Toxic effect of biologically active compound Rutin extracted from Euphorbious plant Codiaeum variegatum against mosquito Culex quinquefasciatus (Diptera: Culicidae) larvae

 

Abhay Deep Johnson, Ajay Singh*

Natural Products Laboratory, Department of Zoology, D.D.U. Gorakhpur University, Gorakhpur – 273 009 (U.P.)

India

 

Abstract:

Laboratory evaluation was made to assess the larvicidal activity of biologically active compound, Rutin extracted from Codiaeum variegatum (family: Euphorbiaceae) leaf obtained through soxhlet apparatus against larvae of Culex quinquefasciatus which is the serious vector of filarial worm, Wuchereria bancrofti causing lymphatic filariasis in human beings. The active compound Rutin extracted through ethyl alcohol solvent from the leaf of Codiaeum variegatum 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 ethyl alcohol extract of Codiaeum variegatum leaf, significantly (P<0.05) altered the level of total protein, total free amino acid, glycogen and enzyme activity i.e. acetyl cholinesterase activity, acid and alkaline phosphates 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. Codiaeum variegatum (Family: Euphorbiaceae) commonly known as croton in India, which is used as traditional medicine for the treatment of various human ailments such as antiamoebic, antibacterial, anticancer, antifungal, antioxidant, emmenagogue, purgative and sedative. A decoction of the crushed leaves is used in the treatment of diarrhea. The root is used in the treatment of gastric ulcers and wound healing activity.

 

 

 

INTRODUCTION:

Mosquitoes are the major public health problem throughout the world. Among the 3492 species of mosquitoes recorded worldwide, more than a hundred species are capable of transmitting various diseases in human and other vertebrates¹. Mosquitoes transmit malaria, dengue fever, yellow fever, filariasis, Japanese encephalitis and chikungunya to humans². Lymphatic filariasis is a mosquito borne disease caused by mosquito transmitted filarial nematodes, including Wuchereria bancrofti. The infected people carry the nocturnally periodic W. bancrofti, which has Culex quinquefasciatus as the main mosquito vector. C. quinquefasciatus is a vector of lymphatic filariasis. Vector mosquitoes are eminent as carrier of many diseases and imposing serious treats to the human health. Several kinds of mosquitoes belonging to different genera are vectors for the pathogen of various diseases³. Filariasis is a vector borne parasitic disease and its global transmission is caused by different species of Culex mosquitoes⁴. Culex quinquefasciatus Say (Diptera: Culicidae) is an urban vector of lymphatic filariasis. This disease is endemic in 80 countries of the world and recognized as one of six potentially eradicable diseases^5-8. Synthetic pesticides are generally used for public health sprays in most parts of the world^9-10. It’s unlimited, uninterrupted 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^11-15. Plant origin pesticides are selectively toxic, do not bio-accumulate and exhibit short term persistence in the environment^16-17 so they can be used as an excellent alternative. Codiaeum variegatum (Family: Euphorbiceae) commonly known as croton in India, which is used as traditional medicine for the treatment of various human ailments such as antiamoebic, antibacterial, anticancer, antifungal, antioxidant, emmenagogue, purgative, amoebiasis and sedative¹⁸ . A decoction of the crushed leaves is used in the treatment of diarrhea. The root is used in the treatment of gastric ulcers and wound healing activity¹⁹.

 

In the present study, the larvicidal activity of Rutin extracted from Codiaeum variegatum 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.

 

MATERIALS AND METHODS:

Collection and maintenance of experimental animal:

Fully fed adult females of Culicines were collected from the different residential areas of Gorakhpur district. 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/WEF²⁰ were atmospheric temperature 30.2°±1.6°C, water temperature 27.6°±1.1°C, pH 7.3-7.5, dissolved oxygen 7.6-8.1mg/L, free CO₂  4.1-5.1mg/L, bicarbonate alkalinity 103.5-105.0 mg/L.     

 

 

 

Collection of plant material:

Plant Codiaeum variegatum (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.

