Journal of Physics: Conference Series
PAPER • OPEN ACCESS
Biological activity of Pomegranate peels Punica granatum Silver
nanoparticles AgNPs extract against Fourth larvae of Culex
quinquefasciatus mosquito (Diptera: Culicidae)
To cite this article: Maallah T. Al-Husseini et al 2020 J. Phys.: Conf. Ser. 1660 012013
View the article online for updates and enhancements.
This content was downloaded from IP address 161.142.231.214 on 24/03/2021 at 16:50
1st International Conference on Pure Science (ISCPS-2020) IOP Publishing
Journal of Physics: Conference Series 1660 (2020) 012013 doi:10.1088/1742-6596/1660/1/012013
Biological activity of Pomegranate peels Punica granatum
Silver nanoparticles AgNPs extract against Fourth larvae of
Culex quinquefasciatus mosquito (Diptera: Culicidae)
Maallah T. AL-Husseini, Harith R. Al-Mousawi, Naser Jawad Kadhim, Ahmed
Abd Al-Redaa Madhloom, Dhifaf Zeki Aziz and Azhar Jewad Kadhim Muha
Faculty of science/ University of Kufa
[email protected]
Abstract. Present study was included the assessment of biological activity of Pomegranate Peels
Punica granaatum plant extract toward fourth Culex quinquefasciatus larvae mosquito (Order:
Diptera, Family: Culicidae) by testing ethanolic and Silver nanoparticles extracts. Results of
present study revealed that Silver nanoparticles extract was more toxic than ethanolic extract and
the mortality rates were reached to 90 % after 72 h exposure time when it treated with 5 μg/ml
compared to 0% at control treatments. Ethanolic extract also was toxic at concentration 5 μg/ml
and mortality rates were reached to 80% after 72 h exposure time. Study of larvae after death
showed that penetration of Silver nanoparticles extract was entered inside mid gut of larvae after
damage of body wall. SEM analysis indicated to that more deformation found in body wall thus
AgNPs were easily penetration through skin of fourth larvae. SEM analysis also was showed
that Silver nanoparticles were clustered and irregular or oval shapes.
1. Introduction
Peels of pomegranate P. granatum contain a wide variety of phytochemical compounds such as
gallotannins, gallic acid, ellagic acid, punicalins and punicalagins [1, 2]. There are several studies
referred to the insecticidal effects of pomegranate juices for instance [3], who tested leaves of
pomegranate as insecticidal activity toward the stored grains Rhyzopertha dominiea (Fabr) under
optimum conditions, the pomegranate powder leaf gave a high incidence of death and development rate
reduction in population.
The pomegranate peels have a several health benefits by their different tannins, alkaloid, flavonoids,
and organic acids. The gallic acid and granatin B exhibit an anti-inflammatory activities [4, 5, 6].
The mosquitoes are one of medical important can spread many of diseases in the world like malaria
diseases and arboviral such as Dengue fever, West Nile Virus and currently Zika Virus [7]. However,
the chemical insecticides dense application to control mosquito vectors after spreading of RVF in the
Arabian Peninsula in 2000 [8] and Dengue Fever in 1997 and 2004 [9] resulted in developed resistance
in mosquitoes.
In Iraq Cx. quinquefasciatus is abundant in middle and southern regions can cause multiple problems
such as biting nuisance and may be serve as carriers for diseases [10]. Nanoparticles are efficient and
Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution
of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI.
Published under licence by IOP Publishing Ltd 1
1st International Conference on Pure Science (ISCPS-2020) IOP Publishing
Journal of Physics: Conference Series 1660 (2020) 012013 doi:10.1088/1742-6596/1660/1/012013
ecofriendly in compart to chemical insecticides especially when they mixed with plant extracts that
provided safe and beneficial methods to synthesis of AgNPs that are simple and large amounts of
nanoparticles can be prepared in a standard time [11]. Present study was attempted to investigate
biological activity of biosynthesis of pomegranate Peels extract nanoparticles (AgNPs) because of their
safety, inexpensive and readily available as alternative method to the traditional chemical insecticides
against Cx. quinquefasciatus.
2. Materials and Methods
2.1. Preparation of Plant Extracts
Peels of pomegranate P. granatum were prepared according [12]. Dried peels of P. granatum were
ground into fine powder via electric grander after drying it at room temperature, then resultant powders
were extracted using 70% ethanol. First primary tested of plant extract was conducted via cool aqueous
extracts at different concentration. 100g of peels powder mixed with 300ml of the ethanol solvent for
24 h, agitation at 24C°. The mixture was filtered through a Whatman filter paper No. 5 and the flow –
throw was dried in a rotary evaporator at 40 C for 2-3h.Then 1g of the extract dissolved in 100ml of
70% ethanol to get 1% as a mother solution.
