Insect growth inhibitors from Machilus japonica

Pergamon

Phyfochemwy, Vol. 35. No 3, pp. 607.610. 1994 Copyrisht 0 1994 Elvv~cr Science Lid Printed in Gmt Brimn All rights reserved 0031-9422/w s66.00+o.m

INSECT GROWTH INHIBITORS FROM MACHILUS

JAPONZCA

AZUCENA GONZALEZ-COLOMA,*~$ PIERRE ESCOUBAS,* JUNYA MIZUTANI* and LABUNMI LAIIDE* *Research and Development Corporation ofJapan (JRDC), Eniwa RBP, Eniwa-Shii Megumino Kita 3-1-1, Hokkaido 061-13, Japan; tHokkaido National Agricultural Experimental Station, Hitsujigaoka,Toyohira-ku, Sapporo 062, Japan (Received in Key Word Index-Machilus

reuisedform

4 Awust 1993)

japonica; Lauraceae; leaves; Spodoptera litwa; insect growth inhibitors;

lignans.

Abstract-The neolignans, licarin A [(2R,3S,4R,5R)-2~3,4dimethoxyphenyl)-3,~dimethyl-5-pi~ronyltetrahydr~ furan], (-)-machilusin and [(2S,3S)-2,3-dihydro-7-methoxy-3-methyl-2-(3,~imethoxyphenyl~S-tra~~1-propenyl)benzofuran], were identified as bioactive constituents in leaves of Japanese Ma&i/us japonica. All compounds exhibited growth inhibition activity against larvae of the insect Spodoptera litura. The most abundant constituent was [(2R,3S,4R,5R)-2-(3,4-dimethoxyphenyl)-3,4-dimethyl-S-pi~ronyltetrahydrofuran] (0.05% dry wt), a new natural product, while [(2S,3S)-2,3-dihydro-7-methoxy-3-methyl-2-(3,4-dimethoxyphenyl)-5-tra~~l-propenyl)-benzofuran] had the highest activity against the test insect (0.01% dry wt, EC,, = 0.13% w/w). A possible synergistic action of these compounds with other M. japonica components is discussed.

INTRODUrXION

As part of a broad study of bioactive constituents of members of the Lauraceae, we conducted an investigation of the anti-insect activity of species present in Japan. This family has a very broad range of distribution. Most of the species are aromatic and many have been used in folk medicine Cl]. Following screening against several insect pest species, we have found that a crude extract of Machilus japonica foliage inhibited the growth of Spodoptera litura larvae [Z]. We have therefore undertaken the chemical analysis of this sample, using bioassay directed fractionation. Although the phytochemistry of various species of Machilus has been previously studied C3-63, there are no reports on any insecticidal components of M. japonica. In this paper, we report on the isolation, identification and bioassay of S. litura larval growth inhibitors present in the leaves of M. japonica. RESULTS ANDDISCUSSION

The crude alcoholic extract of dried M. japonica leaves was found to be active as a growth inhibitor against S. litura larvae when incorporated into their diet. This bioassay was used to guide the fractionation of the plant extract. Table 1 shows the biological activity and the quantities of those fractions which gave significant larval growth : Author lo whom correspondence should be addressed at: Centro de Investigaciones Biol6gicas. CSIC, VelLquez 144, 28006 Madrid, Spain.

Table 1. Growth inhibition effects of M. japonica fractions against S. lifuro larvae (n = 20) at 0.1% (w/w) Average (s.e.)

larval wt*

Fraction

Yield (mg)

Crude extract VLC6 c4 HPLC3 (1) HPLC4 (2) HPLCS (3) HPLC6 (4)

62600 1635 632 15.6 143.7 26.4 28.2

20.83 (3.15) 56.67 (7.93) 48.29 (5.66) 64.15 (7.72) 63.65 (9.14) 67.44 (6.86) 50.01 (6.76)

(% control)

