PHYTOCHEMICAL STUDY AND BIOINSECTICIDAL EFFECT OF THE CRUDE ETHONOLIC EXTRACT OF THE ALGERIAN PLANT ARTEMISIA

to the richness of the plant on phenolics compounds known for their bio-insecticide action.


INTRODUCTION
In Algeria the bean, Vicia faba L., is the crop that is part of farming systems since a long time in different agro-ecological areas of the country. It's the most important food legume with 58000 hectares or 44.3% of the total area reserved for this crop category (Bouznad et al., 2001). In addition to abiotic stresses (cold, frost, heat and salinity), beans crop are exposed to the harmful effects of weeds, fungal and viral diseases, nematodes and insects (Maatougui, 1996). Among subordinate insects, aphids have a special place. The black bean aphid, Aphis fabae, can severely damage bean plants by checking growth, decreasing yield and impairing the quality of the seed (Banks and Macaulay,1967). Honey dew excretion and growth of sooty molds create also an indirect damage through impending some physiological processes in plant (Hurej and Van Der Werf, 1993). Beside the mechanical damage caused, they also serve as the largest group of vectors of plant viruses (Blackman and Eastop, 2006).
However the yields and quality of agricultural products depend largely on the use of synthetic chemical insecticides needed to control the populations of these pests. These compounds are often found as residues in food and pose significant risks to human health and the environment. In the interests of environmental respect and in the context of sustainable development, it should greatly to reduce the amount of synthetic pesticides and develop alternative control strategies. These new approaches should be based on the combined use of biomolecules provided with bioinsecticidal properties. Many molecules that exhibit toxic and defensive action against plant pests have been identified and more than 2000 plant species with insecticidal properties have been identified (Benayad, 2008).
The genus Artemisia is one of the largest and most widely distributed genera of the family Astraceae (Compositae). It is a heterogenous genus, consisting over 500 diverse species distributed mainly in the temperate zones of Europe, Africa, Asia and North America (Kundan and Anupam, 2011).
Artemisia juadaica L. is a perennial fragrant shrub, with pubescent leaves, which grows widely in the deserts (Abd-Elhady, 2012) and is a very common anthelmintic drug in most North African and Middle-Eastern countries where it is known by the Arabic name of "Shih" (Van Wyk and Wink, 2004). The plant has been used also to treat gastro-intestinal disorders, poor eyesight, cardiovascular disease, skin disorders, and weak immune systems as well as to decrease the risk of atherosclerosis, cancer, and arthritis (Liu et al., 2004;Abd-Elhady, 2012).
The aim of this study was to investigate the insecticidal effect of methanolic extract of the aerial parts of A. judaica against the black bean aphid, A. fabae Scop. under laboratory conditions.

MATERIALS AND METHODS
Plant collection and preparation of crude ethanolic extract Aerial parts of A. judaicawere collected during spring seasons 2012 in the Tamnraset region (South of Algeria). The plant was taxonomical identified and confirmed by PrAbdelkrim from the National High College of Agriculture, Algiers, Algeria. The crude ethanolic extract of the above-ground portion of the plant was prepared from leaves, flowers and stems dried in the shade and ground into a fine powder using electrical blender. The extraction was carried out by macerating the powder for 3 days in ethanol, followed by filtration and evaporation at 40°C. The percentage of yield was calculated and the dried extract was kept at 4 °C until further use.

Phytochemical screening
The ethanolic extract was tested for plant secondary metabolites, alkaloids, sugar, phenolic compounds, flavonoids, saponins, tannins, iridois and coumarins. Phytochemical screening of the extract was carried out according to the standard method (Dohou et al., 2003). Visible color change or precipitate formation was taken into consideration for presence (+) or absence (-) of particular active constituents.
Test organisms and bioassays Stock of adults wingless aphids used in this study was collected randomly from infested bean filed in Ain Taya, Algiers area. The bioassays were conducted on Petri dishes under laboratory conditions at temperature of 22 ± 2 °C, 40-80% relative humidity and 16L:8D light regime. Forty aphids were transferred to Petri dishes on fresh leaves of V.fabae serving as a support for the aphids. Wet cotton discs were placed under the bean leaves to keep them fresh during the test period. Four doses of crude ethanolic extract were prepared (12.5, 6.25, 3.12 and 1.56 mgmL -1 ). An appropriate quantity of A. judaica extract was dissolved in ethanol to obtain each test solution. The insecticide solution was applied by topic application (contact) to adult aphids using micropipette. Controls were treated with only absolute ethanol. Mortalities percentages were determined for each treatment after 2, 4, 24, 48, 72 and 96h. The LD50, the concentration that produces 50% mortality, was determined by log probit analysis.

