CYPERENOIC ACD AND (-)-HARDWIGKIIC ACID FROM THE CHLOROFORM EXTRACT OF THE ROOTS OF CROTON AROMATICUS : ISOLATION AND INSECTICIDAL PROPERTIES

: Cyperenoic acid and (-)-hardwickiic acid have been bolrted from the chloroform extract of the roots of Croton aromaticus L. Both compounds displayed moderate insecticidal activity against the groundnut aphi4Apbis cmcciboro. Comparison of antifungal and insecticidal properties exhibited by the extracts of C. ammati-cus L. and C. laccifems L. corroborates previous conclusions of chemouxonomic studies that the two taxa be maintained as two d i i c t Species.


I. Introduction
Pesticides are frequently used in agriculture for the control of pathogens and insects. The contmuous application of synthetic pesticides, however, brings about human health hazards, environmental pollution, lack of speciesspecificity and development of pesticide resistant strains. Investigation of new sources for pesticides is important with a view to developing agrochemicals having specific properties. Compounds of plant origin may have less harmfti'l~effects on mammals than the synthetic pesticides .
The second largest genus of Euphorbiaceac, the spurge family is that of Croton. Some Croton species have shown insecticidal properties c.g; extracts of C. gZandulosus L. and G. tigllium L. have dis layed insecticidal activity against boll weevils and house flies, ~spectively.' The extracts of

B.M. Ratnayake Bandara i t al.
the isolation of (I) and cyperenoic acid (2) from the chloroform extract of the roots of C. aromaticus and evaluation of the insecticidal activity of 2 against A. craccivora. \ C. aromaticus and C. l a c c~e m s which h d application in traditional agriculture and ethnomedicine,' zie commonly known as "keppetiya" in Sinhala and "theppaddi" in T ' a~i l .~ The two texa have somewhat similar morphological characters and consequently Trimen has classified them as varieties of a single species.20 A recent chemotaxonomic study: also supports the contention that they are two distinct species.' p 1 In this paper the question of taxonomy of C. aromaticus and C. lacciferus is also acldressed in terms of their biological activity, particularly antifungal and insecticidal properties.  Further elution of the column with 5% ethyl. acetafe in benzene followed by flash chromatography and thin Iayer chromatography (TLC) gave a white crystalline compound (2) m.p. 163-164O~, in 2.55 x yield. The L W 4 reduction of 2 gave the corresponding alcohol(3) in 9 1% yield: The Physical data of 2 and 3 closely resembled those reported for cyperenoic acid and cgperenol, respectively,' 2 * 1 I' 7 * 1 9 3 1 . ' and the structures were confirmed by a single crystal X-ray study of 2 ( Figure 1). The atomic coordinates of 2 have been reported in an independent X-ray analysis recently.'

Insecticidal properties of the compounds
The insecticidal activity of cyperenoic acid (2) and P-amyrin was.examined, The steafn distillate from the leaves of C. bcciferus recorded a much higher .yield than that fiomC. aromaticus ( Table 1).  The antifungal activity of the leaf volatiles and the root extracts was examined a p s t Cladosporium cladosporioides3 using the TLC-bioassay technique. ' The results are given in Table 1. The chloroform extract of the roots of C. lacciferus displayed a zone of inhibition in the TLC-Cladosporium-bioassay plate, and the corresponding active compound was identified as 2,6-dimetho~~benzo~uinone.~ None of the root extracts of C. aromaticus showed antifungal activity. The zone of inhibition at Rf 0.2 observed for the leaf volatiles of C. aromaticus was absent in the case of

C. lacciferus.
The steam distillate of C. aromaticus caused higher toxic effects to aphids than that of C. laccdferus (Table. 1 ) . The petroleum extract of the roots of C. lacciferus displayed considerably high aphidicidal activity compared to that of C. aromaticus. It is also noted that thq root extracts showed bioactivities, distinctive of each plant, a@inst the cowpea bruchid, Callosobruchus chinensis reared on V i p a radiata seeds in the laboratory.' The observations made with respect to the chemical constitutions and bioactivities of the two taxa strongly support the contention that C. aromatzcus and C. lacciferus be maintained as two distinct species.' ''

Preparation of plant extracts
Plant samples of C. aromaticus were collected near Dambulla and a voucher specimen has been deposited in the University herbarium. The crushed dried roots (4.3 kg) were sequentially and exhaustively extracted with light petroleum (b.p. 60-80'~), chloroform and methanol under reflux conditions. The removal of solvent on a rotavapor gave each extract as a brown semisolid; the yields are given in Table 1. The preparation of leaf volatiles of C. aromaticus and C. lacciferus3 and root extracts of C. lacciferus2 p 7 have been described previously.  -7.7', -18.8').
This compound was identified as cyperenoic acid (2) by comparison with reported spectral data 9'

3'
chemical conversions and X-ray analysis.
Medium pressure chromatography of the'petroleum extract over silica gel (TLC grade, Merck) with 25% CH2C12 -petroleum gave P-amyrin and 1 as described previously.4 closely resembled those reported for cyperenol previo~isly.

~xaminatidn' of insecticidal properties
The protocol used in the bioassay of plant extracts and pure compounds is described elsewhere.' Briefly, the cultures of Aphis cracciiora were maintained in the laboratory on one-week old potted cowpea, Vigna unguiculata (var. Bushztavo) at 25-33'~, 68-84% relative humidity and 12 h photoperiod. One-day old female aphids were used in all experiments. A solution (2000 ppm) of each pure compound was prepared by dissolving in acetone. A sample of each solution (2.5pI, 5 ppm) was directly introduced to aphids using a rnicroapplicator,' and the mortality was counted after 24 h. Forty to fifty insects were used for each sample. The results were analysed by means of a X 2 test. Compoundg 1 and 2 and P-amyrin caused 62% (P < 0.05), 54% (P < 0.05) and 39% mortality to aphids, respectively.