Larvicidal and Repellent Effect of Some Tribulus Terrestris L., (Zygophyllaceae) Extracts 15

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ORIGINAL ARTICLE Larvicidal and repellent eﬀect of some Tribulus terrestris L., (Zygophyllaceae) extracts against the dengue fever mosquito, Aedes aegypti (Diptera: Culicidae)

Tarek M.Y. El-Sheikh *, Zarrag I.A. Al-Fiﬁ, Mohamed A. Alabboud

Department of Biology, College of Science, Jazan University, Jazan, Saudi Arabia

Received 5 May 2012; accepted 29 May 2012 Available online 21 June 2012

KEYWORDS Abstract Aedes aegypti transmits etiologic agents of yellow fever and dengue. Vaccine for dengue Ethanolic extract; virus is not available and vector control is essential to minimize dengue incidence. The larvicidal Acetone extract; and repellent effect of the crude ethanol, acetone and petroleum ether extract leaves of Tribulus Petroleum ether extract; terrestris, against 3rd instar larvae and adults of mosquito, Ae. aegypti the vector of dengue fever

Toxicity; was evaluated. The efﬁcacy of petroleum ether extract seemed to be more effective with LC50 Repellent; 64.6 ppm followed by acetone extract with LC50 173.2 ppm and ﬁnally ethanolic extract with Tribulus terrestris; LC50 376.4 ppm. Moreover, the acetone and petroleum ether extracts exerted a highly delayed toxic Aedes aegypti effect on the pupae and adults resulted from treated larvae, where the pupal mortality was 57.1% and 100% at concentrations 400 and 100 ppm, respectively. Also, the petroleum ether and acetone extracts showed reduction effects on adult emergence. The repellent action of the plant extracts tested was varied depending on the solvent used in extraction and the dose of the extract. The most effective plant extract that evoked 100% repellency or biting deterrence was petroleum ether extract at a dose of 1.5 mg/cm2 compared with 100% repellency for commercial formulation, N,N-diethyl- 3-methylbenzamide (DEET) at the same dose. Hence, these extracts can be used as an effective alternative to the existing synthetic pesticides for the control of Ae. aegypti. ª 2012 King Saud University. Production and hosting by Elsevier B.V. All rights reserved.

1. Introduction

* Corresponding author. Insect-transmitted disease remains a major cause of illness and E-mail address: [email protected] (T.M.Y. El-Sheikh). death worldwide. Mosquitoes alone transmit disease to more Peer review under responsibility of King Saud University. than 700 million people annually (Taubes, 2000). Therefore, the control of mosquitoes is an important public health con- cern around the world. Ae. aegypti (Culicidae) occurs in Asia, Production and hosting by Elsevier Africa and Central and South America and transmits virus of

