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Original article
06 2022
:34;
101960
doi:
10.1016/j.jksus.2022.101960

Chemical composition and mosquitocidal efficacy of panchagavya against Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus

, , , , , , ,
a Nano and Energy Bioscience Laboratory, Department of Biotechnology, Thiruvalluvar University, Serkkadu, Vellore 632115, Tamil Nadu, India
b UNESCO-UNISA Africa Chair in Nanosciences/Nanotechnology Laboratories, College of Graduate Studies, University of South Africa (UNISA), Muckleneuk Ridge, PO Box 392, Pretoria, South Africa
c Nanosciences African Network (NANOAFNET), Materials Research Group (MRG), iThemba LABS-National Research Foundation (NRF), 1 Old Faure Road, 7129, PO Box 722, Somerset West, Western Cape Province, South Africa
d Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia
e Department of Animal Biotechnology, Jeonbuk National University, Jeonju 54896, South Korea
f Department of Animal Biology and Physiology, Faculty of Science, The University of Douala, PO Box 24157, Douala, Cameroon

⁎Corresponding authors at: Nano and Energy Bioscience Laboratory, Department of Biotechnology, Thiruvalluvar University, Serkkadu, Vellore - 632115, Tamil Nadu, India. babukmg@gmail.com (Ranganathan Babujanarthanam)

Received: , Accepted: ,
© The Author(s) 2022
Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Peer review under responsibility of King Saud University.

Abstract

Abstract

Objectives

The current study looks towards reporting the chemical compounds present in the panchagavya (PG), free radicals scavenging and mosquitocidal activity of PG in the laboratory condition.

Methods

The existence of chemical compounds in the PG were studied by GC–MS analysis. Free radicals scavenging activity of PG was studied by using various invitro assays. Mosquitocidal efficacy of PG was studied by the experiment on larvicidal, pupicidal, adulticidal, fecundity, longevity, and ovicidal activity against An. stephensi, Ae. aegypti and Cx. quinquefasciatus.

Results

GC–MS analysis revealed fifteen chemical compounds present in the PG. Free radical scavenging was done by 2,2-diphenyl-1-picrylhydrazyl, 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid), hydroxyl, and superoxide assays, and the IC50 was calculated as 37, 37.5, 35, and 38 μg/mL respectively. PG exhibited better larvae and pupae mortality against I-IV instar of Cx. quinquefasciatus (LC50: 148.765, 162.534, 187.619, 210.835 and 234.624 ppm, LC90: 286.636, 306.390, 350.276, 390.735 and 419.195 ppm). The highest adult mortality was found against An. stephensi (91.10 ± 1.74%) with the IC50 and IC90 values of 128.114 and 260.609 ppm. An. stephensi showed highly decreased fecundity and longevity even at a low concentration of PG. Inhibition of 100% egg hatchability of An. stephensi was obtained at 250 ppm followed by Ae. aegypti, and Cx. quinquefasciatus at 300 ppm respectively. On comparing with other mosquito vectors An. stephensi was effectively inhibited by PG at each stage of their life cycle.

Conclusion

The results provide the first proof that PG could be a successful natural agent for controlling different mosquito vectors. Furthermore, our findings pave the way for more research into the efficacy of natural materials' mosquitocidal activities.

Keywords

Panchagavya
Superoxide
Mosquito vectors
I-IV instar
An. stephensi
PubMed
1

1 Introduction

Mosquitoes are one of the most significant risks to public safety in the world, as they are carriers of many pathogenic micro-organisms that cause various human diseases and those diseases are endangering human life and contributing to multiple morbidities and high mortality (Huang et al., 2019). Mosquito-borne diseases are widespread in more than 100 countries, triggering deaths of about 2 million people worldwide, and at least 1 million children suffer from mosquito-borne diseases per year, endangering as much as 2.1 billion people worldwide. In India, 17 states and 6 territories of the country have been identified as endemic, with about 553 million citizens at risk of mosquito-borne diseases (Elumalai et al., 2016).