Enzymatic assays
The AChE activity was carried out following the method of Ellman et al. (1961) using acetylthiocholine as a substrate. Aphids were sampled from control and treated groups (at low dose 1.56 mgmL -1 ). Pools of twenty adults aphid were homogenized in the solution containing 38.03 mg of ethylene glycol tetraacetic (EGTA), 1mL Triton X-100, 5.845 g NaCl and 80 mL Tris buffer (10Mm, pH 7). The homogenate was centrifuged (5000 g for 5 min at 4°C), and the resulting supernatant was used for enzymatic assay. The AChE activity was measured in aliquots (100µL) of resulting supernatants added to 100 µLof 5-5' dithiobis-(2nitrobenzoic acid) (DNTB) in Tris buffer (0.01 M, pH 8)and 1 mL Tris (0.1 M, pH 8).After 5 min, 100µLof acetylthiocholine was added. Measurements were conducted at a wavelength of 412 nm with a run time of 20 minutes. GST activities were determined with the soluble fraction as enzyme source. GST activities toward 1-chloro-2, 4-dinitrobenzene (CDNB) were measured according to Habig et al. (1974). Treated (at low dose 1.56 mgmL -1 ) and control aphids were homogenized in sodium phosphate buffer (0.1 M, pH 6) and centrifuged (14000 g, 30 min). Two hundred microliter of the resulting supernatant was added to 1.2 mL of reaction mixture containing 1Mm of CDNB and 5 Mm of reduced glutathione (GST) in the homogenization buffer. Changes in absorbance were recorded at 340 nm. Total protein content was determined according to method of Bradford (1976) using bovine serum albuminasa standard. Enzyme activities were expressed as µmolmin -1 mg -1 proteins.

Statistical analysis
Results are expressed as means ± standard deviation (SD). To identify significant effects of the treatments on the variables measured. Data were submitted to a monofactorial ANOVA using XLSTAT 7.5.2. Means were compared using Tukey's HSD test (P< 0.05).

RESULTS AND DISCUSSION Phytochemical screening
The crude ethanolic extract of A. judaica was subjected to qualitative phytochemical screening to identify presence or absence of selected chemical constituents using classical methods of analysis. The results of phytochemical study (Table 1) revealed the presence of phenolic compounds, flavonoids, alkaloids, tannins, saponins and comarins. Antocyans and iridoiswere absents.

Insecticidal activity
The results of the toxicity assay against of the black bean aphid, A.fabae, are given in the figure 1 and the table 2. The test compound showed high insecticidal activity for all tested concentrations. At the highest dose 12.5 mgmL -1 , the 100% of mortality were recorded 2 hours after treatment, and for the lowest dose (1.56 mgmL -1 ) it was after 96 hours. Mortality rates ranged from 50.83 to 71.66% at 2 h after treatment for the average concentration 3.12 and 6.24 mgmL -1 respectively. Total mortality (100%) was achieved 48h and 72h after treatment respectively for the concentrations 6.24 and 3.12 mgmL -1 . The results of probit analysis showed that A. fabae was susceptible to the crude ethanolic extract of A.judaica. The LD50 was obtained 2h after treatment.

