An unusual metabolite, dihydroechinofuran, released from cultured cells of Lithospermum erythrorhizon

Phytochemistry, Vol. 31. No. 2, pp. 519 521, 1992

0031-9422/92 $S.OO+O.oO ‘c, 1992 PergamonPressplc

Printedin Great Britain.

AN UNUSUAL METABOLITE, DIHYDROECHINOFURAN, RELEASED FROM ERYTHRORHIZON CULTURED CELLS OF LITHOSPERMUM HIROSHI FUKUI, MASATO TANI

Faculty

of Pharmaceutical

Sciences, Kyoto (Received

Key Word Index

echinofuran;

-Lithospermum er);throrhizon;

shikonin:

and

MAMORU

University,

TABATA

Yoshida,

Kyoto

606, Japan

11 June 1991)

Boraginaceae;

plant

cell culture;

biosynthesis;

release; dihydro-

benzoquinone.

Abstract-Lithospermum erythrorhizon cell suspension cultures in a growth medium produced no shikonin but an unusual metabolite, dihydroechinofuran (2-[4-(4-methyl-3-pentenyl)-2,5-dihydrofuran-2-yl]-l,4-benzoquinone), which is thought to be derived from geranylhydroquinone, a key intermediate in the biosynthesis of shikonin. This new compound was mostly released from the cells into the medium.

INTRODUCTION

(4-methyl-3-pentenyl)-2,5-dihydrofuranyl group. Thus, 4 could be a benzoquinone derivative of 3. The 13C NMR data (see Experimental) were also in good agreement with the proposed structure. Reductive acetylation of 4 with a mixture of acetic anhydride, pyridine, and zinc powder gave a leucodiacetate that was identical (IR, UV, ‘H NMR and MS) with the acetate obtained from 3 by a treatment with acetic anhydride and pyridine. Therefore, dihydroechinofuran (2-[C(Cmethyl-3-pentenyl)-2,5-dihydrofuran-2-yl]-1,4-benzoquinone) must have the structure shown by formula 4. It is very likely that 4 was derived from geranylhydroquinone (5) via 3 (Scheme 1). The accumulation of 4 in the LS medium was detectable 3 hr after the initiation of culture (Fig. 1A) and its amount increased with time to reach a maximum (40 mg 1-l medium) on day 4 (Fig. 1B). This compound was mostly released from the cells into the medium. After day 5, however, 4 decreased rapidly in quantity with time and was hardly detectable in the medium on day 18, when the cells also contained only a small amount of 4. The release of 4 from cultured cells at day 2 was not inhibited by the administration of Na,VO, (30 and 100 mgl- ’ medium), an inhibitor of plasma membrane-bound ATPase [S], suggesting that it is not due to energy-requiring transport across the plasma membrane (data not shown). When transferred to a fresh medium repeatedly four times at intervals of four days, the cultured cells kept releasing a similar amount of 4 into the medium. Such a quick response of the cells to fresh medium or dilution has also been reported for the activity of phenylalanine ammonia-lyase in parsley cell cultures [9] as well as the biosynthesis of germichrysone in Cassia torosa cell cultures [lo]. Although the biological mechanism is not clear, the rapid synthesis of 4 by Lithospermum cells seems to occur as a result of either a slight change of the cellular environment or osmotic shock, in contrast to the biosynthesis of shikonin which is inducible only under the particular conditions mentioned earlier [2, 33. Our previous investigations [S, 61 showed that shikoninfree cells in LS liquid medium accumulated no 5 but 2 (0.02 mg g- l dry wt) and 3 (0.87 mgg-’ dry wt). However, the accumulation of a substantial amount of 4