The genera Anopheles, Aedes, and Culex mosquito species are carriers of various diseases, such as Japanese encephalitis, filariasis, malaria, chikungunya, dengue, and yellow fever (Benelli and Duggan, 2018). Anopheles mosquitoes are classified as human malaria vectors, filariasis, and some arboviruses and they exist nearly all over the world, except cold temperate areas and there are over 400 known species. Malaria appears to be one of the largest communicable diseases, which results in 300–500 million malaria cases per annum (Kesete et al., 2020). Aedes mosquitoes are painful and persistent biters. These mosquitoes are run sporters of dengue, yellow fever, chikungunya, and Zika viruses. One estimated prediction indicates that 3.9 billion dengue virus infections occurred on an annual basis, and from that, 0.096 billion are clinically expressed with disease severity. Another research on dengue incidence reports that 3.9 billion peoples are at danger of dengue virus infections (Waggoner et al., 2016). Cx. quinquefasciatus is an existing domestic mosquito related to human dwelling and activity. Cx. quinquefasciatus can spread many pathogenic agents like the West Nile virus, lymphatic filariasis, and Saint Louis encephalitis to humans and animals (Guta et al., 2021). Cx. quinquefasciatus may also cause avian malaria to cattle, insects, birds, sheep, cows, and wild animals which results in productivity loss and death (Sutthanont et al., 2019).

For several decades, the transmission chain of vector-borne diseases was broken by using synthetic insecticidal chemicals although, frequent use of synthetic insecticidal chemicals causes less effectiveness and ecological problems such as mosquito tolerance, ecological disturbance, and fallout to mammals (Chareonviriyaphap et al., 2013; Pavela 2008; Rahuman et al., 2008). Therefore, the hazards of synthetic insecticidal chemicals must be stabilized towards humans, wildlife, and non-target organisms. The effects of synthetic insecticidal agents, as well as mosquito tolerance, have prompted the hunt for novel insecticidal agents (Zaim and Guillet 2002; Thomas et al., 2004). To produce new insecticidal substances, we use the novel and effective product with insecticidal properties from the cow namely “panchagavya” (PG) to control various genera of mosquito. PG is an organic formulation, produced by combining five cow products i.e. dung, urine, milk, ghee, and curd. The components like cow dung and cow urine enhance the insecticidal activity of PG (Shailaja et al., 2014). It is already reported that cow urine and cow dung have larvicidal activity (Kumar et al., 2009; Ngugi 2009) and, PG has insecticidal and larvicidal activity (Sayi et al., 2018).

Therefore, the current research focused on preparation of PG, identification of the chemical compounds present in the PG, evaluation of its free radical scavenging activity, and the mosquitocidal efficiency by performing various mosquitocidal assays like larval and pupal toxicity, adulticidal, longevity, fecundity, and ovicidal assays.

2

2 Materials and methods

2.1

2.1 Preparation of PG

By using the Sathiyaraj et al. (2021) method, PG was prepared with some alterations. For the preparation of PG, 1 kg of indigenous cow dung, 300 g of ghee, 1 L of indigenous cow milk, 1 L of indigenous cow urine, 1 L of curd, ½ L of tender coconut water, 1 kg of jaggery, 6 number of ripened bananas, and water were added to the wide-mouthed container. The container was stirred twice a day and kept in the shade until the end of the process. Then the prepared PG was filtered in blotting paper and used for future studies.

2.2

2.2 Gas chromatography - mass spectroscopy (GC–MS) analysis

The components were separated using Helium as the carrier gas at a constant flow rate of 1 ml/min on a Perkin Elmer Clarus 680 GC with a fused silica column packed with Elite-5MS (5% biphenyl, 95% dimethylpolysiloxane, 30 m 0.25 mm ID 250 m df). The injector temperature was set at 260 °C during the chromatographic process. 1 µL of PG was injected into the instrument, and the oven temperature was set to 60 °C for 2 min, followed by 300 °C at a rate of 10 °C for 1 min, and 300 °C for 6 min. The mass detector was set up with the following parameters: 230 °C transfer line, 230 °C ion source, 70 eV electron ionisation mode impact, 0.2 sec scan time, 0.1 sec scan interval, and fragments ranging from 40 to 600 Da. The component spectrums were compared to the GC–MS NIST library's spectra database of known components (2008).

