Biological activity of some steam distillates from leaves of ten species of rutaceous plants.

Some steam distillates collected from leaves of ten Rutaceous plants by Simple Steam Distillation method have been investigated for antimicrobid and insecticidal activity. Murraya paniculata, Toddalia asiatica, Lirnonia acidissima and Glycosmis pentaphylla have shown significant anti fungal activity against Cladosporium cladosponoides. High antibacterial activity was displayed by L. acidissima and M. paniculata against Staphylococcus aureus. Atalantia monophylla and Acronychia pedunculata caused significant mortality of the aphid. Aphis craccivora.


Introduction
Rutaceae is a large family comprising of about 150 genera. Twenty genera have been recorded in Sri Lanka of which the majority are indigenous.' The family is best known for the citrus fruits. Several other species, for example Murraya koenigii (Karapincha or Curry leaf), are grown for their aromatic leaves used in cooking and popular medicine. Some species provide edible fruits, also reputed to have medicinal value, such as the wood apple (Limonia acidissima) and beli fruit (Aegle mamelos).
Compounds having antifungal properties have been isolated from Rutaceae, for example in L. acidissima where the unripe fruit shell, stem and root-bark contain at least four compounds, viz 2-6-dimethoxybemoquinone, psoralen, xanthotoxin and osthenol, inhibitory to a range of fungi. 1 J 6 Euodia lunu-ankenda is one of the several plants in this  antibacterial activity. ' The insecticidal com onent, 'neoherculin', has been .. isolated from Zanthoxylum claua-hercules.
The present study describes the antimicrobial and insecticidal properties of some steam distillates collected by Simple Steam Distillation method9 from leaves of ten species belonging to the family Rutaceae. boxes containing saw dust. Steam was passed into the flasks continuouslyfor about 12 h. and the steaq coming out fiom an outlet was dowed to pass into a separate receiving flask through a condensor unit at room temperature. The resultant aqeous solution was extracted with dichloromethane (1.5-2 litres), dried with Na2S04 and the solvent was evaporated in vacuo (below 40'~).
Plates were then incubated in a moist chamber at 25 k 2 '~ for 48 h. Inhibition areas appeared white against a background of green mycelia. The diameters of zones in which the growth wis inhibited, which were approximately circular, were measured (mm).

Antibacterial Activity
Tests for antibacterial activity6 were carried out using .type strains of Staphylococcus aureas PCTC 6571). Escherichia coli (NCTC 10418) was used as the control organism in antibiotic activity testing. The essential oils (16mg each)were dissolved separately in 4ml aliquots of ethanol and ten fold dilutions were prepared from each with nutrient broth. A series of doubling dilutions were then dispensed in Zrnl aliquots in tubes, with concentrations ranging from 1/10 to 115 120 and inoculated with a fixed bacterial inoculum. The bacterial inoculurn was 0.1 ml of a dilution of bacterial suspension with a density e@ to that of a 2% aqueous suspension of barium sulphate. At thesame time the bacterial inoculum was also dispensed into an identical ( . set of tubes of dilutions made from ethanol alone, and a tube each of nutrient broth, these being used as controls. The tubes were incubated ( 3 7 '~) overnight and observed the next day for turbidity, the highest dilution showing no turbidity being the minimal inhibitory concentration (MIC). A loopful of medium from each of the tubes was then inoculated on nutrient agar and again incubated overnight. The minimal bactericidal concentration (MBC) was considered to be that showing no growth or less than 20 colonies on semisolid medium. In the interpretation of results, the test solutions were compared with those of alcohol dilutions.

Insecticidal Activity
One day old opterous female aphids (Aphis craccivora) obtained from a laboratory culture maintained on cowpea plants (Vigna unguiculata) were used in these experiments. ' Each of the steam distillates of leaves were mixed with homogenate (LF1 R , Ciba-Geigy, Bascle) (1:2 wJw), dissolved in analytical grade acetone and distilled water (1 :9 v/v) to obtain a 1500 ppm emulsion.
Ten aphids were placed on the underside of a detached young cowpea leaf and placed in a glass Petri-dish (9cm diameter) lined with a moistened filter paper. When they were settled, the leaf was tuned upside down and sprayed with 4.0ml of the 1500 ppm emulsion using the Potter's spray tower.' Then the leaf was turned right side up and the base of the petiole was wrapped with a moistened cotton plug. to retard dessication. Each Petridish was covered with a plastic Lid with a circular window of fine gauze. A mixture of distilled water, acetone and th& homogenate was used as the standard control.
The experiments was conducted at a mean temperature 29.5 + 4'~ and huxhidity 80 + 4% RH. Mortality counts were recorded 24 hours after treatment. A completely randomized design (CRD) with four replicates was used.

Results and Discussion
Plant essential oils belong to different classes of organic compounds having acyclic, cyclic, aromatic and heterocyclic structural features. These constituents include terpenes (mono-, sesqui-and diterpenes), aromatic compounds such as eugenol, nitrogen and sulphur containing compounds (ally1 isothiocyanate~) and miscellaneous compounds including unbranched long chain hydrocarbons.' Most of the plants which were investigated are used in ethnomedical preparations4 ( Table 1). The therapeutic valqe of these plants is no doubt due to the prrsence of biologically active natural products. There are several reports on the isolation of chemical constituents from the plants that were studied: alkaloids from M. koenigii3 and G p e n t~p h y l l a ,~ coumarins from L. acidissima, ' T. asiatica' and flavones and coumarins from M. panicu-lata8 are some of them.  Steam distillates of ten plants were tested for antimicrobial activity against Cladosporium cladosporioides, Staphylococcus aureus and Escherichia coli and for insecticidal activity ( Table 3) against Aphis craccivora k o~h .
( Table 2).  Steam distillates of all ten plants showed antifungal activity against C. cladosporioides using the TLC-bioassay technique ( To our knowledge this is the first report of such promising insecticidal activity in 'these plants.
This preliminary study indicates that the members of the family Rutaceae contain volatile substances having antimicrobial and insecticidal properties. Interestingly, the two plants for which there are no reported medicinal uses, 2. rhetsa and C. indica have shown only moderate or low activity compared to the other plants. L. acidksinza and M. paniculata have shown significant antifungal and antibacterial activities while A. monophylla has shown the highest insecticidal activity. The probable role of at least some of these substances would be self-defence against invading pests. Further investigations into their biological importance and role in nature would therefore prove useful.