2.2. Silver Nanoparticles (AgNPs) Extracts Preparation
1 mL of 1mM AgNO3 solution added to 99 mL standard solution of pomegranate Punica granatum
extracts Peels, at room temperature with shaking until changing the color to brown that indicating the
formation of nanoparticles. The shape of the formed nanoparticles was distinguished using scanning
electron microscope (SEM from Central Lab. Faculty of Science/ University of Kufa /Iraq) at an
accelerating voltage of 12.50 KV, [13].
2.3. Identification and Rearing of Mosquito
Rearing of mosquitoes was done according to [14]. The samples of mosquito larvae were collected from
natural pools, then the larvae were identified morphologically and were sent to certify by Natural
Museum/Baghdad as Cx. quinquefasciatus. About (100-150) larvae were placed in (25 x15 x8) cm
plastic container containing water from pool. The larvae were supplied a mixture of yeast and fine bread
at equal amounts as food. Eggs were obtained from a live pigeon that attached to its legs and wings and
placed inside breeding cage during night. The females can take the blood as meal. The received eggs
placed in a small plastic container with a amount of water and a larval culture to obtain the suitable
number of tested larvae.
2.4. Bioassay Experiments
Larvicidal activity of the Peels of pomegranate loaded as Silver nanoparticles (AgNPs) tested on the
fourth larval instars of Cx. quinquefasciatus was assessed by using the standard methods [15]. Different
concentrations (0, 1, 2, 3, 4 and 5) g/ml of (AgNPs) extracts were prepared. For each treatment three
replicates as well as control treatment was prepared. The experiments were carried out under (27 ± 2)
ºC, RH (70± 10) % light time (12-12h). The control treatments were occurred by adding 0.1ml ethanol
70% only with the same amount of water. Mortality rate noted daily, the dead pupae and larvae were
continuously removed until adult appearance.
2.5. Statistical Analysis
The data was analysis as a Factorial Experiments (Completely Randomized Design C.R.D). The results
were compared by using Least Significant Difference Test (L.S.D), P< 0.05 [16].When necessary, the
mortality percentage in the treatments were corrected by using Abbott method [17].
3. Results and Discussions
2
1st International Conference on Pure Science (ISCPS-2020) IOP Publishing
Journal of Physics: Conference Series 1660 (2020) 012013 doi:10.1088/1742-6596/1660/1/012013
3.1. SEM Morphological Characterization
The scanning electron micrographs clearly showed that clustered of irregular or oval shapes of silver
nanoparticles, an average size 40-100 nm, with inter particle distance the magnified done at (1629-1753
X), figure (1).
Figure 1. The SEM image showing the crystalline silver nanoparticles as uniform and aggregates.
3.2. Larvicidal Effects of Peels of Pomegranate Extract and its Silver Nanoparticles on Cx.
quinquefasciatus larvae
The results showed in table (1), the effects of the ethanol pomegranate Peels extract with and without
silver nitrate toward the fourth larvae of Cx. quinquefasciatus. No results findings after 24 h then after
48 the AgNPs extract was highly toxic when treated with high concentration (5) μg/ml, mortality rates
were recorded (85) % and were increased after exposure time 72 h it reached to (90) % compared to (0)
% at control treatments. After 48h ethanolic extract also was recorded mortality rates were (70) % and
were increased to 80% after 72 h. Lower concentration revealed less toxic effects against fourth larvae
at concentrations 1 μg/ml the mortality rates were (26.6) % compared to (0) % at control treatments.
Statistical analysis showed that there were significantly differences at (P 0.05) % between values.