Cultured cells of Lithospermum erythrorhizon Sieb. et Zucc. grown in Linsmaier-Skoog (LS) liquid medium [l] do not synthesize the red naphthoquinone pigment, shikonin (l), but start to produce it when transferred either to the same medium supplemented with certain acidic polysaccharides [a], or to M-9 production medium [3]. Nevertheless, the activity of 4-hydroxybenzoic acid geranyltransferase, a key enzyme in shikonin biosynthesis, was detectable in the logarithmic growth stage of shikonin-free cells in LS medium, although it was much lower than that of shikonin-producing cells in M-9 medium [4]. This suggested that shikonin-free cells might accumulate some biosynthetic intermediates or their derivatives. As might have been expected, we were able to isolate small amounts of 3-geranyl-4-hydroxybenzoic acid (2) and dihydroshikonofuran (3) from shikonin-free cells [S, 61. Because shikonin-producing cells are known to secrete the product to the outside of the cells [7], we examined compounds which were released from these cells into the medium. We report the structure determination of a new quinone derivative (4), dihydroechinofuran, isolated from the LS medium and discuss the possible biosynthetic route leading to the formation of this compound.

RESULTS AND DISCUSSION

Dihydroechinofuran (4) was isolated as an oil from the ether extract of the LS medium used for growing shikonin-free Litbospermum cells. This compound, &H1s03, gave a UV absorption maximum (MeOH) at 247 nm (log E 4.14) and IR bands (CHCI,) at 2900,2840, 1659, 1270, 1050 and 890 cm-‘. These data as well as ‘H NMR data showing signals (CDCI,) at 66.80 (IH, m) and 6.73 (2H, br s) in the aromatic proton region, suggested that 4 is a monosubstituted benzoquinone. In addition, the pattern of additional NMR signals in the upper field at S 5.70 (lH, m), 5.53 (lH, m), 5.07 (lH, m), 4.65 (2H, m) 2.13 (4H, br s), 1.66 (3H, d, J = 1 Hz) and 1.59 (3H, d, J = 1 Hz) was very similar to that of dihydroshikonofuran (3) [6] having a 3519

H.

520

FUKUI et al.

2

CHzCHCOOH :i-;_$BL~_#J$

2 Phenylalanine

5

It COOH

3

4

1

Scheme 1. Biosynthetic pathways leading to dihydroechinofuran

and shikonin in Lithospermum cell cultures.

taining 10v6 M 3-indoleacetic acid and 10e5 M kinetin at 25”in

0

2

4

6

8

10 12 14 16 18 20 22

Culture time (days)

Fig. 1. Time course of cell growth and dihydroechinofuran formation in Lithospermum cell suspension cultures in Linsmaier-Skoog medium. (40 mgl-

’ medium) suggests that this compound is an oxidation product of 5 that could not be transformed to shikonin in LS medium. The biosynthesis of 4 was completely inhibited by white light (6000 lux) which is known to repress 4hydroxybenzoic acid geranyltransferase, but not by the other inhibitors of shikonin biosynthesis such as 2,4dichlorophenoxyacetic acid [ 111, glutamine [ 121, and gibberellin A, [13]. These result suggests that the formation of a naphthoquinone skeleton from 5 is a critical step leading to the biosynthesis of shikonin. EXPERIMENTAL Culture method.

Cell cultures of Lirhospermum erythrorhizon [ll, 14-161 were grown in Linsmaier-Skoog medium [l] con-