2.3

2.3 Free radicals scavenging activity

2.3.1

2.3.1 2,2-Diphenyl-1-picrylhydrazyl (DPPH)

The free radicals scavenging activity of PG against DPPH was performed by the method described previously (Sathiyaraj et al., 2020). 1 mL of PG at various concentrations (0, 10, 20, 30, 40 & 50 µg/mL) was blended with newly prepared DPPH solution (1 mL) and it was placed in the dark at 37 ℃ for 30 min. After incubation, scavenging activity was calculated based on the absorbance of the sample using UV spectrophotometry at 518 nm and it was compared with vitamin C. The scavenging activity of PG was studied by using formula 1.

(1)
% I n h i b i t i o n = Absorbance o f t h e c o n t r o l - A b s o r b a n c e o f t h e s a m p l e Absorbance o f t h e c o n t r o l × 100

2.3.2

2.3.2 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTs) assay

The method previously described was used to investigate ABTs scavenging activity of PG (Suriyakala et al., 2021). The reaction between 7 mM ABTS in distilled H2O and 2.45 mM K2S2O8 creates the ABTs cation radical, which was deposited in the dark for 12–16 h at room temperature before use. After then, the ABTs radical solution was mixed with methanol to obtain a 0.700 absorbance at 734 nm. The absorbance was measured 30 min after the initial mix with the addition of 1 mL of various PG concentrations. Formula 1 was used to get the percent absorbance inhibition at 734 nm. The same was done for vitamin C, which is utilised as a standard control, for comparison.

2.3.3

2.3.3 Hydroxyl radical (OH−) scavenging assay

The approach described by Pavithra and Vadivukkarasi (2015) was used to investigate the PG's OH-scavenging activity. 1.0 mL of PG (0, 10, 20, 30, 40, and 50 µg/mL), iron-EDTA solution (1.0 m), 0.018% EDTA (0.5 ml), DMSO (0.1 ml), and 0.22% vitamin C (1 mL) were included in the reaction mixture. Using a water bath, the reaction was heated to 80–90 °C for 15 min before being stopped by the addition of ice-cold TCA (1 mL). 3.0 ml Nash reagent was added to the reaction mixture and allowed to set at room temperature for 15 min before testing for color production. UV spectroscopy was used to measure the intensity of the yellow colour that developed at 412 nm. Formula 1 was used to calculate the OH− scavenging activity, which was then compared to vitamin C.

2.3.4

2.3.4 Superoxide (O2−) radical scavenging assay

The ability of PG to scavenge superoxide radicals was investigated using the previously established method of depletion of nitro blue tetrazolium (NBT) (Pradhan et al., 2021). 20 mM phosphate buffer (pH 7.4), 73 mM nicotinamide adenine dinucleotide, 50 mM NBT, 15 mM phenazine methosulfate, and various doses of PG (0, 10, 20, 30, 40, and 50 µg/mL) are included in the 1 mL reaction. The reaction mixture was left at room temperature for 5 min and the absorbance was measured at 562 nm. The reaction content's absorbance was compared to that of vitamin C, and the O2− scavenging activity was estimated using formula 1.

2.4

2.4 Mosquitocidal bioassays

All the mosquitocidal bioassays were carried out in the laboratory-reared mosquito vectors, without exposure to pesticides and disease-causing organisms. The selected mosquito species were reared to attain larval hatching by the previously described method (Thomas et al., 2017).

2.4.1

2.4.1 Larvicidal and pupicidal assays

The larvicidal and pupicidal assays using PG were performed by the standard procedures suggested by (Kovendan et al., 2012). Twenty-five numbers of I-IV instar of selected larvae and pupae were added to a beaker containing 2 mL of desired concentration of PG and 248 mL of dechlorinated water and it was checked for mortality. At each desired concentration the experiment was three times replicated. The larvae and pupae were treated with 248 mL of dechlorinated water and 2 mL of acetone which was used as control. Percent mortality was calculated by formula 2,

(2)
Percent M o r t a l i t y = Number o f d e a d l a r v a e o r p u p a e Number o f l a r v a e o r p u p a e i n t r o d u c e d × 100

2.4.2

2.4.2 Adulticidal assay

The adulticidal assay using PG was examined by the method suggested by Ejeta et al. (2021). 2 mL of varying concentrations (50, 100, 150, 200 & 250 ppm) of PG was applied on the filter paper and it could dry completely. The dried filter paper was introduced into an exposure tube in the WHO testing kit. A whole 25 number of 2–5 days old, blood-unfed female mosquitoes were inserted into the holding tube and retained for 1 h to adapt to the condition. Then the mosquitoes were transported in the exposure tube by gentle blowing and retained for an acclimatization period of 1 h. Then the mosquitoes were got back to the holding tube and transferred to a petri-dish containing cotton pad soaked with a 10% C6H12O6 solution for the recovery of mosquitoes. The number of dead mosquitoes was registered at 24-hours of the recovery period, and the percentage mortality was estimated. At the same time control was made by using acetone as well as experiment was replicated three times.

