Green synthesis of silver nanoparticles using Carob leaf extract: Characterization and analysing of toxic effects in model organism Galleria mellonella L. (The Greater Wax Moth)

Green synthesis of silver nanoparticles using Carob leaf extract: Characterization and analysing of toxic effects in model organism Galleria mellonella L. (The Greater Wax Moth) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Green synthesis of silver nanoparticles using Carob leaf extract: Characterization and analysing of toxic effects in model organism Galleria mellonella L. (The Greater Wax Moth) Aslıhan Andırın, Nur Dudu Yaycı, Murat Idikut, Ayse Kara, Mustafa Tuncsoy, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3984885/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Sep, 2024 Read the published version in Environmental Science and Pollution Research → Version 1 posted 5 You are reading this latest preprint version Abstract Silver nanoparticles (AgNP) have been used in many studies due to their inhibitory properties on microorganisms such as bacteria and viruses. In recent years, due to global problems such as environmental pollution, the green synthesis (biosynthesis) method is frequently preferred because it is simple and low cost and does not require the use of toxic substances. In this study, it was determined that the effects on antioxidant enzyme activities (SOD, CAT, GPx, GST), acetylcholinesterase (AChE), and total hemocyte count (THC) as well as phenoloxidase activity to determine their effect on antioxidant defence and the immune system in model organism Galleria mellonella larvae. We observed that green synthesized AgNPs accumulate in the midgut of the larvae and led to the increasing of CAT and SOD activities. GST and AChE activities were increased in the fat body of the larvae otherwise; it was decreased in the midgut. Moreover, increases were found in THC and phenoloxidase activity. Consequently, green synthesized silver nanoparticles led to oxidative stress and immuntoxic effects on G. mellonella larvae. Antioxidant activity Carob Green synthesis Immunotoxicity Silver nanoparticles Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Introduction Nanotechnology, which studies the design and manufacture of metal and metal oxide materials at the nanoscale, is one of the most important sciences of the twenty-first century. Nanoparticles are used in a variety of sectors, including food, agriculture, cosmetics, and medicines (Annu et al. 2018; Shobha et al. 2014 ). Food processing and preservation (nano preservatives, toxin detection, nano encapsulated food additives, etc.) as well as food packaging are examples of NP applications in the food sector (nano coatings, nanosensors, nanocomposites, edible coating NPs, etc.) (Biswas et al. 2022 ). Nanotechnology is employed in agriculture to make nano-fertilizers, insecticides, herbicides, and sensors (Tang et al. 2024). Silver nanoparticles (AgNPs) have been used in different applications due to their different sizes, charges and properties (Wong and Liu, 2010 ). AgNPs can be synthesized by different chemical and physical methods. Due to being expensive and toxicity of physical and chemical methods, nowadays they are not preferred. As an alternative to these methods, biological methods using plants and microorganisms are more utilized (Horsfall, 2014 ). The synthesis of metal nanoparticles with enhanced antioxidant and antimicrobial activities in AgNPs has gained importance in biochemical applications (Chahar et al. 2018 ). It is stated in many studies that this synthesis method opens up new opportunities for easy, inexpensive, fast and large-scale production, and plant-based synthesis of metal nanoparticles has become an alternative to chemical, physical and even microbial methods (Elahi et al. 2018 ). Carob ( Ceratonia siliqua L.) leaves, which have a wide crown, are evergreen, hard and hairy in order to adapt to the harsh conditions of the Mediterranean climate. The length of its leaves, which are always green, is around 3–5 cm; The tree has green, small flowers and these flowers form clusters in groups of 50–60. The most abundant phenolic substance in carob, which is rich in phenolic substances, is gallic acid, which is an effective antioxidant. Studies show that especially gallic acid is very effective in slowing down the oxidation of fats (Kumazawa et al. 2002 ; Owen et al. 2003 ). In other study conducted by Souli et al. ( 2015 ) on mice, it was revealed that carob extract has a protective effect on liver cells against oxidative stress damage caused by ethyl alcohol. In another study with zebrafish, the antioxidant capacity of carob extract against deltamethrin induced oxidative stress was investigated that carob extract effects important antioxidant metabolites and related pathways (Unal et al. 2023 ). Although there are studies regarding the in vivo toxic effects of silver nanoparticles on the antioxidant enzyme activities and immune system, there are almost no studies about green synthesized silver nanoparticles using plant extracts on the antioxidant enzymes and immune system. It is known that the toxic effects of silver nanoparticles occur due to induction of oxidative damage and cell death pathways, so that in this study we aimed to investigate the effects of silver nanoparticles synthesized from carob leaves on antioxidant enzymes and immune system in the larvae of the model organism Galleria mellonella L. Material-Method Ceratonia siliqua leaf collection and preparation of the extract The plant leaves were collected in the campus of Cukurova University. They washed with Milli-Q water and stayed to dry. After drying, the leaves were cut into small pieces. 10 g of Ceratonia silique leaves added into 150 ml of distilled water. Then boiled at 150°C. The obtained extract was filtered with Whatman No.1 filter paper. The pH of the extract and synthesized silver nanoparticle solutions were measured with pH meter. The obtained pH of the solutions was shown in the Table 1 . Table 1 pH values of the solutions. Solution pH 2mM AgNO 3 6.19 Leaf extract of Ceratonia siliqua 6.51 The solution of the mixture 5.95 Green synthesis of silver nanoparticles from the leaf extract of Ceratonia siliqua For the green synthesis of AgNPs, 3 ml of leaf extract was added to 27 ml of 2mM AgNO 3 solution mixed well by heating at 60°C with ultrasonic bath until colour change. The formation of the AgNPs was observed by the change in colour from light yellow to dark brown (Fig. 1 ). It was centrifuged at 12.000 rpm for 45 min. After centrifugations, the pellet was washed with distilled water for three times. Then, the pellet was dried in an incubator for 3 days. Characterization of Silver Nanoparticles Many methods are applied for the characterizing different AgNPs. The morphology of AgSO4 nanoparticles was performed by UV/Vis spectrophotometer, SEM, XRD and FTIR analyses at Cukurova University Central Research Laboratory. UV/VIS Spectrophotometer The AgNP solution synthesized via green synthesis was placed in a quartz cuvette and measured at a wavelength of 200–800 nm using a UV/Vis Spectrophotometer. Fourier Transform Infrared (FTIR) FTIR was used to identify functional groups and various phytochemical components involved in the reduction and stabilization of the synthesized nanoparticles. FTIR analysis of AgNPs was performed in the spectral range of 400–4000 cm − 1 using FTIR spectroscopy (JASCO, FT/IR 6700) at Cukurova University Central Research Laboratory. Scanning Electron Microscope (SEM) The shape and size of the synthesized AgNPs were determined by SEM. The surface of the AgNPs was washed three times with 50% acetone solution. SEM-EDS analysis was then performed with a scanning electron microscope (FEI, Quanta 650 Field Emission). X-Ray Diffraction Pattern (XRD) XRD analysis, copper (Cu Kα, 1.54060 Å) as the radiation source, an XRD system (Polycrystalline XRD, T&T TT-90) with a range of 4–90 degrees and a step width of 0.01 was used to determine the crystal structure of AgNPs. Experimental design G. mellonella larvae was reared as described in Tuncsoy et al. (2019). The last instar in the diet were used for the experiment (n = 20) and divided into two groups as the control and the experimental groups. Ag NP, synthesised from carob leaves at a concentration of 3 µg/ml, was injected into last instar G. mellonella larvae at a rate of 10 microlitres per larva from the proleg using a Hamilton injector. The doses were determined according to the preliminary experiments. Before injection the proleg was sterilized with %70 alcohol by using a swab. After the application, the larvae were placed in Petri dishes and kept at 28 ± 2°C for 72 hours. For the control larvae, distilled water was used. Mean Ag levels in exposure media were determined as 2.960 ± 0.04 mg/L Ag. Experiments were run in triplicate. Antioxidant enzyme activities The midgut and adipose tissue were dissected and homogenised in cold homogenisation buffer for the determination of SOD, CAT and GPx enzyme activities (Fig. 2 ). The methods to be used for the determination of the antioxidant enzyme activities are described in Tuncsoy et al. (2019). Sephadex G-25 was used for the removal of low molecular weight proteins (Gonzalez-Rey et al. 2014 ). GST activity was determined using the method developed by Habig et al. ( 1974 ) based on conjugating CDNB with reduced glutathione (Tuncsoy and Mese, 2021 ). The Bradford method was used to determine total protein content (Bradford, 1976 ). Acetylcholinesterase activity To determine AChE activity, the midgut and fat body were homogenized on ice in five volumes of a Tris–HCl buffer (100 mM, pH 8.0) containing 10% Triton and centrifuged at 12,000×g for 30 min (4°C). The AChE activity was assayed as described by Ellman et al. ( 1961 ). Phenoloxidase activity After 20 µL of hemolymph was obtained from the experimental and control group larvae, it was mixed with 180 µL of cold phosphate buffer. Then, it was centrifuged at 10000 g for 5 min. at 4°C. The supernatant was mixed with 3,4-Dihydroxy L-phenylalanine (L-DOPA) and read at 490 nm absorbance at 5 minute intervals between 0 and 30 minutes in a UV-Spectrophotometer. The data obtained were determined as U/mg protein/min (Brookman et al., 1989 ). Total hemocyte count (THC) Firstly, last instar larvae were kept at -20ºC for 3 minutes to slow down their movements. After the larvae were wiped with 95% ethanol, they were punctured from the proleg with a fine-tipped dissection needle and 5 µl hemolymph was obtained with the help of a microcapillary tube (SIGMA). 