Flavivirus genus, etiologic agents of human diseases like den- 2.2. Plant collection and extraction gue and yellow fever (Roberts, 2002). Yellow fever immunization programs have reduced the risk Fresh leaves of T. terrestris (Family: Zygophyllaceae) were col- of outbreaks in some endemic countries and the disease still oc- lected in the month of November 2011 from the Sabia city (Sa- curs in epidemic patterns only in some countries of Africa and bia Jazan desert road). The plant was identified by Dr. Wael Asia (World Health Organization, 2001). On the other hand, Kassem, Assistant Professor of Plant Taxonomy, Biology there is no vaccine for dengue fever; vector control is the only Department, Faculty of Science, Jazan University, and by com- form to minimize the transmission of the virus. It can be parison with the published plant description in flora of Saudi caused by four serotypes of the DEN arbovirus and clinically Arabia (Migahid, 1987). A voucher specimen has been depos- can happen in asymptomatic forms, classic dengue fever, ited in the herbarium of Biology Department, Faculty of Sci- hemorrhagic dengue fever and other more severe forms. ence, Jazan University. The leaves were washed and dried in Worldwide, 2.5 billion of people are in risk to acquire the dis- the shade at room temperature (27–31 C) for 7 days till they ease and 50 million are infected every year, characterizing a become brittle, then pulverized to powder in a hammer mill. pandemia (World Health Organization, 2002). Dengue The extraction was performed using 70% ethanol, acetone outbreaks in Geddah and Makkah in the Kingdom of Saudi and petroleum ether solvents. One hundred grams of powder Arabia (K.S.A.) in 1994 and re-emerging the second time in for each solvent separately was extracted three times with 2004, 2005, 2006, 2010 with cases of dengue registered by Min- 300 ml of aqueous 70% ethanol, acetone and petroleum ether istry of Health reached 400, 291, 305, 500 and 710, respectively at room temperature. After 24 h, the supernatants were dec- (http://www.aawsat.com). anted, filtrated through a Whatman filter paper No. 5. and Currently, most insecticides are non-selective and can be dried in a rotary evaporator at 40 C for (2–3) hours to ethanol harmful to other organisms and to the environment. There is and (40–60) minutes to other solvents to obtain 14.7 g (etha- an urgent need to develop new materials for controlling mos- nol), 6.3 g (acetone) and 2.2 g (petroleum ether) of a semi solid quitoes in an environmentally safer way, using biodegradable crude extract. The dry extracts were kept in a deep freezer and target-specific insecticides against them (Isman, 2006; (À4 C) until used for experiments. Pavela,2007; Jawale et al., 2010). Bioactive organic compounds produced by plants can act as repellent, oviposition or food 2.3. Larvicidal test deterrents, growth inhibitors, and toxins (Ezeonu et al., 2001; Carlini and Grossi-de-Sa´, 2002). Thus, crude plant extracts In order to study the toxicity of the concerned plant extracts, have been screened as natural and biodegradable forms to con- the tested material of the ethanolic extract was dissolved in trol pests and vectors of infectious diseases (Omena et al., 0.1 ml of 70% ethanol, while the tested material of acetone 2007). Based on the foregoing, the present study aimed to scien- and petroleum extracts was dissolved in 2 drop of Tween. 80 tifically evaluate the larvicidal and repellency or antifeedant as emulsifier to facility the dissolving of tested material in activity of Tribulus terrestris leaf extracts against the larvae water. Different concentrations of each extract were prepared and adults of the mosquito vector, Ae. aegypti. in order to detect mortalities. All tested materials were performed in 100 ml of dechlorinated tap water contained in 2. Materials and methods 200 ml plastic cups. Then, third instar larvae were put immedi- ately into plastic cups that contained different concentrations 2.1. Rearing of Aedes aegypti of extracts. At least three replicates were usually used for each tested concentration. All plastic cups were incubated under The dengue fever mosquito, Ae. aegypti larvae were collected controlled conditions (27 ± 2 C, RH 70 ± 10% and 12–12 in December 2011 through dipping method from the natural light–dark regime). Control larvae received only 0.1 ml of sites located in a village called Sanba at a distance of 10 km 70% ethanol or 2 drop of Tween.80 in 100 ml water. Mortality from Jazan, Kingdom Saudi Arabia to establish the mosquito was recorded daily and the dead larvae and pupae were re- colony in our laboratory in the Department of Biology faculty moved until adult emergence. of science, Jazan University. Larvae were then reared in 250 ml of glass beakers containing 200 ml of distilled water. 2% (w/v) 2.4. Repellent/antifeedant test of brewer’s yeast solutions was routine wise supplied to them as a food source. Pupae were transferred to glass beakers Standard cages (20 · 20 · 20 cm) were used to test the repellent (100 ml) containing 80 ml of distilled water and maintained activity of plant extracts. Different weight from each extract in the standard mosquito cages (30 · 30 · 30 cm) for adult was dissolved in 2 ml (70% ethanol or water + drop of emergence. Adults were maintained by providing with cotton Tween) in glass 4 · 4 cm to prepare different concentrations. circles soaked with 10% honey solution. Two days after emer- One ml from each concentration was directly applied onto gence, female mosquitoes were allowed to blood feed periodi- 5 · 6 cm of ventral surface of pigeon after removing feathers cally from pigeon host. A few days after having a blood meal, from the abdomen to evaluate the repellency against Ae. ae- gravid mosquitoes laid their eggs on the filter paper strips in gypti, compared with commercial repellent (Off!) 15% Deet the glass cups. The filter paper strips with eggs were brought (N,N-diethyl toulamide) (Johnson Wax Egypt) as a positive to dry under laboratory condition for overnight and then kept control. After 10 min of treatment, the treated pigeons were in the same condition until use for larval hatching. Mosquito placed in the cages containing at least 20 Ae. aegypti starved rearing and all experiments were conducted under laboratory females 5–7 d-old for 4 h. Control tests were carried out along- conditions at 27 ± 2 C, relative humidity 70 ± 10% and side with the treatments using ethanol or water. Each test was 12 h L:D phase. repeated thrice to get a mean value of repellent. Larvicidal and repellent effect of some Tribulus terrestris L., (Zygophyllaceae) extracts 15