*All values in this column are significantly different from the control; Mann-Whitney U-test, P
reduction (0.1% w/w). In addition, we observed a reduction in the growth inhibition activity after the first fractionation of the crude extract. Four pure bioactive lignans were separated by reversephase HPLC and identified by ‘H and “C NMR, including one and two dimensional methods (HHCOSY, CHCOSY and NOE), high resolution EI mass spectrometry and also by comparing with data previously reported in the literature. Compound 1 was identified as licarin A. The physicochemical and spectroscopic data were identical in all respects with previous literature reports for this lignan [7, 83. Licarin A (1) has been previously isolated from several Lauraceae species and tissues e.g. MachiIus thunbergii leaves [4], Licaria aritu wood [7-j, fruits of Nectan607

608

A. GONZALEZ-COLOMA er al.

R’T$y R3

Table 2. NOE ‘H NMR spectrum (CDCI,) of 2 Irradiation (d)

Enhanced peak

1.01 (3-Me/4Me)

H-2, H-5, H-3, H-4. H-2’. H-S’, H-6’. H-2”. H-5”. H-6” same as above same as above

1.78 (H-3/H-4) 4.60 (H-2/H-5) 1 4

R’ Me OMe

RZ OMe Me

R3 OH OMc

Table 3. Effective growth inhibition dose of pure lignans against neonate S. liruralarvae in a seven-day diet incorporation bioassay (n = 20)

Compound

95% Confidence limit ECW (% w/w) Lower Upper

1 (HPLC3) 2 (HPLC4) 3 (HPLCS) 4 (HPLC6)

0.20 0.24 0.19 0.13

OMc

OMc

dra glabrescens [9] and the branch wood of Urbanodendron uerrucosum [lo]. It has also been isolated from other plant families e.g. the Krameriaceae (Krameria cystisoides) [ 1 I], Hepaticae (Jackie/la jacanica) Cl23 and Aristolochiaceae [13]. The high resolution mass spectrum of 2 gave the [M] + m/z 356 and the molecular formula C,,H,,OJ. The ‘H NMR spectrum was similar to tetrasubstituted tetrahydrofuran neolignans. The spectrum was similar to machilin-G previously isolated from M. thunbergii [S], except for the high-field chemical shift of the C-3 and C-4 methine protons at 6 1.78 and the specific rotation. The negative specific rotation of 2 ruled out calopiptin, whose absolute stereochemistry had been determined [14]. The ‘%NMR chemical shift for C-3 and C-4 was slightly downfield at 6 50.8 and 5 1.1 when compared to machilinG at 644.5. In order to establish the stereochemistry of 2, NOES were measured (Table 2). Irradiation of either the C-3/C-4 methyl protons at S 1.01 or the C-3/C-4 methine protons at 6 1.78, or the C-2/C-5 methine protons at 64.60 resulted in enhancement of all the respective protons as well as the aromatic protons. The methoxy and the methylenedioxy protons were not affected. This observation suggests an all cis configuration similar to bisnorgalgravin [15]. The cis-configuration of 2 might explain the high-field shift of the C-3/C-4 methine protons which could be due to the shielding effect on these protons by the aryl rings. The downfield shift of the C-3/C-4 carbons by ca 667 when compared with machilin-G, might be due to the deshielding effect of the aryl rings on the carbons. This is the first report of the isolation of an all cis

