Antioxidant and detoxifying enzymes response of stored product insect pests to bioactive fractions of botanical extracts used as stored grains protectant

Recurrent exposure of stored product insects to synthetic insecticides resulted in the development of resistance which occurs due to changes in insect metabolic enzymes. The inhibitory effect of ethyl acetate active fraction of Mitracarpus villosus , Bridelia micrantha, and Clerodendrum capitatum on antioxidant and detoxifying enzymes of stored product insects was investigated in this study. Sitophilus oryazae , Tribolium castaneum , and Rhizopertha dominica were exposed to 5 and 20 μ l concentrations of ethyl acetate active fraction VI 50:50 derived from glass column chromatography in a fumigation chamber and were homogenized separately. The results showed that SOD, CAT, GPx, GSH, and GST activities were dosage-time-dependent. Adult insects exposed to different dosages of C. capitatum active fractions signi ﬁ cantly inhibited GST and GPx activities, and SOD, CAT, and GSH activities were induced in comparison to insects in control. While, M . villosus and B. micrantha extracts exhibited a signi ﬁ cant increase ( p < 0.05) in SOD, CAT, and GSH in contrast to the inhibitory effects on GST and GPx. These results clearly show that stored beetles differ in their response to different enzymatic activities and that the evaluated plant materials may be used as an eco-friendly biopesticide in the IPM strategy for safeguarding stored food grains against stored produced insect pests.


Introduction
Food availability in unindustrialized nations seriously centers on the ability of subsistence farmers to preserve the post-harvest quality of their produce. These resource-poor farmers customarily preserve their food grains in their own small storage facilities with little or no high-tech ideas; thus, infl uencing great quantifi able and qualitative losses due to insect infestation and damage.
Keeping stored grains and milled grain products from insect infestations is a major challenge that causes disquiets in all nations to ensure food security. Tribolium castaneum, Sitophilus oryzae, and Rhyzopertha dominica are amongst agriculturally important insect pests attacking a lot of stored agricultural products. Consequently, it is imperative to explore good protective materials for the stored products against beetle infestation.
Synthetic insecticides and fumigants such as methyl bromide or phosphine play a vital role in controlling this problem, but they have been known to cause serious toxicological and environmental problems. Consequently, there is an urgent need to develop affordable, safe, sound insect pest control agents and techniques [1,2] Several research articles reported that organophosphates induced oxidative stress in non-mammalian systems mainly insects [3]. To protect against the effects of oxidative stress, insects have a variety of detoxifying enzymes at their disposal, such as catalase, glutathione reductase, glutathione peroxidase, and glutathione-S-transferases play an important role in eliminating harmful substances [4,5]. Hence, this study conceptualized the effect of the bioactive fraction of Mitracarpus villosus, Bridelia micrantha, and Clerodendrum capitatum extracts on antioxidant and detoxifying enzyme activities of stored product insects.

Experimental setting
The study was set up at the Institute of Bioresources and

Raising insects
The fully-grown rice weevil Sitophilus oryazae L.

Insecticidal activity of the active fractions of plant extracts
The insecticidal action of the active portions of M. villosus, C. capitatum, and B. micrantha was assessed by fumigation. For all types, ten insects were utilized per treatment. The adult insects were subjected to active fraction dosages ranging from 0 to 100 g/L for 24 hours. Five (5) replications of each dose were used for each species, and there were an equivalent number of untreated control replicates. Injecting the fumigant required the use of gas-tight micro syringes. For each dosage, fi ve replicates were employed and mortality rates for adult insects were calculated 24 hours after exposure. Extract with promising activity was selected for column chromatography.

Column chromatography using glass
The crude extract with encouraging insecticidal action was run through column chromatography by means of a glass column with a diameter of 3 cm and a length of 100 cm, packed with silica gel (60 -120 mesh) and eluted with hexane (100:0, I). Subsequently, a stepwise gradient of ethyl acetate, acetone, chloroform, and methanol was run through the column in the Components that are insecticide-active were combined and concentrated even more and subjected to a second round of column chromatography using a glass column (length 50 cm; diameter 3 cm) before being eluted with ethyl acetate, acetone, chloroform, and methanol [6]. The component with insecticidal potential was combined and concentrated before being used in enzymatic bioassay.

