ABSTRACT- Mosquito-borne diseases have intruded the globe since immemorial time. The present scenario for commanding the mosquitoes is aimed at application of target and stage-specific, cost-effective and biodegradable phytoproducts. Plant extracts are safer for non-target organisms including man. Plant based formulations would be more feasible environmental products with proven potential as insecticide. Therefore, in the present study of larvicidal activity of biologically active compound Apigenin extracted from leaf of Jatropha gossypifolia against the filarial vector, Culex quinquefasciatus was studied. Standard WHO protocols with minor modifications was adopted for the larvicidal bioassay. The active compound Apigenin extracted through ethyl alcohol solvent from the leaf of Jatropha gossypifolia plant of family Euphorbiaceae was administered for 24h or 96h to the larvae of Culex quinquefasciatus. Exposure of larvae over 24h to sub-lethal doses (40% and 80% of LC50) of apigenin, significantly (P<0.05) altered the level of total protein, total free amino acid, glycogen and activity of enzymes acetyl cholinesterase, acid and alkaline phosphatase activity in whole body tissue of Culex quinquefasciatus larvae. The alterations in all these biochemical parameters were significantly (P<0.05) time and dose dependent.
Key-words- Jatropha gossypifolia,Euphorbiaceae, Culex quinquefasciatus, biochemical effects, Wuchereria bancrofti

INTRODUCTION
Mosquitoes transmit several public health problems, such as malaria, filariasis, and dengue causing millions of deaths every year [1]. Mosquitoes in the larval stage are attractive targets for pesticides because they breed in water and, thus, are easy to deal with them in this habitat. The use of herbal products is one of the best alternatives for mosquito control [2]. Mosquitoes are the major vectors for the transmission of malaria, dengue fever, chikungunya, filariasis and Japanese encephalitis affecting humans and domestic animals worldwide, causing millions of deaths every year [3]. Culex quinquefasciatus Say (Diptera: Culicidae) is a predominant house-resting mosquito in many tropical countries [4] breeding in polluted waters such as blocked drains, damaged septic tanks, or soak age pools close to human habitations. It is a pan tropical pest and urban vector of Wuchereria bancrofti, which causes filarial fever [5].
Synthetic pesticides are generally used for public health sprays in most parts of the world [6-7]. It’s unlimited,un-interrupted and indiscriminate use as the principal agent, results in development of insecticide resistance in mosquitoes and also poses a threat to life and our environment [8-12]. Plants are rich source of alternative agents for control of mosquitoes, because they possess bioactive chemicals, which act against a number of species including specific target-insects and are eco-friendly. Plant based pesticides are less toxic, delay the development of resistance and are easily biodegradable [13]. Plant based products do not have any hazardous effect on ecosystem. Plant’s secondary metabolites and their synthetic derivatives provide alternative source in the control of mosquitoes biodegradable. The crude extracts can be effectively used in the control of mosquitoes by replacing the chemical pesticides, which cause environmental pollutions and other burdens [14].
In the present study, the larvicidal activity of Apigenin extracted from Jatropha gossypifolia leaf as well as its biochemical effects on larvae of Culex quinquefasciatus were investigated, these extracts cannot be applied to commercial use without a study of these aspects as well and it can be replace the chemical pesticides which cause environmental pollutions and other health problems [14].

MATERIALS AND METHODS
Collection and maintenance of experimental insects: Fully fed adult females of Culicines were collected from the different residential areas of Gorakhpur district, India. Collections were made from human dwellings with the help of an aspirator supplied by W.H.O. and kept in 30x30x30 cm cages with cotton pads soaked in 10% glucose solution and water containing enamel bowl for egg laying. Experimental conditions of water determined by the method of APHA/AWWA/WEF [15] were atmospheric temperature 30.4°±1.7°C, water temperature 27.5°±1.2°C, pH 7.4-7.6, dissolved oxygen 7.6-8.3mg/L, free CO2 4.2-5.2mg/L, bicarbonate alkalinity 104.5-105.8 mg/L.

Collection of plant material: Plant Jatropha gossypifolia (family: Euphorbiaceae) was collected locally from Botanical garden of Deen Dayal Upadhyay Gorakhpur University, Gorakhpur, and identified by Prof. S. K. Singh, Plant taxonomist, Department of Botany, Deen Dayal Upadhyay Gorakhpur University, Gorakhpur, Uttar Pradesh, India, where a voucher specimen was deposited (Fig. 1).

