Evaluation of Lousicidal Activity of Ziziphus mauritiana Alkaloids and Eucaylptus camaldulensis Terpenoids Leaves Extracts in Chickens: An In Vitro Study

The present study aimed to highlight and assess the in vitro poultry anti-lice properties of Ziziphus mauritiana alkaloids and Eucalyptus camaldulensis terpenoids leaves extracts. In vitro poultry antilouse effects of the extracts of various concentrations (1 mg/mL, 5 mg/mL, and 10 mg/mL) using filter paper contact bioassay was applied. Results revealed that lice mortality was concentration-dependent for both studied extracts. The median lethal concentration (LC50) values were 3.687 and 1.045 for alkaloids and terpenoids extracts respectively. The results indicate that terpenoids extract has strong lousicidal activity. Further steps should be pursued to make these extracts to be tested in vivo then may be used as a novel treatment for poultry lice in future, allowing lice control in a less aggressive way to the environment.

different composition which occur as secondary metabolites [15]. Secondary metabolites and essential oils have various degree of lipophilicity, relative hydrophilicity, and volatility at room temperature [16]. In the current study Ziziphus mauritiana and Eucalyptus camaldulensis were selected because they are commonly available in Iraq.
Ziziphus mauritiana belongs to the family Rhamnaceae [17]. The name Ziziphus is related to an Arabic word and ancient Greeks used the word ziziphon for the jujube [18]. About 40 species of Ziziphus are available, Ziziphus mauritiana is very common [19].
Also Eucalyptus (family Myrtaceae), represented by over 700 species distributed throughout the world. In fact, Eucalyptus oil has been known for hundreds of years as antibacterial, antifungal and antiseptic in nature and it ranks top in quality and has advantages over essential oil from other tree crops [20]. The composition of Eucalyptus essential oils is very dissimilar, so their properties are extremely diverse even conflicting [21].
As known, synthesis of secondary metabolites in plants is determined by certain genetic characteristics (genotype) of the plant, physiological conditions, as well as edaphoclimatic factors. Thus, the chemical composition of plant extracts varies according to the plant origin [22,23], as well as cultivation of plant materials for extraction may imply fluctuation of the results [24].
Relatively, few studies were done on poultry lice available in Middle East [25]. To the best of our knowledge the poultry anti-lice activity of Ziziphus mauritiana alkaloids and Eucalyptus terpenoids of leaves extracts has not been investigated yet. Therefore, this study aimed to evaluate the possible in vitro poultry anti-lice properties of these secondary metabolites.

Materials & Methods
Collection of lice. Poultry lice were collected from naturally infested chickens taken from Najaf bird market. Identification of the lice was done by using insect identification keys [26,27,28]. Lice were collected from the predilection sites of the animals by blunt pointed forceps to avoid any harm to lice and host. Adult lice were examined for activity and morphological integrity under the dissecting microscope, the damaged ones were discarded. Institutional ethical and animal care guidelines were applied during sampling. Collection of plant samples. Ziziphus mauritiana (Rhamnaceae) and Eucalyptus camaldulensis (Myrtaceae) fresh leaves were collected from Najaf governorate/ Iraq in October 2016. The samples were positively identified in the herbarium of college of Education for Girls. In the lab, leaves samples were properly washed with distilled water, then were shed dried at room temperature for several days. The plant samples were ground using blender to obtain leaves powder then stored in refrigerator at 4° C till used. Phytochemical Screening Alkaloids test. Alkaloids were identified by using Dragendroff's reagent. (0.5) mL of Ziziphus aqueous extract in a test tube, equal volume of the reagent were added; appearance of orange precipitate was taken as positive result for presence of alkaloids [29]. Terpenoids test. Two mL of the extract of Eucalyptus were mixed with 2 mL of chloroform and 3 mL of concentrated sulfuric acid then heated for about 2 minutes. Grayish colour indicates to the presence of terpenoids [29]. Extraction Extraction of crude alkaloids. Ten grams of Ziziphus powder were extracted in soxhlet extractor with 200 mL of 95% ethanol for 24 hours then extract was dried by rotary evaporator. Dried material then was dissolved in 5 mL of ethanol, then 30 mL of 20% sulfuric acid were added to the alcoholic extract, rotary evaporator was used again, then little amount of 10% ammonium hydroxide were added to the residual acid solution till pH was equal to 9. Then this solution was extracted by separating funnel further four times with 10 mL of chloroform. The yield of chloroformic extract (inferior layer) was added to 10 grams of anhydrous sodium sulfate for demoisturizing. Finally, extract was filtered then re-evaporated using rotary evaporator again. Alkaloids were collected and kept at -20° C till used [30]. The extraction was repeated to obtain enough alkaloids. Extraction of crude terpenoids. Twenty grams of dry weight of Eucalyptus were added to soxhlet extractor with 200 mL of chloroform as a solvent, for 24 hours at 40° C. Chloroform was evaporated by rotary evaporator, the extract was collected and kept at -20° C till used [31]. The extraction was repeated to obtain enough terpenoids. Preparation of test concentrations. Three concentrations for alkaloids and terpenoids were prepared separately. One gram of each extract was dissolved in 10 mL of dimethyl sulfoxide (DMSO), then the volume was completed to 100 mL by adding distilled water and 0.2 mL of tween-20 to prepare 10mg/mL stock solution. Two diluent concentrations (5, 1) mg/mL were prepared according to the equation (N1V1=N2V2). Control treatment was prepared from 10 mL of DMSO and 90 mL of distilled water and 0.2 mL of tween-20. In vitro filter paper bioassay of lousicidal activity. Lice were divided into groups and were distributed in Petri dishes (6 cm in diameter) lined with Whatman no. 1 filter paper. Three mL of each concentration of extracts were pipetted onto individual filter papers. Filter paper contact bioassay method was used to evaluate the toxicity of different concentrations of present extracts [32]. The experiment was carried out in triplicate, six lice/ replicate (total n=18 for each concentration of the extract). Lice were incubated at 25° C in darkness. Adult mortality were noticed every 5 minutes for 120 minutes by dissecting microscope. The lice were regularly stimulated with a needle to determine their viability, lice showing no movement and complete absence of any vital signs were considered dead [33]. Statistical analysis. All experiments were performed in triplicate. Significant differences among the concentrations and the lethal time for each concentration were analyzed by least significance difference (LSD). Significant differences between the extracts were analyzed by T-test. Median lethal concentration (LC50) were calculated by Probit regression analysis. The results were represented as mean± standard deviation (SD). p-value < 0.05 was considered significant.

