Amarogentin, amaroswerin and four xanthones from hairy root cultures of Swertia japonica

Phylochemistry, Vol. 29, No. 5, pp. 1563-1565, 1990. Printedin Great Britain.

AMAROGENTIN,

KANJI

ISHIMARU,

AMAROSWERIN ROOT CULTURES

HIROSHI

SUDO,*

MOTOYOSHI

TAKEMOTO*

Tsukuba

0

and

AND FOUR XANTHONES OF SWERTIA JAPONICA SATAKE,

YUJI

KOICHIRO

MATSUNAGA,*

YUKI

0031~9422/90 %3.00+0.00 1990 PergamonPress plc

FROM HAIRY

HASEGAWA,*

SHIZUME

SHIMoMuRAt

Plant Research Station, National Institute of Hygienic Sciences, 1 Hachimandai, Tsukuba, Ibaraki, 305, Japan; *Kanebo Ltd, Biochemistry Lab., 3-28, 5-Chome, Kotobuki-cho, Odawara, Kanagawa, Japan

Medicinal

(Receioed in revised form 27 September 1989)

Key Word Index-&e&n japonica; Gentianaceae; Agrobacterium rhizogenes; hairy root; S-O-primeverosylbellidifolin; swertianolin; bellidifolin; methylbellidifolin; amarogentin; amaroswerin.

Abstract-A hairy root culture of Swertia japonica was established and then analysed for xanthone and bitter principle production. Bellidifolin, methylbellidifolin, swertianolin and a new xanthone derivative 8-O-primeverosylbellidifolin were isolated. Two bitter principles, amarogentin and amaroswerin, were also identified.

INTRODUCTION

The whole plant of Swertia juponica (Gentianaceae) is an important bitter stomachic in Japan where it is called ‘senburi’. The plant is also claimed to be effective in the treatment of hepatitis [l]. Components of this plant such as the bitter secoiridoids [2-71, phenyl glucosides [8], flavonoids [9] and xanthones [lO-123 have been intensively studied but, to date, there are few reports of the production of these compounds in vitro. Callus culture [13] produced no bitter principles (secoiridoids), xanthones and flavones but coumarin derivatives, scopoletin and its glucoside were detected. In this paper we describe the isolation of a new xanthone derivative 8-0-primeverosylbellidifolin (1) together with bellidifolin (2) [ 111, methylbellidifolin (3) [ 11) and swetrianolin (4) [12] from a hairy root culture of S. japonica. We also describe the detection of two bitter erinciples amarogentin (5) and amaroswerin (6) from the same culture by HPLC analysis. RESULTS AND DISCUSSION

Fresh shoots of S. japonica (2-year-old-plants), grown in the field, were cultured on hormone free Murashige-Skoog (MS) solid medium [14]. The axenic shoots thus obtained were used for transformation. Agrobacterium rhizogenes strain 15834 harbouring Ri plasmid (pRi 15834) was inoculated by a needle onto the cut ends of these axenic stems. Two to four weeks after inoculation, hairy roots appeared at the infection sites. The hairy roots, after removal of the bacteria on hormone free l/2 MS solid medium containing antibiotic, were cultured on hormone free Root Culture (RC) solid medium [15] containing 3% sucrose. The opines, agropine and mannopine, were detected by paper electrophoresis (data not shown). After several passages, the

tAuthor

to whom correspondence should be addressed.

hairy roots were transferred and cultured in hormone free RC liquid medium. The roots grew satisfactorily in this medium (Fig. 1). The hairy roots, pale yellow in colour, were thought to be enriched in xanthones. In addition, the hairy roots were slightly bitter to the taste indicating the presence of bitter principles. A methanol extract of the hairy roots (73 g dry wt) was partitioned between water and chloroform. The chloroform phase after drying was subjected to silica gel column chromatography (C&H,-EtOAc) to give 2 (136 mg) and 3 (17 mg). The aqueous layer was separated by Sephadex CHP-20P LH-20 (H,O-MeOH) and MCI-gel (H,O-MeOH) to afford 1 (40 mg) and 4 (55 mg). A bitter fraction (fraction A) which contained mainly 5 and 6 was also separated. Compounds 24 were identified as beIlidifolin, methylbellidifolin and swertianolin by comparison of their physical and spectral data with those of authentic samples prepared from the mother plants. Compound 1 was obtained as pale yellow needles and its ‘H NMR spectrum showed methoxyl (6 3.89), meta-coupled aromatic (66.37, 6.58) and ortho-coupled aromatic (6 7.19, 7.28) signals. These signals were closely correlated to those of 4. The ‘%NMR spectrum of 1 (Table 1) also demonstrated the presence of a similar xanthone (1,3,5,8_oxygenated) structure to that of 4. Fur‘HNMR spectrum contained two thermore, the anomeric proton signals CS4.77 (J = 7.5 Hz), 64.21 (J = 7.5 Hz)]; the coupling constant of the former signal and its chemical shift were closely related to the glucose H-l signal of 4. The presence of a glucose moiety was also suggested by the six carbohydrate signals (665.8, 69.8, 73.5, 75.9, 76.5 and 103.8) in the 13CNMR spectrum. Five additional carbohydrate signals (668.7, 69.7, 73.5, 76.3 and 104.3) were also present and the chemical shifts of these signals suggested the presence of a xylose moiety [16]. Acid hydrolysis of 1 with HCI gave D-glucose, D-XylOSe and an aglycone which was identical to 2. The linkage of the glucose moiety to the C-8 position was indicated by the close similarity of the 13CNMR signals for the xanthone moiety and glucose C-l in 1 with

