Chemical Composition and Bioactivity of the Essential Oils Derived from Artemisia annua against The Red Flour Beetle

Triboliumcasteneum is the mostresistant stored grainspest causingmore than 40% grains loss per year.Replacement of the conventional fumigants with an eco-friendly alternative seemed an intelligent move to control the pest which has inclined global research towards the efficacy of pesticidal plants. In the race of finding a better insecticidal candidate, we focused on to the chemical compositionof the essential oils (EO) derived with polar and non-polar solvents from Artemisia annuaand their possible bioactivity against the pest species. GC-MS analyses of Chloroform and n-Hexane derived EOs showed the dominance of Oxygenated Sesquiterpenein the extract. Adults were found more vulnerable to n-Hexane EO (LD50= 0.71 mg adultÉ 1) than to chloroform derived EO (LD50= 0.97mg adultÉ 1) in contact toxicity assays. In the fumigant bioassayboth the adults and larvae were found susceptible towardsn-Hexane EO with LD50 0.66 & 0.53 mg L airÉ1 respectively.Evaluation of the biomolecular profile of adults and larvae at their lethal doses to understand the molecular mechanism underlying oxidative stress has shown significant downfall (pÂ0.01) in the activities of protein, AChE, GST, GSH whereasup regulation of LPOwas distinctly marked. The basic knowledge of employing potential solvent in eluting EOs of A.annua would prove to be an efficient environmental friendly management tool against T.casteneum.

Global demand for food is increasing continuously due to overshooting population.Thisposes great challenge to the sustainable utilization of stored grains which accounts for about more than 70% of their total yield.Unfortunately, stored conditions are a major attraction to different types of infestation mainly by insects due to infinite food resources and favourable abiotic factors (Howe, 1943).Triboliumcasteneum (Herbst, 1797) is the most resistant species among the huge list of stored grains pests and known to exploit a wide range of stored products (Hagstrum, 2017).They aretestified as the primary pest of wheat flour & other milled products and secondary pest of wheat grains (Good, 1933).Studies reported that more than 40% quantitative and qualitativedevastation of wheat ûour is caused by the beetle (Ajayi&Rahman, 2006).The flour beetle is also known to secrete toxic quinones which turns the flour greyish and hence reduces its aesthetic and nutritive values (Ladisch et al., 1967).Moreover, carcinogenic attribute of the quinones areaffirmed thus poses serious risk to human health (El-Mofty et al., 1989).Hence controlling the pest is of utmost importance.
Fumigation through methyl bromide and phosphine is the most dominant measurepracticed in most of the countries to manage stored product pest (Bell, 2000).However, methyl bromide is banned worldwide due to its association in ozone layer depletion (Anbar et al., 1996).Use of phosphine has already triggered much negative impactdue to their fast growing resistance (Benhalimaet al., 2004).Replacement of these conventional fumigants with an eco-friendly alternative seemed an intelligent move to control the pest which has inclined global research towards the efficacy of pesticidal plants.In the race of finding a better insecticidal candidate, different variants of plants are tested andfortunately emerged as a potent solution towards the challenge (Okwute, 2012;Chaudhary et al., 2017).Plants are known to contain a wide range of essential oils (Sasidharan et al., 2011).The Essential Oils (EO) contain plethora of organic compounds which are relatively non-toxic for the environment and can be used as a potent alternative for the synthetic pesticides (Isman, 2000;Caballero-Gallardo et al., 2011).The views were also supported by previous studies whereEOsof Ricinuscommunis seeds and Daturastramonium extracts were found effectivein controlling the red flour beetle (Abbasipour et al., 2011;Babarinde et al., 2011).
Genus Artemisia is the most widely distributed genera of Asteraceae family (Chu et al., 2012) and is extensively used for its medicinal properties in Asian countries (Das, 2012).Artemisia and its EOsare the subject of research interest since decades which is reflected in the wide range of studies conducted across the globe (Bora & Sharma, 2011).The genus was reported to pose toxic effect against pathogens and can be used in human diets and animal fodder (Janssen et al., 1987).It was also testified to possess insecticidal and antifeedant activities (Liu et al., 2006;Gonzalez-Coloma et al., 2012).EOs from the Artemisia sieberiBesser has shown insecticidal properties against three stored grain pest (Negahban et al., 2007).Moreover, EOs from Artemisia princepsare described to be an effective repellent and insecticidal candidate against two major stored grain pests (Liu et al., 2006).
Artemisia annua, the sole producer of Artemisinin, is mainly studied for its efficacy against the malarial parasites, Plasmodium (Dhingra et al., 1999).Artemisinin-based combination therapy is emerged as the most efficient antimalarial drug available in the market against MDR strains (Klayman, 1985;Eastman &Fidock, 2009).However, to the best of our knowledge, studies focusing on the candidature of potent solvents for the extraction of EO from A. annuaare still unknown.Taking into account the serious infestation caused by Tribolium which eventually pose negative impact on human health and on country's economy, the very need for the study was sensed.Hence, authors tried to decipher the chemical compositionof the essential oils (EO)eluted with Chloroform (polar) and n-hexane (non-polar) solvents from Artemisia annuaand testified their possible bioactivity against Triboliumcasteneum.In the present study, fumigant and contact toxicity of A. annua EOsagainst the pest was evaluated.EO obtained by hydro-distillation was analysed through Gas-chromatography to identify major chemical constituents.Additionally, metabolic interference imposed in the treatment sets of T. casteneum (Herbst, 1797) was evaluated.

