Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 75(1): 3-13, 2022 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1353 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Jelili A. Badmus, Samuel A. Oyemomi, John O. Fatoki, Taofeek A. Yekeen, Olaniyi T. Adedosu, Peter I. Adegbola, Musibau A. Azeez, Elijah A. Adebayo, Agbaje Lateef (2022) Anti- haemolytic and cytogenotoxic poten- tial of aqueous leaf extract of Annona muricata (L.) and its bio-fabricated sil- ver nanoparticles. Caryologia 75(1): 3-13. doi: 10.36253/caryologia-1353 Received: June 29, 2021 Accepted: March 31, 2022 Published: July 6, 2022 Copyright: © 2022 Jelili A. Badmus, Sam- uel A. Oyemomi, John O. Fatoki, Tao- feek A. Yekeen, Olaniyi T. Adedosu, Peter I. Adegbola, Musibau A. Azeez, Elijah A. Adebayo, Agbaje Lateef. This is an open access, peer-reviewed arti- cle published by Firenze University Press (http://www.fupress.com/caryo- logia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. ORCID JAB: 0000-0003-4785-2609 JOF: 0000-0003-3246-9172 TAY: 0000-0002-2476-2283 PIA: 0000-0002-4008-1728 MAA: 0000-0002-0059-0309 EAA: 0000-0002-6574-7928 AL: 0000-0001-5302-9892 Anti-haemolytic and cytogenotoxic potential of aqueous leaf extract of Annona muricata (L.) and its bio-fabricated silver nanoparticles Jelili A. Badmus1,3,*, Samuel A. Oyemomi1, John O. Fatoki1, Taofeek A. Yekeen2,3, Olaniyi T. Adedosu1, Peter I. Adegbola1, Musibau A. Azeez2,3, Elijah A. Adebayo2,3, Agbaje Lateef2,3 1 Department of Biochemistry, Ladoke Akintola University of Technology, Ogbomoso, Oyo State, Nigeria 2 Department of Pure and Applied Biology, Ladoke Akintola University of Technology, Ogbomoso, Oyo State, Nigeria 3 Nanotechnology Research Group (NANO+), Ladoke Akintola University of Technology, Ogbomoso, Oyo State, Nigeria *Corresponding author. E-mail: jabadmus@lautech.edu.ng Abstract. Nanotechnology is widely gaining worldwide application in biology and medicine because of its proven efficacy. Annona muricata contains bioactive phyto- chemicals with an inherent ability to bio-fabricate metal ions nanoparticles (NPs). Annona muricata aqueous leaf extract and its green bio-fabricated silver nanoparticles were evaluated on red blood cells (RBC) for anti-haemolytic activity and cytogenotox- icity on Allium cepa cells. The effects of A. muricata extract (Am-E) and its biofabri- cated silver nanoparticles (Am-AgNPs) were observed at 0.7, 7.0 and 70.0 µg/ml on H2O2-induced haemolysis in RBC and cyclophosphamide-induced cytogenotoxicity on A. cepa cells. Results showed significant and concentration dependent anti-haemolytic activity of Am-E relative to Am-AgNPs. Significant (P<0.05) reduction of mitotic index was observed in the groups treated with Am-AgNPs compared with Am-E, which indicates cytotoxic effect of the nanoparticles. The Am-E protected A. cepa meristem root cells from cyclophosphamide-induced mitotic repression better than Am-AgNPs. Different degree of chromosomal abnormalities such as chromosome-bridge, sticky chromosome, and c-mitosis were observed in all the treatment groups with chromo- some-bridge and sticky chromosome being prominent. This study revealed stronger anti-haemolytic efficacy of Am-E at higher concentrations compared with Am-AgNPs. Chromosomal abnormalities observed in this study suggest greater chromosomal insta- bility as influenced by the nanoparticles compared with the extract on onion cells. The protective effect of the extract against cyclophosphamide-induced chromosomal aber- rations may be an indication of its potential as an anti-genotoxic agent. Keywords: green synthesis, anti-haemolytic, Annona muricata, Allium cepa, silver nanoparticles, cytogenotoxicity. 