Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 73(1): 115-123, 2020 Firenze University Press www.fupress.com/caryologiaCaryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/caryologia-136 Citation: H. Ahmad Ganaie, Md. N. Ali, B.A. Ganai (2020) Melissa officinalis: A potent herb against EMS induced mutagenicity in mice. Caryologia 73(1): 115-123. doi: 10.13128/caryologia-136 Received: January 9, 2019 Accepted: February 23, 2020 Published: May 8, 2020 Copyright: © 2020 H. Ahmad Ganaie, Md. N. Ali, B.A. Ganai. This is an open access, peer-reviewed article pub- lished by Firenze University Press (http://www.fupress.com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distri- bution, and reproduction in any medi- um, 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. Melissa officinalis: A potent herb against EMS induced mutagenicity in mice Hilal Ahmad Ganaie1,2,*, Md. Niamat Ali1, Bashir A Ganai2 1 Cytogenetics and Molecular Biology Research Laboratory, Centre of Research for Devel- opment (CORD), University of Kashmir, Srinagar-190 006, J & K, India 2 Phytochemistry Research Laboratory, Centre of Research for Development (CORD), Uni- versity of Kashmir, Srinagar-190 006, J & K, India *Corresponding author. E-mail: hilalganie@hotmail.com Abstract. Melissa officinalis (L) is used traditionally for different medical purposes such as tonic, antispasmodic, carminative, diaphoretic, surgical dressing for wounds, sedative hypnotic, strengthening the memory and relief of stress induced headache. The methanolic extractof Melissa officinalis (Mo-ME) was investigated for antimuta- genic activity. The extraction was done by Soxhlet extraction method and the extract was evaluated for antimutagenic assay against EMS induced mice by micronucleus and chromosomal aberration assay. Briefly, mice were treated with methanolic extract of Melissa officinalis (Mo-ME) (100, 200 300 & 400 mg/kgbw) for 15 days. Without the doses of EMS, no and mutagenic effects were observed in blood and bone mar- row samples of the mice. Micronucleus and chromosomal aberration test revealed the protective effects of Mo-ME when administered at high doses.The reduction profiles in the MN induction of methanolic extract of Melissa officinalis at the concentration (100, 200, 300 and 400 mg/kgbw) with EMS were estimated as 14.5%, 28.0%, 47.7% and 81.5% respectively. The methanolic extract of Melissa officinalis exhibited no cytotoxic and mutagenic effects but only have antimutagenic effects, an effect that can be attrib- uted the presence of majorital compounds, and the antimutagenic property of Mo-ME is an indication of its medicinal relevance. Keywords. Melissa officinalis, GC-MS, EMS, mice, micronucleus test, chromosomal aberration, antimutagenicity. INTRODUCTION Medicinal plants with antioxidant and antimicrobial properties are gain- ing a lot of attention as these properties are commonly assumed to play an important role in preventing diseases caused by oxidative stress, such as cancer, coronary arteriosclerosis and the ageing processes (Haraguchi et al. 2009). Derived forms of medicinal plants (extracts, syrups, etc.) have been the basis of medical therapy for centuries. Traditionally used in the treatment of several human disorders, their pharmacological and therapeutic proper- ties are attributed to various chemical constituents isolated from their crude extracts (Pereira et al. 2009, Kwak and Ju, 2015; Liu et al. 2015). Notwith- 116 Hilal Ahmad Ganaie, Md. Niamat Ali, Bashir A Ganai standing, their correct use requires the manipulation of plants selected for their efficacy and safety, based either on folk tradition or scientific validation (Tovart, 2009). The use of herbal infusions to cure various disorders is very common in folk medicine especially to those who live in upper reaches of Kashmir Himalayas (Dutt et al. 2015). Although the diversity of plant species in Kash- mir Himalayas is a potential source of biologically active compounds, the effects on human health and genetic material are often unknown. There are indications that the protective action on genetic material can lead, not only to its repair, but also the preservation of its integrity (Berhow et al. 2000; Fernandes and Vargas, 2003; Souza et al. 2004). Not all are harmless, some even presenting toxic and mutagenic substances in their phytochemical composition (Bresolin and Vargas, 1993; Sa-Ferreira and Vargas, 1999). Interest in such popular