Insect
Tribolium.casteneum(Herbst,1797) were collected from a small culture maintained in thedivision of Entomology of the Department of Zoology, The Maharaja Sayajirao University of BarodaVadodara, Gujarat, India.Unsexed adults werereared in the defined culture media of wheat flour, wheat grain and yeast in the ratio of 6:3:1.Insects were maintained in the humidity chamber at the suitable temperature and humidity ranges of 27±2ÚC, 70±5 RH respectively.Newly emerged adults of 1-10 days old, were used for the toxicity assays.Final larval stages i.e. 14 days old larvae were used in the experiment.All the experiments were conducted in the dark under the same temperature and humidity ranges.

Plant material
The dried, finely grounded leaves of the A.annuawere procured fromProfessor Neeta Pandya,Department of Botany, The Maharaja Sayajirao University of Baroda, Vadodara, Gujarat, India.Plant powder was then stored in plastic bags at 4ÚC until used for the extraction of EOs.

Extraction of essential oils
Dried plant powder (25 grams) was subjected to hydro-distillationusing a modified Clevenger-type apparatusfor the extraction of EOs (Clevenger, 1928).Onset of distillation was marked with the boiling of the selected solvents contained in the round bottom flask.300 mL of both Chloroform and n-Hexane were used separately for 9& 2 hours respectively for the extraction of EOs.Distillation process continued till the solvent becomes transparent in the extraction chamber.The oil layer was then separated from the aqueous phase using a separating funnel.EOs were then collected and dried over anhydrous sodium sulphate to remove extra water.Crude extracts were further processed inrotary evaporator to remove extra solvents at their boiling ranges.Oil yield was calculated on a dry weight basis employing the Yield (%) formula.

Yield (%)
Where, W EO is the weight of dry EO and W I is the weight of fresh plant powder taken for extraction.Extracts were then stored in the airtight containers in a refrigerator at 4 ÚC until it is used.

Chemical analysis of EO-Gas Chromatographymass spectroscopy (GC-HRMS)
Gas chromatographic analysis was performed on an Agilent 7890N instrument equipped with a flame ionization detector and HP-5MS (30m × 0.25mm × 0.25ìm) capillary column, while the EO components were identified on an Agilent Technologies Jeol mass spectrometer.The GC settings were as follows: the initial oven temperature was held at 60 °C for 1 min and ramped at 10 °C min"1 to 180 °C for 1 min, and then ramped at 20 °C min"1 to 280 °C for 15 min.The injector temperature was maintained at 270 °C.The samples (1 ìL) were injected neat, with a split ratio of 1:10.The carrier gas was helium at flow rate of 1.0 mL min"1.Spectra were scanned from 20 to 550 m/z at 2 scans s-1.Most constituents were identified using gas chromatography by comparing their retention indices with those of the literature and previous studies.The retention indices were determined in relation to a homologous series of n-alkanes (C8-C24) under the same operating conditions.Further identification was made by comparison of their mass spectra on both columns with those stored in NIST library or with mass spectra from literature.Component relative percentages were calculated based on GC peak areas without using correction factors.