4 µl of the obtained hemolymph sample was taken and transferred to Eppendorf tubes kept on ice and containing 36 µl of anticoagulant (0.098 M NaOH, 0.186 M NaCl, 0.017 M Na2EDTA and 0.041 M Citric acid, pH = 4.5). 10 µl of the 1:10 diluted cell suspension was taken and loaded into the Neubauer hemocytometer. Hemocytes were counted under a Leica DM750 microscope and the number of hemocytes in one milliliter of hemolymph was determined. Counted hemocytes were calculated using the Jones ( 1962 ) method. Data analysis The statistical analyses of the data were carried out by a series of Analysis of Variance and Independent Samples T test by using the SPSS programme. Differences were considered as significant when p < 0.05. Results and Discussion Characterization of AgNPs UV-Spectrophotometer analysis For the green synthesis of AgNPs using carob leaf extract, the plant extract was added to 2mM silver nitrate solution and then kept in an ultrasonic bath until the colour of the mixture turned dark brown. When the UV Vis spectrum is examined 5 minutes after synthesis, it is seen that a surface Plasmon resonance band is formed at a wavelength of approximately 440 nm (Fig. 3 ). This peak is the characteristic surface Plasmon resonance band of AgNPs and proves the presence of AgNPs in the medium (Bhakya et al., 2016 ). When the studies were examined, it was determined that AgNPs formed peaks between 400–450 nm wavelengths (Awwad et a., 2013; Beğiç et al., 2021 ; Singh et al., 2023 ). XRD (X-Ray Diffraction) Spectroscopy XRD analysis was performed to determine the crystal and molecular structures of silver nanoparticles synthesized from Ceratonia siliqua leaf extract. Some characteristic peaks at 38.039 [111], 44.210 [200], 64.305 [220], 77.223 [311] and 81.352 [222] were observed in the XRD pattern of Ag NPs synthesized from the Ceratonia siliqua plant. Additionally, the observed silver peaks indicate that Ag crystals have a cubic structure. The [hkl] values of silver are given in Fig. 4 . Fourier Transform Infrared Spectroscopy (FTIR) FTIR analysis method is a method used to understand the relationship of functional groups between biomolecules and nanoparticles. FTIR analyses were performed to identify biomolecules responsible for the synthesis, reduction and stabilization of silver nanoparticles. The peaks obtained in the FTIR measurement of Ag NPs synthesized from Ceratonia siliqua leaf extract, observed at wavelengths of 2979.48, 2350.80, 2114.56 and 1991.14, are alkane (C-H), carbon dioxide (O = C = O), alkyne (C Ξ C) and is thiocyanate, respectively. It corresponds to (N = C = S) functional groups (Fig. 5 ). SEM Analysis The size and shape of the green synthesized Ag NPs were evaluated in University of Cukurova Central Research Laboratory (CUMERLAB). The Ag NPs determined as spherical shape (Fig. 6 ). Mean particle size was determined as 39.653 ± 2.562 nm. When previous studies were examined, it was determined that silver NPs synthesized from plant parts were mostly spherical in shape and 13–75 nm in size (Venkatadri et al., 2020; Baran et al., 2023; Ajlouni et al., 2023; Dua et al., 2023). Bioaccumulation of Ag NPs In present study, it was detected that Ag ions mostly accumulated in the fat body of larvae as well as midgut according to the control group (p < 0.05; Fig. 7 ). The fat body of insects is analogous with liver in vertebrates, and it is known that xenobiotics mostly accumulate in this tissue (Yang et al. 2007). Also it was known that, the digestive track is the route of the entrance of nanoparticles, for direct exposure through intentional ingestion as well as for indirect exposure by means of their dissolution from food containers or by secondary ingestion of inhaled particles (Bergin and Witzmann, 2013 ). After uptake of nanoparticles, this types of nanomaterials cross the gut barrier and reach the immune competent cells in the hemolymph (Mir et al. 2020 ). Antioxidant enzyme activities Despite the advantages of green synthesized nanoparticles, these are not exempt from toxicity toward non-target organisms (Silva Brito et al. 2024 ). According to the previous studies, Ag NPs dissolve to release Ag + and cause the production of reactive oxygen species by transferring silver across the cell membrane (Noga et al. 2023 ). It was well known that the one of the main antioxidant enzyme is SOD for eliminating the reactive oxygen species. In the reaction of dismutation, superoxide anion radicals are eliminated from the cell microenvironment by their conversion to hydrogen peroxide, which is further eliminated by CAT and GPx (Scibior and Czeczot, 2006 ; Jarosiewicz et al., 2019 ). In this study, we observed that green synthesized AgNPs accumulate in the midgut of the larvae and led to the increasing of CAT and SOD activities (p < 0.05; Fig. 7 ; Fig. 8 ). Moreover, we determined that the midgut of the larvae act as the main site of absorption, where the metals mostly accumulated in our previous study (Tuncsoy et al. 2019). In this study, on the other hand, CAT activity was increased, while SOD activity was decreased in the fat body of the larvae (p < 0.05). As for GPx activity, CAT and SOD activities were decreased in all tissues (p < 0.05; Fig. 8 ). A decrease in CAT and GPx enzyme activities may have occurred in fat body due to the oxidant/antioxidant balance being disturbed by increased reactive oxygen levels (Aslankoc et al. 2019). Although there are not enough studies regarding to the effects on GST activity, it is generally known that silver nanoparticles cause toxic effects on mammalian and other non-target species. El-Samad et al. (2022) reported that silver nanoparticle exposure resulted in a decrease in midgut of Blaps polychresta . In the present study, GST activity decreased in the midgut of Galleria mellonella larvae, while GST activity increased in fat body (p < 0.05; Fig. 9 .). This is thought to be due to the fact that silver nanoparticles synthesised using carob leaves may cause an imbalance between oxidation / anti-oxidation processes by causing irregular ROS accumulation in cells (Wang et al. 2020 ). Moreover, previous studies have reported significant decreases in antioxidant enzyme activities in insects exposed to xenobiotic (Yousef et al. 2019 ; El-Samad et al. 2020 ). It is also known that nanoparticles lead to an increase in intracellular ROS and cause inhibition of some enzyme activities (Akter et al. 2018 ). Acetylcholinesterase activity In the present study, AChE activity was significantly decreased in the midgut (2.66-fold), however it was significantly increased in the fat body of the larvae (1.26-fold) (p < 0.05; Fig. 10 ). Similar results are found in our previous study where CuO NP was applied to G. mellonella larvae (Sezer-Tuncsoy et al. 2019). The mechanism by which silver nanoparticles inhibit the AChE enzyme may be due to structural perturbation of the enzyme (Sinko et al. 2014 ). It is also thought that the inhibition caused by nanoparticle exposure may be due to the adsorption of the AChE enzyme to the surface of the nanoparticles. Moreover, Ag NPs are also known to stimulate oxidative stress in nerve cells by changing the permeability of blood brain barrier cells (Tang et al. 2008 ; Baruwati et al. 2013 ; Abdelwahab et al. 2021 ). Total Hemocyte Count (THC) There was an increase in THC in the hemolymph of the Ag NPs exposed groups compared to the control (Fig. 11 ; p < 0.05). The increase of THC may be due to the promotion of haematopoiesis is or the release of hemocytes that adhered on surfaces (sessile hemocytes) within the hemocoel (Jones, 1962 ; Ghasemi et al. 2014 ). Also, the increase of THC may be due to the activation of mitotic division of hemocytes which was activated in response to nanoparticles. Phenoloxidase activity It was determined that PO activity was significantly increased in AgNPs exposed group (p < 0.05; Fig. 12 ). The increase in the number of hemocytes is closely related to the increase in the melanisation innate immunity response where PPO is effective (Kurt and Kayis, 2015). Conclusion Although green synthesized silver nanoparticles are considered to be environmentally friendly, they can be a source of oxidative stress due to their accumulation in tissues as a result of acute use. For this reason, it is necessary to determine the LD 50 values and to determine the correct concentration and application time. Declarations Acknowledge In memory of Yağmur Meşe, who we lost in the earthquake disaster that occurred in our region on February 6, 