usage has recently gained strength, through recent knowledge that chemi- cals, such as proteases and antioxidants may prevent or reduce the development of cancer by blocking genetic damage (Hernandez-Ceruelos et al. 2002). Melissa officinalis belongs to Lamiaceae family, a large group of medicinal plants. M. officinalis is native to southern Europe and northern Africa; although, over the last several centuries it has been successfully cul- tivated all over the world. Today it can be found grow- ing wildly throughout North America, Europe, Asia, and in the Mediterranean. The leaves of M. officinalis have been used in folk medicine especially in Turkey and Iran, for the treatment of some disease (Sadraei et al., 2003). Also, the leaves of M. officinalis are often used as herbal teas. M. officinalis contains some phenolic and flavonoid compounds such as rosmarinic acid (Herodez et al., 2003). The phenolic contents in plants have some antioxidant properties (Chen et al., 2001). Essential oils and extracts of this plant have been reported to have antiviral (Schnitzler et al., 2008), antimicrobial and anti- oxidant properties (Dastmalchi et al., 2008). As little has been done on the antimutagenicity of Melissa officinalis, therefore, the purpose of this study was to determine the antimutagenic activities of methanolic extract of Melissa officinalis. MATERIAL AND METHODS Collection and air drying of plant material Aerial parts of M. officinalis were collected from Bandzoo area of Pulwama from the garden of IIIM, Srinagar and from SKUAST-K in the month July, 2013. The plant was identified at the Centre of Biodiversity and Plant Taxonomy, Department of Botany, Univer- sity of Kashmir, Srinagar, J&K and a voucher specimen (JKASH/CBT/227 Dated 08. 08. 2014) was deposited there. The parts were allowed to dry under shade (30 °C) for 8-10 days. Preparation of extracts After shade drying, the aerial parts were macer- ated to fine powder, 1 kg of leaves were extracted suc- cessively with hexane for defatenning and methanol for 16 h using Soxhlet apparatus. The extracts were filtered through a Buchner funnel using Whatman No. 1 filter paper, and all the extracts were concentrated to dry- ness under vacuum using a Heidolph rotary evaporator, yielding hexane, and methanol crude extracts of 65 and 48g respectively. All the extracts were stored at 4°C in air tight glass bottles before use. GC-MS analysis GC-MS analysis was carried out with GCMS-QP2010 Plus, Shimadzu, Japan fitted with programmable head space auto sampler and auto injector. The capillary col- umn used was DB-1/RTX-MS (30 metre) with helium as a carrier gas, at a flow rate of 3 mL/min with 1 µL injec- tion volume. Samples were analysed with the column held initially at 100°C for 2 min after injection, then increased to 170°C with 10°C/min heating ramp without hold and increased to 215°C with 5°C/min heating ramp for 8 min. Then the final temperature was increased to 240°C with 10°C/min heating ramp for 15 min. The injections were performed in split mode (30: 1) at 250°C. Detector and injector temperatures were 260°C and 250°C, respectively. Pressure was established as 76.2 kPa and the sample was run for 70 min. Temperature and nominal initial flow for flame ionization detector (FID) were set as 230 °C and 3.1 mL/min, correspondingly. MS parameters were as follows: scan range (m/z): 40-650 atomic mass units (AMU) under the electron impact (EI) ionization (70 eV). The constitu- ent compounds were determined by comparing their retention times and mass weights with those of authentic samples obtained by GC and as well as the mass spectra from the Wiley libraries and National Institute of Stand- ards and Technology (NIST) database. Experimental Animals Both sex of albino mice, Balb/c strain useful for research in cancer and immunology, age of 6 weeks, weigh- ing 25-35 g were obtained from the Indian Institute of Inte- 117Melissa officinalis: A potent herb against EMS induced mutagenicity in mice grative Medicine (IIM), Canal Road Jammu-India, kept in plastic cages in an animal room under controlled condi- tions of temperature (22 ± 2°C), humidity (55 ± 10%), 12 h light/dark cycles and access to food and water. They were randomized at the beginning of the experiment.The study design was approved by the Institutional Animal Ethical Committee, and the experiments undertaken in accordance with the ethical principles of the CPCSEA norms. Treatment