Preparation of homogenates for enzymatic activity
Twenty (20) adult insect species were exposed to fi ve (5) replicates with ethyl acetate active fraction of the designated plant extracts at varied concentrations (5 and 20 μl) in a desiccator that functions as a fumigant chamber. Injecting the fumigant required the use of gas-tight micro syringes. The insects were removed after 3, 6, and 12 hours post-exposure.
Additionally, untreated insects were used as a control. To remove insect tissue debris, the insects were normalized in 2 ml of 0.1 M phosphate buffer (7.4 pH) in a glass tissue grinder set in an icebox. The homogenate was then centrifuged at 1000 rpm for 5 min at 4 °C in a chilled centrifuge. To stop the enzyme from becoming inactive, the supernatant was divided into fresh tubes and stored in an ice box until use. However, due to the dissimilarity in the homogenization buffer, insects used to measure glutathione S-transferase activities were normalized with 2 ml Tris-HCl [7].

Superoxide dismutase activity (SOD): According to
Marklund and Marklund [8], pyrogallol (2 mM) auto-oxidation was used to assess Superoxide Dismutase (SOD) activity. The enzyme was combined with pyrogallol in 0.1 M tris buffer (pH 8.2) in the reaction mixture (whole insect homogenate). Results were expressed as units/mg protein for the reaction, which was started by adding the substrate, and were read at 420 nm for 3 minutes at intervals of 1 minute. One unit of enzyme activity is the quantity of the enzyme that reduces auto-oxidation by 50%. Centrifuging at 2000 rpm for 10 minutes after preparing 10% whole insect homogenates in 5% w/v TCA, the supernatant (GSH) was combined with 10 mM DTNB in 0.1 M phosphate buffer (pH 8.0). The combination was allowed to stand at room temperature for 10 minutes while the color was measured at 412 nm. A standard curve was used to determine the glutathione content, which was then expressed as g/mg protein.
Catalase activity (CAT): CAT activity was measured using the Aebi [9] technique. The reaction mixture contains 0.05 M of phosphate buffer and 3% H 2 O 2 (pH 7.0). About 100μl of the enzyme (whole insect homogenates) was added to start the reaction. After three minutes, the change in absorbance at 240 nm was recorded, and the activity was reported as n mole H 2 O 2 / min/mg protein.
Glutathione peroxidase: The modifi ed Rotruck, et al. [10] method was used to quantify glutathione peroxidase (GPx) activity. The reaction mixture contains 2 ml of pure water, 0.05 Glutathione-S-transferase: The Warholm, et al. [11] method was used to measure the glutathione-S-transferase

Experimental design and data analysis
Five replications of the enzymatic activity studies, each containing 10 unsexed insects, were set up in a completely randomized designed manner. To assess if there were signifi cant differences in the treatment means, a one-way analysis of variance (ANOVA) was used, followed by Tukey's multiple comparison tests. The SAS computer software was used for analysis and to express the data as the mean Standard Error of the Mean (SEM). At p < 0.05, the results were deemed signifi cant.

Insecticidal activity of the active fractions of plant extracts
Though not presented, the fumigant activity of the crude plant extract showed that ethyl acetate exerted signifi cant adult mortality and was selected for column chromatography. In the second round of column chromatography fraction II 80:20 exhibited promising insecticidal activity and was selected for the antioxidant and detoxifying enzymatic activities.

Effect of bioactive fractions of botanical extracts on insect antioxidant and detoxifying enzyme system
The activities of GST and GPx in adults S. oryzae exposed      Octenal were the lowest peak phytochemicals obtained from C. capitatum hexane extract (Figure 9).