Fig. 1: Jatropha gossypifolia plant
Extraction of active compounds: The Apigenin was isolated from the leaves of Jatropha gossypifolia by the method of Subramanian [16]. The leaves of these plants were washed properly in tap water and the leaves were cut by scissors then dried in shady place and finally dried in an incubator at about 350C temperature; dried leaves were powdered by electric Grinder. About 50 g powder of leaves was subjected in Soxhlet extraction unit with about 250-300 mL ethyl alcohol for about 72h at 30-400C. In case of compound Apigenin after extraction, the aqueous layer was collected and left to stand in a cold place for 72 hours; a yellow precipitate separated out from the solution. The precipitate was filtered and washed with a mixture of chloroform: ethyl acetate: ethanol (2:1:1). The un-dissolved part of the precipitate was dissolve in hot methanol and filtered, the filtrate was evaporating to dryness to give 280 mg yellow powder of Apigenin. Confirmation of the compound (Fig 2) was made through IR and Rf values data of Dordevice [17] and was also confirmed by comparing it with the authentic sample obtained from Sigma Chemical Company, USA.



Fig 2: Chemical structure of Apigenin
(Source: https://en.wikipedia.org/wiki/Apigenin)
Toxicological experiment: Toxicity experiment was performed by using the method of W.H.O. [18]. Twenty late, third instar larvae of Culex quinquefasciatus mosquito were exposed to four different concentrations of apigenin. Doses were maintained in 500 ml of de-chlorinated tap water in glass beakers (15 cm in diameter and 7.5cm in height) containing twenty mosquito larvae in each test concentration. Six replicates were maintained for each concentration. Control larvae were kept in similar conditions without treatment. Culex larvae were exposed for 24hr to 96hr at four different concentrations (70 mg, 80 mg, 90 mg and, 100 mg) of apigenin. Mortality was recorded after every 24hr up to 96hr exposure periods. LC values, upper and lower confidence limits, slope value, t-ratio and heterogeneity were calculated by POLO computer programme of Robertson [19].

Biochemical experiment: The late third instar larvae were treated with 40% and 80% of 24h LC50 of Apigenin obtained from Jatropha gossypifolia leaf for 24h. Six beakers were set up for each dose and each beaker contained 50 larvae in 1L de-chlorinated tap water. The LC50 value of Apigenin was 93.11 mg/L for 24h against Culex quinquefasciatus larvae. 40% and 80% of 24h, LC50 of ethyl alcohol extract was selected as sub-lethal dose to analyze its time and dose dependent effects in this present study and at that dose there was no mortality were observed in the treated larvae. After the stipulated time (24h), the dead larvae were removed from the beaker and washed with water and the whole body tissue stored in deep freezer for biochemical analysis. Control larvae were held in the same condition without any treatment. Each experiment was replicated six times and the values are expressed as mean ±SE of six replicates. Student’s ‘t’ test was applied to locate significant changes with controls[20-21].

Total protein: Total protein level was estimated by the method of Lowry [22]. Homogenates (10mg/mL) was prepared in 10% tri-chloroacetic acid (TCA). Bovine serum albumin was used as a standard.

Total free amino acids: Total free amino acids level was estimated by the method of Spies [23]. Homogenates (10mg/mL) were prepared in 95% ethanol. Glycine was used as a standard.

Glycogen: Glycogen level was estimated by the method of Van der Vies [24]. Homogenate (10mg/mL) was prepared in 5% TCA. Glucose was used as a standard.

Acetylcholinesterase activity: Acetylcholinesterase activity was measured by the method of Ellman [25]. Homogenate (50 mg/ml, w/v) was prepared in 0.1 M-phosphate buffer, pH 8.0 for 5 min in an ice bath. The change in optical density at 412nm caused by the enzymatic reaction was monitored for 3 min at 25°C.

Acid and alkaline phosphatase activity: Acid and alkaline phosphatase activity was determined by the method of Andersch and Szcypinski[26]. Homogenates (2% w/v) were prepared in ice-cold 0.9% NaCl solution and centrifuged at 5000 xg at 0?C for 15 min.

Statistical analysis: Each experiment was replicated at least six times and data has expressed as mean ±SE. Student’s t-test as applied for locating significant differences Sokal and Rohlf [20].