2023. Ethical Approval No ethical approval is required for this study. Consent to Participate and Publish This study is not a clinical study, the living organism used is an invertebrate. Therefore, there is no need for any permission to participate or to publish. Author Contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Aslıhan Andırın, Nur Dudu Yaycı, Murat İdikut, Ayse Kara, Benay Tuncsoy, Mustafa Tuncsoy and Pınar Ozalp. The first draft of the manuscript was written by Benay Tuncsoy and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Funding The authors declare that there is no funding during the preparation of this manuscript. Competing Interests There is no conflict of interest between the authors. References Abdelwahab GM, Mira A, Cheng YB, Abdelaziz TA, Lahloub MFI, Khalil AT. (2021). Acetylcholine esterase inhibitory activity of green synthesized nanosilver by naphthopyrones isolated from marine-derived Aspergillus niger. PLoS One, 16(9):e0257071. doi: 10.1371/journal.pone.0257071. Akter, M., M.T. Sikder, M.M. Rahman, A.K.M.A. Ullah, K.F.B. Hossain, S. Banik, T. Hosokawa, T. Saito, M. Kurasaki (2018). A systematic review on silver nanoparticles- induced cytotoxicity: physicochemical properties and perspectives, J. Adv. Res. 9:1–16. Annu. Ahmeda, S. Kaurb, G. Sharmac, P. Singhc, S. Ikrama, S. (2018). Fruit waste (peel) as bio-reductant to synthesize silver nanoparticles with antimicrobial, antioxidant and cytotoxic activities. Journal of Applied Biomedicine, 175, 1-11. Aslankoç R, Demirci D, İnan Ü, Yıldız M, Öztürk A, Çetin M, Savran EŞ, Yılmaz B. (2019). The Role Of Antioxidant Enzymes İn Oxidative Stress - Superoxide Dismutase (Sod), Catalase (Cat) And Glutathione Peroxidase (Gpx). Med J SDU; 26(3): 362-369. Awwad, A.A., Salem, N.M., Abdeen, A.O. (2013). Green synthesise of silver nanoparticles using carob leaf extract and its antibacterial activity. International Journal of Industrial Chemistry, 4:29. Baruwati B, Simmons SO,Varma RS,Veronesi B. (2013). “Green”synthesized and coated nano silver alters the membrane permeability of barrier (intestinal, brain endothelial) cells and stimulates oxidative stress pathways in neurons. ACS Sustainable Chemistry&Engineering. 1(7):753–9. Beğiç, N, Bener, M, Apak, R. (2021). Development of a green synthesized silver nanoparticle based antioxidant capacity method using carob extract. Journal of Nanostructure in Chemistry. 11:382-394. Bergin, I. L., Witzmann, F. A. (2013). Nanoparticle toxicity by the gastrointestinal route: evidence and knowledge gaps. International Journal of Biomedical Nanoscience and Nanotechnology. 3, 163–210. Bhakya, S, Muthukrishnan, S., Sukumaran, M., Muthukumar, M. 2016. “Biogenic synthesis of silver nanoparticles and their antioxidant and antibacterial activity,” Appl. Nanosci., 6(5): 755–766. Biswas R, Alam M, Sarkar A, Haque MI, Hasan MM, Hoque M. (2022) Application of nanotechnology in food: processing, preservation, packaging and safety assessment. Heliyon. 2022 Nov 21;8(11):e11795. doi: 10.1016/j.heliyon..e11795. PMID: 36444247; PMCID: PMC9699984. Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254 Brito, R.S., Bebianno, M.J.,Rocha, T.L. (2024). Plant-based silver nanoparticles ecotoxicity: Perspectives about green technologies in the One Health context, Critical Reviews in Environmental Science and Technology, DOI: 10.1080/10643389.2024.2303298 Brookman, J. L., Ratcliffe, N. A. and Rowley, A. F. (1989). Studies on the activation of the prophenoloxidase system of insects by bacterial cell wall components. Insect Biochemistry, 19 (1): 47-57. Chahar, H. S., Corsello, T., Kudlicki, A. S., Komaravelli, N., Casola, A. (2018). Respiratory syncytial virus infection changes cargo composition of exosome released from airway epithelial cells. Scientific reports, 8(1), 1-18. Elahi, N., Kamali, M., Baghersad, M. H. (2018). Recent biomedical applications of gold nanoparticles: A review. Talanta, 184, 537-556. Ellman GL, Courtney KD, Andres V, Featherstone RM (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 7:88–95 El-Samad, L.M., M. El Hassan, D.A. Kheirallah, K.K. Abdul-Aziz, N.A. Toto, (2020) Biochemical, molecular, and histological markers in Pimelia latreillei (Coleoptera: Tenebrionidae) for evaluating soil pollution, Fresenius Environ. Bull. 29 4224–4239. Ghasemi V, Yazdi AK, Tavallaie FZ, Sendi JJ (2014) Effect of essential oils from Callistemon viminalis and Ferula gummosa on toxicity and on the hemocyte profile of Ephestia kuehniella (Lepidoptera:Pyralidae). Arch Phytopathol Plant 47(3):268–278 Gonzalez-Rey M, Mattos JJ, Piazza CE, Bainy ACD, Bebianno MJ (2014) Effects of active pharmaceutical ingredients mixtures in mussel Mytilus galloprovincialis. Aquat Toxicol 153:12–26 Habig WH, Pabst MJ, Jakoby WB (1974) Glutathione S-transferases: the first enzymatic step mercapturic acid formation. J Biol Chem 249:7130–7139 Horsfall, L. 2014. Biological Synthesis of Metallic Nanoparticles by Bacteria, Fungi and Plants. Journal of Nanomedicine & Nanotechnology, 5. Jarosiewicz, M., Anita Krokosz, Agnieszka Marczak, Bożena Bukowska, Changes in the activities of antioxidant enzymes and reduced glutathione level in human erythrocytes exposed to selected brominated flame retardants, Chemosphere, Volume 227, 2019, Pages 93-99, Jones JC (1962) Current concepts concerning insect hemocytes. Am Zool 2:209–246. Kumazawa S., Taniguchi M., Suzuki Y., Shimura M., Kwon M.S., Nakayama T. Antioxidant activity of polyphenols in carob pods. J. Agric. Food Chem. 2002;50:373–377. Kurt D, Kayış T (2015) Effects of the pyrethroid insecticide deltamethrin on the hemocytes of the Galleria mellonella . Turk J Zool 39:452–457. Lamia M. El-Samad, Nahed R. Bakr, Saeed El-Ashram, Eman H. Radwan, Karoline K. Abdul Aziz, Hussein K. Hussein, Abeer El Wakil, Mohamed A. Hassan, Silver nanoparticles instigate physiological, genotoxicity, and ultrastructural anomalies in midgut tissues of beetles,Chemico-Biological Interactions,Volume 367,2022,110166, Mir, A.H., Qamar, A., Qadir, I. et al. 2020. Accumulation and trafficking of zinc oxide nanoparticles in an invertebrate model, Bombyx mori , with insights on their effects on immuno-competent cells. Sci Rep 10 , 1617. https://doi.org/10.1038/s41598-020-58526-1 Noga, M.; Milan, J.; Frydrych, A.; Jurowski, K. Toxicological Aspects, Safety Assessment, and Green Toxicology of Silver Nanoparticles (AgNPs)—Critical Review: State of the Art. Int. J. Mol. Sci. 2023, 24 , 5133. https://doi.org/10.3390/ijms24065133 Owen R.W., Haubner R., Hull W.E., Erben G., Spiegelhalder B., Bartsch H. Isolation and structure elucidation of the major individual polyphenols in carob fibre. Food Chem. Toxicol. 2003;41:1727–1738. Scibior D, Czeczot H. Katalaza--budowa, właściwości, funkcje [Catalase: structure, properties, functions]. Postepy Hig Med Dosw (Online). 2006;60:170-80. Polish. PMID: 16618987. Sezer Tuncsoy B, Tuncsoy M, Gomes T, Sousa V, Teixeira MR, Bebianno MJ, Ozalp P (2019) Effects of copper oxide nanoparticles on tissue accumulation and antioxidant enzymes of Galleria mellonella L.. B Environ Contam Tox 102(3):341–346 Shobha, G., Moses, V., & Ananda, S. (2014). Biological synthesis of copper nanoparticles and its impact. Int. j. pharm. Sci. Invent, 3(8), 6-28. Singh, C., Anand, S.K., Upadhyay, R., Pandey, N., Kumar, P., Singh, D., Tiwari, P., Saini, R., Tiwari, K.N., Mishra, S.K., Tilak, R. 2023. Green synthesis of silver nanoparticles by root extract of Premna integrifolia L. and evaluation of its cytotoxic and antibacterial activity, Materials Chemistry and Physics, 297: 127413, Sinko G,VrčekIV, GoesslerW, LeitingerG, Dijanos ićA, Miljanić S.Alteration of cholinesterase activity as possible mechanism of silver nanoparticle toxicity. Environmental Science and Pollution Research. 2014;21(2):1391–400.https://doi.org/10.1007/s11356-013-2016-zPMID:23904256 Souli A, Sebai H, Chehimi L, Rtibi K, Tounsi H, Boubaker S, Sakly M, El-Benna J, Amri M. Hepatoprotective effect of carob against acute ethanol-induced oxidative stress in rat. Toxicol Ind Health. 2015;31(9):802–810. doi: 10.1177/0748233713475506. Tang J, Xiong L,, Wang S,, Wang J,, Liu L,, Li J, e tal. Influence of silver nanoparticles on neurons and blood-brain barrier via subcutaneous injection in rats. Applied Surface Science.2008;255(2):502–4. Tang, Y.; Zhao, W.; Zhu, G.; Tan, Z.; Huang, L.; Zhang, P.; Gao, L.; Rui, Y. Nano-Pesticides and Fertilizers: Solutions for Global Food Security. Nanomaterials 2024, 14, 90. https://doi.org/10.3390/nano14010090 Tuncsoy, B., Mese, Y. (2021). Influence of titanium dioxide nanoparticles on bioaccumulation, antioxidant defense and immune system of Galleria mellonella L.. Environ Sci Pollut Res 28, 38007–38015. https://doi.org/10.1007/s11356-021-13409-4 Unal, E., Sulukan, E., Senol, O., Baran, A., Nadaroğlu, H., Kankaynar, M., Kızıltan, T., Ceyhun, S.B. 2023. Antioxidant/protective effects of carob pod ( Ceratonia siliqua L.) water extract against deltamethrin-induced oxidative stress/toxicity in zebrafish larvae, Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 267: 109584. https://doi.org/10.1016/j.cbpc.2023.109584. Wang, D., M. Dan, Y. Ji, X. Wu, X. Wang, H. Wen, Roles of ROS and cell cycle arrest in the genotoxicity induced by gold nanorod core/silver shell nanostructure, Nanoscale Res. Lett. 15 (2020) 224. Wong, K. and Liu, X. 2010. Silver nanoparticles - The real "silver bullet" in clinical medicine? Med. Chem. Comm., 1. Yousef, H.A. , E.A. Abdelfattah, M. Augustyniak, Antioxidant enzyme activity in responses to environmentally induced