Repellency test
Repellency in insects was evaluated according to Cosimi et al. (2009) where pests were exposed to different concentrations of chloroformand n-hexane derived EOs dissolved in acetone.Filter papers measuring 7 cm in diameter were cut into two equal halves where one half was treated with the desired concentration of EO and other half with acetone.After drying for two minutes, both the halves of filter paper were attached underside with the cellotape and fixed to the petriplate.10 unsexed adults were released into the centre of the plate.Five replicates were maintained for each concentration.Readings were taken at the interval of 1, 2, 3, 4, 5, 6, 12 and 24 hours.Insects were then transferred to the plastic vials containing media and checked regularly for 3 days if any mortality is recoded.Raw data was converted to calculate percentage repellency using the following formula: PR = 2(C -50) Where C is the percentage of insects recorded on the untreated half of the disc.Positive values expressed repellency and negative values attractancy.Results of PR were analysed using ANOVA and Tukey's pairwise comparison test.

Contact toxicity
To evaluate contact toxicity following the method of Huang et al. (1998), adults and larvae were treated with the desired concentrations of chloroform and n-hexane derived EOs. 10 unsexed adults were taken in plastic vials and kept in the freezer for one minute.This made them unconscious and hence their handling became easy.An aliquot of 5µl of EO was then topically applied on the meso-thoracic region.After 2 minutes they were transferred in the plastic vials containing media.The mortality was recoded till 3 days at the interval of 12 hours.

Fumigant toxicity
Fumigant toxicity was assessed according to López et al. (2008).Filter papers (Whatman No.1, 7 cm in diameter) were impregnated with the different concentrations of chloroform and n-hexane derived EOs.Insects were exposed to concentrations ranged from 0.24 to 2.37 mg L airÉ 1 of polar solvent and 0.14 to 1.42 mg L airÉ 1 of nonpolar solvent.Concentrations were decided after of 12 hours.Loss of antennal and leg movement was considered as an indication for mortality.
In the toxicity assays, data obtained at the end of the third day was considered as final and processed further for statistical analysis.Probit analysis (Finney, 1971) using Medcalc software was employed in analysing the dosage-mortality response in both the acute toxicity assays.

Biochemical analysis
Quantitative analyses of biochemical constituents in treated (LC     Pairwise Comparison Test were employed using Sigma plot 13.0 statistical software package to compare means.
In both the EOs, higher % of Oxygenated Sesquiterpene was recorded with 20.08% and 36.02% in case of chloroform and n-Hexane extracts respectively.The second largest group identified was ketones in chloroform EO and alcohol in n-Hexane EO.Chemical groups like aldehydes and carboxylic acids have contributed in the total composition of n-Hexane distillate EO but absent in chloroform distillate (Table 1 & 2).Interestingly, lesser amount of compounds like Caryophyllene oxide and Squalene known for their antimicrobial and anticancer properties were also detected in both the extracts (Falowo et al., 2019;Smith, 2000).

Repellency test
EOsofA.annua showed strong repellent activity against the adult beetles.In the present study, repellency was more evident in the treatment sets ofn-hexane derived EO than the chloroform EO.Highest concentration of the EO (0.90mg cmÉ 2 ) had demonstrated 93.5% and 88.25% of repellency by n-hexane and chloroform derived EOs respectively (Table 3).However, repellency was recorded to increase insignificantly (pÃ0.01) with the increase in concentration.

Contact toxicity
Topical application was employed to evaluate whether the insecticidal activity of the EO of A.annuaagainst T. casteneumadults and 14 days old larvae was attributable to contact toxicity (Table 4).No mortality was recorded in the control sets.When LD 50 and LD 90 values were compared, T.castaneum adults were recorded to be more susceptible than its larval stage to chloroform derived EOs (No overlap in 95% confidence interval).Moreover, no significant (pÃ0.01)difference in contact toxicity was seen between larvae and adult beetle when treated with n-HexaneEO as overlap in 95% confidence interval is marked.On the basis of LD 90 values, adult beetles were found more vulnerable to chloroformderived EO whereas larvae were more responsive towards n-Hexane derived EO.

Fumigant toxicity
Fumigant toxicity was more pronounced in n-hexane eluted EO treated sets than to Chloroform derived EO (Table 8).Zero mortality was found in control set.Results testified the susceptibility of larval stagestowards solvents derived EOs when compared with the adults.While comparing the LD 90 values, both the stages were found sensitive towards the n-hexane extracted EO treated sets.