4 Jelili A. Badmus et al. 1. INTRODUCTION Nanotechnology has captured a great scientific interest worldwide due to its wider objectives cum applications in biology and medicine (Shaniba et al. 2017). Its fundamen- tal building block resides in the synthesis of Nanoparticles (NPs) which are products of creation, production, charac- terization, and manipulation of materials at nano-scale. It enables the amendment of materials at the atomic level with a view to obtain unique properties, which can be annexed for desired applications (Gleiter, 2000). The distinct optical, electrical, catalytic properties of metal nanoparticles such as Ag, Zn, Pt, Au and Pd, and their roles in biological and pharmaceutical applications are being studied intensively due to their unique ame- nability (Jacob et al. 2012; Firdhouse and Lalitha, 2015; Shaniba et al. 2017). Silver nanoparticles (AgNPs) have found extensive use in pharmaceutical and cosmetic industries among other metal nanoparticles owing to their broad utility (Sathishkumar et al. 2012; Patil et al. 2017; Annu et al. 2018; Patra et al. 2018). Biological syn- thesis of AgNPs from natural products viz. bacterial, fun- gi, yeast and plant extract, and their applications in biol- ogy and medicine have tagged them eco-friendly (Lokina et al. 2014; Shaniba et al. 2017; Adebayo et al. 2019a,b). Annona is a genus of flowering plants of Annonace- ae family known for its exotic fruits. Four species of the genus such as A. muricata, A. squamosa, A. senegalensis and A. cherimola have been reported to have compel- ling pharmacological activities (Santos-Sánchez et al. 2018). The pharmacological activities of the genus have been related to considerable quantity of bioactive princi- ples such as phenolic compounds (flavonoids and phe- nolic acids) (Perrone et al. 2022). A. muricata is the one of the most studied species of the genus Annona (Santos- Sánchez et al. 2018). A. muricata also known as sour- soup is a typical tropical evergreen tree with heart shaped edible fruits and it is ubiquitous in most tropical coun- tries (Gavamukulya et al. 2017). Pharmacological and tra- ditional uses of the leaf, bark, root, stem, fruit, and seed extracts include hypoglycemic, anti-cough and analgesic (Hardoko et al. 2015; Coria-Tellez et al. 2018). It has also been found useful as antispasmodic, sedative (Mishra et al. 2013; Moghadamtousi et al. 2015), anti-malarial (Som- sak et al. 2016), antioxidant (Balderrama-Carmona et al. 2020), anti-inflammatory (Abdul Wahab et al. 2018) and anticancer (Yang et al. 2015; Najmuddin et al. 2016; Coria- Tellez et al. 2018). It contains phytochemicals such as fla- vonoids, cardiac glycosides, saponins, alkaloids, tannins, phytosterol, and terpenoids giving it the ability to reduce metal ions (Vijayameena et al. 2013). Previous study from our laboratory have indicated that the physicochemi- cal property of silver nanoparticles synthesized using A. muricata aqueous leaf extract is within a normal range (Badmus et al. 2020). The nanoparticles displayed robust biomedical applications such as antidiabetic, antioxidant, antimicrobial and anti-proliferative potential. Generally, AgNPs have wide applications in house- hold materials, food, pharmaceutical and cosmetic industries. The increase and unregulated disposal of the nanoparticles will elevate environmental availability and bioaccumulation (McGillicuddy et al. 2017). There is a dearth of scientific evaluation of toxicological capability and implication of some identified nanoparticles with strong biomedical presentations. Therefore, this research was designed to study the anti-haemolytic, and cytogen- otoxic potential of silver nanoparticles synthesized using an aqueous leaf extract of A. muricata on red blood cell and A. cepa cell chromosomes respectively. 2. MATERIALS AND METHODS 2.1 Collection of Plant Materials The leaves of Annona muricata were collected from Ologundudu, Ondo State, Nigeria and identified by a taxonomist at the Department of Pure and Applied Biol- ogy, Ladoke Akintola University of Technology, Ogbo- moso, Oyo State, Nigeria. A sample of the plant was deposited in Herbarium Unit of the Department with voucher number LHO 250. 