RESULTS AND DISCUSSION
In the present study exposure to the apigenin extracted from Jatropha gossypifolia leaf caused significant behavioural changes in the larvae of mosquito Culex quinquefasciatus. Behavioural changes appear after 4-5 hours of exposure. Larvae were incapable of rising to the surface shown restlessness, loss of equilibrium, lethargic and finally death. No such behavioural symptoms and mortality occurred in the control groups indicating that the plant moieties were actual factors responsible for altered behavior and larval mortality.
Percent mortality produced by apigenin for the periods ranging from 24 to 96hr is shown in Table 1. The toxicity of ethyl alcohol extract was time and dose dependent for Culex quinquefasciatus larvae. The LC50 values of apigenin are shown in Table 1. There was a significant negative correlation between LC values and exposure periods. i.e. LC50 values of ethyl alcohol extract of Jatropha gossypifolia leaf decreased from 93.11 mg/L (24h)> 86.26 mg/L (48h)> 77.81mg/L (72h)> 71.92 mg/L (96h) in case of Culex quinquefasciatus larvae (Table 1).

Table 1: Toxicity (LC values) of different concentrations of Apigenin extracted from leaf of Jatropha gossypifolia plant against Culex quinquefasciatus larvae at 24h to 96h exposure period

Exposure Period (hours)	Effective dose (mg/L)	Limits (mg/L)		Slope value	't' ratio	Heterogeneity
		LCL	UCL
24	LC10= 67.06	37.75	75.83	8.99±6.13	2.84	0.02
	LC50=93.11	85.33	117.43
	LC90=129.29	108.01	324.75
48	LC10=64.02	40.26	72.49	9.90±5.98	3.20	0.09
	LC50=86.26	78.80	96.33
	LC90=116.21	101.52	194.46
72	LC10=57.83	31.76	67.28	9.95±6.03	3.16	0.01
	LC50=77.81	66.41	84.29
	LC90=104.69	93.76	156.36
96	LC10=52.92	21.15	63.79	9.62±6.35	2.90	0.07
	LC50=71.92	53.24	78.63
	LC90=97.74	88.32	147.03

Batches of twenty mosquito larvae were exposed to four different concentrations of the extract. Concentrations given are the final concentration (w/v) in the glass beaker containing de-chlorinated tap water. Each set of experiment was replicated six times.
Mortality was recorded after every 24h.
Regression coefficient showed that there was significant (P<0.05) negative correlation between exposure time and different LC values. LCL: Lower confidence limit; UCL: Upper confidence limit.
There was no mortality recorded in the control group.

After exposure to sub-lethal doses of 40% and 80% of LC50 of apigenin extracted from Jatropha gossypifolia leaf for 24h or 96h caused significant (P<0.05) alterations in total protein, total free amino acids and glycogen metabolism in whole body tissue of the larvae of Culex quinquefasciatus (Table 2). Total protein and glycogen levels were significantly reduced, while free amino acid level was significantly enhanced after the exposure to sub-lethal doses. Total protein levels were reduced to 92% of control after exposure to (24h) of apigenin extracted from Jatropha gossypifolia leaf. The maximum decrease in protein level (86% of control) was observed in larvae treated with 80% of LC50 (24h). Total free amino acid levels were induced to 104% of controls after treatment with 40% of LC50 (24h) and maximum increase in total free amino acids level (110% of control) was observed in larvae treated with 80% of LC50 (24h) of ethyl alcohol extract of Jatropha gossypifolia leaf and the glycogen level was reduced up to 83% and 76% respectively (Table 2).

Table 2: Changes in total protein, glycogen and total free amino acid activity in whole body tissue of Culex quinquefasciatus larvae after 24h exposure to sub-lethal doses (40% and 80% of LC50 of 24h) of active compound Apigenin extracted from leaf of Jatropha gossypifolia plant

Parameters	Control	40% of LC50 (+, £) (37.24 mg/L, 24h LC50)	80% of LC50 (+, £) (74.49 mg/L, 24h LC50)
Protein	24
	1.90±0.003 (100)	1.75±0.003 (92)	1.64±0.004 (86)
Glycogen	24
	1.20±0.003 (100)	1.00±0.004 (83)	0.91±0.006 (76)
Amino acid	24
	0.50±0.005 (100)	0.52±0.004 (104)	0.55±0.004 (110)

values are mean ±SE of six replicates. Values in brackets indicate percent biochemical activity with control taken as 100%.
Doses are 40% and 80% of LC50 for period for which animals were exposed.
+, significant (P<0.05) when two way variance was applied to see whether protein, glycogen and amino acid alterations was time and dose dependent.
£, significant (P<0.05) when Student ‘t’ test was applied between control and treated groups.