the dark. These cultures were agitated in 100 ml conical flasks containing 30 ml medium on a reciprocal shaker (100 strokes min-‘), and subcultured at intervals of 3 weeks. For the production of dihydroechinofuran (4), 4g of fresh cells were inoculated in 300 ml conical flasks containing 100 ml medium. In the time course experiments, 1 g of fresh cells tiere transferred to 30 ml medium in a 100 ml conical flask (3 replicates). Isolation of dihydroechinofuran (4). The culture medium (a total of 2 1)was separated from the cells by filtration 4 days after inoculation, and extracted with EtzO, twice. The Et,0 extract was coned in uacuo to give a pale yellow oil (95 mg), which was then subjected to TLC (silica gel, CHCI,) to isolate 4 (76 mg) as an oil. [a]o (MeOH, 25”)- 172” (MeOH; ~0.5); UVnz:z”: 247 nm (log E 4.14); IR vale” cm-i: 2900, 2845, 1650 br, 1270, 1050, 890; ‘H NMR (CDCl,, 300 MHz): 6 1.59 (3H, d, J = 1 Hz, H-S’ or H-6”), 1.68 (3H, d, .I = 1 Hz, H-6’ or H-5”), 2.13 (br s, 4H, H-l” and H-2”), 4.65 (2H, m, H-S), 5.07 (lH, m, H-3”), 5.53 (lH, m,H-3’),5.70(1H,m,H-2’),6.73(2H,brs,H-3andH-5),6.80(1H, m, H-6), ‘zC NMR (CDCI,, 75 MHz): 6 17.729 (4, C-5” or C-6”) 25.646 (4, C-6” or C-5”), 26.182 (t, C-l” or C-2”), 27.194 (t, C-2” or C-l”), 77.352 (t, C-5’), 82.368 (d, C-2’), 120.330(d, C-3’), 123.240(d, C-3”), 130.085(d, C-3), 132.541(s, C-4”), 136.485(d, C-5 and C-6), 142.015 (s, C-4’), 148.655 (s, C-2), 187.643 (s, C-l or C-4), 188.145 (s, C-4 or C-l). The assignments were confirmed by the CHCOSY spectrum. Found [M]’ 258.1264, Cr6H1s03 requires [M]’ 258.1257. MS (75 eV) m/z (rel. int.): 258 [M]’ (25), 243 (lo), 202 (30), 189 (SO), 171 (20), 137 (25), 69 (lOO),41 (90). Reductive acetylation of compound 4. A mixt. of 4 (24 mg), Zn powder (30 mg), AczO (1.5 ml), and pyridine (1.5 ml) was allowed to stand at room temp. for 2 hr. Ice-Hz0 was poured onto the mixt. and the whole soln was extracted with Et,0 (15 ml x 3). The Et,0 layer was washed with 1 M HCl, 5% NaHCO, and

Quinone

from Lithospermum

H,O, successively, dried (Na,SO,) and coned in vacua. The crude product was purified by prep. TLC (CHCl,, R, 0.6) to yield a leucodiacetate (19 mg) as an oil. [a]n (MeOH) - 110 (MeOH; c 0.45); UV 1,~$“‘: 270 nm (log E 3.03). IR v~~~~ cm-‘: 2900,2840,1740br, 1490,1360,1160,1140,1050,1ooO. ‘H NMR (CHCl,, 300 MHz): 61.53 (3H, s, H-5” or H-6”) 1.61 (3H, s, H-6” or H-5”), 2.08 (4H, br s, H-l” and H-2”), 2.19 (3H, s, OAc-1 or OAc-4) 2.22 (3H, s, OAc-4 or OAc-1), 4.59 (2H, M, H-5’), 5.02 (1H,m,H-3”),5.40(1H,brs,H-3’),5.77(1H,m,H-2’),6.93(1H,dd, 8 Hz, H-6), 7.03 (br d, .I 5=2, 8 Hz, H-5), 6.97 (lH, dd, J=l, =2 Hz, H-3). Found [M]’ 344.1629, CZOHZ405 requires [M]’ 344.1624. MS (75 eV) m/z (rel. int.) 344 [M]’ (6) 301 (20) 285 (100) 260 (14) 243 (14) 229 (lo), 190(1 l), 179 (22), 69 (60), 43 (76). Quantitative determination ofcompound 4. An aliquot (10 ml) of the culture medium was extracted with n-pentanol (2 ml). The pentanol extract was subjected to HPLC on ODS (TSK gel 120A) and eluted with MeOH-H,O-HOAc (70:30:1). The quantity of 4 was estimated from the peak area detected at 254 nm. Acknowledgements-We are grateful to Dr N. Akimoto and Ms. S. Funakoshi of our Faculty for MS and NMR measurements, respectively. REFERENCES

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