2.5. Criteria studied aegypti. Complete larval mortality percent (100.0%) was caused at the highest concentration (1000 ppm). Meanwhile, After larval treatment, the larvae were observed daily until the larval mortality % decreased to 23.3% at the lowest con- pupation and adult emergence to estimate the following centration (200 ppm) (compared to 6.7% for the untreated lar- parameters: vae). A negative correlation between the pupation % and the Larval mortality was indicated by a failure to respond to concentration was observed, where the pupation % was mechanical stimulation (Williams et al., 1986). Larval mortal- 0.0% at the highest concentration (1000 ppm) compared to ity percent was estimated using the following equation (Briggs, 93.3% for the untreated group. 1960): larval mortality % = A–B/A · 100 (where: A = num- The lethal effect of ethanolic extract was not extended ber of tested larvae, B = number of tested pupa). to the pupal stage. Also, the adult emergence percent was Pupation rate was estimated using the following equation: not affected at all concentrations used as compared with the Pupation % = A/B · 100 (where: A = number of pupae, control. B = number of tested larvae). Pupal mortality was indicated by a failure to respond to 3.2. Acetone extract mechanical stimulation or failure to metamorphose into the adult stage. The pupal mortality percent was estimated using Arranged data in Table 2 indicated the biological activity of the following equation: Pupal mortality % = A–B/A · 100 acetone extract from T. terrestris (Leaves) against the 3rd in- (where: A = number of produced pupae, B = number of ob- star larvae of Ae. aegypti. As shown in table, the highest mor- served adults). tality percent (100%) was recorded at the concentration Adult emergence of males and females was counted and cal- (600 ppm) and the lowest mortality percent (13.3%) was ob- culated using the following equation: Adult emergence served at the lowest concentration (50 ppm) (compared to % = A/B · 100 (where: A = number of emerged adults, 6.7% for the controls). The pupation % of treated larvae de- B = number of tested pupae). creased as the concentration increased (86.7% at 50 ppm com- Repellency % was calculated according to Abbott (1925). pared to 93.3% for the controls). Repellency % = [%AÀ%B/100À%B] · 100 Where: A = per- The acetone extract exhibited a toxic effect against the pu- cent of unfed females in treatment, B = percent of unfed fe- pae resulted from the treated larvae at all the concentrations males in control. (400,200, 100 and 50 ppm) (the pupal mortality percents were 57.1%, 30.8%, 23.8% and 7.7%, respectively, compared to 2.6. Statistical analysis 0.0% at the control). The total larval and pupal mortality percents were 90.0%, 70.0%, 46.7% and 20.0% at 400, 200, 100 Statistical analysis of the data was carried out according to the and 50 ppm, respectively, compared to 00.0% for the untreated group. method of Lentner et al. (1982).LC50 was calculated using multiple linear regression (Finney, 1971). A remarkable reduction in the adult emergence was observed. At 400 ppm the adult emergence % was 42.9%, this percent increased to 69.2% at the next concentration 3. Results (200 ppm) and gradually increased reaching to the highest value (92.3%) at 50 ppm, while the adult emergence percent for The biological activity of ethanolic, acetone and petroleum the untreated group was 100.0%. The highest percent of adult ether extracts of leaves against the 3rd instar larvae of Ae. ae- mortality (66.7%) was recorded at 400 ppm, this percent de- gypti has been studied. The biological activity included the lar- creased to 22.2 and 12.5% at 200 and 100 ppm, respectively, vicidal activity, pupal rate, pupal mortality, total larval and compared to 0.0% for the untreated insects. pupal mortality and adult emergence rate. The present results can be arranged as follows: 3.3. Petroleum ether extract

