Larvicidal Activity of Essential Oil of Syzygium aromaticum (Clove) in Comparison with Its Major Constituent, Eugenol, against Anopheles stephensi.

Background
In this study, larvicidal activity of clove essential oil (EO), as a green and relatively potent larvicide, was compared with its main constituent, Eugenol, against Anopheles stephensi.


Methods
High-performance liquid chromatography (HPLC) was used to determine the amount of eugenol, major constituent of clove EO. In addition, larvicidal activity of clove EO and eugenol was evaluated against An. stephensi.


Results
The amount of eugenol in clove EO was determined as 67% using HPLC analysis. LC50 and LC90 of clove EO (57.49 and 93.14ppm, respectively) were significantly lower than those of eugenol (86.96 and 128.18 ppm, respectively).


Conclusion
EO showed more effective than its major component. Considering the lower cost of the essential oil and lower risk in occurrence of resistance in larvae, use of clove EO is preferred as larvicide in comparison with eugenol, against An. stephensi.


Introduction
More than 17% of all infectious diseases around the world are vector-borne diseases, such as dengue fever, yellow fever, and malaria (1). The number of death for such diseases is more than 1 million annually e.g. malaria caused 429000 death just in 2015 (1,2). In order to control malaria, WHO recommends control of larva which now used in 55 countries (2). Unfortunately, due to frequent use of synthetic larvicides (such as Temephos), not only environmental pollution have appeared, but also many cases of resistance has occurred in mosquitoes around the world (3)(4)(5)(6). Essential oils (EOs) have been suggested as alternative sources for insect control as repellents, insecticides or larvicides and they offer ad-vantages such as biodegradability and negligible effects on non-target specious and environment (7, 8).
Recently, comparisons of larvicidal activity of EOs with their major components have been reported (9, 10). However, there is no conclusion so fare, EOs are better or their major constituent(s) in terms of larvicidal activity.
Syzygium aromaticum (Clove) belongs to the Myrtaceae family which considers as an important medicinal plant with wide range of biological activities such as anti-bacterial or anti-oxidant activities (11,12). Clove EO has also shown larvicidal activity against field collected larva of Ae. Aegypti with LC50 of 92.56 and 62.3ppm in two different reports (13,14). Eugenol, as the major constituent of clove EO, has also indicated larvicidal activity against laboratory reared and field collected of Ae. aegypti (LC50: 33 and 93.3ppm, respectively) (14,15). Its larvicidal activity against other population of mosquito has been documented, with LC50 of 25.4, 28.14 and 30.8ppm against An. subpictus, Ae. albopictus and Cx. tritaeniorhynchus (16).
Anopheles stephensi is an important malaria vector with wide distribution in the Arabian Peninsula, the Indian subcontinent, Afghanistan and Iran (17)(18)(19). In this research, for the first time, larvicidal activity of clove EO and eugenol against An. stephensi was evaluated and compared.

Materials
High-performance liquid chromatography (HPLC) grade methanol, pure eugenol (99%) and Ethanol were supplied by Merck (Germany). Clove EO was purchased from Green Plants of Life Co (Iran).

Determining of eugenol contents in clove EO oil by HPLC
HPLC analysis was used to determine the amount of eugenol in clove EO. The apparatus consisted of a 30cm× 3.9mm reverse phase C18 column (Waters, Milford, USA), a pressure less injection pump (Model L-6200, Hitachi, Japan) to drive solvent and loading of samples, a UV visible detector (Model L-4000, at 280nm, Hitachi, Japan) for detection, a chromato-integrator (Model D-2500, Hitachi, Japan) for analyses. A mixture of methanol and distilled water was used as mobile phase with flow-rate of 1mL/min. Analytical procedure was started with dissolving 50µL of eugenol or 0.5mL of clove EO in 10mL of methanol, then, 20µL of this solution was injected into system at a flow-rate of 0.7mL/ min. The optimum mobile phase with a methanol: water ratio of 80:20 was used for elu-tion. By comparing peak areas of eugenol with solution of clove EO, its amount in clove EO was determined.

Evaluation of larvicidal activity
Third and fourth instar larvae of An. stephensi were used, they obtained from insectarium of Tehran University of Medical Sciences. They reared in special condition: 28± 2 °C, 12:12 dark and light periods and relative humidity of 65±5%. Larvicidal bioassay was performed in line with WHO recommendation test in lab, with some modifications (20). Standard solutions were prepared by dissolving in ethanol at appropriate concentrations (i.e. eugenol 60 µL/mL and clove EO 30µL/ mL). By adding 1mL from each sample (0.5%v/v) to cups containing 199mL of no chlorine water, desired concentrations of samples were prepared. Using separated nets, 25 larvae of An. stephensi were added slowly to all containers. Dead and moribund larvae (unable to respond to stimulating agent) were counted after 24h of exposing in all cups. Larvicidal bioassays were performed in 16 repetitions at 4 different replicates at concentrations (ppm) of 12.5, 25, 50, 75, 100, 150 for clove EO and 12.5, 25, 50, 100, 150, 200 and 300 for eugenol. In each replicate, two control groups were considered having ethanol (0.5%v/v) with similar treatments. Lethal concentrations of each sample (i.e. LC50 and LC90), were determined using a probit regression model in SPSS ver. 19 (Chicago, IL, USA).

