Journal of Ethnopharmacology 79 (2002) 261– 263 www.elsevier.com/locate/jethpharm
Larvicidal activity of extracts from Quercus lusitania var. infectoria galls (Oliv.) A. Redwane a, H.B. Lazrek a,*, S. Bouallam b, M. Markouk a, H. Amarouch c, M. Jana a a
Laboratory of Medicinal Plants and Phytochemistry, Department of Biology, Faculty of Sciences — Semlalia POB, 2390 Marrakech, Morocco b Laboratory of Hydrobiology, Department of Biology, Faculty of Sciences — Semlalia POB, 2390 Marrakech, Morocco c Laboratory of Microbiology, Department of Biology, Faculty of Sciences Aı¨n Chock, Casa I, Morocco Accepted 2 November 2001
Abstract The present study indicates the efficacy of extracts and fractions of Quercus lusitania var. infectoria galls (Oliv.) as larvicidal agents and their possible use in biological control of Culex pipiens, the urban nuisance mosquito. Extracts and fractions were tested against second and fourth instar larvae. The LC50 values of gallotannins were 335 and 373 ppm, respectively for the 2nd and 4th instar period. The most interesting value of LC50 (24 h) is obtained with the fraction F2 (60 ppm). © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Quercus lusitania var. infectoria galls; Methyl gallate; Tannins; Culex pipiens; Larvicidal activity
1. Introduction Researchers are now looking for natural insecticides which do not have any ill effects on non-target population and are easily degradable. The search is underway to find out newer insecticides which will be effective and safe, and also easily available at low cost. It has been pointed out that there is an urgent need to control vector population because the incidence of malaria (WHO, 1973), dengue fever and filariasis is increasing. Due to spiralling costs of insecticides and labour, paucity of funds and due to resistance developed by plasmodia or anophelines to chemicals, diseases carried by mosquitoes are back since 1980. Increased irrigation resulting in increased humidity, and congenital conditions for the growth of mosquitoes have further been created by bad sanitation, lack of adequate disposal facilities, and unplanned growth in urban, and rural sectors. So far only a few compounds of plant origin are used as insecticides. The best known and widely used insecticide extracted from flowers of Chrysanthemum cinnerar* Corresponding author. Fax: + 212-4-443-7408. E-mail address:
[email protected] (H.B. Lazrek).
iaefolium is pyrethrum (Leonard and Bruce-Chwatt, 1970). It is used in control of adult mosquitoes; 10– 25% extract of crushed (Hill, 1960) dry flowers in kerosene or other organic solvents yields pyrethrum. In continuation of our valorisation program of natural regional resources (Redwane et al., 1996; Larhsini et al., 1996, 1997; Markouk et al., 2000), the aim of this work is to investigate the insect growth inhibitory properties of the extracts and fractions of Quercus lusitania var. infectoria galls (Oliv.) (Fagaceae). In a previous study, methyl gallate and extracts of Q. infectoria galls isolated from ethyl acetate extract have presented interesting molluscicidal activity (Redwane et al., 1998a,b). The extracts (aqueous, methanol, ethyl acetate, n-butanol, acetone and gallotannins) and fractions of Q. lusitania var. infectoria galls were tested against Culex pipiens mosquito larvae.
2. Material and methods
2.1. Plant extracts Q. lusitania var. infectoria (Oliv.) (Fagaceae) is a shrub from the Mediterranean area. The galls result
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A. Redwane et al. / Journal of Ethnopharmacology 79 (2002) 261–263
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Table 1 LC50 (24 h) values (ppm) of extracts of Q. lusitania var. infectoria galls against C. pipiens larvae Instar period
2nd instar 4th instar
Extract Aqueous
Methanol
Ethyl acetate
n-Butanol
Acetone
Gallotannins
256 1532
365 1698
497 1531
570 \2000
312 556
335 373
from the tumours provoked by the bite of female Cynips gallae tinctoria insect in the buds of this plant. The gall particles of Q. lusitania var. infectoria are globulous, hard, gray brown and of astringent savour. Their diameter is 1–2.5 cm. The galls were purchased from a herbalist in Marrakech (Morocco), identified at the Laboratory of Plant Ecology and by Pr. Jana (Laboratory of Medicinal Plants and Phytochemistry), Faculty of Sciences — Semlalia, Marrakech (Morocco). Galls were extracted by three methods: 1. Aqueous extraction: The aqueous extract is obtained by warming an infusion of the powder during 24 h. 2. Extraction by organic solvents: The pulverised galls (100 g) are extracted in a Soxhlet extractor with chloroform. The defatted galls were then extracted with methanol. The methanolic solution was concentrated under reduced pressure, below 40 °C, to dryness. The residue obtained was suspended in hot distilled water and successively extracted with ethyl ether, ethyl acetate and n-butanol (Netien and Lebreton, 1964). 3. Extraction of gallotannins: Powdered galls (40 g) were extracted twice with acetone at room temperature for 12 h. After evaporation of the solvent, the residue was dissolved in water and partitioned with ethyl acetate. The combined organic phases were dried over Na2SO4 and evaporated to give a mixture of gallotannins (Nishizawa and Yamagishi, 1983; Nonaka et al., 1990).