Table 1. The effect of pomegranate peels P. granatum ethanol extract and its Silver nanoparticle
extract AgNPs after 24, 48h exposure against Cx. quinque fasciatus larvae
Ethanol extract Silver nanoparticle extract AgNPs
Conc. L.S.D
μg/ml Larval Mortality (%) Larval Mortality (%) after P ˃0.05
(24) h (48)h (72)h (24)h (48)h (72)h
Control 0.0± 0.0± 0.0± 0.0± 0.0± 0.0± 00
1.0 0.0± 0.0± 10± 0.0± 26.6± 30± 2
2.0 0.0± 10± 30± 0.0± 30± 50± 2
3.0 0.0± 20± 40± 0.0± 60± 70± 2
4.0 0.0± 50± 70± 0.0± 70± 90± 2
5.0 0.0± 70± 80± 0.0± 85± 90± 2
L.S.D
00 3.5 2.5 00 3 2 2
P ˃0.05
3
1st International Conference on Pure Science (ISCPS-2020) IOP Publishing
Journal of Physics: Conference Series 1660 (2020) 012013 doi:10.1088/1742-6596/1660/1/012013
From data reviewed above apparently showed that mixing of the extract and the prepared AgNPs
were found more active as insecticide against the 4th larvae of Cx. quinquefasciatus. Several previous
studies indicated that silver nanoparticles could provide powerful larvicidal effects. The biological
activity of the AgNPs may be contributed to penetrate of AgNPs through the larval membrane which
leads to the death of the delicate larvae [18] as well as, the increased activity might be attributed to small
size of the particles that facilitate its passage through the body wall to inside the cells which might
reverse negatively the ecdysis addition to other physiological processes [19].
The AgNPs as a larvicidal activity may be due to denaturation of the sulfur-containing proteins, or
phosphorus containing compounds like DNA that leads to the denaturation of organelles and enzymes
[20], and reduces the cellular membrane permeability and reduction in ATP synthesis which finally
causes cell death [21]. Similar findings [22] were revealed that mosquito larvicidal bioassay with the
AgNPs synthesized by dried green fruits of Drypetes roxburghii against two species of mosquito Cx.
quinquefasciatus and Anopheles stephensi. Study [23] who indicated that the larvicidal activity of silver
nanoparticles synthetize by Swietenia mahagoni (L.) Jacq. Leaf extract against An. stephensi larvae and
group of Cx. vishnui and Cx. quinquefasciatus by concentration 80 ppm was exhibit a significantly
higher than the mortality rates by 20 ppm, 40 ppm and 60 ppm of AgNPs of S. mahagoni leaf extract.
From other side the biological activity of ethanolic plant extract might be due to multiple compounds
including phenolic, terpenoids, and alkaloids especially tannins were identified in ethanol extract by
study of [24]. The tannin formed irreversible complex with the proteins containing proline lead to inhibit
cell protein synthesis, this might be attributed for the mortality in Cx. quinquefasciatus larvae [25].
3.3. Histological Examination
Results of present study clearly showed that the penetration by AgNPs plant extract of mid gut along
the elementary canal after 72 h when dead fourth larva Cx. quinquefasciatus was treated with 5 μg/ml
of AgNPs plant extract which tested by light microscope at (40X) (figure 2).
40 X
Figure 2. Fourth larva of Cx. quinque fasciatus treated with 5 μg/ml of AgNPs plant extract of
pomegranate Peels P. granatum.
Results of SEM also indicated more damaged and clearly changes in body wall of fourth
larva Cx. quinquefasciatus revealed more shrinkage when it treated at 5 μg/ml of AgNPs plant
4
1st International Conference on Pure Science (ISCPS-2020) IOP Publishing
Journal of Physics: Conference Series 1660 (2020) 012013 doi:10.1088/1742-6596/1660/1/012013
extract after 72 h compared to control treatments that appeared more distended tissues figure
(3).
Figure 3. Fourth larva of Cx. quinquefasciatus tested by SEM (A and B) treated with 5 μg/ml of
AgNPs plant extract of pomegranate Peels P. granatum. (C and D) control treatments.
From data of present study mentioned above demonstrated that larvicidal effects, included multiple
alterations in body wall and then penetration of AgNPs plant extract inside alimentary canal. If the body
wall was damaged or injured penetration by AgNPs plant extract would be easier passage that proved
the plant extract mixing with silver nitrate has led to penetration and this was agreed with many previous
studies. The study of [26] reported the silver particles processing from Melia dubia leaves extracts were
higher effective than the crude extract against mosquito larvae Cx. quinquefasciatus. The alterations
were found in the mid gut and body wall may be explained according to [26] because the midgut which
responsible for digestion process of larva which direct interaction with the toxic compounds, and so
initiating death. Also progressive damage of the intestinal tissue of larvae, finally, resolve the gut cells
content with hemolymph, which is responsible for larval mortality. The study [27] confirmed the activity
of aqueous extract of Eucalyptus leaves acts on Cx pipiens larvae and hypertrophy was observed in
intestinal cells which began to pull away from each other. The results of present study agreed with [28],
that reported clear damage of the epithelial cells of the midgut and intestinal tissue muscles and cuticle
5
1st International Conference on Pure Science (ISCPS-2020) IOP Publishing
Journal of Physics: Conference Series 1660 (2020) 012013 doi:10.1088/1742-6596/1660/1/012013
of Cx pipiens larvae were the most severely damaged when treatment by the Citrus limon and Allium
sativum oils. The study [29] appeared aqueous leaves extract of Ricinus communis induces
histopathological changes at different levels of the body leading to disorganization of movements
followed by immobilization and subsequent death of the fourth larvae of Cx pipiens.