protocol The mice were divided into 8 groups, with 5 animals per group. Ethyl methane sulfonate (EMS, SigmaAldrich) was used to induce mutations. Just before use, the EMS was diluted in 0.9% NaCl. The exposure route was by gavage(1/4thof LD50 of EMS; 117.5 mg/kgbw). Evaluation of either DNA damage or protection by the methanolic extracts of Melissa officinalis was according to protocol developed by Azevedo et al. (2003), with the some adap- tations. The mice in group 1 received only distilled water (10 mL/kg bw. per day by gavage) for 2 weeks and acted as negative control (Table 1). Mice in group 2 were exposed to EMS (1/4thof LD 50) for 24 h and this group acted as positive control. Group 3 and 4 were given different dos- es (100 & 400 mg/kgbw) of the extract to see the cyto- toxic and mutagenic potential of M. officinalis and served as positive control of plant extracts. Group 5, 6, 7 and 8 were treated with dose of 100, 200, 300 and 400 mg/kgbw respectively for 15 days after treatment with EMS. The mice were killed by cervical dislocation on 16thday for evaluation of micronucleus and chromosomal aberrations. The micronucleus test The method of MacGregor et al. (1987) was used for micronucleus test. Mice were sacrificed by cervical dis- location. Slides were prepared with blood collected from the jugular vein. The slides were air-dried, fixed in abso- lute methanol, stained in 10% Giemsa and then coded for blind analysis. One thousand polychromatic erythro- cytes (PCE) were analysed per mouse. The proportion of PCE and normochromatic erythrocytes (NCE) in 1000 erythrocytes/group was calculated, to detect possible cytotoxic effects. The slides were scored blindly, using a light microscope with a 45x and 65x objectives. Photog- raphy was done using 100x immersion objective. Chromosomal aberration Mice were injected intraperitoneal with 0.5 ml of 0.06% colchicine and two hours later, were sacrificed by cervical dislocation. Both the femurs were fleshed out from the muscles and kept in HBSS (Hank ’s balanced salt solution). The femurs were then rinsed with 3 ml 0.056% KCl solution in a centrifuge tube. The tube was then incubated at 37oC for 20 minutes. After incubation, centrifugation at 800 rpm for 4 minutes was carried out. Supernatant was discarded and fresh Carnoy’s fixa- tive was added (3:1 methanol: acetic acid). The process of centrifugation was repeated three times. Then slides were prepared, stained with 4% Giemsa, air dried and studied under compound microscope. Statistical analysis Variable normality was assessed using the Kolmogo- rov-Smirnov test. Micronucleus testing and chromosom- al aberration involved multiple pair-wise comparison between experimental groups and positive and negative controls, with the Mann Whitney U test at a significance level of <0.05. Lower the Mann Whitney statistic value and Z score value, higher the difference. Table 1. Grouping, dose (distilled water, EMS and Ab-ME in concentrations of 100, 200, 300 and 400 mg/kg bw) and duration of experi- ment. Group Dose Purpose of group Duration Group 1 Distilled water Negative control 15 days Group 2 1/4th LD50 EMS Positive control EMS 24 h Group 3 Mo-ME 100 mg/kg bw Positive control Melissa officinalis 24 h Group 4 Mo-ME 400 mg/kg bw Positive control Melissa officinalis 24 h Group 5 Mo-ME 100 mg/kg bw + EMS Treated Group 15 days Group 6 Mo-ME 200 mg/kg bw + EMS Treated Group 15 days Group 7 Mo-ME 300 mg/kg bw + EMS Treated Group 15 days Group 8 Mo-ME 400 mg/kg bw + EMS Treated Group 15 days Mo-ME = Methanolic extract of Melissa officinalis. 118 Hilal Ahmad Ganaie, Md. Niamat Ali, Bashir A Ganai RESULTS GC-MS analysis In order to find out the phytocomponents of Melis- sa officinalis, the methanolic extract was subjected to GC-MS analysis. The active principals present in the methanolic fraction of Melissa officinalis along with their retention time (RT), molecular formula, molecu- lar weight (MW) and peak area (%) are presented in Table 2. The chromatograms of methanolic extract of Melissa officinalis (Mo-ME) showed three major peaks (Fig. 1): 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4H- pyran-4-one (51.62%), 5-(hydroxymethyl)-2-furan car- boxaldehyde (29.46%), hexadecanoic acid, methyl ester (8.24%), constituting 89.32% of the total peak area. The minor fractions of Mo-ME include octadecanoic acid (3.26%), stigmast-5-en-3-ol (1.44%), tetradecanoic acid (1.43%), 2, 4- cresotaldehyde (1.17), comprising 