oxidative stress in the 5th instar nymphs of Aiolopus thalassinus (Orthoptera: Acrididae), Environ. Sci. Pollut. Res. Int. 26 (2019) 3823–3833. Cite Share Download PDF Status: Published Journal Publication published 18 Sep, 2024 Read the published version in Environmental Science and Pollution Research → Version 1 posted Editorial decision: Major Revision 23 Aug, 2024 Reviewers agreed at journal 14 May, 2024 Reviewers invited by journal 08 Apr, 2024 Editor assigned by journal 01 Mar, 2024 First submitted to journal 26 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3984885","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":288760999,"identity":"9589ff41-cf66-402c-8662-36042fc47478","order_by":0,"name":"Aslıhan Andırın","email":"","orcid":"","institution":"Adana Alparslan Türkeş Bilim ve Teknoloji Üniversitesi: Adana Alparslan Turkes Bilim ve Teknoloji Universitesi","correspondingAuthor":false,"prefix":"","firstName":"Aslıhan","middleName":"","lastName":"Andırın","suffix":""},{"id":288761000,"identity":"0aab66fc-ba5a-47c5-9e5c-1046ca4154d4","order_by":1,"name":"Nur Dudu Yaycı","email":"","orcid":"","institution":"Adana Alparslan Turkes Science and Technology University: Adana Alparslan Turkes Bilim ve Teknoloji Universitesi","correspondingAuthor":false,"prefix":"","firstName":"Nur","middleName":"Dudu","lastName":"Yaycı","suffix":""},{"id":288761001,"identity":"1d3ce5dd-1a6e-4641-87ba-ba99c202d5c5","order_by":2,"name":"Murat Idikut","email":"","orcid":"","institution":"Adana Alparslan Türkeş Bilim ve Teknoloji Üniversitesi: Adana Alparslan Turkes Bilim ve Teknoloji Universitesi","correspondingAuthor":false,"prefix":"","firstName":"Murat","middleName":"","lastName":"Idikut","suffix":""},{"id":288761002,"identity":"b7296bf8-bde3-46c8-8ba4-456105445365","order_by":3,"name":"Ayse Kara","email":"","orcid":"","institution":"Cukurova University: Cukurova Universitesi","correspondingAuthor":false,"prefix":"","firstName":"Ayse","middleName":"","lastName":"Kara","suffix":""},{"id":288761003,"identity":"3c793ab7-55aa-4ebf-8661-ba0189065d64","order_by":4,"name":"Mustafa Tuncsoy","email":"","orcid":"","institution":"Cukurova University: Cukurova Universitesi","correspondingAuthor":false,"prefix":"","firstName":"Mustafa","middleName":"","lastName":"Tuncsoy","suffix":""},{"id":288761004,"identity":"1426cdfa-b63d-4b8f-baa5-f4e071730777","order_by":5,"name":"Benay Tuncsoy","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0ElEQVRIiWNgGAWjYBACAxiDH0IxE6GFDcqQbGCGamHDoxxFi8EBYrWYy/eYPfjxxyZx8/HzxyQYKqwTG+R7H+DVYtnGY27Y25aWuO1MMpsEw5n0xAY2dgO8WgyO8ZhJMzYcTtx2AKiFse0wUAsBl4G1MPw5nLi5/zFQyz+itbAdTtwgAbKlgQgtlm1pZZJAvxjPuPHY2CLhWLpxG1safi3mzIe3SQBDTLa/P/HhjQ811rL9zMfwa4EBxwYQmcBAOFrgwJ5YhaNgFIyCUTACAQDrEz6lbxMdpQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-4361-3475","institution":"Adana University of Science and Technology: Adana Alparslan Turkes Bilim ve Teknoloji Universitesi","correspondingAuthor":true,"prefix":"","firstName":"Benay","middleName":"","lastName":"Tuncsoy","suffix":""},{"id":288761005,"identity":"35bdc9c2-2721-431a-b334-4ebbb0076bab","order_by":6,"name":"Pınar Ozalp","email":"","orcid":"","institution":"Cukurova University: Cukurova Universitesi","correspondingAuthor":false,"prefix":"","firstName":"Pınar","middleName":"","lastName":"Ozalp","suffix":""}],"badges":[],"createdAt":"2024-02-24 12:03:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3984885/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3984885/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11356-024-34996-y","type":"published","date":"2024-09-18T15:57:53+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":54516705,"identity":"579ce632-8c06-4f69-9481-25d955b7cd2d","added_by":"auto","created_at":"2024-04-11 16:41:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":842684,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eCeratonia siliqua\u003c/em\u003e leaf extract, AgNO\u003csub\u003e3\u003c/sub\u003e solution and synthesized silver nanoparticle, respectively\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3984885/v1/da2aeccfea8709d999af63bf.png"},{"id":54516693,"identity":"393b1803-522d-4aba-8f74-8bdd3c042075","added_by":"auto","created_at":"2024-04-11 16:41:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":755325,"visible":true,"origin":"","legend":"\u003cp\u003eDissection of \u003cem\u003eG. mellonella\u003c/em\u003e larvae\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3984885/v1/3bc21d804f14b83cf3db4ec6.png"},{"id":54516691,"identity":"9ce7a2eb-15f5-4326-8911-6be2043f024f","added_by":"auto","created_at":"2024-04-11 16:41:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":138765,"visible":true,"origin":"","legend":"\u003cp\u003eUV-Vis Spectrum of AgNP biosynhtesized from \u003cem\u003eCeratonia siliqua\u003c/em\u003eleaf extract.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3984885/v1/d38f45d6cbc0684c77dea2ae.png"},{"id":54516698,"identity":"9ebe7a3e-9a72-49b8-b36c-a2a655494a86","added_by":"auto","created_at":"2024-04-11 16:41:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":34900,"visible":true,"origin":"","legend":"\u003cp\u003eXRD analysis of AgNPs synthesized from \u003cem\u003eCeratonia siliqua\u003c/em\u003eplant\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3984885/v1/aed08b3e2a18fc14b9a29bc1.png"},{"id":54516706,"identity":"f5e55258-c341-4933-8979-e0fdf7bc8195","added_by":"auto","created_at":"2024-04-11 16:41:13","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":137417,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectrum of Ag NPs synthesized from \u003cem\u003eCeratonia siliqua\u003c/em\u003e leaf extract\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3984885/v1/a397b1ca8a42df1bd701bef9.png"},{"id":54516701,"identity":"7569e599-8096-4639-bacb-abb95ff5defd","added_by":"auto","created_at":"2024-04-11 16:41:12","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":524333,"visible":true,"origin":"","legend":"\u003cp\u003eSEM image of Ag NPs biosynthesized from \u003cem\u003eC. siliqua\u003c/em\u003e leaf extract.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-3984885/v1/ca0d8f1ae9d567c79ea54086.png"},{"id":54516702,"identity":"7d396ed2-5035-4832-aeed-b4a65e7ee2bb","added_by":"auto","created_at":"2024-04-11 16:41:13","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":33432,"visible":true,"origin":"","legend":"\u003cp\u003eAg accumulation in the tissue of \u003cem\u003eG. mellonella \u003c/em\u003elast instar larvae. t test; “*” represents statistical differences for each treatment of exposure (p\u0026lt;0.05)\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-3984885/v1/645af72f10ece020bc52a366.png"},{"id":54516704,"identity":"1f8d9491-a86a-47ba-a6e3-4a6442d59ce5","added_by":"auto","created_at":"2024-04-11 16:41:13","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":52686,"visible":true,"origin":"","legend":"\u003cp\u003eA. CAT activity B. SOD activity C. GPx activity in the tissues of \u003cem\u003eG. mellonella \u003c/em\u003elarve\u003cem\u003e \u003c/em\u003eexposed to green synthesized Ag NPs. t test; “*” represents statistical differences for each treatment of exposure (p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-3984885/v1/8f4b948d37ac707e4b55605c.png"},{"id":54516692,"identity":"de2a2b19-6139-40ce-91b7-cec9ad9043e2","added_by":"auto","created_at":"2024-04-11 16:41:12","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":31598,"visible":true,"origin":"","legend":"\u003cp\u003eGST\u003cstrong\u003e \u003c/strong\u003eactivity in the tissues of \u003cem\u003eG. mellonella \u003c/em\u003eexposed to green synthesized Ag NPs. t test; “*” represents statistical differences for each treatment of exposure (p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-3984885/v1/9b6b27451fc887ac23c2fa75.png"},{"id":54517086,"identity":"d4ab334d-44d8-47eb-9957-20f6efe1434a","added_by":"auto","created_at":"2024-04-11 16:49:12","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":28551,"visible":true,"origin":"","legend":"\u003cp\u003eAChE activity in the tissues of \u003cem\u003eG. mellonella \u003c/em\u003eexposed to green synthesized Ag NPs. t test; “*” represents statistical differences for each treatment of exposure (p\u0026lt;0.05)\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-3984885/v1/45d7e216d7c9ef32e8dbf40b.png"},{"id":54516700,"identity":"2b98596b-8442-4122-9d12-112fcf851a89","added_by":"auto","created_at":"2024-04-11 16:41:12","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":31135,"visible":true,"origin":"","legend":"\u003cp\u003eTotal hemocyte number in hemolymph of \u003cem\u003eG. mellonella\u003c/em\u003e exposed to green synthesized Ag NPs. t test; “*” represents statistical differences for each treatment of exposure (p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-3984885/v1/91c1280c40c01409947a0895.png"},{"id":54516703,"identity":"0bfe36bb-7bdc-4609-b125-5069393a2faa","added_by":"auto","created_at":"2024-04-11 16:41:13","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":28264,"visible":true,"origin":"","legend":"\u003cp\u003ePhenoloxidase activity in hemolymph of \u003cem\u003eG. mellonella\u003c/em\u003e exposed to green synthesized Ag NPs. t test; “*” represents statistical differences for each treatment of exposure (p\u0026lt;0.05)\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-3984885/v1/ae134adbee8862fffdf3b05e.png"},{"id":65104092,"identity":"b479bc42-fb09-4995-b0fa-5e8cca7fd66a","added_by":"auto","created_at":"2024-09-23 16:11:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2974918,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3984885/v1/7e847e0f-a325-41e4-9a66-3f97f32eb913.pdf"}],"financialInterests":"","formattedTitle":"Green synthesis of silver nanoparticles using Carob leaf extract: Characterization and analysing of toxic effects in model organism Galleria mellonella L. (The Greater Wax Moth)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNanotechnology, which studies the design and manufacture of metal and metal oxide materials at the nanoscale, is one of the most important sciences of the twenty-first century. Nanoparticles are used in a variety of sectors, including food, agriculture, cosmetics, and medicines (Annu et al. 2018; Shobha et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Food processing and preservation (nano preservatives, toxin detection, nano encapsulated food additives, etc.) as well as food packaging are examples of NP applications in the food sector (nano coatings, nanosensors, nanocomposites, edible coating NPs, etc.) (Biswas et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Nanotechnology is employed in agriculture to make nano-fertilizers, insecticides, herbicides, and sensors (Tang et al. 2024).