Agr. Biol. Chem., 40 (11), 2283•`2287, 1976

Chavicol, as a Larva-growth Inhibitor, from Viburnum japonicum Spreng.

Hajime OHIGASHI and Koichi KOSHIMIZU

Department of Food Science and Technology. Kyoto University, Kyoto Japan Received July 22, 1976

Chavicol was isolated as a drosophila larva-growth inhibitor from the leaves of Viburnum japonicum Spreng. The inhibitory activities of chavicol and its related compounds against drosophila larvae and adults were examined.

To obtain biologically active substances assignable to allylic, terminal olefinic, a hydrox

against insects from plants, we recently devised yl, an olefinic and 1, 4-di-substituted benzene a convenient bio-assay using drosophila larvae. ring protons, respectively. The double The assay is of great advantage to judge easily resonance experiment clarified that the protons

the effects of compounds on the growth of the at ƒÂ 3.26 coupled with both the proton at ƒÂ insects at each stage from the larvae to the 5.7•`6.2 (with J=7Hz) and protons at ƒÂ 4.9•`

adults. In the screening of plant extracts by

this method, we found that the methanol ex tract of the leaves of Viburnum japonicum

Spreng. inhibited remarkably the growth of the larvae. We report here the isolation,

identification of the active component of V. japonicum, and also report the activities of the component and the related compounds against the adults as well as the larvae.

An ethyl acetate-soluble part of the methanol

extract was chromatographed on silicic acid- Celite 545 eluted with benzene of an increasing ratio of ethyl acetate. The larva-killing activi

ty was found in a fraction eluted with 5% ethyl acetate in benzene. Further chromato

graphy of the active fraction on Florisil, fol lowed by preparative thin-layer chromato

graphy on silica gel afforded an active com

ponent (1) as a colorless oil. The active component (1) showed UV absorp

tion maximum at 259nm (in EtOH, ƒÃ=1200) and IR absorption bands (in CHCl3) at 3450

(-OH), 1645, 990 and 920 (-HC=CH2), 1600, 1510 and 1440cm-1 (benzene ring), respective-

ly. The PMR spectrum in CDCl3 gave the signals at ƒÂ 3.26 (2H, broad d., J=7 Hz),

4.9•`5.2 (2H, m.), 5.6 (1H, broad, disappeared in addition of D2O), 5.7•`6.2 (1H, m.) and 6.71

(2H, d., J=8Hz), 7.00 (2H, d., J=8Hz), 2284 H. OHIGASHI and K. KOSHIMIZU

5.2 (long-range coupling). The spectral TABLE I. THE EFFECTOF CHAVICOL(1) evidences indicated the active component (1) ON THE LARVA-GROWTHOF to be a 4-allyl-phenol (chavicol) and the identity D. melanogaster was confirmed by direct comparison with an authentic chavicol derived from an estragole (2).1) Chavicol has been known to occur in spice- plants such as Piperaceae,2) Myrtaceae,3) Zingiberaceae,4) Rutaceae5) and Labiatae,6) although neither the larva-growth inhibitory property nor the occurrence in V. japonicum have been reported. The larva-growth inhibitory assay was only slight inhibition of the adult-emergence carried out by the following method. Ten was observed. larvae of Drosophila melanogaster in 2nd The larva-growth inhibitory activities of the instar were cultivated on a Pearl's synthetic related compounds such as eugenol (5), isoeu medium (3ml)7) in a test tube containing a genol (6), safrole (7), isosafrole (8), o-allyl certain amount of a sample to be tested. The - phenol (9) and their derivatives, 10•`14 were development of the larvae was observed every examined and the results are shown in Table 24hr up to the time of emergence of control III. Eugenol (5), isoeugenol (6), safrole (7), adults. The larva-growth inhibitory activity isosafrole (8), o-allyl-phenol (9) and their can be judged by either inhibition of the dihydro-derivatives, 10, 12 and 13 exhibited pupation (larva-killing activity) or of adult the larva-killing activity at a concentration of emergence from the pupae formed. either 1700 ppm or 3500 ppm, whereas acetyl- As shown in Table I, chavicol (1) exhibited derivatives, 11 and 14, were relatively inac the larva-killing activity at a concentration of tive and only inhibited the adult-emergence at ca. 1700ppm. The activities of the derivatives a higher concentration. of 1 such as chavicol methyl ether (estragole) The remarkable activity was not found in

(2), acetyl chavicol (3) and dihydrochavicol (4) phenol (15), o-cresol (16) and other simple are summarized in Table II. Dihydrochavicol phenols (17•`21) containing two or three