2.4.3

2.4.3 Fecundity and longevity studies

The fecundity and longevity of the selected mosquito species were studied by the methodology suggested by (Nareshkumar et al., 2012). The mosquito fecundity was checked by picking an equivalent number of male and female mosquito larvae from the control and treated using PG. From each concentration, these mosquitoes were separately placed in 30 × 30 cm cages. Three days followed by the blood meal; eggs were collected regularly from tiny plastic bowls that hold water contained in an ovitrap in the cages. Fecundity was determined from the number of eggs laid in an ovitrap, separated by the individuals of females mated. Consideration has been granted to adult mortality in such studies. Adult longevity of male and female mosquitoes was calculated according to the number of days the adult mosquitoes lived. A total number of days were recorded from adult emergence to death and the means were calculated to give the average longevity within days.

2.4.4

2.4.4 Ovicidal activity

The method from Torawane et al. (2021) was used and applied with some modification for ovicidal activity. A total of 100 Eggs of the chosen mosquito species were subjected to different concentrations of PG (50, 100, 150, 200, 250 & 300 ppm). After 24 h of examination, the eggs were counted using a microscope and the eggs were separately taken, then transferred to a beaker containing demineralized water for egg hatching evaluation. The process was triplicated along with proper control. After 48 h of PG exposure, the egg hatchability was calculated by formula 3,

(3)
Percent h a t c h a b i l i t y = Number o f h a t c h e d l a r v a e Total n u m b e r o f e g g s × 100

2.5

2.5 Data analysis

The LC50, LC90, and other statistics were calculated using the mean mortality data at 95% upper and lower fiducial limits. The chi-square values were calculated using the statistical software SPSS (version 25). Statistically significant results are those with a P value of less than 0.05.

3

3 Results

3.1

3.1 GC–MS analysis

In the GC–MS analysis, 15 different compounds were obtained, and the chromatogram was given in Fig. 1. The compounds Retention Time (RT), Peak Area (%), and biological activities have been presented in Table 1.

GC–MS chromatogram of PG.
Fig. 1
GC–MS chromatogram of PG.
Table 1 Compounds identified in the PG and their biological activities.
S. No Retention Time % Area Compound Biological activity Reference
1 9.951 3.628 Paraldehyde Anticonvulsant activity, Anti-HIV activity Rowland et al., 2009; Flavin et al., 1996
2 10.041 14.483 1,2-propanediol diformate No biological activity reported –
3 10.176 10.233 Acetaldehyde, tetramer No biological activity reported –
4 10.236 8.017 Paraldehyde Anticonvulsant activity, Anti-HIV activity Rowland et al., 2009; Flavin et al., 1996
5 10.291 4.617 Ethene, 1,1′-[oxybis(2,1-ethanediyloxy)]bis No biological activity reported –
6 10.371 10.385 Butanoic acid, 3-hydroxy-, ethyl ester, (.+/-.)- Antimicrobial, anti-inflammatory, antioxidant, Anticancer, antiarthritic, antihistimic, antieczemic, and anticoronary activities Kumar et al., 2010
7 10.477 13.685 2-butanol, 3-chloro No biological activity reported –
8 10.632 2.551 Methoxyacetic acid, butyl ester Antimicrobial activity Ganesh and Mohankumar 2017
9 14.723 2.325 1-methyl-1-(2-hydroxyethyl)-1-silacyclobutane No biological activity reported –
10 15.519 3.070 Butanamide, 3,n-dihydrox- No biological activity reported –
11 15.699 5.301 Propanoic acid, 3-methoxy-, methyl ester Antibacterial, antifungal, antioxidant, nematicide, pesticidal activities Mahalakashmi and Thangapandian 2019
12 26.758 2.594 Cholest-5-en-3-ol (3.beta.)-, acetate No biological activity reported –
13 27.353 2.546 2,3-o-benzal-d-mannosan No biological activity reported –
14 27.629 10.816 Di-n-propylmalonic acid No biological activity reported –
15 27.999 5.751 Pseduosarsasapogenin-5,20-dien methyl ether No biological activity reported –