2.2 Extract Preparation Aqueous extraction of A. muricata leaf was carried out using the method as earlier reported by Yekeen et al. (2017a) with slight modification. The leaves were pulver- ized and 6 g of it was soaked in 100 ml distilled water. The soaked sample was heated with continuous stirring for 30 min at 40 °C. The mixture was filtered with Whatman No. 1 filter paper and stored in a refrigerator at 4 °C until use. 2.3 Green Synthesis of Silver Nanoparticles (AgNPs) A. muricata aqueous extract (1 ml) was added to 40 ml of 1 mM AgNO3 in glass container while 40 ml of Am-E and 1 mM AgNO3 solutions were separately kept in containers as controls. The controls and the reacting mixture of the extract and AgNO3 were placed in sun- light for a complete synthesis of nanoparticles (Yekeen et al. 2017a). A complete change of colour of reacting mixture, an indicator of synthesized nanoparticles was 5Anti-haemolytic and cytogenotoxic potential of aqueous leaf extract of Annona muricata observed after 30 min. 2.4 Determination of Anti-haemolytic Activity Anti-haemolytic activities of Am-E and Am-AgNPs were carried out using the method of Joujeh et al. (2017). The blood sample collected from a male Wistar rat through heart puncture was spun at 5000 rpm for 5 min. The plasma was discarded and the precipitate was washed 3 times with phosphate buffer saline (pH 7.4). Five percent of erythrocyte was prepared in phos- phate buffer saline. Samples (biosynthesized nanoparti- cles and the extract) (500 µl) at different concentrations (700, 350 and 175 µg/ml) were added to 1 ml of 5% erythrocyte and incubated for 20 min at room temper- ature (25 °C). Next, 500 µl of H2O2 was added and spun at 5000 rpm for 5 min. The absorbance of free haemo- globin content in the supernatant was read at 540 nm while the percentage inhibitions of H2O2-induced hae- molysis of the nanoparticles and the extract were cal- culated using the equation 1. % 𝑖𝑖𝑖𝑖ℎ𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖 𝑖𝑖𝑜𝑜 ℎ𝑎𝑎𝑎𝑎𝑎𝑎𝑖𝑖𝑎𝑎𝑎𝑎𝑎𝑎𝑖𝑖𝑎𝑎 = 𝐴𝐴𝑖𝑖𝑎𝑎(𝐶𝐶𝑖𝑖𝑖𝑖𝑖𝑖𝐶𝐶𝑖𝑖𝑎𝑎–𝑆𝑆𝑎𝑎𝑎𝑎𝑆𝑆𝑎𝑎𝑎𝑎) 𝐴𝐴𝑖𝑖𝑎𝑎(𝐶𝐶𝑖𝑖𝑖𝑖𝑖𝑖𝐶𝐶𝑖𝑖𝑎𝑎) × 100 (1) 2.5 Allium cepa Cytogenotoxicity Assay Onion bulbs (240) of approximately the same size were bought at a local market, Owode-Egba, Ogun State, Nigeria. The method earlier reported by Badmus et al. (2013) and Yekeen et al. (2017a, b) was adopted in this study and the experimental set up as described in Table 1. The onions were sundried for two weeks to minimize moisture and aid root growth. The outer scales of the onion bulbs were carefully peeled without affecting the primordial root ring. Fifteen onions were used for each group as indicated in Table 1. The base of each onion bulb was suspended in each container (100 ml beaker) sepa- rately containing the control and test solutions at different concentrations. All samples were placed in a dark cup- board at 25 ± 2 °C to reduce the fluctuation of dividing cells. The controls and test solutions were changed at 24 h intervals. Five onions per group were respectively harvest- ed at 48 h and 72 h of growth, and their roots were fixed in ethanol: acetic acid (3:1, v/v) for microscopic evaluation. 2.5.1 Microscopic Evaluation The fixed roots were hydrolyzed in 1 N HCl at 65 °C for 3 min. The tip of two roots was squashed on each of the six slides per group and chopped carefully to ease the scoring process. Aceto-orcein was used to stain the prepared slides for 15 min. Five slides were analyzed per group, in which 1000 cells were scored per slide at x1000 magnification for normal and abnormal chromosome behaviour during cell division using various template as earlier reported (Badmus et al. 2013; Yekeen and Ade- boye 2013; Yekeen et al. 2017a,b). 2.5.2 Macroscopic Evaluation After 72 h, the length of the roots of five onions with best growth selected from each of the concentrations was measured with ruler in cm. 2.6 Statistical Analysis All the data obtained in this study were expressed as mean ± SD. Comparison between treatments was done by analysis of variance (ANOVA) on Statistical Package for Social Sciences (SPSS) 21.0. Software Duncan’s mul- tiple range post hoc test was performed to measure vari- ation between the mean with significant difference con- sidered at p<0.05. 