References
[1] Farag, R. S. and Emam, S. S. (2016). Insecticidal activities of Pomegranate Peels and leave crude
Juices: Bull. Env. Pharmacol. Life Sci., Vol 5 [3] February p: 68-75.
[2] Farag, R.S.; Abdel-Latif, M.S.; Emam,S.S. and Tawfeek, L.S. (2015).Phytochemical screening
and polyphenol constituents of pomegranate peels and leave crude juices. Int. J. Med. Med.
Sci., 48:1584-1591.
[3] Ghandi, N. and Pillai, S. (2011). Control of Rhyzopertha dominica (Coleoptera: Bostrichidae) by
pulverized leaves of Punica granatum (Lythraceae) and Murraya koenigii (Rutaceae). Int. J.
Agric. Biol., 13(4): 535-540.
[4] Satomi, H.; Umemura, K.; Ueno, A.; Hatano, T.; Okuda, T. and Noro, T. (1993). Carbonic
anhydrase inhibitors from the pericarps of Punica granatum L. Biol. Pharma. Bull. 16:787–
790
[5] Amakura, Y.; Okada, M. ; Tsuji, S. and Tonogai, Y. (2000). High performance liquid
chromatographic determination with photodiode array detection of ellagic acid in fresh and
processed fruits. J Chromat 896:87–93
[6] Malviya,S.; Arvind,A.J. and Hettiarachchy,N.(2014). Antioxidant and antibacterial potential of
pomegranate peel extracts. J. Food Sci. Technol. 51(12):4132–4137.
[7] WHO.(2016).Malariareport.Availableatathttp://www.who.int/malarai/puplications/world -
malaria20%-report-2012.
[8] Himeidan, Y.E ; Kweka, E.J.; Mahgoub, M.M;El Rayah,EA; and Ouma, J.O.(2014).Recent
outbreaks of rift valley fever in East Africa and the Middle East. Frontiers in Public Health
2:169.
[9] Alhaeli, A.; Bahkali, S.; Ali, A.; Househ, M.S. and El-Metwally, A.A. (2016).The epidemiology
of dengue fever in Saudi Arabia: a systematic review. Journal of Infection and Public Health
9:117-124.
[10] Shirzadi, M.R. (2019) Entomological Studies on the Mosquito Borne Diseases in Iran: An Up-
To-Date. Insights Biomed Vol.4 No.2:10.
[11] Afrah,E.M. (2015). Green synthesis, antimicrobial and cytotoxic effects of silver nanoparticles
mediated by Eucalyptus camaldulensis leaf extract, vol-5(5), Pp no-382-386.
[12] Ghramh, H. A.; Al-Ghamdi,K.M.; Mahyoub,J.A. and Ibrahim,E.H. (2018). Chrysanthemum
extract and extract prepared silver nanoparticles as biocides to control Aedes aegypti (L.), the
vector of dengue fever. Journal of Asia-Pacific Entomology. Vol: 21:205–210.
[13] Ebrahiminezhad, A.; Taghizadeh, S.;Berenjian, S. ; Rah, A. and Ghasemi,Y.(2016).Synthesis
and Characterization of silver nanoparticles with natural carbohydrate capping using Zataria
multiflora.Adv.Mater.Lett.7, 939-994.
[14] Santos CS, Pie MR, da Rocha TC, Navarro-Silva MA (2019) Molecular identification of blood
meals in mosquitoes (Diptera, Culicidae) in urban and forested habitats in southern Brazil.
PLoS ONE 14(2): e0212517.
[15] WHO. (2005). Guidelines for Laboratory and field testing of Mosquito Larvicides. C
DS/WHOPES/GCDPP/p.13.
[16] Al-Rawi, K.M and Khalafalla,A.A (2000).Agricultural and experimental design and analysis. 2nd
edit. Mosul. University Iraq p. 285.