1563

K. ISHIMARU rt al.

1564

1

R, =H,

2

R,,

R,=-Glc-Xyl

RI=”

3 R, =Me, R,=H 4 R, =H, Rz=-Glc

Weeks Fig.

Table

1.

1. ‘-‘C NMR spectra of 14 at 67.5 MHz (dvalues) in DMSO-d, + D,O 1

2

3

4

2 3 4 4a 5 6 7 8 8a 9 9a 10a --OMe

162.7 97.5 166.6 92.6 156.7 140.9 121.8 112.6 149.7 112.0 181.3 103.0 145.3 56.4

161.9 97.5 167.1 92.9 157.4 137.3 123.8 109.5 151.8 107.5 184.0 102.1 143.3 56.2

161.8 97.8 167.2 93.1 157.4 139.7 120.7 109.2 152.7 107.6 183.9 102.3 144.6 56.4 56.7

162.7 97.2 166.3 92.2 156.4 141.0 121.1 112.3 149.4 111.9 181.1 103.1 145.0 56.1

GlC 1 2 3 4 5 6

103.8 73.5 75.9 69.8 76.5 65.8

XYl 1 2 3 4 5

104.3 73.5 76.3 69.7 68.7

C I

103.5 73.5 76.1 69.7 77.4 60.8

5

R=H

6

R=OH

those of 4. The glucose C-6 signal was observed relatively downfield (665.8) compared to that of 4 (660.8) which indicated that the xylose C-l position was connected to the glucose C-6 position. The configuration of the anomeric centres of glucose and xylose was concluded to be /I based on the J value (7.5 Hz) of their H-l signals. Together with further support from FAB-mass spectrometry of 1 which showed a prominent ion peak at m/z 1 was concluded to be 8-O-pri591 [M + Na]+, meverosylbellidifolin. The aqueous layer was analysed by HPLC and two bitter principles amarogentin (5) and amaroswerin (6) were detected. The HPLC spectrum of fraction A which was prepared by further column chromatographies of the aqueous layer showed more clearly the production of these bitter principles. The R,s and UV spectra of these bitter compounds were identical with those of the authentic samples isolated from the mother plants, TLC of these compounds could not be carried out due to the low contents [O.OOOOl-0.0001% dry wt]. This is the first report of bitter principle production by a tissue culture of S.japonica. It is interesting biosynthetitally that the hairy roots produce only 1,3,5,8-oxygenated xanthones although in LGW plants produce both 1,3,5,8-and 1,3.7,8-oxygenated derivatives (presumed to be biogenetically equivalent) [ 111. The hairy roots of this plant also produce several new phenyl glucosides, of which details of their structural elucidation will be presented elsewhere.

EXPERIMENTAL Mps: uncorr. ‘H and 13CNMR were measured at 270 and 67.5 MHz, respectively, locked to the major deuterium resonance of the solvent (DMSO-d,). TLC was conducted on silica gel and spots were visualized by spraying with 10% H,SO,. HPLC was performed on apparatus equipped with a photodiode array detector with detection at 254 nm at 35‘. Plant materiul. Snertiu japonica Makino (2-year-old-plant), grown in the field at Tsukuba Medicinal Plant Research Station in Japan, was collected in May 1987. The deleaved fr. shoots were dipped in 75% EtOH for 30 set and rinsed once with sterilized H,O. After surface sterilization for 10 min in 2% NaOCl with Tween 20 (1 drop in 40 ml), followed by washing x 3 with sterilized H,O, the axenic shoots were used as explants for shoot culture. Aymhucterium rhizrxpw.c strain 15834 harboring Ri plasmid (pRi 15834) grown on YEB agar medium [17] was inoculated by a needle on to the cut ends of sterile stems (1 cm length) obtained from the shoot cultures. Two lo 4 weeks after inoculation, hairy roots appeared at the inoculated sites. The