3. RESULTS 3.1 Biosynthesis of Silver Nanoparticles The colour change from colourless to brown of the reaction mixture after 30 min exposure to sunlight (UV Table 1. Experimental Design of Cytogenotoxic Evaluation. Groups Treatments 1 Distilled water only (Negative Control) 2 100 µg/ml cyclophosphamide (Positive) 3 0.17 µg/ml AgNO3 4 0.7 µg/ml Am-AgNPs 5 7.0 µg/ml Am-AgNPs 6 70.0 µg/ml Am-AgNPs 7 100 µg/ml cyclophosphamide + 0.7 µg/ml A.m-AgNPs 8 100 µg/ml cyclophosphamide + 7.0 µg/ml A.m-AgNPs 9 100 µg/ml cyclophosphamide + 70.0 µg/ml A.m-AgNPs 10 0.7 µg/ml Am-E 11 7.0 µg/ml Am-E 12 70.0 µg/ml Am-E 13 100 µg/ml cyclophosphamide. + 0.7 µg/ml Am-E 14 100 µg/ml cyclophosphamide. + 7.0 µg/ml Am-E 15 100 µg/ml cyclophosphamide. + 70.0 µg/ml Am-E 16 100 µg/ml of cyclophosphamide. + 1 mM AgNO3 6 Jelili A. Badmus et al. rays) is an indication of bio-reduction of silver ion to AgNPs 3.2 Anti-haemolytic Activity of Aqueous leaf Extract of A. muricata and its fabricated silver nanoparticles The results in Table 2 show that the bio-fabricat- ed nanoparticle and extract exhibited anti-haemolytic activity in an inverse concentration dependent manner. The activities of the extract and silver nanoparticles were stronger at lower concentration with the extract show- ing significantly (P<0.05) higher activity compared with their biosynthesized silver nanoparticles. 3.3 Effects of Aqueous leaf Extract of A. muricata, its fab- ricated silver nanoparticles and cyclophosphamide on root length of A. cepa The root lengths assessed after 72 h of exposure revealed that the average root length of the treated groups Am-AgNPs (Groups 4, 5, 6), Cyclo + Am-AgNPs (7, 8 and 9) decreased in a concentration dependent manner and significantly (P<0.05) lower than that of the control group. Non-significant (P>0.05) increase in the mean root length was observed in the groups treated with 0.7 and 7.0 µg/ml Am-E only (Group 10, 11), while at 70 µg/ml Am-E only (Group 12) significant (p<0.05) increase was observed compared to the control group and cyclophosphamide treated group (Group 2) (Table 3). Whereas non-significant (P>0.05) difference in aver- age root length was observed in the Cyclo + Am-E (13, 14 and 15) treated groups relative to the Control (Group 1). The mean root length of Am-AgNPs and Cyclo + Am-AgNPs at 0.7 and 7.0 µg/ml decreased significantly (p<0.05) relative to the group treated with cyclophos- phamide alone. No significant difference was observed between the root lengths of the cyclophosphamide (Group 2) treated group and the control. No growth of roots was observed in the onions treated with AgNO3 alone and Cyclophosphamide + AgNO3. 3.4 Cytogenotoxic effects of Aqueous leaf Extract of A. muricata, its fabricated silver nanoparticles and Cyclophos- phamide on A. cepa cells The cytogenotoxic effects of Am-AgNP and A. muri- cata extract on A. cepa cells are, respectively revealed in Tables 4 and 5 after 48 and 72 h exposure. At 48 h, a concentration dependent reduction in the total num- ber of dividing cells was observed in each of the treated groups compared with the negative control group. The mitotic index (MI) value of the treatment groups was lower than that of the control group whereas, a complete cell growth arrest was observed in groups treated with AgNO3 solution alone and the highest concentration of Am-AgNPs only and in combination with cyclophos- phamide. Furthermore, the lesser MI value was observed for both Am-E and Am-AgNPs singly and when com- bined with cyclophosphamide compared to group treat- ed with cyclophosphamide only. Mitotic index values lesser than the half of the negative control were recorded Table 2. Anti-haemolytic Activity of Am-AgNP and Am-E. Concentration (µg/ ml) Am-E (%) Am-AgNPs (%) 175 65.44 ± 0.6 49.22 ± 0.9 350 61.64 ± 0.6 45.92 ± 0.7 700 49.64 ± 0.8 34.77 ± 1.7 Data were Mean ± SD of triplicate experiments conducted at differ- ent time. Am-E (aqueous leaf extract of A. muricata); Am-AgNPs (A. muricata-fabricated silver nanoparticles). Table 3. Effects of Aqueous leaf Extract of A. muricata and its fabri- cated silver nanoparticles on A. cepa root growth. Groups Root length (cm) Mean ±SD 1 (Distilled water only (Negative Control)) 1.60±0.80 a 2 (100 µg/ml cyclophosphamide (Positive)) 1.42±0.50 a 3 (0.17 µg/ml AgNO3) 0 4 (0.7 