3.1. Ethanolic extract As seen in Table 3, data indicated the biological activity of petroleum ether extract from leaves against the 3rd instar lar- Data given in Table 1 indicated the biological activity of etha- vae of Ae. aegypti. The highest concentration (100 ppm) nolic extract of T. terrestris against the 3rd instar larvae of Ae. caused complete mortality, meanwhile the lowest mortality

Table 1 Effect of ethanolic extract of Tribulus terrestris (leaves) on mortality percent of different stages of Aedes aegypti. Conc. Larval Pupation Pupal Larval and pupal Adult Adult ppm mortality (%) (%) mortality (%) mortality (%) emergence (%) mortality (%) 1000 100.0 ______800 86.7 13.3 0.0 86.7 100.0 0.0 600 70.0 30.0 0.0 70.0 100.0 0.0 400 53.3 46.7 0.0 53.3 100.0 0.0 200 23.3 76.7 0.0 23.3 100.0 0.0 Control 6.7 93.3 0.0 6.7 100.0 0.0 No. of tested larvae = 30; Conc. = Concentration; ppm = particle per million. 16 T.M.Y. El-Sheikh et al.

Table 2 Effect of acetone extract of Tribulus terrestris (leaves) on mortality percent of different stages of Aedes aegypti. Conc. Larval Pupation Pupal Malformed Larval and pupal Adult Adult ppm. mortality (%) mortality pupae (%) mortality (%) emergence mortality (%) (%) (%) (%) 600 100 ______400 76.7 23.3 57.1 28.6 90 42.9 66.7 200 56.7 43.3 30.8 15.4 70 69.2 22.2 100 30 70 23.8 14.3 46.7 76.2 12.5 50 13.3 86.7 7.7 0 20 92.3 0 Control 6.7 93.3 0 0 6.7 100 0 No. of tested larvae, Conc., ppm: see footnote of Table 1.

Table 3 Effect of petroleum ether extract of Tribulus terrestris (leaves) on mortality percent of different stages of Aedes aegypti. Conc. Larval Pupation Pupal Malformed Larval and pupal Adult Adult ppm mortality (%) mortality pupae (%) mortality (%) emergence mortality (%) (%) (%) (%) 300 100.0 ______200 90.0 10.0 100.0 100.0 100.0 0.0 ___ 100 73.3 26.7 100.0 100.0 100.0 0.0 ___ 50 43.3 46.7 82.3 64.3 90.0 17.7 66.7 25 26.7 73.3 54.5 25.0 66.7 45.5 20.0 Control 6.7 93.3 0.0 0.0 6.7 100.0 0.0 No. of tested larvae, Conc., ppm: see footnote of Table 1.