(4) showed the larva-killing activity at an equal phenolic hydroxyls as summarized in Table IV. concentration to 1. On the other hand , the Since myristicin (5-allyl-l-methoxy-2, 3- activity was not found in both 2 and 3, where methylenedioxybenzene) (22), one of the re-

TABLE II. THE EFFECTS OF CHAVICOL-DERIVATIVES ON THE LARVA-GROWTH OF D. melanogaster Chavicol, as a Larva-growth Inhibitor, from V. japonicum 2285

TABLE III. THE EFFECTS OF CHAVICOL-RELATED COMPOUNDS ON THE LARVA-GROWTH OF D. melanogaster

TABLE IV. THE EFFECTS OF PHENOLS ON THE LARVA-GROWTH OF D. melanogaster

TABLE V. THE EFFECTS OF CHAVICOL AND ITS RELATED COMPOUNDS ON THE ADULTS OF D. melanogaster 2286 H. OHIGASHI and K. KOSHIMlZU

lated compounds, was isolated as a drosophila emerged, which were transferred into a new culture adult-killing component from Pastinica sativum bottle. In this procedure, continuous supply of the adults and larvae was possible. L.,8) the effects of chavicol (1) and the related In the larva-growth inhibitory assay, 1.5ml of the compounds, 2, 3 and 5, on the adults were solution A was added to a certain amount of a sample tested by a paper disk method. As shown in to be tested in a test tube (1.5cm i.d.•~3.0cm height). Table V, chavicol (1) and eugenol (5) showed Then 1.5ml of the solution B, in which the agar was knocked down effect. Chavicol methyl ether previously dissolved, was added to the solution thus pre pared with shaking. After the medium was cooled to (2) and acetyl chavicol (3), however, were inac room temperature, 20mg of dry yeast was added to the

tive against the adults. medium. Ten larvae, which were 48•`72 hr old after The results through the experiments on the hatching (2nd instar) were placed on the medium and activities of chavicol and its related compounds cultivated in a usual way. A control experiment was against the larvae and adults suggested carried out on a medium without the sample. that both an alkyl side chain and a free phenolic The effects of compounds on the adults were tested by a paper disk method. A Toyo-filter paper (No. hydroxy or methylenedioxy group (in the case 2) disk (1.3cm diameter) which had been dipped in a

of safrole) play significant roles to exhibit the chloroform solution of a sample at a certain concentra activities. tion, was placed on the bottom of a test tube (1.5cm Further study on the structure/activity rela i.d.•~3.0cm height). A control experiment was run tionship on chavicol and the related compounds on a paper disk without the sample. The solvent was evaporated under reduced pressure and ten adults were is in progress. introduced into the tube. After 15 min, 30min, 3hr

and 24hr, the adults, either motionless or unable to

stand, were registered as knocked down. EXPERIMENTAL

PMR spectra were obtained on a Hitachi Model R-22 Extraction and isolation of the active component

spectrometer (90MHz) in CDCl3 and chemical shifts (1). The fresh leaves (500g) of V. japonicum were are expressed in ppm from tetramethyl silane as an extracted with methanol. Evaporation of methanol

internal standard. IR and UV spectra were recorded on gave an aqueous extract which was extracted with a Hitachi EPI-G3 infrared spectrometer and a Shi ethyl acetate. The ethyl acetate layer was dried over

madzu UV-200 spectrophotometer, respectively. The anhydrous sodium sulphate and the solvent was re following chromatographic materials were used: moved to give a dark-green viscous matter. It was

silicic acid (Mallinckrodt, 100mesh, U. S. A.), Florisil chromatographed on silicic acid-Celite 545 (1:1)

(Floridin Company, 100•`200 mesh), silica gel PF254 which was eluted with benzene of an increasing ratio (Merck, for preparative TLC) and Celite 545 washed of ethyl acetate. The fraction eluted with 5% ethyl successively with distilled water and acetone and then acetate in benzene was further chromatographed on dried for 5 hr before use. Florisil by a stepwise elution from benzene to ethyl acetate. The active fraction eluted with 5% ethyl Bio-assay. The larvae and the adults of D. melano acetate in benzene was purified by preparative TLC

gaster (Oregon R) were used for the bio-assay. The on silica gel PF254 to give a colorless oil (20mg) as an drosophilae were cultivated on a Pearl's synthetic active component (1). medium, made up as follows: solution A; water (300

ml), KNaC4H4O6•E4H2O (5g), MgSO4•E7H2O (0.3g), Chavicol from estragole (2). To a solution of methyl CaCl2 (1.5g), (NH4)2SO4 (1.2g), sucrose (50g), and iodide (7.2g) in dry ether (20ml) was added magnesium

solution B; water (300ml), tartaric acid (3g), KH2 ribbon (1.3g) step by step with ice-cooling and stirring. PO4 (0.6g), agar (13.5g). After dissolving of magnesium, the solvent was removed