µg/ml Am-AgNPs) 1.57±0.71 a 5 (7.0 µg/ml Am-AgNPs) 1.26±0.64 b 6 (70.0 µg/ml Am-AgNPs) 0.16±0.07 b 7 (100 µg/ml cyclophosphamide + 0.7 µg/ml A.m- AgNPs ) 1.58±0.64 a 8 (100 µg/ml cyclophosphamide + 7.0 µg/ml A.m- AgNPs) 0.67±0.32 b 9 (100 µg/ml cyclophosphamide + 70.0 µg/ml A.m- AgNPs) 0.27±0.13 b 10 (0.7 µg/ml Am-E) 1.63±0.71 a 11 (7.0 µg/ml Am-E) 1.79±0.98 a 12 (70.0 µg/ml Am-E) 2.48±1.14 b 13 (100 µg/ml cyclophosphamide. + 0.7 µg/ml Am-E) 1.55±0.68 a 14 (100 µg/ml cyclophosphamide. + 7.0 µg/ml Am-E) 1.75±0.74 a 15 (100 µg/ml cyclophosphamide. + 70.0 µg/ml Am-E) 1.61±0.73 a 16 (100 µg/ml of cyclophosphamide. + 0.71 µg/ml AgNO3) 0 Data are presented as Mean ± SD of triplicate experiment. Mean ±SD with different superscript are significantly different at P<0.05. AgNO3: Silver Nitrate, A.m-E: Annona muricata Extract, Am- AgNPs: Annona muricata- Silver Nanoparticles, Positive control: Cyclophosphamide, Negative control: Distilled water, SD: Standard Deviation. 7Anti-haemolytic and cytogenotoxic potential of aqueous leaf extract of Annona muricata for both the 70.0 µg/ml Am-E treated group and in com- bination with cyclophosphamide. Higher mitotic inhibi- tion values were observed in Am-E, cyclophosphamide + Am-E and cyclophosphamide + AgNO3 treated groups relative to the other groups. The mitotic inhibition val- ues were lower in the groups treated with 0.7 µg/ml of AgNPs and AgNPs + cyclophosphamide relative to the group treated with cyclophosphamide only. The high- est percent proportion of prophase was observed in the negative control group whereas the least was observed in the group treated with 70.0 µg/ml Am-E alone and when combined with cyclophosphamide. The 7.0 µg/ml Am-E + cyclophosphamide treated group had higher percentage proportion of prophase compared to the neg- ative control. A reduction was observed in the percent- age of metaphase in all the treated groups relative to the negative control group. Anaphase stage was higher in the group treated with 0.7 µg/ml AgNPs relative to the other groups and the control. Telophase in the 0.7 µg/ ml AgNPs + cyclophosphamide and Am-E only treat- ment groups was higher than the control and the other groups, whereas it was higher in the control group than the other treated groups. At 72 h, reduction in the total number of dividing cells of the treatment groups relative to negative con- trol was observed. Cumulative numbers of dividing cells were lower in the 0.7 µg/ml Am-E treated group than half of the positive and negative control. The MI values at 72 h and 48 h were found to be significantly reduced in the treated groups relative to the negative control group. The reduction in mitotic index values was con- centration dependent except in the 0.7 µg/ml Am-E and Table 4. Cytogenotoxic Effect of A. muricata Extract-Mediated Silver Nanoparticles on Allium cepa roots meristerimatic cells at 48 h com- pared with control (positive and negative) Conc (µg ml-1) No of Dividing Cells Mitotic Index (%) Mitotic Inhibition (%) Prophase Metaphase Anaphase Telophase CM SB CB VC F No. of A/D % Aberrant per cell scored Control 412 8.24 - 227 106 35 44 - - - - - - - Cyclo 100 288 5.76 30.10 136 50 35 36 1 16 14 - - 0.11 0.62 AgNO3 0.17 - - - - - - - - - - - - - - Am-AgNPs 0.7 303 6.06 26.46 134 47 37 31 - 38 16 - - 0.18 1.08 7 285 5.70 30.83 127 39 33 38 - 30 18 - - 0.17 0.96 70 - - - - - - - - - - - - - - Cyclo + Am-AgNPs 100 + 0.7 315 6.30 23.54 125 74 32 47 - 27 10 - - 0.12 0.74 100 + 7 283 5.66 31.31 147 63 12 16 - 36 9 - - 0.16 0.90 100 + 70 - - - - - - - - - - - - - - Am-E 0.7 267 5.34 35.19 144 38 33 46 - 1 5 - - 0.02 0.12 7 249 4.98 39.56 132 46 27 36 - 1 7 - - 0.03 0.16 70 224 4.48 45.63 105 43 35 37 - 2 2 - - 0.02 0.08 Cyclo + Am-E 100 + 0.7 271 5.42 34.22 135 56 27 30 - 16 7 - - 0.08 0.46 100 + 7 246 4.92 40.29 121 57 21 35 1 11 - - - 0.05 0.24 100 + 70 199 3.98 51.70 105 36 15 37 - - 6 - - 0.03 0.12 Cyclo + AgNO3 100 + 170 - - - - - - - - - - - - - - Cyclo: Cyclophosphamide, Conc: Concentration, CM: C-mitosis, SC: sticky chromosome, CB: chromosome bridge, VC: vagrant chromosome, F: fragmentation, No. of A/D: number of aberration per dividing cell, Positive control: Cyclophosphamide, Negative control: Distilled water. 