3.2

3.2 Free radicals scavenging activity

DPPH, ABTs, OH− and O2− are the typical reactive oxygen species (ROS), which trigger diseases linked to oxidative stress. PG effectively scavenged all four ROS and the percent inhibition was raised in a concentration-dependent way. The IC50 of PG was found to be 37, 37.5, 35, and 38 μg/mL respectively on DPPH, ABTs, OH− and O2−. The results of PG were compared with standard control at each concentration (Figs. 2 & 3).

Free radicals scavenging at different concentrations A) DPPH B) ABTs.
Fig. 2
Free radicals scavenging at different concentrations A) DPPH B) ABTs.
Free radicals scavenging at different concentrations A) OH− B) O2−.
Fig. 3
Free radicals scavenging at different concentrations A) OH− B) O2−.

3.3

3.3 Mosquitocidal bioassays

Under the in-vitro condition, PG was found to be toxic towards I-IV instar of larvae and pupae of An. stephensi, Ae. aegypti and Cx. Quinquefasciatus (Table 2). The LC50 of I-IV instar of An. Stephensi was found to be 121.991, 146.723, 166.471, 192.303, and 216.687 ppm (Pupae) respectively. The LC90 of I-IV instar of larvae and pupae of An. stephensi was found to be 246.063, 286.663, 327.514, 375.741, and 411.457 ppm (Pupae) respectively. The LC50 of I-IV instars of Ae. aegypti recorded as follows: 129.754, 153.531, 179.644, 203.369 and 298.276 ppm (pupae) and LC90 was found to be 258.552, 297.332, 344.920, 391.304 and 424. 610 ppm (pupae) respectively. The LC50 of I-IV instar of Cx. quinquefasciatus was noted as follows: 148.765, 162.534, 187.619, 210.835, and 234.624 ppm (Pupae), and the LC90 was found to be 286.636, 306.390, 350.276, 390.735, and 419.195 ppm (Pupae) respectively. After 24 h exposure of adulticidal mosquitoes to 50, 100, 150, 200, and 250 ppm concentration of PG, it was found that PG exhibited high toxicity to the selected adult mosquito vectors however, the highest mortality was found against An. stephensi (91.10 ± 1.74%) with the IC50 and IC90 values of 128.114 and 260.609 ppm followed by Ae. aegypti (88.86 ± 1.50%) and Cx. quinquefasciatus (85.70 ± 1.41%) with the IC50 and IC90 values of 135.179, 144.336 ppm and 272.575, 283.066 ppm respectively (Table 3).

Table 2 Larvicidal and pupicidal activities of PG against selected mosquito species.
Mosquito species Target LC50 (LC90) (ppm) 95% confidence Limit Regression equation χ2 (df = 4)
LC50 (LC90)
LCL UCL
Anopheles stephensi I instar 121.991
(246.063)		 109.007
(226.351)		 133.813
(273.033)		 y = 1.260 + 0.010x 4.311n.s.
II instar 146.723
(286.663)		 133.517
(261.212)		 159.837
(322.918)		 y = 1.344 + 0.009x
 4.887n.s.
III instar 166.471
(327.514)		 151.830
(293.769)		 182.445
(378.308)		 y = 1.325 + 0.008x
 2.727n.s.
IV instar 192.303
(375.741)		 175.078
(330.398)		 213.970
(448.581)		 y = 1.343 + 0.007x
 2.530n.s.
Pupa 216.687
(411.457)		 196.404
(356.910)		 245.310
(502.949)		 y = 1.426 + 0.007x
 1.128n.s.
Aedes aegypti I instar 129.754
(258.552)		 116.783
(237.292)		 141.845
(287.965)		 y = 1.291 + 0.010x 3.823n.s.
II instar 153.531
(297.332)		 140.173
(270.208)		 167.179
(336.351)		 y = 1.368 + 0.009x
 3.928n.s.
III instar 179.644
(344.920)		 164.446
(307.920)		 197.346
(401.489)		 y = 1.393 + 0.008x
 2.802n.s.
IV instar 203.369
(391.304)		 184.969
(342.179)		 227.793
(471.601)		 y = 1.387 + 0.007x
 1.803n.s.
Pupa 228.276
(424.610)		 206.449
(366.883)		 260.330
(522.613)		 y = 1.490 + 0.007x
 2.162n.s.
Culex quinquefasciatus I instar 148.765
(286.636)		 135.789
(261.533)		 161.757
(322.244)		 y = 1.383 + 0.009x 4.942n.s.
II instar 162.534
(306.390)		 149.272
(278.244)		 176.597
(347.053)		 y = 1.448 + 0.009x
 2.053n.s.
III instar 187.619
(350.276)		 172.326
(312.912)		 206.000
(407.287)		 y = 1.478 + 0.008x
 2.978n.s.
IV instar 210.835
(390.753)		 192.462
(343.182)		 235.590
(467.437)		 y = 1.502 + 0.007x
 2.716n.s.
Pupa 234.624
(419.195)		 213.096
(364.682)		 266.167
(509.933)		 y = 1.629 + 0.007x
 1.807n.s.

