Bangladesh Journal of Pharmacology Volume: 15; Number 1; Year 2020 Cite this article as: Banerjee A, Firdous SM. In vitro antimutagenic activity of Ipomoea staphylina. Bangla- desh J Pharmacol. 2020; 15: 41-43. In vitro antimutagenic activity of Ipomoea staphylina Sir, The study of antimutagenic activity gives information which discovers the possibilities for combating the genotoxic risk of mutagens. Genotoxicity includes DNA damage, gene mutation, chromosomal aberration, and formation of micronucleus (Swift, Golsteyn, 2014). Genotoxicity commonly occurs with anti-cancer drugs which causes a high level of DNA damage that activa- tes cell cycle checkpoints leading to cell cycle arrest or cell death (Helleday et al., 2008). Several studies on medicinal plants have revealed antimutagenic and antigenotoxic properties mainly due to antioxidants that scavenge reactive oxygen species (ROS) (Zani et al., 1993; González-Avila et al., 2003; Park et al., 2004). Ipomoea staphylina leaves extract possesses mainly anti- inflammatory (Firdous and Koneri, 2013), antiulcer (Banerjee and Firdous, 2015), anti-diabetic (Firdous and Singh, 2016), hepatoprotective, nephroprotective (Bag and Mumtaz, 2013), cytotoxic (Padmashree et al., 2018a) and antibacterial (Padmashree et al., 2018b) activities. There is no report on the antimutagenic activity of this plant. Hence, an attempt was taken to evaluate the antimutagenic property of the hydroalcoholic extract of I. staphylina leaves. The leaves were collected from the forest area in Karnataka and taxonomically authenticated by Dr. K Karthigeyan of Central National Herbarium, Botanical Garden, Howrah. A voucher specimen was conserved with a reference No. SMF-01. The dried leaves were powdered and defatted with petroleum ether (bp 60-80°C) for 72 hours and then extracted with a mixture of ethanol : distilled water (7:3) to get a yield of 11.6% w/w. The dried extract was then stored at 4°C. Ames test was performed using two nonvirulent strains of Salmonella typhi viz., TA 1535 and TA 1538 (Maron and Ames, 1983). Sodium azide and 2-nitrofluorene were used as standard mutagens at a concentration of 50 µg/mL. To determine the mutagenic effect of I. staphylina extract, 5 mg of the extract per plate was given along with both strains of S. Typhi differently. Besides, three different concentrations of the extract viz., 5, 10, and 20 mg/plate along with standard mutagen were exposed to both strains of S. typhi differently. After 48 hours of incubation number of bacterial colonies was counted. The mutagenicity of sodium azide and 2-nitrofluorene in absence of the extract was by the formula given below; %Inhibition = [1-T/M]×100 where T = number of revertants per plate in the presence of mutagen and extract and M = number of revertants per plate in the presence of mutagen only Allium cepa chromosomal aberration test (Ping et al., 2012) was carried out using onions (20-25 g) which were grown in reverse osmosis water for 3-4 days until the length of the roots reaches up to 1-1.5 cm. Then onions were exposed in different concentrations (250 and 500 µg/mL) of extract and cyclophosphamide (50 µg/mL) which was considered as a positive control. To deter- mine the antimutagenic property of extract, a few onions were exposed to cyclophosphamide (50 µg/mL) in the presence of extract (250 and 500 µg/mL). After 24 hours (at least one mitotic cycle) of chemical exposure roots of deferent onions were collected in glass vials containing fixative solution [methanol : glacial acetic acid (3:1)] and stored overnight at 4°C. On the next day, roots were taken into clear glass vials containing a mixture of 2% aceto-orcein and 1N HCl in a ratio of 5:1 and heated gently for 2-5 sec and kept for 1-1.5 hours. Then, a small drop of 45% acetic acid was added in a clear microscopic glass slide and root tip (about 1-2 mm) was placed onto the acetic acid and left for few sec for washing the excess stain. A coverslip was mounted on it in such a way that no air bubble was formed and the root tip was squashed a little with a gentle tap of thumb or a match stick. At last the open interface of coverslip and slide was sealed with dibutylphthalate polystyrene xylene to prevent the drying of cells. Three slides were made and analyzed for each treatment group for the determination of mitotic index and various chromosomal abnormalities. In mitotic index about 1,000 cells were counted and the mitotic index was calculated as given below; [(number of dividing cells/total number of cells) × 100] The Ames test was performed without metabolic activation (addition of S9 mix) and the numbers of revertant colonies per