8 Jelili A. Badmus et al. Table 5. Cytogenotoxic Eff ect of A. muricata Extract-Mediated Silver Nanoparticles on Allium cepa root meristerimatic cells at 72 h com- pared with control (positive and negative) Concentration (µg ml-1) No of Dividing Cells Mitotic Index (%) Mitotic Inhibition (%) Prophase Metaphase Anaphase Telophase CM SB CB VC F No. of A/D % Aberrant per cell scored Control 244 4.88 - 144 46 17 37 - - - - - - - Cyclo 100 207 4.14 15.16 112 35 19 32 - 5 4 - - 0.04 0.18 AgNO3 0.17 - - - - - - - - - - - - - Am-AgNPs 0.7 216 4.32 11.48 121 43 11 29 - 1 11 - - 0.06 0.24 7 142 2.84 41.80 75 29 3 31 - - 4 - - 0.03 0.08 70 - - - - - - - - - - - - - - Cyclo + Am-AgNPs 100 + 0.7 198 3.96 18.85 100 39 9 34 1 4 11 - - 0.08 0.32 100 + 7 167 3.34 31.56 78 37 9 16 - 19 8 - - 0.16 0.54 100 + 70 - - - - - - - - - - - - - - Am-E 0.7 83 1.66 65.98 45 22 9 6 - - 1 - - 0.01 0.02 7 200 4.00 18.03 104 36 17 38 1 - 4 - - 0.03 0.10 70 175 3.50 28.28 95 20 9 43 - 2 6 - - 0.05 0.16 Cyclo + Am-E 100 + 0.7 213 4.26 12.70 121 29 24 37 - - 2 - - 0.01 0.04 100 + 7 229 4.58 6.15 157 18 20 33 - - 1 - - 0.00 0.02 100 + 70 - - - - - - - - - - - - - Cyclo + AgNO3 100 + 170 - - - - - - - - - - - - - - Cyclo: Cyclophosphamide, Conc: Concentration, CM: C-mitosis, SC: sticky chromosome, CB: chromosome bridge, VC: vagrant chromosome, F: fragmentation, No. of A/D: number of aberration per dividing cell, Positive control: Cyclophosphamide, Negative control: Distilled water. Figure 1. Representative photomicrographs of normal stages of mitotic cell divisions in treated Allium cepa root cells and observed chromo- somal aberrations. 9Anti-haemolytic and cytogenotoxic potential of aqueous leaf extract of Annona muricata Am-E+ cyclophospahamide treatment groups where lower values were observed. Complete cell growth arrest was demonstrated in 70.0 µg/ml Am-AgNPs alone, 70.0 µg/ml Am-AgNPs + Cyclo and AgNO3 solution treated groups at 48 h. Chromosomal aberrations, including chromosome- bridge, sticky chromosome, and c-mitosis were observed in different degrees in all the treated groups with chro- mosome-bridge and sticky chromosome being promi- nent in most of the treated groups (Figure 1). 4. DISCUSSION 4.1 Biosynthesis and Characterization of Silver Nanoparti- cles A change in colour of silver nitrate solution in the presence of the aqueous leaf extract of Annona muricata from colourless to brown that confirms the synthesis of Am-AgNPs nanoparticles has been previously reported (Badmus et al. 2020). Physicochemical characteriza- tion results of Am-AgNPs similar to the previous study (Badmus et al. 2020) indicated that the nanoparticles absorbed maximally at 420 nm and FTIR showed that the synthesized silver nanoparticles was possible because of amide and hydroxyl groups of the aqueous leaf extract. Zeta potential of the nanoparticles was -27.2 mV, DLS indicated 86.8 nm size with polydispersity index of 0.329 and XRD/SAED presented crystalline nature of the nanoparticles with face centre cubic (FCC) phase (Santhosh et al. 2015; Gavamukulya et al. 2020; Badmus et al. 2020) 4.2 Anti-haemolytic Effects of Aqueous leaf Extract of A. muricata and its fabricated silver nanoparticles The erythrocyte model for assessing the anti-haemo- lytic activity of test compounds can reveal the toxicity of an agent and can serve as an indicator of membrane toxicity (Zohra and Fawzia, 2014). Blood cells are eas- ily isolated and the test method using the blood cell can mimic other cell membrane (Farag and Alagawany, 2018). The haemolytic ability of the test compound is proportional to the concentration, chemical constitu- ent and potency of the compound (Zohra and Fawzia, 2014). In this study, the Am-E and Am-AgNPs demon- strated a concentration dependent anti-haemolytic activ- ity. The extract showed a better protection against H2O2- induced haemolysis of the red cell membrane compared to Am-AgNPs. This implies that Am-AgNPs is best used at lower concentration because a high concentration as used in this