LC50 = Lethal concentration that kills 50% of the exposed mosquito species.

LC90 = Lethal concentration that kills 90% of the exposed mosquito species.

LCL = Lower Confidence Limit.

UCL = Upper Confidence Limit.

χ2 = chi-square.

n.s. = not significant (α = 0.05).

Table 3 The adulticidal activity of PG against selected mosquito species.
Mosquito species Concentration (ppm) Mortality (%) (mean ± SD) LC50 ppm (LCL-UCL) LC90 ppm (LCL-UCL) χ2 (df = 3)
Anopheles stephensi Control
50
100
150
200
250		 0.00 ± 0.00
26.84 ± 1.59e
33.98 ± 1.33d
58.64 ± 1.21c
72.68 ± 1.47b
91.10 ± 1.74a 		128.114
(114.674–140.511)		 260.609

(238.694–291.155)		 3.615n.s
Aedes aegypti Control
50
100
150
200
250		 0.00 ± 0.00
25.70 ± 1.46e
32.38 ± 1.62d
54.52 ± 1.04c
70.38 ± 1.67b
88.86 ± 1.50a 		135.179

(121.690–147.932)		 272.575

(248.926–305.943)		 3.190n.s
Culex quinquefasciatus Control
50
100
150
200
250		 0.00 ± 0.00
22.94 ± 1.73e
29.92 ± 1.85d
50.62 ± 1.66c
68.84 ± 1.62b
85.70 ± 1.4a 		144.336

(131.129–157.285)		 283.066

(258.283–318.191)		 2.148n.s

The findings were expressed as mean ± standard deviation. According to Duncan's technique, the values and a-e superscripts are significantly different from each other (p = 0.05). n.s. = not significant (α = 0.05).

Adult longevity and fecundity of the selected mosquito vectors using PG were given in Table 4. When compared with control, a great significant result was observed in the longevity and fecundity of the selected mosquito vectors. Longevity of male mosquitoes of An. Stephensi was found to be 22.2 days (d), 18.4 d, 14.6 d, 10.2 d, 07.4 d, and in the longevity of male mosquitoes of An. Stephensi was found to be 26.3 d, 22.2 d, 14.1 d, 12.4 d, and 08.1 d respectively on 50, 100, 150, 200, and 250 ppm, whereas control showed 26.0 d in males and 35.0 d in females for adult longevity. After exposure of PG, the fecundity of An. stephensi also reduced as follows: 126.2, 107.2, 96.4, 77.2, and 55.8 at 50, 100, 150, 200, and 250 ppm respectively.