(26.7%) was recorded at the lowest concentration (25 ppm), Table 4 LC50 values (ppm) of ethanolic, acetone and petro- compared to 6.7% for the untreated insects. The pupation leum ether extracts of Tribulus terrestris leaves against Aedes percent was 73.3% at the lowest concentration (25 ppm). Fur- aegypti larvae. thermore, no pupation was observed at concentration Extract LC (ppm) Slop (b) Correlation coefficient (r) 300 ppm, (compared to 93.3% for the controls). 50 The lethal effect of petroleum ether extract was extended to Ethanol 376.4 0.107 0.954 the pupal stage at the all concentrations used: 200, 100, 50 and Acetone 173.2 0.184 0.863 25 ppm, where the pupal mortality percent was 100.0%, Pet. ether 64.6 0.406 0.369 100.0%, 82.3% and 54.5%; respectively, vs. 0.0% for the control. The total larval and pupal mortality were found to be highly affected by petroleum ether extract. The highest mortality (100.0%) was noticed at the concentrations 200 and Table 5 Repellency/antifeedant effect of ethanolic, acetone 100 ppm, the lowest mortality (66.7%) was noticed at the con- and petroleum ether extract leaves of Tribulus terrestris on centration 25 ppm; respectively compared to 6.7% at the con- Aedes aegypti. trol group. 2 There was a remarkable reduction in the percentage of Plant part Dose (mg/cm ) Repellency (%) adult emergence from pupae produced by treated larvae with Ethanol Acetone Petroleum the petroleum ether extract. The adult emergence percent extract extract ether extract (0.0%) occurred at the concentrations 200 and 100 ppm, Leaves 3.0 44.4 84.6 100.0 meanwhile the percent increased to 17.7% and 45.5% at the 1.5 32.0 73.0 100.0 concentrations 50 and 25 ppm compared to 100.0% at the con- 0.75 12.0 65.2 80.7 trol group. The petroleum ether extract had extended toxic Deet 1.5 100.0 100.0 100.0 activity on the survival adults resulted from treated larvae at Control ___ 0.0 0.0 0.0 the two concentrations 50 and 25 ppm because the adult mortality was recorded as 66.7% and 20.0%, respectively, compared to 0.0% for the controls. Recalling to the aforementioned results, the toxicity %s of 3.4. Repellency/antifeedant action tested extracts of T. terrestris (leaves) based on LC50 values (Table 4) can be arranged in a descending order as Generally, the repellency of the different plant extracts tested follows: petroleum ether extract > acetone extract > etha- and Deet gave a variable degree of repellency (Table 5). The nolic extract. efficacy of petroleum ether extract from T. terrestris and Deet Larvicidal and repellent effect of some Tribulus terrestris L., (Zygophyllaceae) extracts 17 at the dose (1.5 mg/cm2) produced the highest protection ether extracts induced reductions of the adult emergence. (100%) during the entire research period of 4 h post-treatment. The reduction was found to be concentration–dependent. While the other two plant extracts exhibited < 75% repellency These results are comparable to earlier results of Sharma within the 4 h post-treatment. The acetone and ethanolic ex- et al. (2006) using petroleum ether extract of A. annua against tracts showed repellency % of 73% and 32% at the same do- An. stephensi and C. quinquefasciatus larvae, respectively, se1.5 mg/cm2. Also the result showed that the relative Wiesman and Chapagain (2006) using one fraction obtained repellency increased as the dose was increased. The repellent from the silica gel column chromatography of the methanol activity of the present extracts against starved Ae. aegypti fe- extract against Ae. aegypti mosquito larvae and Pavela males varied according to the solvent and the doses used. (2009) using essential oils obtained from Thymus vulgaris, Satureja hortensis and Thymus satureioides plants, Gunaseka- ran et al. (2009) using Neem Azal against Ae. Aegypti, Patil 4. Discussion et al. (2011) Plumbago zeylanica (Plumbaginaceae) and Ces- trum nocturnum (Solanaceae) plant extracts against Ae. ae- Several diseases are associated to the mosquito-human inter- gypti. and Kovendan et al. (2012) using Carica papaya action. Mosquitoes are the carriers of severe and well-known (Caricaceae) leaf extract against, Aedes aegypti. Gang et al. illnesses such as malaria, arboviral encephalitis, dengue fever, (2000) concluded that, some plants such as T. terresteis, Eup- chikunguniya fever, West Nile virus, and yellow fever. These atorium fortunei and Datura stramouium have value for devel- diseases produce significant morbidity and mortality in hu- opment as botanical insecticides against C. pipiens. mans and livestock around the world. The plant tested in Results obtained in the present study indicated that the the present study is known to be ecofriendly and is not toxic toxicity of plant extracts against the 3rd instar larvae of to vertebrates but used as a medicinal plant. This plant is Ae. aegypti was extended to the adults causing mortality grown widely as herbal plant in Jazan region in Saudi Ara- reached to 66.7% for acetone and petroleum ether extracts bia. Moreover, it is clearly proved that crude or partially at the concentrations 400 and 50 ppm. Similar results were purified