[17] Abbott, W.S. (1925).A method for computing effectiveness of an insecticide .J.Econ. Entomol.
18:265-267.
[18] Rawani,A. (2017). Mosquito larvicidal activity of green silver nanoparticle synthesized from
extract of bud of Polianthus tuberosa L. International J. of Nanotechnology and Applications
6
1st International Conference on Pure Science (ISCPS-2020) IOP Publishing
Journal of Physics: Conference Series 1660 (2020) 012013 doi:10.1088/1742-6596/1660/1/012013
ISSN 0973-631X Volume 11, Number 1, pp. 17-28.
[19] Alyaha, H. S. ; Mahyoub, J. A. ; Al Ghamdi,K. ; Ghramh, H. A. and Alkenani ,N. A.(2018).
Synthesis of silver nanoparticle using Juniperus procera and it's larvicidal activity against
dengue and zika viruses vector Aedes aegypti mosquito. Life Sci. J. Vol.:15(1):12-16].
[20] Choi, O.; Deng, K.K.; Kim, N.J.; Ross, L. J. and Surampalli, R.Y. (2008) The inhibitory effects
of silver nanoparticles, silver ions, and silver chloride colloids on microbial growth. Water
Res. Vol. 42: 3066-3074.
[21] Sap-iam, N.; Homklinchan, C.; Larpudomlert, R.; Waris-noicharoen, W. and Sereemaspun, A.
(2010) .UV irradiation induced silver nanoparticles as mosquito larvicides. J. Appl. Sci. Vol.
10; 3132-3136.
[22] Haldar, K.M., Haldar, B., Chandra, G., 2013, “Fabrication, characterization and mosquito
larvicidal bioassay of silver nanoparticles synthesized from aqueous fruit extract of putranjiva,
Drypetes roxburghii (Wall)”, Parasitol. Res., 112(4), pp. 1451–1459.
[23] Adhikari,U. ;Bhattacharya, K.; Mitra, P. and Chandra, G.(2018). Larvicidal efficacy of silver
nanoparticles synthesized biologically using Swietenia mahagoni (L.) Jacq. Leaf extract
against larvae of An. stephensi and Cx. quinquefasciatus and Cx. vishnui group.Indian J. of
Experiments Biology. Vol: 56.pp.14-19.
[24] Govindarajulu,B.; Srimathi,A. ; Bhuvana, R. and Karthikeyan, J.(2015). Mosquito Larvicidal
Efficacy of the Leaf Extracts of Annona reticulata Against Aedes aegypti.
Int.J.Curr.Microbiol.App.Sci. Vol. 4(8): 132-140.
[25] Hayatie, L.; Biworo, A. and Suhartono, E. (2015). Aqueous Extracts of Seed and Peel of Carica
Papaya A gainst Aedes Aegypti. J. of Med. and Bioengineering Vol. 4:No. 5, pp. 417-421.
[26] Raccaud, J.S. (1980).Les Insectes, physiologie, développement. Masson, Paris., 276 Rajakumar,
G.; Rahuman, A.A.; Roopan, S.M.; Chung, I.M.; Anbarasan, K.and Karthikeyan, V.( 2015).
Efficacy of larvicidal activity of green synthesized titanium dioxide nanoparticles using
Mangifera indica extract against blood-feeding parasites. Parasitol. Res. 114, 571–581.
http://dx.doi.org/10.1007/s00436-014-4219-8.
[27] Sarra, Z.; Amel, A. and Selima, B. (2017). Histopathology of Culex pipiens (Linée, 1753)
(Diptera, Culicidae) larvae exposed to the aqueous extract of Eucalyptus globulus Hér, 1789
(Myrtaceae). J.of Entomol. and Zool.Studies Vol. 5 No.(3): 759-765.
[28] Zayed, A.A.; Rokaya, M.A.; El Namaky, H.A.; Ismail, H.M. and Mady, H.Y. (2009). Influence
of Allium sativum and Citrus limon Oil Extracts and Bacillus thuringiensis Israelensis on some
Biological Aspects of Culex pipiens Larvae (Diptera:Culicidae). World Journal of
Zoology.Vol. 4 No. (2):109-121.
[29] Aouinty, B.; Chennaoui, M.; Mahari, S.; Rihane, A. and Mellouki, F. (2018). Larvicidal effects
of aqueous extract from Ricinus communis L. leaves against mosquito Culex pipiens :
mortality and histopathology of treated larvae. J. Mater. Environ. Sci., 9 (2), pp. 619-623.