1615, 1530, 1442, 1329 (br s), 1250, 1208, 1000, 860, 761 cm − 1. 1 H NMR (250 MHz, DMSO-d6): d 9.2 (3H, s, 3OH), 6.9 (2H, s, Ar–H), 3.7 (3H, s, OCH3). 13 C NMR (250 MHz, DMSO-d6): d 166.34 (1C, C-1), 145.59 (2C, C-3 and C-5), 138.42 (1C, C-4), 119.31 (1C, C-1), 108.51 (2C, C-2 and C-6), 51.61 (1C, CH3). MS (%): m/z 184 (M + , 56), 153 (100), 125 (21). The spectral data were according to those of methyl gallate given in literature (Larjis and Khan, 1994). The fraction F3 was a mixture of two bands after characterisation by TLC. The fractions F2, F3 and F4 are presently under chemical investigation.
2.2. Bioassay test Standard methods for testing larvicidal action of chemicals suggested by WHO (1963) were followed in all the experiments. They were carried out in laboratory on C. pipiens larvae (2nd and 4th instar period) at the rate of 10 larvae per pillox containing 80 ml of well water. Larvae of C. pipiens were collected from a stream in Agadir Tachraft (country side of Marrakech) and maintained at a temperature of 259 3 °C and relative humidity of 7595%. Mortality was recorded after 24 h of exposure during which no food was offered to the larvae. The LC50 was determined by Probit analysis program.
3. Results
2.1.1. Separation of the ethyl acetate extract The ethyl acetate extract (10 g) was chromatographed over Merck 60 silica gel (230– 400 mesh). Four fractions were obtained: F1 (2.268 g); hexane/ethyl acetate (50:50); F2 (200 mg); hexane/ethyl acetate (40:60); F3 (6.2 g); hexane/ethyl acetate (0:100); F4 (771 mg); ethyl acetate/methanol (50:50). The fraction F1 was a pure slightly brown solid m.p. 197–200 °C. UV (MeOH) (umax =274 nm, umin =240 nm), HCl 0.1 N (umax =270 nm, umin =238 nm), NaOH (umax = 291 nm, umin = 267 nm). -IR (KBr): 3350 (br s), 1680,
The susceptibility level of C. pipiens larvae to extracts (Table 1) and fractions (Table 2) of Q. lusitania var. infectoria galls was determined. It is evident that only Table 2 LC50 (24 h) values (ppm) of fractions of ethyl acetate of Q. lusitania var. infectoria galls against C. pipiens larvae Instar period
2nd instar 4th instar
Fraction F1 (methyl gallate)
F2
F3
F4
206 293
60 389
208 325
253 529
A. Redwane et al. / Journal of Ethnopharmacology 79 (2002) 261–263
the 2nd instar period was more sensitive at all extracts and fractions than the 4th instar period. The aqueous extract was the most active on the 2nd instar period of C. pipiens larvae (LC50 (24 h)=256 ppm) (Table 1). The gallotannins were the most active products for the 4th instar (LC50 (24 h)=373 ppm) (Table 2).
4. Discussion and conclusion The same study was carried out by Girdhar et al. (1984) and Markouk et al. (2000) using Calotropis procera extracts against Anophelinae larvae. Other attempts have been carried out on Aedes aegypti larvae, the vector of yellow fever. Thus, Azadiracta indica, Melia 6olkensii and Lippia stoechadifolia appeared to be toxic (Grundy and Still, 1985; Mwangi and Rembold, 1988; Mwangi and Mukiama, 1988). The aqueous extract of Balanites aegyptiaca, Gardenia lutea and Randia nilotica at a dose of 1000 ppm is active on A. arabiensis larvae. Our results showed that the LC50 (24 h) values of methanol and n-butanol extracts of Q. lusitania var. infectoria galls were, respectively, of 365 and 570 ppm for the 2nd instar. The fractionating of ethyl acetate has led to four fractions and were tested against C. pipiens. The first one was a pure compound which was characterized and identified as methyl gallate based on comparison of its spectral data to the values reported in literature (Larjis and Khan, 1994). This compound appears to be active on the 4th instar period of C. pipiens with a LC50 value of 293 ppm. The other fractions of this extract are a mixture of 2–3 compounds. The fraction F2 proved to be very active on C. pipiens larvae, mainly on the 2nd instar period, with LC50 value 60 ppm. This result is similar to that found by Kumar and Dutta (1987) using oils extracted from 10 plant species on A. stephensi larvae. Amongst the plants which appeared to be the most active: Cedrus deodora, La6andula officinalis, Mentha ar6ensis, with LC50 (24 h), respectively, of 63.2; 83.6 and 83 ppm. The acetone extract of Q. infectoria galls showed the same LC50 value=300 ppm as that of Ipomoca carnea on A. stephensi larvae (Saxena and Sumitra, 1985). The lethal toxicity of extracts and fractions differs according to the larval stages of C. pipiens. The 2nd instar period is more sensitive to all extracts and fractions of galls of Q. lusitania var. infectoria. The LC50 varies according to the extracts and fractions, due to the difference in their chemical composition. In conclusion, at the 2nd instar period, the aqueous extract was the most active on C. pipiens larvae (LC50 (24 h) =256 ppm). In the light of this finding, the larvicide activity of galls is probably due to the fraction F2 (LC50 (24 h) value is 60 ppm). This result may offer a great potential as new control agent against C. pipiens. Further research into their mode of action, effect on non-target
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organisms, and field evaluation are presently under investigation.