The agar in solution B was dissolved by heating, and from the reaction mixture on a water bath and then an

equal parts (20ml) of solution A and B were mixed in a oil bath successively to give a solid. After cooling of

milk bottle (200ml). After the medium was cooled to the solid to room temperature, a commercial estragole

room temperature, dry yeast (1g) was added to the (6g) was added to the solid. The reaction mixture was medium on which a paper toweling (3•~10cm) was kept for 3hr at 160•`170•Ž. To the mixture was

placed to absorb surplus moisture and decrease the added ice water under cooling, followed by addition of danger of drowning the adults. The adults were water and 1N HCI (10ml), successively. The mixture introduced into the bottle, which was then stoppered was extracted with ether and the ether layer was further with a sponge plug, and cultivated under 27•Ž, 60% extracted with 10% NaOH solution (50ml). The

humidity. After about two weeks, the new adults aqueous layer thus obtained was made acid to Congo Chavicol, as a Larva-growth Inhibitor, from V. japonicum 2287 red with dilute HCI solution and extracted with ether anhydride-pyridine or over Adams' Pt. (70ml). The ether layer was washed with water, dried over anhydrous sodium sulphate and the solvent was Acknowledgement. This work was supported in evaporated to give a chavicol (1) (3.9g) as a colorless part by a grant for the scientific research from the Mini oil. stry of Education. The authors wish to express their sincere thanks to Acetylation of chavicol. A chavicol (1) (20mg) in National Institute of Genetics for the generous supply acetic anhydride (3ml) and pyridine (1.5ml) was al of D. melanogaster, to Dr. Fumiki Takahashi, an lowed to stand overnight at room temperature. The Assistant Prof. of Kyoto University, for the helpful reaction mixture was poured into ice water and extract suggestion for the bio-assay and to Dr. Yataro Segawa, ed with ethyl acetate. The ethyl acetate layer was an Assistant Prof. of Kyoto University, for the identi dried over anhydrous sodium sulphate and the solvent fication of the plant. Thanks are also due to the late was removed to give an acetyl chavicol (3) (18mg) Prof. Tetsuo Mitsui for his encouragement throughout as a colorless oil, PMR ƒÂ (CDCl3): 2.26 (3H, s.), 3.29 the study. (2H, d. t., J=7 Hz and 2 Hz), 4.9•`5.2 (1H, m.) and 6.9•`7.4 (4H, m.). REFERENCES Catalytic hydrogenation of chavicol. A chavicol (1)

(20mg) in EtOH (5ml) was hydrogenated over Adams' 1) G. Zemplen and A. Gerecs, Ber., 70, 1098 (1937). Pt (5mg). The catalyst was filtered off and the filtrate 2) J. F. Eykman, ibid., 22, 2736 (1889). was concentrated to give a dihydrochavicol (4) (20mg) 3) "The Essential Oils," Vol. IV, ed. by E. Guenther, as a colorless oil, PMR ƒÂ (CDCl3): 0.86 (3H, t., J= D. Van Nostrand Co. Inc., New York, 1950, pp.

7Hz), 1.53 (2H, sextet, J=7Hz), 2.44 (2H, t., J=7 392•`395.

Hz), 5.78 (1H, -OH), 6.71 (2H, d., J=8Hz) and 4) "Koryo Kagaku Soran," Vol. I, ed. by O. Okuda, 6.98 (2H, d., J=8Hz). Hirokawa Publishing Co., Tokyo, Japan, 1967, p. 157.

Preparations of chavicol-related compounds and 5) E. Goulding and O. Roberts, J. Chem. Soc., 105, other phenols. Estragole (2), eugenol (5), isoeugenol 2613 (1914).

(6), safrole (7), isosafrole (8), o-allyl-phenol (9), phenol 6) S. Dutt, Indian Soap J., 21, 12 (1955). (15), o-cresol (16), hydroquinone (17), resolcinol (18), 7) "Culture Methods for Invertebrate Animals," pyrogallol (19), phloroglucinol (20) and gallic acid (21) ed. by P. Galtsoff, Comstock Publishing Co. used were commercial ones, which were purified by pre Inc., Ithaca, New York, 1937, pp. 437•`445. parative TLC, if necessary. Acetyl and dihydro 8) E. P. Lichtenstein and J. E. Casida, Agr. and Food. derivatives such as 10, 11, 12, 13 and 14 were obtained Chem., 11, 410 (1963). from the usual treatments of 5, 7 and 9 with acetic