Table 4 Longevity and Fecundity effects of PG.
Treatment (ppm) Adult longevity (days) Fecundity (no. eggs)
Male Female
Control 26.0 ± 0.61a 35.0 ± 0.70a 146.0 ± 0.64a
Anopheles stephensi 50 21.4 ± 1.14b 29.2 ± 1.48b 131.7 ± 1.70b
100 18.6 ± 1.51c 23.7 ± 0.95c 128.2 ± 0.83c
150 17.4 ± 2.50d 18.2 ± 0.83d 115.2 ± 1.78d
200 12.6 ± 0.89e 15.4 ± 1.14e 101.8 ± 1.09e
250 9.2 ± 0.44f 11.2 ± 1.30f 99.4 ± 1.51f
Aedes aegypti Control 26.0 ± 0.61a 35.0 ± 0.70a 146.0 ± 0.64a
50 25.6 ± 1.67b 32.8 ± 1.64b 136.8 ± 1.30b
100 22.8 ± 1.48c 25.8 ± 0.44c 132.5 ± 0.91c
150 19.2 ± 1.09d 22.7 ± 0.44d 128.9 ± 1.67d
200 13.8 ± 1.48e 18.6 ± 1.51e 117.4 ± 1.51e
250 10.1 ± 0.93f 13.8 ± 1.09f 103.0 ± 1.87f
Culex quinquefasciatus Control 26.0 ± 0.61a 35.0 ± 0.70a 146.0 ± 0.64a
50 27.2 ± 1.92b 34.2 ± 0.44b 133.6 ± 1.81b
100 24.4 ± 2.07c 29.6 ± 1.51c 130.4 ± 2.50c
150 20.4 ± 1.81d 25.0 ± 0.57d 129.4 ± 1.51d
200 17.4 ± 1.14e 21.2 ± 0.44e 125.8 ± 0.83e
250 13.1 ± 0.77f 19.6 ± 0.89f 108.8 ± 1.64f

The findings were expressed as mean ± standard deviation. According to Duncan's technique, the values with different superscripts are significantly different from each other (p = 0.05).

Adult longevity of Ae. aegypti (males) was noted as follows 23.8 d, 19.2 d, 15.4 d, 12.5 d, 09.3 d, and the longevity of Ae. aegypti in females was noted as 30.2 d, 24.6 d, 18.9 d, 15.4 d, 10.2 d at 50, 100, 150, 200, and 250 ppm respectively. Here 27.0 d in males and 35.0 d in females was observed for adult longevity of control. After exposure of PG, the fecundity of Ae. aegypti also reduced as follows: 127.8, 112.2, 95.2, 73.6, and 57.4 at 5, 10, 15, 20, and 25 ppm respectively.

Adult longevity of Cx. quinquefasciatus in males, after exposure of PG, was found to be 23.3 d, 20.5 d, 18.7 d, 12.1 d, 10.1 d and in females exhibited as follows 31.2 d, 23.3, 17.0 d, 14.4 d, 12.3 d at 50, 100, 150, 200 and 250 ppm whereas control showed 26.0 d in males and 35.0 d in females. After exposure of PG, the fecundity of Cx. quinquefasciatus also reduced as follows: 130.8, 111.2, 92.0, 74.6, and 59.6 at 5, 10, 15, 20, and 25 ppm respectively. The ovicidal activity of different concentrations of PG (50, 100, 150, 200, 250, and 300 ppm) on tested mosquito species was presented in Table 5. PG efficiently inhibits the 100% egg hatchability of An. stephensi, Ae. aegypti and Cx. Quinquefasciatus at 250 ppm, 300 ppm, and 300 ppm respectively.

Table 5 Ovicidal activity of PG.
Mosquito species Egg hatchability (%)
Concentration (ppm)
Control 50 100 150 200 250 300
Anopheles stephensi 100
±0.0a 		58.8
±1.92b 		39.6
±1.14c 		25.6
±1.51d 		11.4
±1.81e 		NH NH
Aedes aegypti 100
±0.0a 		60.2
±1.78b 		41.4
±1.81c 		27.6
±1.94d 		13.4
±1.14e 		07.0
±1.58f 		NH
Culex quinquefasciatus 100
±0.0a 		61.6
±1.67b 		40.0
±1.58c 		28.8
±1.30d 		15.2
±1.78e 		09.6
±1.14f 		NH

The findings were expressed as mean ± standard deviation. Significantly differed values were mentioned with a-e superscripts according to Duncan's method (p = 0.05).

NH – no hatchability.