Table 3 was clearly shown that sub-lethal exposure of apigenin at 40% and 80% of LC50 the AChE activity decreases 82% , 71% at 24h with respect to control but at 96h exposure the AChE activity also decreases as 78%, 64% at 40% and 80% of LC50 respectively with respect to control.
According to Table 3 at sub-lethal treatment of apigenin of 40% and 80% of LC50 (24h), Acid phosphatase activity decreases by 88% to 82% respectively with respect to control. At longer duration (96h) exposure, 40% and 80% of LC50 of apigenin also decreases the activity of acid phosphatase by 73%, 71% respectively with respect to control. In the case of enzyme alkaline phosphatase, exposure of 40%, 80% LC50 of apigenin also decreases the enzyme activity by 90% to 80% and 71% to 60% at 24h or 96h respectively with respect to control (Table 3).

Table 3: Changes in acetylcholinesterase, acid and alkaline phosphatase activity in whole body tissue of Culex quinquefasciatus larvae after 24h or 96h exposure to sub-lethal doses (40% and 80% of LC50 of 24h) of active compound Apigenin extracted from leaf of Jatropha gossypifolia plant

Parameters	Control	40% of LC50 (+, £) (37.24 mg/L, 24h LC50)	80% of LC50 (+, £) (74.49 mg/L, 24h LC50)
AChE	AChE activity (µm SH hydrolyzed/min/mg protein
	24h
	0.072±0.006 (100)	0.059±0.004 (82)	0.051±0.004 (71)
	96h
	0.072±0.006 (100)	0.056±0.004 (78)	0.046±0.004 (64)
Acid phosphatase	µm p-nitrophenol formed/30 min/mg protein
	24h
	0.170±0.003 (100)	0.150±0.003 (88)	0.140±0.003 (82)
	96h
	0.180±0.003 (100)	0.132±0.004 (73)	0.127±0.003 (71)
Alkaline phosphatase	µm p-nitrophenol formed/30 min/mg protein
	24h
	0.400±0.004 (100)	0.360±0.003 (90)	0.320±0.004 (80)
	96h
	0.380±0.004 (100)	0.270±0.004 (71)	0.228±0.004 (60)

Values are mean ±SE of six replicates.
Values in brackets indicate percent biochemical activity with control taken as 100%.
Doses are 40% and 80% of LC50 for period for which animals were exposed.
+, significant (P<0.05) when two way variance was applied to see whether enzyme inhibition was time and dose dependent.
£, significant (P<0.05) when Student ‘t’ test was applied between control and treated groups.