Comparison of larvicidal activity of clove EO with eugenol
Evaluation of overlaps between confidence intervals (CI) of two groups is an easy and common approach to compare various LC values. If no overlap is observed, the difference is considered as significant (21). LC50 and LC90 of clove EO and eugenol were calculated and compared using independent sample test by SPSS.

Determining content of eugenol in clove EO
Results of HPLC analysis of eugenol and clove EO samples are shown in Fig. 1. Comparing the graphs, the peak related to eugenol is observed at retention time of 6.43 and 6.41min, for eugenol and clove EO, respectively. By comparing peak areas of eugenol and clove EO, percentage of eugenol in clove EO was calculated as 67%, considered relatively high.

Evaluation of larvicidal activity of clove EO and eugenol
Results of larvicidal activities of clove EO and eugenol against An. stephensi are shown in Fig. 2. Calculated LC50 and LC90 values were 57.49 and 86.96ppm for clove EO and 93.14 and 158.2ppm for eugenol, respectively (Table  1).
Larvicidal activity in both samples (i.e. clove EO and eugenol) appeared at 25ppm and enhanced with increasing the concentration of those. Nevertheless, perfect larvicidal activities were achieved at 100 and 200ppm for clove EO and eugenol, respectively. Furthermore, no overlap in CI of LC50 and LC90 for clove EO and eugenol are observed, thus, larvicidal activity of clove EO is significantly better than eugenol (Table 1). Moreover, Probit regression line of the both clove EO and eugenol are illustrated in Fig. 3. A= intercept; B±SE= slope and standard error of the line; CI= confidence interval (0.05), UCL= Upper Confidence Limit, LCL= Lower Confidence Limit, χ2 (df)= Chi 2 and degree of freedom. *Since the significance level is less than 0.15, a heterogeneity factor is used in the calculation of confidence limits.

Determining content of eugenol in clove EO
In this study, amount of eugenol in clove EO was found as 67%, which is comparable with other reports. Reviewing other reports, content of eugenol has been reported in values more or less similar to this value: e.g. 58.29% (22), 59.29% (23), 76.8% (24), 86.61% (25) and 88.58% (26).
Larvicidal activity of either of clove EO and eugenol, against other species of mosquito, has already been reported. Ae. aegypti is shown to be more susceptible than Cx. quinquefasciatus when using clove EO (i.e. LC50: 92.56 vs. 124.42ppm) (13). Larvicidal activity of synthetic derivatives of eugenol against Ae. aegypti has been shown (LC50~ 62.3ppm or higher) (14).
Recently, many reports have been released about comparison of larvicidal activity of EOs with their major components. However, there is no conclusion so far whether EOs outperforms or their major constituent in terms of larvicidal efficacy. LC50 in Allium tuberosum EO against Aedes albopictus was found to be 17.9ppm, lower than that of its two major components, allyl methyl trisulfide and dimethyl trisulfide, with LC50 of 27.5 and 36.4ppm, respectively (38). LC50 of Ruta chalepensis EO has been evaluated against Anopheles quadrimaculatus (14.9 ppm) and Aedes aegypti (22.2ppm). While, 2-undecanone, its major component, showed similar LC50 to that of total EO against An. quadrimaculatus (14.2ppm), significantly lower values against Ae. aegypti (14.37ppm) were obtained (21).
Synergistic effects of constituents of some EOs are nowadays well-known when they are used as anti-fungal or anti-bacterial agents (39,40). Our findings in this research and our previous study also show that An. stephensi is more susceptible to the clove EO or EO of Kelussia odoratissima (with LC50 of 57.49 and 4.77ppm, respectively), compared with their major constituents, eugenol (93.14ppm) and Z-ligustilide (8.73ppm), respectively (28). A type of synergism may have occurred in larvicidal activity of the EOs too.
EOs are mixtures of many constituents such as flavonoids, alkaloids, and monoterpenes (41,42). Modes of action of mentioned constituents are different e.g. main site action of flavonoids is acetylcholinesterase (43), while alkaloids and monoterpenes target Na-K-ATPase or Na + and K + channels (44)(45)(46). This could be the main reason for occurring synergism in larvicidal activity in our study. Having mentioned that resistance against larvicides is most-ly observed when a single active agent is used compared with those having multi components (47)(48)(49).

Conclusion
Use of clove EO as a green larvicide against An. stephensi is preferred compared with its major constituent (Eugenol). Considering the fact that the EO is a lot cheaper than eugenol and is composed of several components, thus, has lesser chance of occurring resistance, the whole EO may be suggested as a proper larvicide.