Results & Discussion
Biological control means destruction or elimination of pest or unwanted populations by natural means; phytotherapy represents one of them. The present study focuses on lousicidal effect of alkaloids of Ziziphus, and terpenoids of Eucalyptus extracts. To our knowledge the lousicidal effects of these extracts has not been previously reported on poultry.
The chicken louse Menacanthus stramineus under the dissecting microscope (40x) shown in Figure (1.A). Large amount of lice on the feathers of infested chicken during sampling (Fig.1.B). Filter paper contact bioassay to assess the toxicity of extracts ( Fig.1.C). Finally, the two dried extracts, alkaloids of Ziziphus, and terpenoids of Eucalyptus (Fig.1.D).
The current study appeared that Eucalyptus terpenoids extracts caused higher lice mortality than Ziziphus alkaloids extracts for all concentrations as shown in Table  (1). The statistical analysis observed that the concentration 10 mg/mL was significantly different for both extracts (p< 0.05). The higher extract concentration, the better mean mortality of lice. Also, terpenoids revealed significant differences at the concentrations (1 and 5) mg/mL as shown in Table (1). Terpenoids extract of Eucalyptus revealed higher mortality rates in all concentrations. This result was in agreement with the data noticed by [34] that proved pediculocidal activity of Eucalyptus essential oils. Terpenes of Eucalyptus showed to be effective against head lice [35]. Monoterpenes (1,8-Cineole and α-Pinene) were the most effective essential oils of Eucalyptus globulus against head lice [36]. Eucalyptus spp have been shown to release large amounts of terpenes into the atmosphere [37], some of these terpenes are insects repelling, for example, 1,8-cineole, α-terpineol and bicyclogermacrene [38]. Limonene demonstrated insecticidal activity in a mechanical way by penetrating the cuticle of the insect, by respiration, and through the digestive system [39]. Their low molecular weight can pass through the cuticle of louse up to the trachea causing the death by suffocation [16].
Other Eucalyptus terpenoids were Vinidiflorol and α-Penine, which have certain characteristics to be used as insecticides [40], these compounds prevent transmitting of nervous impulses in adults beetles Callosobruchus maculatus by inhibition of acetyl choline hydrolysis [41]. As suggested by [42], the mode of delivery of the essential oils and compounds was likely by vapor mode via respiratory system or spiracles blocking. Other study showed that fumigation tests of some monoterpenoids against female lice were more effective in closed cups than in open ones, indicating to the vapor phase action [36] .The cytotoxic activity of terpenoids is due to the containment of phenols, aldehydes and alcohols in their structure [43]. In many cases, terpenoids have been shown to act synergistically to get the overall pesticidal effect [44].
The lipophilicity of the products and the rate of diffusion through the cuticle are important factors which could affect the contact toxicity results [45,46]. The density and the molecular structure also affect the penetration rate. Essential oils with higher densities are generally more toxic than those with low densities [47]. Also, degradation of the essential oils component, movement of the compound to target site, and the ability of the insect to excrete the compound [48]; all these factors may cause the insecticidal effect.
Alkaloids display antiparasitic and antimicrobial properties. Recent research has proven that they are not toxic to the organisms that produce them, but toxic toward foreign organisms or cells and it is selective. Alkaloids can alter DNA, selectively deform cells [49]. More than 100 cyclopeptide alkaloids have been isolated from various plants of the genus ziziphus [50]. The cyclopeptide alkaloids of Ziziphus showed antibacterial and antifungal activity [51]. Phyto-chemically, six alkaloid compounds were demonstrated in Ziziphus mauritiana. The highest concentration was Iusiphine followed by Coclaurine [52]. These two compounds may be mainly responsible for the lice mortality in the present study.
Lethal time for the each concentration of the applied extracts significantly differed. In control group, the lice still alive even after the 120 min treatment period. The highest concentration of both extracts (10 mg/mL) demonstrated strong lice toxicity, and its activity tended to be time-dependent (Table 2). With regard to the concentration-response data, the median lethal concentration values (LC50) were 3.687 and 1.045 of alkaloid and terpenoid extracts respectively (Table 3). This observation indicates that terpenoid extract is more effective than alkaloid extract against poultry lice.

Conclusions and Recommendations
Eucalyptus camaldulensis terpenoids and Ziziphus mauritiana alkaloids showed potential lousicidal activity. Eucalyptus camaldulensis terpenoids was more effective lousicide, since its LC50 was less. The mortality of lice was observed within relatively short period of time; these data suggested them to be promising bio-control candidates against poultry lice. Further in vivo studies are needed to confirm these findings.