plate were counted (Table I). In this study, sodium azide and 2-nitrofluorene (50 µg/ plate) were been able to cause gene mutation in histidine operon of Salmonella strains TA 1535, and TA A Journal of the Bangladesh Pharmacological Society (BDPS) Bangladesh J Pharmacol 2020; 15: 41-43 Journal homepage: www.banglajol.info Abstracted/indexed in Academic Search Complete, Agroforestry Abstracts, Asia Journals Online, Bangladesh Journals Online, Biological Abstracts, BIOSIS Previews, CAB Abstracts, Current Abstracts, Directory of Open Access Journals, EMBASE/Excerpta Medica, Global Health, Google Scholar, HINARI (WHO), International Pharmaceutical Abstracts, Open J-gate, Science Citation Index Expanded, SCOPUS and Social Sciences Citation Index ISSN: 1991-0088; DOI: 10.3329/bjp.v15i1.43446 Letter to the Editor This work is licensed under a Creative Commons Attribution 4.0 International License. You are free to copy, distribute and perform the work. You must attribute the work in the manner specified by the author or licensor https://www.ncbi.nlm.nih.gov/pubmed/?term=Swift%20LH%5BAuthor%5D&cauthor=true&cauthor_uid=24573252 https://www.ncbi.nlm.nih.gov/pubmed/?term=Golsteyn%20RM%5BAuthor%5D&cauthor=true&cauthor_uid=24573252 1538 respectively, that was confirmed by colony counter. The number of colonies in the extract (5 mg/ plate) treated group was almost same to that of control groups of both the strains. From this study, it was clear that extract does not possess any mutagenic nature. When extract was given along with mutagen (sodium azide), the decrease in the number of colonies was not found in TA 1535. But extract in TA 1538 was found effective to reduce the number of revertant colonies in the presence of 2-nitrofluorene. The extract produced 79.8% protection at the dose of 20 mg per plate. Thus, I. staphylina possesses antimutagenic activity against 2- nitrofluorene induced base-pair mutation. Besides, the extract (500 µg/mL) causes a considerable decrease in the mitotic index when compared to the control group (Table II). Cyclophosphamide (50 µg/ mL) produced extensive chromosomal abnormalities. When the extract (250 µg/mL) was combined with cyclophosphamide (50 µg/mL), a slight decrease in chromosomal abnormalities was observed which was indicated by an increase in mitotic index. Spindle fiber disruption occurs due to plant alkaloids (Fasola and Egunyomi, 2005), and I. staphylina in the preliminary phytochemical study showed the presence of alkaloids. In conclusion, antimutagenic potential of I. staphylina is dose-dependent. Amartya Banerjee1 and Sayeed Mohammad Firdous2 1Krish Biotech Research Private Limited, T-1, QK-17, Part, WBIDC, Kalyani, Phase III, West Bengal 741235, India; 2Department of Pharmacology, Calcutta Institute of Pharmaceutical Technology and AHS, Uluberia, Howrah 711316, West Bengal, India. Corresponding author: email: firdous.cology@gmail.com References Bag AK, Mumtaz SMF. Hepatoprotective and nephroprotec- tive activity of hydroalcoholic extract of Ipomoea staphylina leaves. Bangladesh J Pharmacol. 2013; 8: 263-68. 42 Bangladesh J Pharmacol 2020; 15: 41-43 Table I Effect of I. staphylina on TA 1535 and TA 1538 along with mutagens Treatment Treatment Number of colonies (mean ± SD) %Inhibition TA 1535 control 27.7 ± 1.0 - TA 1535 (sodium azide 5 µg/plate) 776.3 ± 13.9 - TA 1535 (HEIS 5 mg/plate) 26.3 ± 0.9 - TA 1535 (sodium azide 5 µg/plate) Extract (5 mg/plate) 803.3 ± 12.5 -3.5 TA 1535 (sodium azide 5 µg/plate) Extract (10 mg/plate) 788.7 ± 16.4 -1.6 TA 1535 (sodium azide 5 µg/plate) Extract (20 mg/plate) 792.7 ± 10.2 -2.1 TA 1538 control 10.3 ± 0.7 - TA 1538 (2-nitrofluorene 5 µg/plate) 461.0 ± 7.4 - TA 1538 Extract (5 mg/plate) 12.3 ± 0.5 - TA 1538 (2-nitrofluorene 5 µg/plate) Extract (5 mg/plate) 298.7 ± 11.5 35.2 TA 1538 (2-nitrofluorene 5 µg/plate) Extract (10 mg/plate) 180.5 ± 8.7 60.8 TA 1538 (2-nitrofluorene 5 µg/plate) Extract (20 mg/plate) 93.3 ± 4.6 79.8 Data are mean ± SD; Experiment was performed in triplicate Table II Effect of I. staphylina on mitotic index in presence of mutagen in onion Treatment Dividing cells (mean) Non-dividing cells (mean) Interphase Mitotic index (mean ± SD) Prophase Metaphase Anaphase Telophase Water (control) 35 13 7 20 925 8.1 ± 1.0 Extract (250 µg/mL) 30 13 6 21 930 7.5 ± 0.8 Extract (500 µg/mL) 16 6 2 7 969 3.2 ± 0.4 Cyclophosphamide (50 µg/mL) 7 3 2 2 986 1.4 ± 0.3 Cyclophosphamide (50 µg/mL) plus extract (250 µg/mL) 10 5 3 3 979 2.1 ± 0.5 Cyclophosphamide (50 µg/mL) plus extract (500 µg/mL) 8 4 2 2 984 1.6 ± 0.3 Experiment was performed in triplicate Banerjee A. 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