\u003c/p\u003e \u003cp\u003eSilver nanoparticles (AgNPs) have been used in different applications due to their different sizes, charges and properties (Wong and Liu, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). AgNPs can be synthesized by different chemical and physical methods. Due to being expensive and toxicity of physical and chemical methods, nowadays they are not preferred. As an alternative to these methods, biological methods using plants and microorganisms are more utilized (Horsfall, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The synthesis of metal nanoparticles with enhanced antioxidant and antimicrobial activities in AgNPs has gained importance in biochemical applications (Chahar et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). It is stated in many studies that this synthesis method opens up new opportunities for easy, inexpensive, fast and large-scale production, and plant-based synthesis of metal nanoparticles has become an alternative to chemical, physical and even microbial methods (Elahi et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCarob (\u003cem\u003eCeratonia siliqua\u003c/em\u003e L.) leaves, which have a wide crown, are evergreen, hard and hairy in order to adapt to the harsh conditions of the Mediterranean climate. The length of its leaves, which are always green, is around 3\u0026ndash;5 cm; The tree has green, small flowers and these flowers form clusters in groups of 50\u0026ndash;60. The most abundant phenolic substance in carob, which is rich in phenolic substances, is gallic acid, which is an effective antioxidant. Studies show that especially gallic acid is very effective in slowing down the oxidation of fats (Kumazawa et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Owen et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). In other study conducted by Souli et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) on mice, it was revealed that carob extract has a protective effect on liver cells against oxidative stress damage caused by ethyl alcohol. In another study with zebrafish, the antioxidant capacity of carob extract against deltamethrin induced oxidative stress was investigated that carob extract effects important antioxidant metabolites and related pathways (Unal et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough there are studies regarding the in vivo toxic effects of silver nanoparticles on the antioxidant enzyme activities and immune system, there are almost no studies about green synthesized silver nanoparticles using plant extracts on the antioxidant enzymes and immune system. It is known that the toxic effects of silver nanoparticles occur due to induction of oxidative damage and cell death pathways, so that in this study we aimed to investigate the effects of silver nanoparticles synthesized from carob leaves on antioxidant enzymes and immune system in the larvae of the model organism \u003cem\u003eGalleria mellonella\u003c/em\u003e L.\u003c/p\u003e"},{"header":"Material-Method","content":"\u003cp\u003e \u003cb\u003eCeratonia siliqua\u003c/b\u003e \u003cb\u003eleaf collection and preparation of the extract\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe plant leaves were collected in the campus of Cukurova University. They washed with Milli-Q water and stayed to dry. After drying, the leaves were cut into small pieces. 10 g of \u003cem\u003eCeratonia silique\u003c/em\u003e leaves added into 150 ml of distilled water. Then boiled at 150\u0026deg;C. The obtained extract was filtered with Whatman No.1 filter paper. The pH of the extract and synthesized silver nanoparticle solutions were measured with pH meter. The obtained pH of the solutions was shown in the Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003epH values of the solutions.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSolution\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2mM AgNO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6.19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLeaf extract of \u003cem\u003eCeratonia siliqua\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6.51\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThe solution of the mixture\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.95\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eGreen synthesis of silver nanoparticles from the leaf extract of\u003c/b\u003e \u003cb\u003eCeratonia siliqua\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFor the green synthesis of AgNPs, 3 ml of leaf extract was added to 27 ml of 2mM AgNO\u003csub\u003e3\u003c/sub\u003e solution mixed well by heating at 60\u0026deg;C with ultrasonic bath until colour change. The formation of the AgNPs was observed by the change in colour from light yellow to dark brown (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). It was centrifuged at 12.000 rpm for 45 min. After centrifugations, the pellet was washed with distilled water for three times. Then, the pellet was dried in an incubator for 3 days.\u003c/p\u003e\n\u003ch3\u003eCharacterization of Silver Nanoparticles\u003c/h3\u003e\n\u003cp\u003eMany methods are applied for the characterizing different AgNPs. The morphology of AgSO4 nanoparticles was performed by UV/Vis spectrophotometer, SEM, XRD and FTIR analyses at Cukurova University Central Research Laboratory.\u003c/p\u003e\n\u003ch3\u003eUV/VIS Spectrophotometer\u003c/h3\u003e\n\u003cp\u003eThe AgNP solution synthesized via green synthesis was placed in a quartz cuvette and measured at a wavelength of 200\u0026ndash;800 nm using a UV/Vis Spectrophotometer.\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eFourier Transform Infrared (FTIR)\u003c/h2\u003e \u003cp\u003eFTIR was used to identify functional groups and various phytochemical components involved in the reduction and stabilization of the synthesized nanoparticles. FTIR analysis of AgNPs was performed in the spectral range of 400\u0026ndash;4000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e using FTIR spectroscopy (JASCO, FT/IR 6700) at Cukurova University Central Research Laboratory.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eScanning Electron Microscope (SEM)\u003c/h2\u003e \u003cp\u003eThe shape and size of the synthesized AgNPs were determined by SEM. The surface of the AgNPs was washed three times with 50% acetone solution. SEM-EDS analysis was then performed with a scanning electron microscope (FEI, Quanta 650 Field Emission).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eX-Ray Diffraction Pattern (XRD)\u003c/h2\u003e \u003cp\u003eXRD analysis, copper (Cu Kα, 1.54060 \u0026Aring;) as the radiation source, an XRD system (Polycrystalline XRD, T\u0026amp;T TT-90) with a range of 4\u0026ndash;90 degrees and a step width of 0.01 was used to determine the crystal structure of AgNPs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eExperimental design\u003c/h2\u003e \u003cp\u003e \u003cem\u003eG. mellonella\u003c/em\u003e larvae was reared as described in Tuncsoy et al. (2019). The last instar in the diet were used for the experiment (n\u0026thinsp;=\u0026thinsp;20) and divided into two groups as the control and the experimental groups. Ag NP, synthesised from carob leaves at a concentration of 3 \u0026micro;g/ml, was injected into last instar \u003cem\u003eG. mellonella\u003c/em\u003e larvae at a rate of 10 microlitres per larva from the proleg using a Hamilton injector. The doses were determined according to the preliminary experiments. Before injection the proleg was sterilized with %70 alcohol by using a swab. After the application, the larvae were placed in Petri dishes and kept at 28\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C for 72 hours. For the control larvae, distilled water was used. Mean Ag levels in exposure media were determined as 2.960\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 mg/L Ag. Experiments were run in triplicate.