plant extracts are less expensive and highly effica- obtained by Shalaby et al. (1998) using peel oils of lemon, cious for the control of mosquitoes rather than the purified grapefruit and naval orange against C. pipiens larvae, Jeyaba- compounds or extracts (Jang et al., 2002; Cavalcanti et al., lan et al. (2003) using methanol extract of Pelargonium citros- 2004; Maurya et al., 2009; Kovendan et al., 2012). The pres- a leaf against An. stephensi, Nathan et al. (2005) using the ent study showed high bioactivity of the different extracts neem A. indica extract against An. Stephensi, Nathan et al. from T. terrestris against Ae. Aegypti. Such results may offer (2006) using methanolic extracts of leaves and seeds from an opportunity for developing alternatives to rather expen- the chinaberry tree Melia azedarach against An. stephensi sive and environmentally hazardous organic insecticides. and El-Sheikh et al. (2011) using leaf and stem extracts from Toxicity of the tested plant extracts against the 3rd larval Cestrum nocturnum (solanaceae) against Culex pipiens. instar varied according to the solvent used and the extract All the concentrations of plant extracts used in the present concentration. The larval mortality percent increased as ex- study exhibited repellency activity against the starved female tract concentration increased for all plant extracts. The toxic- adults of Ae. aegypti. The repellent action of the plant ex- ity values of tested extracts of T. terrestris based on LC50 tracts tested varied depending on the solvent used in extrac- values may be arranged in a descending order as follows: tion and the dose of the extract. The present study petroleum ether extracts > acetone extracts > ethanolic ex- indicates that the petroleum ether extraction of the plant used tracts. These results agree, to some extent, with the previously was more effective in exhibiting the repellent action against mentioned suggestions of (Maurya et al., 2009; Kovendan et the mosquito tested as compared with the ethanol and ace- al., 2012). Extracts from several other plant species were tone extraction and showed same repellency percent (100%) tested on different species of mosquitoes by many authors as a commercial formulation, N,N-diethyl-m-methylbenza- worldwide. The activity of the plant extracts on larval mor- mide (DEET). tality of Ae. aegypti, in the present study, was in agreement Many plant extracts and essential oils manifest repellency with the results obtained by Coria et al. (2008), Maurya activity against different mosquito species. The present re- et al. (2009), Govnidarajan (2010), Patil et al. (2010 and sults are in accordance with such results obtained by Shar- 2011), Kovendan et al. (2012). The evaluation of T. terrestris ma et al. (1995) using neem oils against mosquito bites of Linn (Zygophyllaceae) acetone extract for larvicidal activity Anopheles spp., Culex spp. and Ae. spp., Kim et al. (2002) against mosquito vector Ae. aegypti was tested by Singh using ethanol extract of fruits from Foeniculum vulgarea et al. (2008), The LC50 value of leaf acetone extract estimated against hungry Ae. aegypti, Yang et al. (2004) using metha- for 3rd-instars larvae Ae. aegypti was 185 ppm. In the present nol extracts from 23 aromatic medicinal plant species against study, also, acetone extract showed the same effect against female blood – starved Ae. aegypti, Choochote et al. (2007) Ae. aegypti with LC50 173.2 ppm but the petroleum ether ex- using repellent activity of selected essential oils from ten tract seemed to be more effective than other extracts with plant species against Ae. Aegypti, Chio and Yang (2008) LC50 values of 64.6 ppm. using neem tree (Azadirachta indica) oil against the Asian A remarkable decrease in the pupation percent was in- tiger mosquito (Ae. albopictus), Singh et al. (2008) using ace- duced by all plant extracts in the present study. The pupation tone leaves extract of T. terrestris against Ae. Aegypti. and % decreased as the concentration of the plant extract in- El-Sheikh (2009) using 16 ethanolic and petroleum ether ex- creased. Moreover, the pupation rate depended on the sol- tracts of 4 indigenous plants as a repellent in the field vent used in extraction. The present study showed that the against wild mosquitoes. Govnidarajan and Sivakumar toxic effect of acetone and petroleum ether extracts had been (2011) tested the repellent activities of crude hexane, ethyl extended to the pupae. In addition, acetone and petroleum acetate, benzene, chloroform, and methanol extracts of leaf 18 T.M.Y. El-Sheikh et al. of Eclipta alba and Andrographis paniculata at three different Govnidarajan, M., 2010. Larvicidal efficacy of Ficus benghalensis L. concentrations of 1.0, 2.5, and 5.0 mg/cm2 against important plant leaf extracts against Culex quinquefasciatus Say, Aedes vector mosquito Ae. aegypti and they suggested that the leaf aegypti L. and Anopheles stephensi L. (Diptera: Culicidae). Eur. solvent plant extracts have the potential to be used as an Rev. Med. Pharmacol. Sci. 14 (2), 107–111. 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