0.10 0.13 0.11 0.08

0.5 0.65 0.45 0.28

isomer of machilin-G and its other isomers. The relative configuration of this compound may thus be represented as (2R*, 3S*, 4R*, SR*) -2-(3,4_dimethoxyphenyl) -3, 4dimethyl-5-piperonyl tetrahydrofuran. High resolution mass measurement of 3 gave the CM] + m/z 356 and the molecular formula C,,H,,O,. The physicochemical characteristics and the ‘H and i3CNMR spectra of 3 coincided with (-)-machilusin, previously isolated from ,Vf. japonica [3]. The chemical shifts and coupling constants (J = Hz) of the C-2, C-3, C4, C-5 methine protons and the specific rotation were identical with those of (- )-machilusin and machilin-I previously isolated from M. thunbergii [S], and thus the stereochemistry of 3 was assumed to be similar. The relative configuration of 3 may be represented as (2S*, 3R*, 4S*, SR*)-2-(3,4-dimethoxyphenyl)-3,4-dimethyl-5piperonyltetrahydrofuran. High resolution mass measurement of 4 gave the [M] + m/z 340 and the molecular formula Cl,Hz40.,. The ‘H and i3C NMR spectra were similar to licarin A, except for the presence of an extra methoxy proton signal. The absolute configuration was determined as 2S* and 3S* by comparing the specific rotation of 4 ([z]n - 6 I ‘} with the previously reported licarin A ([z]n - 59”) [S]. This is the first isolation of this compound from M. japonica. The comparative insect growth reduction activity of compounds l-4 is shown in Table 3. Compound 4 was the most active one; the other three produced similar effects. Lignans are abundant in the plant kingdom and have a broad range of biological activities. Amongst those with anti-insect action are podophyllotoxin analogues, which are effective insect growth inhibitors, sesamin and sesamolin with weak juvenile hormone activity , p-benzolactone, an insect feeding inhibitor [16] and neolignans e.g. magnolol and a biphenyl ether, toxic to non-adapted insects [17]. None of the lignans described here has been previously isolated as insect growth inhibitors.

Insect growth inhibitors from hfclchilusjaponica In the course of our experiments, we also observed a decrease in the insect growth reduction activity from the crude extract to the semi-purified fractions or the pure components. We propose a potential synergistic action of either all or some of these compounds along with other plant components present in the crude extract of M. japonica, since the biological activity of the fractions containing the four lignans together (fractions VLC6 and C4 of Table 1) was similar to the activity of each one individually and significantly lower than the activity of the crude extract. Plants include synergists in their chemical defences e.g. mixed-function oxidase (PSMO) inhibitors [18]. Machilus japonica contains other lignans e.g. galbacin, galbegin, licarin B, calopeptin and veragensin [3] as well as many other components that have not been investigated and could account for the overall insect growth inhibition activity of the plant extract. Furthermore, an isomer of licarin A (dehydrodiisoeugenol) has been isolated from Myristicajiagans, along with licarin B, as an inhibitor of hepatic PSMO enzymes [19, 203. We conclude that the larval growth inhibition activity of M. japonica leaves against S. litura can be partially explained by the presence of lignans l-4. The biological activity observed for these compounds is greater in combination with other plant components of the crude extract. We propose a synergistic action of either all or some of these lignans against S. litura when ingested by this insect along with other plant metabolites. EXPERIMENTAL

Plant material. Leaves of M. japonica Sieb. et &cc. were collected in Kyushu, Japan, in 1992. A dry voucher sample has been deposited in the Department of Botany, Miyazaki University, Japan. General. Mps are uncorr. ‘H and 13CNMR spectra were obtained at 270 and 67 MHz, respectively, with TMS as int. standard. Insect bioassay. To test for chronic growth inhibition of S. Iitura we incorporated the crude plant extract or frs from it into a commercial artificial diet (Insecta LF, Nihon Nosan Kogyo) using cellulose as an inert carrier (10% dry wt). Samples dissolved in an appropriate solvent were applied to 1 g of cellulose powder and were vacuumdried until complete evapn of solvent. The cellulose was then thoroughly mixed with the dry, powdered diet. The final diet was prepd by adding distilled Hz0 (70%) to the dry components. Control diets had the solvent carrier alone added to the cellulose. Twenty neonate larvae were fed on the treated and control diets at 27” in the darkness for 7 days; their live weights were then recorded. Differences in larval weights between treatment and control were analysed by the non-parametric MannWhitney U-test. Five concns of each pure compound were tested (1000, 750, 500, 250 and 100 ppm) as described above to calculate the EC,, values (the effective concentration for 50% growth inhibition) which were determined from log probit analysis [21].