study is toxic to RBC membrane (Raja et al. 2016; Hamouda et al. 2019). Anti-haemolytic activ- ity of plant extract has been shown to be related to the constituent antioxidant agents such as polyphenolic compound (Ramchoun et al. 2015; Karim et. 2020). The bioactive compounds of the plant are responsible for the synthesis of the nanoparticles and also confer the bio- medical property such as anti-haemolytic on the synthe- sized nanoparticles (Kuppusamy et al. 2016; Badmus et al. 2020). The toxic influence of RBC membrane at high concentration by Am-AgNPs could be attributed to the Ag component of the nanoparticles as earlier reported by Choi et al. (2011) and Hamouda et al. (2019) to cause the death of red blood cells, even at low concentration. Clinical outcome of haemolysis can cause anaemia and contribute to blood coagulation abnormalities. However, nanoparticles including sliver have been shown to pro- tect blood against coagulation (Lateef et al. 2018; Eleg- bede and Lateef, 2019; Azeez et al. 2020). These activities are indicative of biomedical applications of nanoparticles in blood disorder. 4.3 Effects of Aqueous leaf Extract of A. muricata and its fabricated silver nanoparticles and Cyclophosphamide on root length Macroscopic evaluation helps to determine the root sprouting or root growth inhibition effect exerted by the test solution on the onion roots while microscopic evaluation helps to study the harmful qualitative and quantitative effect (cytotoxic effect on onion meristem cells) (Yekeen et al. 2017a). Observations of A. cepa root growth inhibition after 48 and 72 h were used as indi- cator of the cytotoxic nature of Am-AgNPs and Am-E. The extract did not show inhibition of root length, but did protect the roots from cyclophosphamide-induced root length reduction. The protective effect of Am-E on the root growth inhibition imposed by cyclophospha- mide and increased mean root length when Am-E was used alone may be credited to the ability of the extract to induce root sprouting (Yekeen et al. 2017a). Contra- rily, the Am-AgNPs reduced the root length in a concen- tration dependent manner attesting to its mitodepressive capability. The reduction of root length was augmented in the presence of both Am-AgNPs and cyclophos- phamide at both 48 and 72 h. Root growth inhibition observed in this experiment at both 48 and 72 h expo- sure to cyclophosphamide together with Am-AgNPs and Am-AgNPs alone is an indication of the genotoxic- ity nature which can be linked to the presence of heavy metals constituent (Yekeen et al. 2017a). Stunted growth, hardness, and colouration of the roots observed at 72 h 10 Jelili A. Badmus et al. exposure in addition to the aforementioned observations confirm the mitodepressive effect on the onion roots meristem cells. 4.4 Cytogenotoxic Effect of Aqueous leaf Extract of A. muricata and its fabricated silver nanoparticles and Cyclo- phosphamide The study of chromosome behavior during cell divi- sion in order to establish health safety status of a given compound has been the focus of Scientists employing A. cepa test. A. cepa assay is widely used to study nor- mal and the abnormal chromosome response when the onions base is suspended in a test solution. The assay reveals the effect of a test substance at a minute level of interaction with genetic material, which makes it a robust tool for effective assessment of genotoxic com- pounds (Bonciu et al. 2018). It is reproducible, sensitive, fast, cheap, and effective in monitoring genetic materi- als response on exposure to environmental pollution and mutagenic compounds (Badmus et al. 2013; Bhat et al. 2017). Prophase stage of cell division dominated the other cell division stages in all the treated and control groups. An increase in prophase number compared to other stages of cell division has been related to delay in the breaking down of its nuclear membrane (Pankaj et al. 2014). Cell division at the root tip of the onion mer- istematic region was assessed using mitotic index (Bad- mus et al. 2013). The reduction in the mitotic index values of the treated groups compared with