 

Extraction of active compounds:

The Rutin was isolated from the leaves of Codiaeum variegatum respectively by the method of Subramanian et al²¹ . The leaves of these plants were washing properly in tap water and cut the leaves by scissors then dried in shady place and finally dried in an incubator at about 35⁰C temperature; dried leaves were powder 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 Rutin 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 (Rutin), and its melting point was measured as 194-196⁰C.Confirmation of the compound was also made through IR and Rf values when compared to the authentic sample obtained from Sigma Chemical Company, USA.

 

Toxicological experiment:          

Toxicity experiment was performed by using the method of  W.H.O²². Twenty late third instar larvae of Culex quinquefasciatus mosquito were exposed to four different concentrations of rutin. 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 of rutin. Mortality was recorded after every 24hr upto 96hr exposure periods. LC values, upper and lower confidence limits, slope value, t-ratio and heterogeneity were calculated by probit log analysis method using POLO computer programme of  Russel et al²³.

 

Biochemical experiment:

The late third instar larvae were treated with 40% and 80% of 24h LC₅₀ of  Rutin obtained from Codiaeum variegatum 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 Rutin was 75.53 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 the 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^24-25.

 

Total protein: Total protein level was estimated by the method of  Lowry et al²⁶. Homogenates (10mg/mL) was prepared in 10% tri-chloroacetic acid (TCA). Bovine serum albumin was used as standard.

 

Total free amino acids: Total free amino acids level was estimated by the method of  Spies²⁷. Homogenates (10mg/mL) were prepared in 95% ethanol. Glycine was used as standard.

 

Glycogen: Glycogen level was estimated by the method of Van der Vies²⁸. Homogenate (10mg/mL) was prepared in 5% TCA. Glucose was used as standard.

 

Acetylcholinesterase activity: Acetylcholinesterase activity was measured by the method of Ellman et al²⁹. 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 monitor for 3 min at 25°C.

 

Acid and alkaline phosphatase activity: Acid and alkaline phosphatase activity was determined by the method of Andersch and Szcypinski³⁰ . 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 differences²⁴.

 

RESULT:

Exposure to the rutin extracted from Codiaeum variegatum 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, show 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 behaviour and larval mortality.  

 

Percent mortality produced by Rutin 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  Rutin 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 Codiaeum variegatum leaf decreased from 75.53mg/L (24h)> 59.86mg/L (48h)> 43.33mg/L (72h)> 32.13mg/L (96h) in case of Culex quinquefasciatus larvae (Table 1).

 

 

Table 1: Toxicity (LC values) of different concentrations of rutin extracted through ethyl alcohol from the leaf of Codiaeum variegatum 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=27.60	4.39	40.45	2.904±1.869	2.803	0.057
	LC50=75.53	57.82	155.89
	LC90=206.06	118.69	3858.76
48	LC10=23.66	5.24	34.98	3.179±1.783	3.147	0.215
	LC50=59.86	45.01	84.47
	LC90=151.43	99.32	794.61
72	LC10=16.95	2.46	27.45	3.144±1.733	3.148	0.124
	LC50=43.33	25.97	55.74
	LC90=110.75	78.28	396.43
96	LC10=11.91	0.50	22.30	2.984±1.786	2.858	0.172
	LC50=32.13	10.78	43.28
	LC90=86.38	62.84	310.42

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 rutin extracted from Codiaeum variegatum 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 81% of control after exposure to (24h) of rutin extracted from Codiaeum variegatum leaf. The maximum decrease in protein level (68% of control) was observed in larvae treated with 80% of LC₅₀ (24h). Total free amino acid levels were induced to 116% of controls after treatment with 40% of LC₅₀ (24h) and maximum increase in total free amino acids level (128% of control) was observed in larvae treated with 80% of LC₅₀ (24h) of ethyl alcohol extract of Codiaeum variegatum leaf and the glycogen level was reduced up to 65% and 51% respectively (Table 2). 