Biochemical analysis
Quantitative analysis of biomolecules was performed to assess the changes in their normal range on exposure to the plant EOs.The protein concentrations of the whole body homogenate in the adult and 14-days old larvae were found to be in the ranges of 318-955 µg mlÉ 1 .However, there was a significant (pÂ0.01)downfall in the protein concentration of the treated sets of contact and fumigant bioassays.In chloroform EO treated sets, protein level of adults decreased from 954µg mlÉ 1 (control)to 833µg mlÉ 1 (LD 90 ) and in larvae 407µg mlÉ 1 in control to 326 µg mlÉ 1 in LD 90 (Fig. 1 i).Results of n-Hexane EO treated sets were similar (Graph 1 ii).However, reduction in protein concentration was more pronounced in fumigant toxicity assays.
As explained in the Graph2, level of AChE was reduced significantly (pÂ0.01) in the lethal doses when compared with the control.However, insignificant (pÃ0.01)reduction is marked between the LD 50 & LD 90 .In contact toxicity bioassays, AChE level in the adults treated with chloroform EO decreased from 0.223 to 0.045µmoles/min/ ml of enzyme and 0.136 to 0.077µmoles/min/ ml of enzyme in larvae.Similar downfall was documented from the n-HexaneEO treated sets (Graph 2 ii).AChE reduction was more pronounced in fumigant toxicity assays from 0.293 to 0.038 µmoles/min/ml of enzyme in adult beetles and 0.14 to 0.069 µmoles/min/ml of enzyme in larvae.
The GST activity was declined significantly (pÂ0.01) in the treated sets while comparing their normal range recorded in the control group.But, slight and insignificant (pÃ0.01)decrease was noticed in the enzyme activity between the LD 50 & LD 90 groups.However, as depicted in the Graph3 i, adults treated topically with the chloroform derived EO has shown significant (pÂ0.01)decrease between the sub-lethal (0.115 µmoles/min/ml of enzyme) and lethal group (0.077 µmoles/min/ ml of enzyme).Similar results were documented from n-Hexane eluted EO treated sets of adult beetles (Graph3 ii) where values decline from 0.047 µmoles/min/ml of enzyme in LD 50 to 0.038 µmoles/min/ml of enzyme in LD 90 groups.
A significant (pÂ0.01)reduction in GSH activity was well marked in all the treatment sets from their normal range (209-179µmoles/mg protein in larvae; 237-181µmoles/mg protein in adults).Moreover, the enzymatic activity was found to be highly downregulated in n-hexane EO treated sets (Graph4 i) than the chloroform EO treatment sets (Fig. 4 ii).
LPO activity was significantly (pÂ0.01)upregulated in the treated sets when compared with the control.Moreover, the enzymatic activity was found to be dose-dependent and increased significantly from the LD 50 to the LD 90 groups (Graph 5).Adults exposed to fumigation of both the EOs, has shown a steep increase in the LPO level (17.983 nmole of MDA/gm of tissue in control to 40.25 nmole of MDA/gm of tissue in larvae).Similar downfall is attained in the fumigant assays as well.