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eAntioxidant enzyme activities\u003c/h2\u003e \u003cp\u003eThe midgut and adipose tissue were dissected and homogenised in cold homogenisation buffer for the determination of SOD, CAT and GPx enzyme activities (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The methods to be used for the determination of the antioxidant enzyme activities are described in Tuncsoy et al. (2019). Sephadex G-25 was used for the removal of low molecular weight proteins (Gonzalez-Rey et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). GST activity was determined using the method developed by Habig et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1974\u003c/span\u003e) based on conjugating CDNB with reduced glutathione (Tuncsoy and Mese, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The Bradford method was used to determine total protein content (Bradford, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1976\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eAcetylcholinesterase activity\u003c/h2\u003e \u003cp\u003eTo determine AChE activity, the midgut and fat body were homogenized on ice in five volumes of a Tris\u0026ndash;HCl buffer (100 mM, pH 8.0) containing 10% Triton and centrifuged at 12,000\u0026times;g for 30 min (4\u0026deg;C). The AChE activity was assayed as described by Ellman et al. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1961\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePhenoloxidase activity\u003c/h2\u003e \u003cp\u003eAfter 20 \u0026micro;L of hemolymph was obtained from the experimental and control group larvae, it was mixed with 180 \u0026micro;L of cold phosphate buffer. Then, it was centrifuged at 10000 g for 5 min. at 4\u0026deg;C. The supernatant was mixed with 3,4-Dihydroxy L-phenylalanine (L-DOPA) and read at 490 nm absorbance at 5 minute intervals between 0 and 30 minutes in a UV-Spectrophotometer. The data obtained were determined as U/mg protein/min (Brookman et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1989\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eTotal hemocyte count (THC)\u003c/h2\u003e \u003cp\u003eFirstly, last instar larvae were kept at -20\u0026ordm;C for 3 minutes to slow down their movements. After the larvae were wiped with 95% ethanol, they were punctured from the proleg with a fine-tipped dissection needle and 5 \u0026micro;l hemolymph was obtained with the help of a microcapillary tube (SIGMA). 4 \u0026micro;l of the obtained hemolymph sample was taken and transferred to Eppendorf tubes kept on ice and containing 36 \u0026micro;l of anticoagulant (0.098 M NaOH, 0.186 M NaCl, 0.017 M Na2EDTA and 0.041 M Citric acid, pH\u0026thinsp;=\u0026thinsp;4.5). 10 \u0026micro;l of the 1:10 diluted cell suspension was taken and loaded into the Neubauer hemocytometer. Hemocytes were counted under a Leica DM750 microscope and the number of hemocytes in one milliliter of hemolymph was determined. Counted hemocytes were calculated using the Jones (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1962\u003c/span\u003e) method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eThe statistical analyses of the data were carried out by a series of Analysis of Variance and Independent Samples T test by using the SPSS programme. Differences were considered as significant when p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of AgNPs\u003c/h2\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003eUV-Spectrophotometer analysis\u003c/h2\u003e \u003cp\u003eFor the green synthesis of AgNPs using carob leaf extract, the plant extract was added to 2mM silver nitrate solution and then kept in an ultrasonic bath until the colour of the mixture turned dark brown. When the UV Vis spectrum is examined 5 minutes after synthesis, it is seen that a surface Plasmon resonance band is formed at a wavelength of approximately 440 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This peak is the characteristic surface Plasmon resonance band of AgNPs and proves the presence of AgNPs in the medium (Bhakya et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). When the studies were examined, it was determined that AgNPs formed peaks between 400\u0026ndash;450 nm wavelengths (Awwad et a., 2013; Beği\u0026ccedil; et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Singh et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eXRD (X-Ray Diffraction) Spectroscopy\u003c/h2\u003e \u003cp\u003eXRD analysis was performed to determine the crystal and molecular structures of silver nanoparticles synthesized from \u003cem\u003eCeratonia siliqua\u003c/em\u003e leaf extract. Some characteristic peaks at 38.039 [111], 44.210 [200], 64.305 [220], 77.223 [311] and 81.352 [222] were observed in the XRD pattern of Ag NPs synthesized from the \u003cem\u003eCeratonia siliqua\u003c/em\u003e plant. Additionally, the observed silver peaks indicate that Ag crystals have a cubic structure. The [hkl] values of silver are given in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eFourier Transform Infrared Spectroscopy (FTIR)\u003c/h2\u003e \u003cp\u003eFTIR analysis method is a method used to understand the relationship of functional groups between biomolecules and nanoparticles. FTIR analyses were performed to identify biomolecules responsible for the synthesis, reduction and stabilization of silver nanoparticles. The peaks obtained in the FTIR measurement of Ag NPs synthesized from \u003cem\u003eCeratonia siliqua\u003c/em\u003e leaf extract, observed at wavelengths of 2979.48, 2350.80, 2114.56 and 1991.14, are alkane (C-H), carbon dioxide (O\u0026thinsp;=\u0026thinsp;C\u0026thinsp;=\u0026thinsp;O), alkyne (C Ξ C) and is thiocyanate, respectively. It corresponds to (N\u0026thinsp;=\u0026thinsp;C\u0026thinsp;=\u0026thinsp;S) functional groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eSEM Analysis\u003c/h2\u003e \u003cp\u003eThe size and shape of the green synthesized Ag NPs were evaluated in University of Cukurova Central Research Laboratory (CUMERLAB). The Ag NPs determined as spherical shape (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Mean particle size was determined as 39.653\u0026thinsp;\u0026plusmn;\u0026thinsp;2.562 nm. When previous studies were examined, it was determined that silver NPs synthesized from plant parts were mostly spherical in shape and 13\u0026ndash;75 nm in size (Venkatadri et al., 2020; Baran et al., 2023; Ajlouni et al., 2023; Dua et al., 2023).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eBioaccumulation of Ag NPs\u003c/h2\u003e \u003cp\u003eIn present study, it was detected that Ag ions mostly accumulated in the fat body of larvae as well as midgut according to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The fat body of insects is analogous with liver in vertebrates, and it is known that xenobiotics mostly accumulate in this tissue (Yang et al. 2007). Also it was known that, the digestive track is the route of the entrance of nanoparticles, for direct exposure through intentional ingestion as well as for indirect exposure by means of their dissolution from food containers or by secondary ingestion of inhaled particles (Bergin and Witzmann, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). After uptake of nanoparticles, this types of nanomaterials cross the gut barrier and reach the immune competent cells in the hemolymph (Mir et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eAntioxidant enzyme activities\u003c/h2\u003e \u003cp\u003eDespite the advantages of green synthesized nanoparticles, these are not exempt from toxicity toward non-target organisms (Silva Brito et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). According to the previous studies, Ag NPs dissolve to release Ag\u003csup\u003e+\u003c/sup\u003e and cause the production of reactive oxygen species by transferring silver across the cell membrane (Noga et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). It was well known that the one of the main antioxidant enzyme is SOD for eliminating the reactive oxygen species. In the reaction of dismutation, superoxide anion radicals are eliminated from the cell microenvironment by their conversion to hydrogen peroxide, which is further eliminated by CAT and GPx (Scibior and Czeczot, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Jarosiewicz et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In this study, we observed that green synthesized AgNPs accumulate in the midgut of the larvae and led to the increasing of CAT and SOD activities (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Moreover, we determined that the midgut of the larvae act as the main site of absorption, where the metals mostly accumulated in our previous study (Tuncsoy et al. 2019). In this study, on the other hand, CAT activity was increased, while SOD activity was decreased in the fat body of the larvae (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). As for GPx activity, CAT and SOD activities were decreased in all tissues (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). A decrease in CAT and GPx enzyme activities may have occurred in fat body due to the oxidant/antioxidant balance being disturbed by increased reactive oxygen levels (Aslankoc et al. 2019).