathways of 68, 1261–1266 (2007). phenylpropanoids are quite different as between plant- 8) Shao B, Guo H, Cui Y, Ye M, Han J, and Guo D, Phytochemistry, 68, 623–630 (2007). cultured cells and green algae. Plant-cultured cells of 9) Vincken JP, Heng L, Groot A, and Gruppen H, Phytochemistry, E. perriniana would be useful in the preparation of 68, 275–297 (2007). phenylpropanoid glycosides. This is the first report on 10) Imaida K, Hirose M, Yamaguchi S, Takahashi S, and Ito N, the glycosylation and hydroxylation of phenylpropa- Cancer Lett., 55, 53–59 (1990). noids by plant-cultured cells. Further studies of the 11) Heilmann J, Calis I, Kirmizibekmez H, Schuhly W, Harput S, physiological properties of phenylpropanoid glycosides and Sticher O, Planta Med., 66, 746–748 (2000). and of the that catalyze the glycosylation of 12) Tominaga H, Kobayashi Y, Goto T, Kasemura K, and Nomura M, Yakugaku Zasshi, 125, 371–375 (2005). phenylpropanoids are now in progress. 13) Tripathi U, Rao SR, and Ravishankar GA, Process Biochem., 38, 419–426 (2002). References 14) Shimoda K, Harada T, Hamada H, Nakajima N, and Hamada H, Phytochemistry, 68, 487–492 (2007). 1) Srinivasan M, Sudheer AR, and Menon VP, J. Clin. Biochem. 15) Shimoda K, Kwon S, Utsuki A, Ohiwa S, Katsuragi H, Nutr., 40, 92–100 (2007). Yonemoto N, Hamada H, and Hamada H, Phytochemistry, 68, 2) Feng Y, Lu YW, Xu PH, Long Y, Wu WM, Li W, and Wang R, 1391–1396 (2007). Biochim. Biophys. Acta, 1780, 659–672 (2008). 16) Tanguy J and Martin C, Phytochemistry, 11, 19–28 (1972). 3) Ivanauskas L, Jakstas V, Radusiene J, Lukosius A, and 17) Ibrahim RK and Shaw M, Phytochemistry, 9, 1855–1858 Baranauskas A, Medicina, 44, 48–55 (2008). (1970). 4) Ishihara K, Hamada H, Hirata T, and Nakajima N, J. Mol. Cat. 18) Filippo I, Phytochemistry, 15, 1786 (1976). B: Enzymatic, 23, 145–170 (2003). 19) Hatem B, Zine M, Hichem BJ, Susan M, and Pedro MA, J. Nat. 5) Moyer BG and Gustine DL, Phytochemistry, 26, 139–140 Prod., 68, 517–522 (2005). (1987). 20) Jin-Lan Z, Guo-Dong Z, and Tong-Hui Z, J. Asian Nat. Prod. 6) Tabata M, Umetani Y, Ooya M, and Tanaka S, Phytochemistry, Res., 7, 49–58 (2005). Advance27, 809–813 (1988). View

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