4

4 Discussion

4.1

4.1 Compounds in the PG

The compounds identified in the PG are; paraldehyde; 1,2-propanediol diformate; acetaldehyde, tetramer; paraldehyde; ethene, 1,1′-[oxybis(2,1-ethanediyloxy)]bis; butanoic acid, 3-hydroxy-, ethyl ester, (.+/−.)-; 2-butanol, 3-chloro; methoxyacetic acid, butyl ester; butanamide, 3,n-dihydrox-; propanoic acid, 3-methoxy-, methyl ester; cholest-5-en-3-ol (3.beta.)-, acetate; 2,3-o-benzal-d-mannosan; di-n-propylmalonic acid; pseduosarsasapogenin-5,20-dien methyl ether. These compounds possess various biological activities including anti-HIV, anti-cancer, antioxidant, anti-inflammatory, nematicide, and pesticidal activities (Rowland et al., 2009; Flavin et al., 1996; Kumar et al., 2010; Ganesh and Mohankumar 2017; Mahalakashmi and Thangapandian 2019).

4.2

4.2 Free radicals scavenging activity

Based on free radicals scavenging results, the PG scavenges toxic free radicals, and it can protect the free radicals from adverse effects. Both hydrophilic and hydrophobic antioxidants are present among naturally occurring antioxidants. Ascorbic acid, α-lipoic acid, bioflavonoids, polyphenols, and glutathione are the main hydrophilic antioxidants while vitamin E, α- tocopherol, co-enzyme Q10, Total carotenoids, and Lycopene are the hydrophobic antioxidants (Lazzarino et al., 2019). Free radicals are produced in both water and fat portions in intracellular and extracellular environments; hence, a mixture of hydrophilic and hydrophobic antioxidants is essential for the body to achieve the maximum spectrum of protection. Certain antioxidants are synthesised by the body, while others are obtained from external sources such as milk and nutraceuticals. Panchagavya is a unique blend of water-based (fat-free colloidal milk, curd, urine, and dung) and fat-based materials (ghee, fat-particulate milk). This is likely to provide natural antioxidants which are both polar and non-polar. The data of the free radicals scavenging activity of PG reveals that the PG act as an excellent antioxidant. Athavale et al. (2012) evaluated the free radicals scavenging activity of PG and the results of the DPPH assay and superoxide assay were agreed with our findings. Previous studies confirmed that cow urine and cow dung alone have free radical scavenging activity (Jarald et al., 2008; Jirankalgikar et al., 2014), when it is used in a combination it exhibits an exceptional free radical scavenging activity.

4.3

4.3 Mosquitocidal activity

Synthetic insecticides have been employed against mosquito vectors over the last 5 decades. Therefore, side effects such as environmental contamination and toxic hazards towards humans and other non-target species were developed. Such side effects of synthetic insecticides provided recognition of the requirement for mosquito control which is eco-friendly, biodegradable, target-specific, and indigenous material (Reegan et al., 2015; Amerasan et al., 2015). The development cycle of a mosquito represents the egg, larvae, pupae, and adult stage. To control the mosquito population, kill them at egg and larvae stages, which is more effective compared to targeting the free-flying adult mosquitoes (Carvalho et al., 2003). Here we are using PG, formulated from indigenous material for mosquito control. PG potentially acts against mosquitoes not only at the larval stage but also includes each development stage of mosquitoes. Our findings of larvicidal, pupicidal, adulticidal, fecundity, longevity, and ovicidal activity of PG on I to IV instar of selected mosquito vectors showed proof of a lethal effect, and mortality was dose dependent. Kumar et al. (2009) tested the larvicidal activity of cow urine against Cx. quinquefasciatus at 5% and they obtain 95% mortality on the 3rd day. There are few reports were available on cow dung as a mosquito repellent (Sharma et al., 2017; Mandavgane et al., 2005). There are no references available in the literature related to mosquitocidal studies on PG and a few cow products. Hence, this study will be established as a future reference for further investigations on PG and cow products.

5

5 Conclusion

In the present study, fifteen chemical compounds have been identified from PG by GC–MS analysis and these compounds have various biological activities. Further, PG possesses excellent free radical scavenging and mosquitocidal properties. Our results confirm that the PG is toxic towards An. stephensi, Ae. aegypti and Cx. Quinquefasciatus at each stage of their life cycle. It was concluded that PG is often very effective against a specific target, less costly, effortlessly biodegradable and it could be a substitute for synthetic insecticides for mosquito vector control.

Acknowledgement

The authors acknowledge King Saud University, Riyadh, Saudi Arabia for funding this research through Researchers Supporting Project No. (RSP2022R465). Also authors thank the University of Douala, Cameroon, for the support and encouragement.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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