\u003c/p\u003e \u003cp\u003eAlthough there are not enough studies regarding to the effects on GST activity, it is generally known that silver nanoparticles cause toxic effects on mammalian and other non-target species. El-Samad et al. (2022) reported that silver nanoparticle exposure resulted in a decrease in midgut of \u003cem\u003eBlaps polychresta\u003c/em\u003e. In the present study, GST activity decreased in the midgut of \u003cem\u003eGalleria mellonella\u003c/em\u003e larvae, while GST activity increased in fat body (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e.). This is thought to be due to the fact that silver nanoparticles synthesised using carob leaves may cause an imbalance between oxidation / anti-oxidation processes by causing irregular ROS accumulation in cells (Wang et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Moreover, previous studies have reported significant decreases in antioxidant enzyme activities in insects exposed to xenobiotic (Yousef et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; El-Samad et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). It is also known that nanoparticles lead to an increase in intracellular ROS and cause inhibition of some enzyme activities (Akter et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eAcetylcholinesterase activity\u003c/h2\u003e \u003cp\u003eIn the present study, AChE activity was significantly decreased in the midgut (2.66-fold), however it was significantly increased in the fat body of the larvae (1.26-fold) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e). Similar results are found in our previous study where CuO NP was applied to \u003cem\u003eG. mellonella\u003c/em\u003e larvae (Sezer-Tuncsoy et al. 2019). The mechanism by which silver nanoparticles inhibit the AChE enzyme may be due to structural perturbation of the enzyme (Sinko et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). It is also thought that the inhibition caused by nanoparticle exposure may be due to the adsorption of the AChE enzyme to the surface of the nanoparticles. Moreover, Ag NPs are also known to stimulate oxidative stress in nerve cells by changing the permeability of blood brain barrier cells (Tang et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Baruwati et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Abdelwahab et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eTotal Hemocyte Count (THC)\u003c/h2\u003e \u003cp\u003eThere was an increase in THC in the hemolymph of the Ag NPs exposed groups compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The increase of THC may be due to the promotion of haematopoiesis is or the release of hemocytes that adhered on surfaces (sessile hemocytes) within the hemocoel (Jones, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1962\u003c/span\u003e; Ghasemi et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Also, the increase of THC may be due to the activation of mitotic division of hemocytes which was activated in response to nanoparticles.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003ePhenoloxidase activity\u003c/h2\u003e \u003cp\u003eIt was determined that PO activity was significantly increased in AgNPs exposed group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e). The increase in the number of hemocytes is closely related to the increase in the melanisation innate immunity response where PPO is effective (Kurt and Kayis, 2015).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eAlthough green synthesized silver nanoparticles are considered to be environmentally friendly, they can be a source of oxidative stress due to their accumulation in tissues as a result of acute use. For this reason, it is necessary to determine the LD\u003csub\u003e50\u003c/sub\u003e values and to determine the correct concentration and application time.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledge\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn memory of Yağmur Meşe, who we lost in the earthquake disaster that occurred in our region on February 6, 2023.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo ethical approval is required for this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate and Publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study is not a clinical study, the living organism used is an invertebrate. Therefore, there is no need for any permission to participate or to publish.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Aslıhan Andırın, Nur Dudu Yaycı, Murat İdikut, Ayse Kara, Benay Tuncsoy, Mustafa Tuncsoy and Pınar Ozalp. The first draft of the manuscript was written by Benay Tuncsoy and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there is no funding during the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere is no conflict of interest between the authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbdelwahab GM, Mira A, Cheng YB, Abdelaziz TA, Lahloub MFI, Khalil AT. (2021). Acetylcholine esterase inhibitory activity of green synthesized nanosilver by naphthopyrones isolated from marine-derived Aspergillus niger. PLoS One, 16(9):e0257071. doi: 10.1371/journal.pone.0257071.\u003c/li\u003e\n\u003cli\u003eAkter, M., M.T. Sikder, M.M. Rahman, A.K.M.A. Ullah, K.F.B. Hossain, S. Banik, T. Hosokawa, T. Saito, M. Kurasaki (2018). A systematic review on silver nanoparticles- induced cytotoxicity: physicochemical properties and perspectives, J. Adv. Res. 9:1\u0026ndash;16.\u003c/li\u003e\n\u003cli\u003eAnnu. Ahmeda, S. Kaurb, G. Sharmac, P. Singhc, S. Ikrama, S. (2018). Fruit waste (peel) as bio-reductant to synthesize silver nanoparticles with antimicrobial, antioxidant and cytotoxic activities. Journal of Applied Biomedicine, 175, 1-11.\u003c/li\u003e\n\u003cli\u003eAslanko\u0026ccedil; R, Demirci D, İnan \u0026Uuml;, Yıldız M, \u0026Ouml;zt\u0026uuml;rk A, \u0026Ccedil;etin M, Savran EŞ, Yılmaz B. (2019). The Role Of Antioxidant Enzymes İn Oxidative Stress - Superoxide Dismutase (Sod), Catalase (Cat) And Glutathione Peroxidase (Gpx). Med J SDU; 26(3): 362-369.\u003c/li\u003e\n\u003cli\u003eAwwad, A.A., Salem, N.M., Abdeen, A.O. (2013). Green synthesise of silver nanoparticles using carob leaf extract and its antibacterial activity. International Journal of Industrial Chemistry, 4:29.\u003c/li\u003e\n\u003cli\u003eBaruwati B, Simmons SO,Varma RS,Veronesi B. (2013). \u0026ldquo;Green\u0026rdquo;synthesized and coated nano silver alters the membrane permeability of barrier (intestinal, brain endothelial) cells and stimulates oxidative stress pathways in neurons. ACS Sustainable Chemistry\u0026amp;Engineering. 1(7):753\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eBeği\u0026ccedil;, N, Bener, M, Apak, R. (2021). Development of a green synthesized silver nanoparticle based antioxidant capacity method using carob extract. Journal of Nanostructure in Chemistry. 11:382-394.\u003c/li\u003e\n\u003cli\u003eBergin, I. L., Witzmann, F. A. (2013). Nanoparticle toxicity by the gastrointestinal route: evidence and knowledge gaps. International Journal of Biomedical Nanoscience and Nanotechnology. 3, 163\u0026ndash;210.\u003c/li\u003e\n\u003cli\u003eBhakya, S, Muthukrishnan, S., Sukumaran, M., Muthukumar, M. 2016. \u0026ldquo;Biogenic synthesis of silver nanoparticles and their antioxidant and antibacterial activity,\u0026rdquo; Appl. Nanosci., 6(5): 755\u0026ndash;766.\u003c/li\u003e\n\u003cli\u003eBiswas R, Alam M, Sarkar A, Haque MI, Hasan MM, Hoque M. (2022) Application of nanotechnology in food: processing, preservation, packaging and safety assessment. Heliyon. 2022 Nov 21;8(11):e11795. doi: 10.1016/j.heliyon..e11795. PMID: 36444247; PMCID: PMC9699984.\u003c/li\u003e\n\u003cli\u003eBradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248\u0026ndash;254\u003c/li\u003e\n\u003cli\u003eBrito, R.S., Bebianno, M.J.,Rocha, T.L. (2024). Plant-based silver nanoparticles ecotoxicity: Perspectives about green technologies in the One Health context, Critical Reviews in Environmental Science and Technology, DOI: 10.1080/10643389.2024.2303298\u003c/li\u003e\n\u003cli\u003eBrookman, J. L., Ratcliffe, N. A. and Rowley, A. F. (1989). Studies on the activation of the prophenoloxidase system of insects by bacterial cell wall components. Insect Biochemistry, 19 (1): 47-57.\u003c/li\u003e\n\u003cli\u003eChahar, H. S., Corsello, T., Kudlicki, A. S., Komaravelli, N., Casola, A. (2018). Respiratory syncytial virus infection changes cargo composition of exosome released from airway epithelial cells. Scientific reports, 8(1), 1-18.\u003c/li\u003e\n\u003cli\u003eElahi, N., Kamali, M., Baghersad, M. H. (2018). Recent biomedical applications of gold nanoparticles: A review. Talanta, 184, 537-556.\u003c/li\u003e\n\u003cli\u003eEllman GL, Courtney KD, Andres V, Featherstone RM (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 7:88\u0026ndash;95\u003c/li\u003e\n\u003cli\u003eEl-Samad, L.M., M. El Hassan, D.A. Kheirallah, K.K. Abdul-Aziz, N.A. Toto, (2020) Biochemical, molecular, and histological markers in \u003cem\u003ePimelia latreillei\u003c/em\u003e (Coleoptera: Tenebrionidae) for evaluating soil pollution, Fresenius Environ. Bull. 29 4224\u0026ndash;4239.\u003c/li\u003e\n\u003cli\u003eGhasemi V, Yazdi AK, Tavallaie FZ, Sendi JJ (2014) Effect of essential oils from \u003cem\u003eCallistemon viminalis \u003c/em\u003eand \u003cem\u003eFerula gummosa\u003c/em\u003e on toxicity and on the hemocyte profile of \u003cem\u003eEphestia kuehniella\u003c/em\u003e (Lepidoptera:Pyralidae). Arch Phytopathol Plant 47(3):268\u0026ndash;278\u003c/li\u003e\n\u003cli\u003eGonzalez-Rey M, Mattos JJ, Piazza CE, Bainy ACD, Bebianno MJ (2014) Effects of active pharmaceutical ingredients mixtures in mussel Mytilus galloprovincialis. Aquat Toxicol 153:12\u0026ndash;26\u003c/li\u003e\n\u003cli\u003eHabig WH, Pabst MJ, Jakoby WB (1974) Glutathione S-transferases: the first enzymatic step mercapturic acid formation. J Biol Chem 249:7130\u0026ndash;7139\u003c/li\u003e\n\u003cli\u003eHorsfall, L. 2014. Biological Synthesis of Metallic Nanoparticles by Bacteria, Fungi and Plants. Journal of Nanomedicine \u0026amp; Nanotechnology, 5.