Enzymathic effects
The effect of the crude ethanolic extract of A. judaica on enzymatic activities (GST and AchE) is presented in figures 2 and 3. The results showed an inhibition of AchE activity in treated aphids at high concentration of the crude extract of A. judaica. However, an activation of GST activity was observed on treated aphids.  Certain natural products can be suitable alternatives to synthetic pesticides owing to their generally reduced negative impacts on humans, beneficial insects and the environment. Higher plants constitute a diverse source of highly bioactive agents that include some that have contributed significantly to the successful use of natural products and analogues for crop protection (Akhtar and Isman, 2013). In this study, phytochemical screening and the insecticidal activity of the crude ethanolic extract of the Algerian plant A. judaica were studied. The results of phytochemical screening revealed the presence of phenolic compounds, flavonoids, alkaloids, tannins, saponins and comarins. These results are in agreement with knows compositions of many Artemisia species (Masotti et al., 2012). Kundan and Anupam (2011) reported that the Artemisia species comprise mainly terpenoids, flavonoids, coumarins, caffeoylquinic acids, sterols and acetylenes.
Many researchers have reported on the effectiveness of plant extracts against insects (Acheuk et al., 2012;Abdellaoui et al., 2013Abdellaoui et al., , 2016Pavela,2004 andNathan et al., 2006). In the present work, the crude ethanolic extract of A. judaica showed potent insecticidal effects against the black aphid, A.fabae. Total mortality (100%) was achieved 2h after treatment with the higher concentration. This toxic effect of the extract might be due to the various bioactives compounds that exist in the aerial part of the plant. Indeed, the crude extract of plant is a mixture of potentially bioactive substances which may act synergically (Acheuk et al., 2012) or independently (Kabir, 2013). Previous studies demonstrated the insecticidal effects of Artemisia plant extracts against other insects pest. Abd-Elhady (2012) reported that volatile oils from the aerial parts of A. judaica, were found to have an insecticidal effect against Callosobruchus maculatus (Fab.). A concentrated extraction of 50% was shown to have the highest mortality. As our results, Masotti et al. (2012) reported that ethanolic extracts of A. molinieri and A. campestris Varglutinosa showed larvicidal activity against mosquito Culex pipiens. However, extracts of A. molinieri revealed a higher larvicidal activity than those of A. campestris. The biocide differences found for the tested extracts can be explained by their different chemical compositions, with aromatic polyacetylene dominant fraction for A.molinieri ethanolic extracts. In the same way, Dane et al. (2016) reported that the methanolic extract from A. absinthium showed potent toxicity for Sitophilus oryzae and causing 100% in 24h at a dose of 60 mgcm -2 .
Many natural plant compounds used in the control of insect pest are known to exhibit effects in the enzymatic profiles (Smirle et al., 1996;Zhang et al., 2013). The finding of our study showed that, the crude ethanolic extract of A. judaica inhibited AchE activity on treated aphids. It's known that AchE regulate nerve impulse transmission across cholinergic synaps. Monoterpenoids tested by López and Pascual-Villalobos (2010), showed inhibitor effect on acethylcholinesterase enzyme. From the all compounds tested by these authors, fenchone, S-carvone and linalool produced the highest inhibition. Similarly, the study conducted by Abdel-Aziz et al. (2015), demonstrated that, rosacide treatment showed the highest AchEpercent of inhibition (72.06%) on Aphis craccivora. This inhibition is due to the presence of high amount of the monoterpenoid 1, 8-cineolein rosacide, which is known for its insecticidal, feeding, deterrent and repellent properties. It inhibits acetylcholinesterase by occupying the hydrophobic site of enzyme's active centre. In agreement, Nathan et al.(2008) found that Azadirachtin significantly inhibits the activity of AchE in Nilaparvata lugens.
In insects, allelochemical defense system include P450, gluthathione Stransferase and esterases which are typically concentrated in midgut allowing rapid elimination of ingested toxic substances (Rattan, 2010). Elevated detoxification enzymes activity in insects tissues are often associated with enhanced detoxification of allelochemicals (Valles et al., 1999). GST play a pivotal role in detoxification and antioxidant defense of insects against natural and synthetic exogenous xenobiotics including insecticides, allelochemicals and endogenously activated compounds (Papadopoulos et al., 2004). GSTs catalyse the addition of a tripeptide glutathione to a wide variety of electrophilic substrates including host plant secondary compounds (Yu and Hsu, 1993). In our study, the GST activity in A. fabae was significantly stimulated by the low dose of the crude ethanolic extract of A. judaica. Similarly, different plant secondary compounds; catechol (phenolics), gramine (alkaloid) and L-ornithine-HCl (non protein amino acid), tested in the artificial diet activated the GST activity in the English grain aphid Sitobion avenae and a significant correlation was also observed between the concentration of each compound in diet and GST activity in aphids (Zhang et al., 2013). Our findings are also in agreement with those of Abdel-Aziz et al. (2015) who reported that the green insecticide Cura (curcuma oil mixed with mineral and vegetable oils) increased the GST activity with (10.16%) than control in Aphis craccivora.

CONCLUSIONS
The results of our study demonstrate the potent aphicidal activity of the crude ethanolic extract of the Algerian Asteraceae A. judaica. However, further studies for guided isolation of the insecticidal compounds and their effects on the non target organisms are strongly needed.