Table 2 clearly shows that sub-lethal exposure of Rutin at 40% and 80% of LC₅₀ the AChE activity decreases 73% , 56% at 24h with respect to control but at 96h exposure the AChE activity also decreases as 51%, 34% at 40% and 80% of LC₅₀ respectively with respect to control.

 

According to Table 2 at sub-lethal treatment of Rutin of 40% and 80% of LC₅₀ (24h), Acid phosphatase activity decreases by 78% to 67% respectively with respect to control. At longer duration (96h) exposure, 40% and 80% of LC₅₀ of Rutin also decreases the activity of acid phosphatase by 62%, 50% respectively with respect to control. In the case of enzyme alkaline  phosphatase, exposure of 40%, 80% LC₅₀ of Rutin also decreases the enzyme activity by 73% to 59% and 63% to 46% at 24h or 96h respectively with respect to control (Table 2).

 

 

 

Table 2: Changes in total protein, glycogen, total free amino acid, acetylcholinesterase activity, 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 compound extracted through ethyl alcohol solvent from leaf of Codiaeum variegatum.

Parameters	Control	40% of LC50 (+, £) (30.21 mg/L, 24h LC50)	80% of LC50 (+,£) (60.42 mg/L, 24h LC50)
Protein	24h
	1.80±0.003 (100)	1.45±0.003 (81)	1.22±0.004 (68)
Glycogen	24h
	1.100±0.003 (100)	0.72±0.003 (65)	0.56±0.004 (51)
Amino acid	24h
	0.50±0.003 (100)	0.58±0.004 (116)	0.64±0.003 (128)
AChE	AChE activity (μm SH hydrolyzed/min/mg protein
	24h
	0.085±0.0004 (100)	0.062±0.0005 (73)	0.048±0.0004 (56)
	96h
	0.082±0.0004 (100)	0.042±0.0004 (51)	0.028±0.0004 (34)
Acid phosphatase	μm p-nitrophenol formed/30 min/mg protein
	24h
	0.180±0.003 (100)	0.140±0.003 (78)	0.120±0.003 (67)
	96h
	0.200±0.003 (100)	0.124±0.0003 (62)	0.100±0.0003 (50)
Alkaline phosphatase	μm p-nitrophenol formed/30 min/mg protein
	24h
	0.410±0.004 (100)	0.300±0.003 (73)	0.240±0.004 (59)
	96h
	0.460±0.003 (100)	0.290±0.003 (63)	0.210±0.003 (46)

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 analysis of variance was applied to see whether enzyme inhibition was time and dose. £, significant (P<0.05) when Student ‘t’ test was applied between control and treated groups.

 

 

 

 

DISCUSSION:

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 control³¹.

 

In the present study the rutin extracted with ethyl alcohol from Codiaeum variegatum leaf has potent larvicidal activity of Culex quinquefasciatus mosquitoes. Exposure to sub-lethal doses of compound rutin of Codiaeum variegatum 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 Codiaeum variegatum 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 still³². 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 chains³³. The decreased protein content attributed to the destruction or necrosis of cells and consequent impairment in protein synthesis machinery³⁴. 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 Codiaeum variegatum 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 acids³⁵ and their involvement in the maintenance of an acid base balance³⁶. 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 pathway³⁷. The transamination reaction is probably the most important pathway in the metabolism of many amino acids³⁸.  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 Codiaeum variegatum 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 treatment³⁹. Larvae also secrete catecholamine in excess amount, during stress, which depletes glycogen reserves⁴⁰. 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 cycle⁴¹.

 

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. Vorbrodt⁴² 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 enzymes⁴³. Acid phosphatase is the lysosomal enzyme and plays an important role in catabolism, pathological necrosis, autolysis and phagocytosis⁴⁴.

 

CONCLUSION:

The study on the larvicidal activity of the rutin extracted through ethyl alcohol from Codiaeum variegatum leaf is highly toxic to larvae of C. 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 C. quinquefasciatus mosquitoes in aquatic stage.

 

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