609

Extraction and isolation. Oven-dried (500,48 hr) leaves (313 g dry wt) were ground in a Wiley mill to pass a 2 mm sieve and extracted with EtOH in a Soxhlet apparatus. The extract was coned in uacuo (62.6 g, 19.2% w/w yield) and fractionated by silica gel vacuum liquid chromatography (VLC column 9 x 5 cm, packed with TLC grade silica gel, 5-40 pm mesh) using a n-hexane-EtOAcMeOH gradient, which resulted in the identification of one biologically active fr. (1.63 g, 400 ml, nhexane-EtOAc, 9: 1). This fr. was further chromatographed on a silica gel 63-200 pm mesh) to provide another biologically active fr. (632 mg, 15 ml, nhexane-EtOAc, 4: 1) which was then analysed by prep. reverse-phase HPLC. The column was a 250 x 20 mm C18 (Gasukuro Kogyo prep-ODS, 5 pm pore size) and an isocratic system of 60% aq. MeCN was used at a flow rate of 18 ml min- ‘. Peaks were detected at 254 nm. Four pure bioactive compounds were obtained as a result of HPLC purification. Licarin A (1). Needles, mp 115-117”. [rr]:: -62” (MeOH; c 0.002), lit [aID -59”. Found m/z 326.1478, CM]‘, C,,Hz20, calcd 326.1504. Data identical in all respects with those previously reported [7, 83. (2R,3S,4R,5R)-2-(3,4-Dimethoxyphenyl)-3,4-dimethyl-5piperonyltetrahydrojiran (2). Oil. [aID -39” (MeOH; c 0.002). Found m/z 356.1625, [Ml’, C2,Hz405, calcd 356.1635. ‘H NMR (CDCI,): 61.01 (6H, d , 2 x Me), 1.78 (2H m, H-3, H-4), 3.86 (3H, s , OMe), 3.90 (3H, s, OMe), 4.59 (lH, dd, J=6.8 Hz, H-2 or H-5), 4.61 (lH, dd, J =6.8 Hz, H-2 or H-5), 5.95 (2H, s, OCH,,), 6.72-6.91(6H, m, aromatic-H). 13C NMR (CDCI,): 6 13.8 (3-Me), 13.8 (4Me), 50.8 (C-3 or C-4), 51.1 (C-3 or C-4), 55.9 (OMe), 55.9 (OMe), 88.2 (C-2), 88.2 (C-5), 100.9 (OCH,O), 106.5 (C2”), 107.9 (C-2’), 109.1 (C-S”), 110.8 (C-5’), 118.6 (C-6’), 118.7 (C-6”), 124.7 (C-l’), 136.5 (C-l”), 146.9 (C-4”), 147.7 (C-3”), 148.5 (C-4’), 149.0 (C-3’). (-)-Machilusin(3). Crystals, mp 120-121”. [a]u - 121” (MeOH; c 0.003) lit. [alo - 130.6”. Found m/z 356.1620 [Ml’, C,,H,,O,, 356.1635. Data identical in all respects with (- kmachilusin isolated from M. japonica [3]. (2S,3S)-2,3-Dihydro-7-methoxy-3-m.ethyl-2-(3,4-dim&oxypheny&5-trans-(l-propenyf)-benzofiran (4). Oil. [a]n -61” (MeOH; c 0.0025). Found m/z 340.1655 CM]‘, C,,Hz~O~ calcd 1660. ‘H NMR (CDCI,):6 1.38 (3H, d, J = 6.6 Hz, H-9), 1.87 (3H, dd, J = 1.9, 6.6 Hz, H-9’), 3.46 (lH, m, H-8), 3.87 (3H, s, OMe), 3.88 (3H, s, OMe), 3.89 (3H,s,OMe),5.l1(1H,d,J=9.5H7.,H-7),6.10(1H,ddd,J =1.5,6, l5.5Hz,H-8’),6.38(1H,dd,5=1.5, 15.5 H&H-7’, 6.76-6.96 (5H, m, ArH). Acknowledgements-This work was partially supported by the Research and Development Corporation of Japan (JRDC) under the ERATO program and the Science and Technology Agency of Japan (STA) through a fellowship to A.G.C. We acknowledge Dr 0. Saito (Hokkaido Nat1 Agric. Exp. Stn.) for his cooperation, Drs K. Hasegawa (JRDC) and E. Tsuzuki (Miyazaki Univ.) for their assistance in plant collection, Y. Matsuzawa and S. Carlin for technical support, and Dr M. Reina for his comments on the manuscript.

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