the nega- tive control revealed the cytotoxicity potential of cyclo- phosphamide, Am-AgNPs and Am-E at both 48 and 72 h. The reduction of MI by any agent compared with the untreated control is known to relate to cytotoxicity of the tested compound (Asita and Matebest, 2010; Yekeen et al. 2017a). The depression of MI could be linked to the inhibition of DNA synthesis due to the blockage of G2 phase of the cell cycle, which prevents the cell from entering M-phase during the cell cycle (Badmus et al. 2013; Obute et al. 2016; Yekeen et al. 2017a). Inhibition of mitotic activities is employed for tracing cytotoxic substance (Singh and Roy, 2016). As earlier reported, MI reduction might be as a result of the adverse effects of the extract and Am-AgNPs on the microtubule (Yekeen et al. 2017a). This was corroborated by the total cell arrest obtained when A. cepa was treated with AgNO3 and the highest concentration of AgNPs with or with- out cyclophosphamide. However, the ability of AgNPs to induce cell arrest could be an indication of its capability as an agent of antiproliferation against uncontrolled cell division in cancer cell (Chukwujekwu and Van Staden, 2014). In addition, reduction of mitotic activity in this study could be because of impaired synthesis of nucleo- protein coupled with low level of ATP to power spindle elongation, movement of chromosome and microtubule dynamics (Yekeen et al. 2017a). The structural changes of chromosome due to an exchange or a break of chromosomal materials are termed chromosome aberration (Preston, 2014). Chro- mosome aberration (CA) could be as a result of improp- er or unrepair oxidation of DNA deoxyribose sugar and a nitrogenous base leading to the breaking of the dou- ble strand (Badmus et al. 2013). CA observed in cells could be either lethal or viable and can induce somatic or inherited genetic effects (Chang-Hui, 2019). Vari- ous chromosomal abnormalities such as chromosome- bridge, sticky chromosome, and c-mitosis were observed in different degrees in all the treatment groups with chromosome-bridge and sticky chromosome being prominent in most of the treatment groups (Figure 1). Kuchy et al. (2016) reported that bridge formation could be linked to chromosome breaks, stickiness, or a reun- ion of already broken ends of chromosomes. Olorunfe- mi et al. (2012) reported that sticky chromosome effect is irreversible and ultimately result in cell death. There- fore, total growth inhibition observed with AgNO3 and the highest concentration of Am-AgNPs with or without cyclophosphamide treatment may be due to sticky chro- mosome formation. The total root growth inhibition as observed in AgNO3 treated group shows that the pres- ence of Ag in Am-AgNPs is responsible for root inhi- bition and chromosomal aberration observed in Am- AgNPs treated groups. 5. CONCLUSION The green fabricated NPs using plants have been shown by several studies to have robust biomedical applications. Their actions have been linked to increase surface area due to the reduced size. This study estab- lished the anti-haemolytic activity of Am-E and Am- AgNPs. Am-E demonstrated a better anti-haemolytic activity relative to Am-AgNPs at tested concentrations suggesting the toxic potential of biosynthesized AgNPs to RBC at high concentration. The cytotoxicity of Am- AgNPs was revealed through reduction of MI value and increased root growth inhibition of the treatments, sug- gesting the possibility of employing the biogenic parti- cles as anti-proliferative agent in cancer study. Induction of CA observed at both 48 and 72 h in this study shows the genotoxic potential of both Am-E and Am-AgNPs. While considering the possible influence of Am-AgNPs in disease therapy, its cytogenotoxic potential should 11Anti-haemolytic and cytogenotoxic potential of aqueous leaf extract of Annona muricata be robustly evaluated before its exposure to human. In addition, there should be restrain in disposing any syn- thesized nanoparticles into the environment because their toxicity could be far reaching at high concentra- tion. 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