Hairy

root culture

hairy roots were removed and cultured on hormone free half-strength MS solid medium (Gelrite 2 g/l) containing the antibiotic (Claforan 0.5 mg/ml) in the dark at 25”. After removal of bacteria the axenic hairy roots were transferred and maintained on hormone free RC solid medium containing 3% sucrose. The opines (agropine and mannopine) of the hairy roots were extd and detected by the method of ref. [18]. Voucher specimens are deposited at the Herbarium of Breeding and Physiology Laboratory in the Research Station. Growth experiments. About 0.02 g (fr. wt) of hairy roots were inoculated into hormone free liquid RC medium containing 3% sucrose (50 ml in 100 ml Erlenmeyer flask) and cultured at 25” on a rotary shaker at 80 rpm in the dark. The growth rate was determined by the increase of fr. wt between 2 and 8 weeks of culture. Extraction and isolation. Lyophilized hairy roots (73 g dry wt), cultured in RC liquid medium for 6 weeks, were mashed and extracted at room temp. with MeOH (2 x 400 ml). The extract, after concn under red. pres. to ca 50 ml, was poured into H,O (200 ml) and extracted with CHCI, (3 x 100 ml). The CHCI, layer, after evapn to dryness (1.7 g), was applied to a silica gel CC (C&H,-EtOAc, 10: l-5: 1) to give 2 (136 mg) and 3 (17 mg). The aq. layer, after concn, was chromatographed over Sephadex LH-20 (60% MeOH) and MCI-gel CHP-2OP (H,O-MeOH, 1:o-O: 1) to afford 1 (40 mg), 4 (55 mg) and fr A (5 mg) (mainly 5 and 6). 8-0-Primeuerosylbellidifolin (1). Pale yellow needles, mp 265”, [a]? -82.8” (pyridine; ~0.3) ‘H NMR (DMSO-d,): 62.95400 (tn. sugar-H), 3.89 (3H, s, OMe), 4.21 (lH, d, J = 7.5 Hz, xyl H-l), 4.77 (lH, d, J = 7.5 Hz, glc H-l), 6.37 (lH, d, .I = 2 Hz, H-2), 6.58 (lH, d, J = 2 Hz, H-4), 7.19 (lH, d, J = 9 Hz, H-7), 7.28 (lH, d, J = 9 Hz, H-6), i3CNMR: see Table 1, FABMS m/z (rel. int.): 591 [M + Na]+(22), (Found: C, 50.15; H, 5.04. C,,H,,0,,.3/2 H,O requires: C, 50.42; H, 5.24 %). Acid hydrolysis ofl. A mixt. of 1 (5 mg) and cone HCI (1 ml) was heated (90”) for 10 min. The reaction mixt, after cooling and diln with H,O (1 ml), was extracted with CHCl, (1 ml). The CHCI, layer was evapd to dryness and recrystallized from EtOH to yield 2 (1 mg). The aq. layer, after concn, was subjected to TLC analysis on silica gel in CHCl,-MeOH-H,O (14:6: 1); R, 0.19 (D-ghCOSe),

R, 0.33 (D-XylOSe).

Be[lidi$olin (2). Pale yellow needles, mp 261”, ‘HNMR (DMSO-d,): 63.89 (3H, s, OMe), 6.38 (lH, d, J = 2 Hz, H-2), 6.60 (lH, d, J = 2 Hz, H-4), 6.63 (lH, d, J = 9 Hz, H-7), 7.25 (lH, d, J = 9 Hz, H-6), i3C NMR: see Table 1. Methylbellidifolin (3). Pale yellow needles, mp 185”, ‘H NMR (DMSO-d,): 63.89 (3H, s, OMe), 3.91 (3H, s, OMe), 6.43 (lH, d, J = 2 Hz, H-2), 6.70 (lH, d, J = 2 Hz, H-4), 6.75 (lH, d, ./ = 9 Hz, H-7), 7.48 (IH, d, J = 9 Hz, H-6), i3CNMR: see Table 1. Swertianolin (4). Pale yellow needles, mp 203”, ‘HNMR (DMSO-I,): 63.10-3.80 (m, sugar-H), 3.89 (3H, s, OMe), 4.81 (IH, d, J = 7.5 Hz, glc H-l), 6.37 (lH, d, J = 2 Hz, H-2), 6.58 (lH, d, J = 2 Hz, H-4), 7.13 (lH, d, J = 9 Hz, H-7), 7.26 (lH, d, J = 9 Hz, H-6), “CNMR: see Table 1.

of Swertia japonica

1565

HPLC analysis. Samples (aq. layer and fr. A) were dissolved in MeOH and subjected to HPLC on a Nucleosil lOO-5C18 (4.6 mm id. x 250 mm) column, mobile phase MeOH-H,O (9: IO), flow rate 0.8 ml/min, column temp. 35”: 5; R, 13.1 min. UV::p nm: 263,308,6; R, 11.1 min, UVf:$ nm: 270,308. UV spectra were measured from 200 to 400 nm using a photodiode array detector.

Acknowledgements~The authors thank Dr Andrew D. Powell for previewing this manuscript. They are also indebted to Dr Y. Yamakawa (Department of Agricultural Chemistry, University of Tokyo) and Dr K. Saito (Faculty of Pharmaceutical Sciences, Chiba University) for the measurement of FABMS and elemental analysis, respectively. This work was supported in part by Ministry of Health and Welfare, Science Research Fund Subsidy granted to Japan Health Science Foundation.

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