\u003c/li\u003e\n\u003cli\u003eJarosiewicz, M., Anita Krokosz, Agnieszka Marczak, Bożena Bukowska, Changes in the activities of antioxidant enzymes and reduced glutathione level in human erythrocytes exposed to selected brominated flame retardants, Chemosphere, Volume 227, 2019, Pages 93-99,\u003c/li\u003e\n\u003cli\u003eJones JC (1962) Current concepts concerning insect hemocytes. Am Zool 2:209\u0026ndash;246.\u003c/li\u003e\n\u003cli\u003eKumazawa S., Taniguchi M., Suzuki Y., Shimura M., Kwon M.S., Nakayama T. Antioxidant activity of polyphenols in carob pods. J. Agric. Food Chem. 2002;50:373\u0026ndash;377.\u003c/li\u003e\n\u003cli\u003eKurt D, Kayış T (2015) Effects of the pyrethroid insecticide deltamethrin on the hemocytes of the \u003cem\u003eGalleria mellonella\u003c/em\u003e. Turk J Zool 39:452\u0026ndash;457.\u003c/li\u003e\n\u003cli\u003eLamia M. El-Samad, Nahed R. Bakr, Saeed El-Ashram, Eman H. Radwan, Karoline K. Abdul Aziz, Hussein K. Hussein, Abeer El Wakil, Mohamed A. Hassan, Silver nanoparticles instigate physiological, genotoxicity, and ultrastructural anomalies in midgut tissues of beetles,Chemico-Biological Interactions,Volume 367,2022,110166,\u003c/li\u003e\n\u003cli\u003eMir, A.H., Qamar, A., Qadir, I. \u003cem\u003eet al.\u003c/em\u003e 2020. Accumulation and trafficking of zinc oxide nanoparticles in an invertebrate model, \u003cem\u003eBombyx mori\u003c/em\u003e, with insights on their effects on immuno-competent cells. \u003cem\u003eSci Rep\u003c/em\u003e\u003cstrong\u003e10\u003c/strong\u003e, 1617. https://doi.org/10.1038/s41598-020-58526-1\u003c/li\u003e\n\u003cli\u003eNoga, M.; Milan, J.; Frydrych, A.; Jurowski, K. Toxicological Aspects, Safety Assessment, and Green Toxicology of Silver Nanoparticles (AgNPs)\u0026mdash;Critical Review: State of the Art. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e 2023, \u003cem\u003e24\u003c/em\u003e, 5133. https://doi.org/10.3390/ijms24065133\u003c/li\u003e\n\u003cli\u003eOwen R.W., Haubner R., Hull W.E., Erben G., Spiegelhalder B., Bartsch H. Isolation and structure elucidation of the major individual polyphenols in carob fibre. Food Chem. Toxicol. 2003;41:1727\u0026ndash;1738.\u003c/li\u003e\n\u003cli\u003eScibior D, Czeczot H. Katalaza--budowa, właściwości, funkcje [Catalase: structure, properties, functions]. Postepy Hig Med Dosw (Online). 2006;60:170-80. Polish. PMID: 16618987.\u003c/li\u003e\n\u003cli\u003eSezer Tuncsoy B, Tuncsoy M, Gomes T, Sousa V, Teixeira MR, Bebianno MJ, Ozalp P (2019) Effects of copper oxide nanoparticles on tissue accumulation and antioxidant enzymes of \u003cem\u003eGalleria mellonella\u003c/em\u003e L.. B Environ Contam Tox 102(3):341\u0026ndash;346\u003c/li\u003e\n\u003cli\u003eShobha, G., Moses, V., \u0026amp; Ananda, S. (2014). Biological synthesis of copper nanoparticles and its impact. Int. j. pharm. Sci. Invent, 3(8), 6-28.\u003c/li\u003e\n\u003cli\u003eSingh, C., Anand, S.K., Upadhyay, R., Pandey, N., Kumar, P., Singh, D., Tiwari, P., Saini, R., Tiwari, K.N., Mishra, S.K., Tilak, R. 2023. Green synthesis of silver nanoparticles by root extract of Premna integrifolia L. and evaluation of its cytotoxic and antibacterial activity, Materials Chemistry and Physics, 297: 127413,\u003c/li\u003e\n\u003cli\u003eSinko G,VrčekIV, GoesslerW, LeitingerG, Dijanos ićA, Miljanić S.Alteration of cholinesterase activity as possible mechanism of silver nanoparticle toxicity. Environmental Science and Pollution Research. 2014;21(2):1391\u0026ndash;400.https://doi.org/10.1007/s11356-013-2016-zPMID:23904256\u003c/li\u003e\n\u003cli\u003eSouli A, Sebai H, Chehimi L, Rtibi K, Tounsi H, Boubaker S, Sakly M, El-Benna J, Amri M. Hepatoprotective effect of carob against acute ethanol-induced oxidative stress in rat. Toxicol Ind Health. 2015;31(9):802\u0026ndash;810. doi: 10.1177/0748233713475506.\u003c/li\u003e\n\u003cli\u003eTang J, Xiong L,, Wang S,, Wang J,, Liu L,, Li J, e tal. Influence of silver nanoparticles on neurons and blood-brain barrier via subcutaneous injection in rats. Applied Surface Science.2008;255(2):502\u0026ndash;4.\u003c/li\u003e\n\u003cli\u003eTang, Y.; Zhao, W.; Zhu, G.; Tan, Z.; Huang, L.; Zhang, P.; Gao, L.; Rui, Y. Nano-Pesticides and Fertilizers: Solutions for Global Food Security. Nanomaterials 2024, 14, 90. https://doi.org/10.3390/nano14010090\u003c/li\u003e\n\u003cli\u003eTuncsoy, B., Mese, Y. (2021). Influence of titanium dioxide nanoparticles on bioaccumulation, antioxidant defense and immune system of \u003cem\u003eGalleria mellonella\u003c/em\u003e L.. Environ Sci Pollut Res 28, 38007\u0026ndash;38015. https://doi.org/10.1007/s11356-021-13409-4\u003c/li\u003e\n\u003cli\u003eUnal, E., Sulukan, E., Senol, O., Baran, A., Nadaroğlu, H., Kankaynar, M., Kızıltan, T., Ceyhun, S.B. 2023. Antioxidant/protective effects of carob pod (\u003cem\u003eCeratonia siliqua\u003c/em\u003e L.) water extract against deltamethrin-induced oxidative stress/toxicity in zebrafish larvae, Comparative Biochemistry and Physiology Part C: Toxicology \u0026amp; Pharmacology, 267: 109584. https://doi.org/10.1016/j.cbpc.2023.109584.\u003c/li\u003e\n\u003cli\u003eWang, D., M. Dan, Y. Ji, X. Wu, X. Wang, H. Wen, Roles of ROS and cell cycle arrest in the genotoxicity induced by gold nanorod core/silver shell nanostructure, Nanoscale Res. Lett. 15 (2020) 224. \u003c/li\u003e\n\u003cli\u003eWong, K. and Liu, X. 2010. Silver nanoparticles - The real \u0026quot;silver bullet\u0026quot; in clinical medicine? Med. Chem. Comm., 1.\u003c/li\u003e\n\u003cli\u003eYousef, H.A. , E.A. Abdelfattah, M. Augustyniak, Antioxidant enzyme activity in responses to environmentally induced oxidative stress in the 5th instar nymphs of \u003cem\u003eAiolopus thalassinus\u003c/em\u003e (Orthoptera: Acrididae), Environ. Sci. Pollut. Res. Int. 26 (2019) 3823\u0026ndash;3833. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"environmental-science-and-pollution-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"espr","sideBox":"Learn more about [Environmental Science and Pollution Research](https://www.springer.com/journal/11356)","snPcode":"11356","submissionUrl":"https://submission.nature.com/new-submission/11356/3","title":"Environmental Science and Pollution Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Antioxidant activity, Carob, Green synthesis, Immunotoxicity, Silver nanoparticles","lastPublishedDoi":"10.21203/rs.3.rs-3984885/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3984885/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSilver nanoparticles (AgNP) have been used in many studies due to their inhibitory properties on microorganisms such as bacteria and viruses. In recent years, due to global problems such as environmental pollution, the green synthesis (biosynthesis) method is frequently preferred because it is simple and low cost and does not require the use of toxic substances. In this study, it was determined that the effects on antioxidant enzyme activities (SOD, CAT, GPx, GST), acetylcholinesterase (AChE), and total hemocyte count (THC) as well as phenoloxidase activity to determine their effect on antioxidant defence and the immune system in model organism \u003cem\u003eGalleria mellonella\u003c/em\u003e larvae. We observed that green synthesized AgNPs accumulate in the midgut of the larvae and led to the increasing of CAT and SOD activities. GST and AChE activities were increased in the fat body of the larvae otherwise; it was decreased in the midgut. Moreover, increases were found in THC and phenoloxidase activity. Consequently, green synthesized silver nanoparticles led to oxidative stress and immuntoxic effects on \u003cem\u003eG. mellonella\u003c/em\u003e larvae.\u003c/p\u003e","manuscriptTitle":"Green synthesis of silver nanoparticles using Carob leaf extract: Characterization and analysing of toxic effects in model organism Galleria mellonella L. (The Greater Wax Moth)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-11 16:41:05","doi":"10.21203/rs.3.rs-3984885/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revision","date":"2024-08-23T08:58:39+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-05-14T15:12:14+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-08T10:12:40+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-01T06:20:07+00:00","index":"","fulltext":""},{"type":"submitted","content":"Environmental Science and Pollution Research","date":"2024-02-27T03:27:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"environmental-science-and-pollution-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"espr","sideBox":"Learn more about [Environmental Science and Pollution Research](https://www.springer.com/journal/11356)","snPcode":"11356","submissionUrl":"https://submission.nature.com/new-submission/11356/3","title":"Environmental Science and Pollution Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b8c73fbc-d87b-4f6c-8c09-fcc2a68d8267","owner":[],"postedDate":"April 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-23T16:04:40+00:00","versionOfRecord":{"articleIdentity":"rs-3984885","link":"https://doi.org/10.1007/s11356-024-34996-y","journal":{"identity":"environmental-science-and-pollution-research","isVorOnly":false,"title":"Environmental Science and Pollution Research"},"publishedOn":"2024-09-18 15:57:53","publishedOnDateReadable":"September 18th, 2024"},"versionCreatedAt":"2024-04-11 16:41:05","video":"","vorDoi":"10.1007/s11356-024-34996-y","vorDoiUrl":"https://doi.org/10.1007/s11356-024-34996-y","workflowStages":[]},"version":"v1","identity":"rs-3984885","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3984885","identity":"rs-3984885","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}