Discussion
Medicinal plants are rich sources of secondary metabolites with antioxidant properties. Many secondary metabolites from medicinal plants have diverse biological effects, including the inhibition and induction of several important enzymes [12].
The toxicity of natural products particularly plant extracts has been implicated in the alterations of biochemical parameters [13].
Determining the inhibitory abilities of exogenous compounds on the activities of enzymes in the insect body is an important method for evaluating insecticidal activities to oxygen and hydrogen peroxide [18,19]. The ability of SOD to scavenge O 2 -is tempo rary and limited [20]. SOD is an antioxidant enzyme that can protect normal cells from ROS.
The ability of this enzyme to overcome the toxic effects of ROS in insects has been documented [21]. It is also plausible to suggest that at higher con centration of the herbal insecticides, more O 2 -is generated, which may accumulate to an extent that may overwhelm the scavenging ability of the SOD enzyme, SOD activity for all the herbal insecticide formulationtreated insects increases with increased concentration and exposure period. The increase in SOD might be connected to multivarious chemical components of the plant's active fractions. The increased active fractions concentration in all the treated samples stimulated the synthesis of SOD, resulting in higher dismutation of superoxide anion (O2-). Thus, preventing the production of hydroxyl radical (OH-) -a highly reactive species [22].
GSH (glutathione) is a thiol group-containing major nonenzymatic antioxidants that play a very important role in the defense against ROS. GSH participates in the protection against oxidative stress by its involvement in the ascorbate-GSH cycle, regulation of protein thiol-disulfi de redox status, and reduction of H 2 O 2 to water [23]. It is a tripeptide (-glutamylcysteinyl glycine), ubiquitously found in cells. The antioxidant activity of glutathione protects the cells against oxidative damage by free radicals and represents an important cellular defense mechanism. GSH pool is maintained in the cells by the restoration of the oxidized form of glutathione (GSSH) [24]. This fi nding shows that GSH increased in treated insects with both doses indicating higher oxidative stress brought about by the active fraction.
The exposure of the storage beetles to the active fractions might have brought about the induction of the GSH synthesis and the effect of herbal insecticides formulation could be a result of the medley of allelochemicals that characterized the active fractions [25]. The response of the beetle GSH to the insecticides could demonstrate the insect sensitivity to xenobiotics and might be connected to enhance the utilization of GSH as co-sub strate for GSH transferase or cofactor for GSH POX [26]. Glutathione and glutathione-dependent enzymes have been known to play a central role in the protection and detoxifi cation of peroxides and hydroperoxides. Marked depletion of GSH and its dependent enzymes in the insects by active fractions indicates a major deleterious effect on the insects. This fi nding revealed that the oxidative imbalance may be involved in the toxic effects of active fractions from C. capitatum, M. villosus, and B. micrantha. GST belongs to the phase II detoxifi cation system involved in conjugation reactions and may also detoxify a number of toxic ligands by acting as a non-catalytic intracellular binding protein (Kostaropoulos, et al. 2004). GST plays an important role in protecting cells against ROS-mediated injury by detoxifi cation of lipid hydroperoxides formed due to oxidative damage [27,28]. It is believed that these enzymes play an essential role in the survival of insects exposed to endogenous or exogenous xenobiotics [29] as they are involved in the detoxifi cation of various plant xenobiotics [30]. They usually catalyze the conjugation of the thiol group of reduced glutathione to electrophilic toxic xenobiotics and endogenously activated compounds and molecules, thereby increasing their solubility and promoting rapid excretion or facilitating degradation [14,31,32]. GSTs are potential drug targets and plant extracts enter tissues and organs of target insects and affect the activity of various detoxifying enzymes. Several secondary plant metabolites may inhibit GSTs activity, whereas others can activate GSTs activity [30,33,34]. Bhattacharya and Chandra [38] stated that botanicals are an important source of chemical compounds and proved to be effi cient biopesticides for the control of insect pests. The effi cacy of these plant products (extracts) could be accredited to the manifestation of phytochemical secondary metabolites, thus accountable for the different actions comprising insecticidal, enzyme detoxifi cation, and antioxidant properties [39].
This study has established that extraction of active biochemical from plants is directly proportional to the polarity of the solvents used. The ethyl acetate effectiveness as observed in these fi ndings aligned with the observation of Adesina [40] who stated that ethyl acetate is a reasonably polar solvent (polarity index of 4.4) that largely extracts steroids, alkaloids, etc., and was seen to give good results. He went further to claim that lethal secondary metabolites present in botanicals have the tendency to block ion channels, inhibit enzymes, or interfere with neurotransmission. This might have accounted for the varying degree of enzyme detoxifi cation exhibited in this study. The observed activities of the plant volatiles in the study may not be restricted only to its major bioactive chemical components; it could also be due to some minor constituents or a synergistic effect of several constituents. The isolated compounds from the three evaluated plants have been previously implicated for antifeedant, larvicidal, oviposition deterrent, and ovicidal activities in various classes of insects [41][42][43].

Conclusion
The inhibitory effect of insecticides on enzymatic activities indicated that these enzymes are not responsible for the decontamination of the insecticide and might be accountable for the increase in insect susceptibility to these insecticides.
The results from these fi ndings evidently show that stored beetles differ in their response to different enzymatic activities and that the evaluated plant materials may be used as an ecofriendly biopesticide in the IPM strategy for safeguarding stored food grains against stored produced insect pests' infestation.