DISCuSSIOn
Our results are quite different from the previous reports.A.annua is well established for possessing Artemisininwhich is a potent antimalarial component.However, 1,8-cineole has emerged as a major insecticidal candidate in various studies (Tripathi et al., 2001).Conversely, GC-MS analyses of the present study have clearly depicted the presence of some novel component in the chloroform and n-Hexane derived EOs of A. annuain excessively high amount.Oxygenated sesquiterpene, unique to A. annuais responsible for pharmacological activity is reported to be the major chemical group present in both the EOs (Martínez et al, 2012;Brown, 2010).The group include compounds like 3,4-Hexadienal,2butyl-2-ethyl-5-methyl and Deoxyqinghaosu in different proportions (Figure 1) which can be attributable to the insecticidal properties of both the EOs.Insecticidal compounds like2H-1-Benzopyran-2-one (Xiaorong& Taiping, 2008) and Squalene (Chauhan, 2015) were also detected in the EOs which ascertains the insecticidal candidature of plant EOs derived with chloroform and n-hexane.
A.annua showed potent contact, fumigant and repellent activity against T.casteneum with chloroform and n-Hexane derived EOs.However, the insecticidal properties of the EOsvaried with solvents and the life stages of the red flour beetle.Presence of Deoxyqinghaosu in the extract was an obvious outcome (Li, 2012;Ni et al., 2012).Correlated to the fact, relatively higherPercent composition of Deoxyqinghaosu in n-hexane EO can be attributable to the better repellent activityand was reported for the first time in the present study from filter paper arena tests.
Results of contact toxicity bioassays have clearly depicted the susceptibility of the adults (LD 50 = 0.71mg adultÉ 1 ) andlarvae (LD 50 = 0.47mg insectÉ 1 ) towards the n-hexane EO.Similarly, Fumigant toxicity has represented n-hexane a better eluent of EO for both the life stages.This could be due to the presence of high percentage of 3,4-Hexadienal,2-butyl-2-ethyl-5-methyl in the crude extract.Moreover, higher Percent composition of Squalene whose insecticidal properties (Chauhan et al., 2015) were already described could have surplused the bioactivity of the plants EOs.
Evaluation of the biomolecular profile of T. casteneum adults and 14-days' old larvae at their lethal doses were carried out to understand the molecular mechanism underlying oxidative stress following exposure to plant EOs.Decline in protein level with the increase in concentration has been seen in case of both contact and fumigant toxicity bioassays.The results were continuous with a number of earlier investigations where scientists recorded significant downfall in protein level (Smirle et al., 1996;Huang et al., 2004;Macedo et al., 2007).Spectrometric quantification of primary detoxifying enzymes like AChE, GST, GSH and LPO has demonstrated the plants mode of action.Gradual Reduction in the level of AChE, GST and GSH were marked at the lethal doses when compared with the control.In a similar study conducted with the extract of soapnutSapindusemarginatus against Aedesaegypti has shown significant reduction in larval AChE concentration (Koodalingam et al., 2011).GST is the key cytosolic enzyme for resistance development in insect's acts by catalysing the conjugation of reduced glutathione to the toxic molecule thus transforms it to less toxic end product (Grant & Matsumura, 1989).Our results on GST level were continuous with a number of previous investigations (Yu, 1982;Vanhaelen et al., 2001).On the contrary, Zibaee&Bandani (2010) has reported a positive correlation between the concentration of plant extract and GST activity after 24 h of exposure.However, reduction in the level of the enzyme was marked with the increase in exposure time.Correlated with GST, GSH level also experienced a significant downfall in the treated sets (Vontas et al., 2001).Upregulation of LPO levelwas an obvious outcome due to sudden increase in oxidative stress.Similar results were reported by Hasspieler (1990) where an increase in LPO activity was found in mosquito larvae.

COnCluSIOn
The present study hasestablishednon-polar solvents as the potent candidate for derivingEOs of A.annuawith better insecticidal and repellent activity against T. casteneum.Moreover, newly identified allelochemicals have emerged as an efficient in vivo suppressor of life supporting biomolecules except LPO in the toxicity assays.Hence, these compounds can be used as a potential synergist in pest management by interfering with enzyme mediated detoxification.Moreover, by analysing the gene expression of bioassay survived individuals, future research can focus on modulation of bioassays to channelize the toxicity of Artemisia annuawith potent non-polar solvents against other stored grain pests.

Graph 1 .
(i & ii) Protein activity (Mean±SE) in T. casteneum exposed to solvent derived EOs of A. annua at lethal doses.Columns marked with different letters are significantly different (PÂ0.01;ANOVA and Tukey's honest posthoc test) Graph 2. (i & ii) AChE activity (Mean±SE) in T. casteneum exposed to solvent derived EOs of A. annua at lethal doses.Columns marked with different letters are significantly different (PÂ0.01;ANOVA and Tukey's honest posthoc test)

Table 1 .
Chemical composition of the essential oils of A.annua extracted with chloroform The filter papers were allowed to air dry for 2 minutes post-treatment to evaporate the solvent.Impregnated paper was then sealed on the screw cap of the plastic vials (25mL).10 unsexed adults and larvae were tested separatelyfor each concentration.Five replicates were maintained for each concentration.Mortality was determined regularly for 3 days at the interval b Identification of volatile components was carried out by comparing Mass spectrum (MS) and Retention indices (RI) of components with those of the authentic standards in NISTlibraryandpreviousstudy.c Resultsobtainedbypeak-areanormalization standardisation of the process.

Table 2 .
Chemical composition of the essential oils of Artemisia annua extracted with n-hexane b Retention indices reported in previous studies c Identification of volatile components was carried out by comparing MS spectrum and RIs of components with those of the authentic standards in NIST 5 library and previous study.d Results obtained by peak-area normalization.
50, LC 90 ) and control sets were assessed.Protein profiling by Biurate kit method (HiMedia Laboratories Pvt.

Table 3 .
Repellency of solvent derived essential oils of A. annua against T. castaneum adults using Filter paper arena test Means (±SEM) followed by * indicate no significant difference (p < 0.01) according to the ANOVA.

Table 5 .
Fumigant toxicity of essential oils of Artemisia annuato *= Signi?cant (P ?0.01) Lethal values are expressed as mean of ?ve replicates.