Statistical analysis of the data on toxicity brings out several important points. The X2 test for goodness of fit (heterogeneity) demonstrated that the mortality counts were not found to be significantly heterogeneous and other variables, e.g. resistance etc. do not significantly affect the LC50 values, as these were found to lie within the 95% confidence limits. The dose mortality graphs exhibit steep values. The steepness of the slope line indicates that there is a large increase in the mortality of the larvae of Culex quinquefasciatus with relatively small increase in the concentration of the toxicant. The slope is, thus an index of the susceptibility of the target animal to the plant origin pesticides used.
The mosquito larval control using larvicidal agents is a major component in the control of vector borne diseases. Plant as potential larvicides is considered as viable and preferred alternative in the control of the mosquito species at the community level. A large number of plant extracts have been reported to have mosquitocidal or repellent activities against mosquito vectors, but few plant products have shown practical utility for mosquito control [27].
In the present study the apigenin extracted with ethyl alcohol from Jatropha gossypifolia leaf has potent larvicidal activity of Culex quinquefasciatus mosquitoes. Exposure to sub-lethal doses of compound apigenin of Jatropha gossypifolia leaf against larvae of Culex quinquefasciatus significantly altered the level of total protein, total free amino acid, glycogen and enzyme activity of acetylcholinesterase, acid and alkaline phosphatase activity. Significant exceptional changes as given in result section of Culex quinquefasciatus larvae like ecdysial failure, abnormalities during intermediate stages, prolongation of the life span of treated instars, emergence of adultoids after treatment with ethyl alcohol extract of Jatropha gossypifolia leaf may be due to the effect of active moiety present in the plant extract. The effect of compound depends on the synthesis or release of ecdysone and in absence of it, the insect lapses into a state of developmental stand still [28]. It resulted into ecdysial failure. The male and female emerged from treated groups were unable to feed on sugar solution as well as on mammal blood ultimately they died sooner. Laboratory observations revealed that, their mouth parts were undeveloped, legs were paralysed and the females were incapable of egg laying after treatment, eventually they died sooner.
Carbohydrates are the primary and immediate source while the protein acts as the next alternative source of energy to meet the increase energy demand. The depletion of the protein fraction in treated mosquito larvae of Culex quinquefasciatus may have been due to their degradation and the possible utilization for metabolic purposes. The protein content is depends on the rate of protein synthesis and its depletion might have been due to their degradation and possible utilization for metabolic purposes. The quantity of protein may also be affected due to impaired incorporation of amino acids into polypeptide chains [29]. The decreased protein content attributed to the destruction or necrosis of cells and consequent impairment in protein synthesis machinery [30]. The total free amino acids content showed a significant increase in whole body tissue of mosquito larvae exposed to sub-lethal doses of ethyl alcohol extract of Jatropha gossypifolia leaf. The augmentation in total free amino acids level in the whole body tissue suggests high proteolytic activity. The accumulation of free amino acids can also be attributed to lesser use of amino acids [31] and their involvement in the maintenance of an acid base balance [32]. Another possibility for enhancement of free amino acid level might be due to transamination and amination to keto acids. Stress conditions induce elevation in the transamination pathway [33]. The transamination reaction is probably the most important pathway in the metabolism of many amino acids [34]. In stress condition, carbohydrate reserve depleted to meet energy demand. In the present study, the diminished glycogen content in body tissues of Culex larvae indicates its rapid utilization for energy generation; a demand caused by rutin extracted from Jatropha gossypifolia leaf as a consequence toxic stress during the experiment.
Finally, glycogenolysis seems to be the result of increased secretion of catecholamine due to stress of plant extracts treatment [35]. Larvae also secrete catecholamine in excess amount, during stress, which depletes glycogen reserves [36]. Anaerobic and aerobic segments are two important components of carbohydrate metabolism. In first case, breakdown of glucose or glycogen through Embden-Meyerhof pathway (glycolysis) takes place while the next one consists oxidation of pyruvate to acetyl co-A to be utilized through citric acid cycle [37].
Effect of toxicants on enzymatic activity is one of the most important biochemical parameters, which Affect physiology of body. When an organ is diseased due to the effect of a toxicant, enzyme activity appears to be increased or it may be inhibit due to the active site being either denature or destroyed. Acetylcholinesterase, or acetyl-hydrolase, is a serine protease that hydrolyses the neurotransmitter acetylcholine. AChE found mainly at neuromuscular junctions and brain synapse, where its activity serves to terminate synaptic transmission. It belongs to carboxyl esterase family of enzymes.
Enzyme alkaline phosphatase plays an important role in animal metabolism. Vorbrodt [38] has reported that the role of this enzyme is in the transport of metabolites across the membrane. The enzyme has been shown to be intimately associated with protein synthesis and is thus involved in the synthesis of certain enzymes [39]. Acid phosphatase is the lysosomal enzyme and plays an important role in catabolism, pathological necrosis, autolysis and phagocytosis [40].

CONCLUSIONS
In conclusion, the larvicidal activity of the apigenin extracted through ethyl alcohol from Jatropha gossypifolia leaf is highly toxic to larvae of Culex quinquefasciatus mosquito. This extract significantly suppresses the population build up of the mosquito by morphogenetic action on insect. Sub-lethal doses of ethyl alcohol extract significantly alter the protein, amino acids, glycogen, enzyme activity like acetylecholinesterase, acid, and alkaline phosphatase activity of Culex larvae. We therefore believe that the plant extracts may eventually be of great value for the control of Culex quinquefasciatus mosquitoes in aquatic stage.

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How to cite this article: Johnson AD, Singh A: Larvicidal activity and Biochemical Effects of Apigenin against Filarial Vector Culex quinquefasciatus. Int. J. Life. Sci. Scienti. Res., 2017; 3(5):1315-1321. DOI:10.21276/ijlssr.2017.3.5.9 Source of Financial Support:Nil, Conflict of interest: Nil