References Girdhar, G., Deval, K., Mittal, P.K., Vasudevan, P., 1984. Mosquito control by Calotropis latex. Pesticides, 26 – 29. Grundy, D.L., Still, C.C., 1985. Isolation and identification of the major insecticide compound of Polio (Lippia stoechadifolia). Pesticide Biochemistry and Physiology 23, 3778 – 3782. Hill, S., 1960. Pyrethrum cultivation in East Africa. World Crops 12, 216 – 218. Kumar, A., Dutta, G.P., 1987. Indigenous plants oils as larvicidal agent against Anopheles stephensi mosquitoes. Current Science 56 (18), 959 – 960. Larhsini, M., Lazrek, H.B., Amarouch, H., Jana, M., 1996. Investigation of antifungal and analgesic activities of extracts from Sium nodiflorum. Journal of Ethnopharmacology 53, 105 – 110. Larhsini, M., Bousaid, M., Lazrek, H.B., Amarouch, H., Jana, M., 1997. Evaluation of antifungal and molluscicidal properties of extracts of Calotropis procera. Fitoterapia 68, 371 – 373. Larjis, N.H., Khan, M.N., 1994. Extraction, identification and spectrophotometric determination of second ionization constant of methyl gallate, a constituent present in the fruits shells of Pinthecollobium jiringa. Indian Journal of Chemistry 33, 609 –612. Leonard, J., Bruce-Chwatt, C.M.G., 1970. Essential Malariology. Heinemann, London. Markouk, M., Bekkouche, K., Larhsini, M., Bousaid, M., et al., 2000. Evaluation of some Moroccan medicinal plants extracts for larvicidal activity. Journal of Ethnopharmacology 73, 293 –297. Mwangi, R.W., Mukiama, K.T., 1988. Evaluation of Melia 6olkensi extract as mosquito larvicides. Journal of the American Mosquito Control Association 4, 442 – 447. Mwangi, R.W., Rembold, H., 1988. Growth inhibiting and larvicidal effects of Melia 6olkensi extracts on Aedes aegypti larvae. Entomologia Experimentalis Applicata 46, 103 – 108. Netien, G., Lebreton, P., 1964. Les flavonoı¨des et autres substances polyphe´ noliques du millpertuis (Hypericum nummularium). Annales Pharmaceutiques Franc¸ aises 22, 69 – 79. Nishizawa, M., Yamagishi, T., 1983. Tannins and related compounds. Part 9. Isolation and characterisation of polygalloylglucoses from Turkish galls (Quercus infectoria). Journal of the Chemical Society Perkin Transactions I, 961 – 965. Nonaka, G.I., Sakai, T., Nakayama, S., Nishioka, I., 1990. Tannins and related compounds, 101. Isolation and structures of C-glycosyl hydrolysable tannins from Turkish galls. Journal of Natural Products 53 (5), 1297 – 1301. Redwane, A., Lazrek, H.B., Amarouch, H., Jana, M., 1996. Tannins des galles de Quercus lusitania var. infectoria: Isolement et e´ tude de quelques aspects pharmacologiques. Polyphenols communications, Bordeaux (France), July 15 – 18, 1, 141 – 142. Redwane, A., Lazrek, H.B., Amarouch, H., Jana, M., 1998a. Molluscicidal activity of methyl gallate. Fitoterapia LXIX, 169 –170. Redwane, A., Markouk, M., Lazrek, H.B., Amarouch, H., Jana, M., 1998b. Laboratory evaluation of molluscicidal activity of extracts from Cotula cinerea and Quercus lusitania var. infectoria galls. Annales Pharmaceutiques Franc¸ aises 56, 274 – 276. Saxena, S.C., Sumitra, L., 1985. Laboratory evaluation of leaf extract of a new plant to suppress the population of Malaria vector Anopheles stephensi. Current Science 54, 201 – 202. WHO, 1963. Treizie`me rapport du comite´ OMS d’experts des insecticides, 163. Organisation Mondiale de la Sante´ Series de Rapports Techniques No. 265. WHO, 1973. Manual on larval control operations in Malaria programmes, WHO Offset Publications, No. 1, Geneva.