7.30% of the total peak area. The phytoconstituents identified in the methanolic fraction of Melissa officinalis along with their retention time (RT), molecular formula, molecular weight (MW) and peak area (%) are present- ed in Table 3.6. Micronucleus test According to MN testing of mouse blood cells the low frequencies of micronucleated cells presumes the too little effects of methanolic extract of Melissa officinalis (Mo-ME) 100 and 400mg/kg (Table 3, Fig. 2), thereby indicating the virtual absence of mutagenic or cytotoxic effects. In other words, no statistically significant differ- ence in the frequency of MN polychromatic erythrocytes (PCE) or the ratio of PCE to normochromatic eryth- rocytes (NCE), between the negative control and the groups that ingested extracts could be detected. When evaluating antimutagenicity inMo-ME, a significant decrease in the frequency of EMS-induced MNPCE was observed only in mice that had received 100, 200, 300 and 400 mg/kg ofMo-ME (p = 0.05 –Mann Whitney U test). In the present study, the methanolic extract of M. officinalis showed antimutagenic activities by reducing the % age of micronuclei with increase in the dose of the extract (Fig. 3). Table 2. Phytocomponents identified in the methanolic extract of Melissa officinalis (Mo-ME) by GC-MS. S. No. Compound RT % Area MF MW 1 2,4-Cresotaldehyde 18.52 1.17 C8H8O2 136 2 D-allose 19.41 0.56 C6H12O6 180 3 Dodecanoic acid 21.32 0.58 C12H24O2 200 4 Tetradecanoic acid 25.79 1.43 C14H28O2 228 5 2,6,10-Trimethyl,14-ethylene-14-pentadecne 27.37 0.49 C20H38 278 6 Hexadecanoic acid, methyl ester 29.13 8.24 C38H68O8 652 7 5- (hydroxymethyl)-2-furan carboxaldehyde 30.07 29.46 C6H6O3 126 8 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4H-pyran-4-one 33.47 51.62 C6H8O4 144 9 Octadecanoic acid 33.76 3.26 C18H36O2 284 10 Malonic acid, 3-hexyl tridecyl ester 34.05 0.38 C22H42O4 370 11 9-Octadecenoic acid 34.22 0.12 C18H34O2 282 12 9,12-Octadecadienoic acid 34.77 0.50 C18H32O2 280 13 1,2-Benzenedicarboxylic acid 40.36 0.34 C24H38O4 390 14 Tochopherol 51.97 0.41 C29H50O2 430 15 Stigmast-5-en-3-ol 57.19 1.44 C29H50O 414 Figure 1. GC-MS chromatogram of methanolic extract of Melissa officinalis. 119Melissa officinalis: A potent herb against EMS induced mutagenicity in mice Chromosomal aberrations The chromosomal aberrations induced by ethyl methanesulphonate (EMS 117.5 mg / kg body weight; positive control) were significant (p<0.05) to that of the control group. The frequency of breaks per cell in the EMS treated group at 24 h was 0.12 ± 0.010, which was significantly higher when compared to that of total number of breaks per cell in the control group (0.014 ± 0.002) (p<0.05). Our results also showed that in the Mo- ME and EMS combined treatment group, the frequency of chromosomal aberrations was significantly lower in comparison to those observed for the EMS only treated group at 24 h. The methanolic extract of Melissa offici- nalis reduced the chromosomal aberrations by 38.1%, 60.1%, 74.5% and 91.5% at 100, 200, 300 and 400 mg/ kgbw (Table 4, Fig. 4). The chromosomal aberrations induced by EMS are mainly chromatid breaks, exchang- es, gaps, fragments and rings (Fig. 5). DISCUSSION From ancient times, medicinal plants are being used as remedies for various diseases in human. In today’s industrialized society, the use of medicinal plants has been traced to the extraction and development of sev- eral drugs as they were used traditionally in folk medi- cine (Shrikumar and Ravi, 2007). Medicinal plants have potent phytoconstituents which are important source of compounds and are responsible for the therapeutic properties (Jeeva et al., 2011; Florence et al., 2014; Sum- athiet al., 2014, Ganaie et al, 2016; 2017). These phyto- constituents endow them with medicinal properties. Many plants possess antioxidant properties because of the presence of phenolic compounds (Brown and Rice- Table 3. Frequency profile of micronuclei induced alone by ethyl methanesulphonate and Melissa officinalis methanolic extract and their simultaneous exposure for different doses to evaluate antimutagenicity in Mus musculus. Treatment Total No. of cells analysed per mice No. of cells with micronuclei % age of MN % Reduction Group 1 Negative Control (Distilled water) 1000 2.35 ± 0.12 - - Group 2 Positive control (EMS) 1000 7.23 ± 0.89 - - Group 3 Mo- ME 100 mg/kgbw 1000 2.28 ± 0.10 - - Group 4 Mo- ME 400 mg/kgbw 1000 2.27 ± 0.09 - - Group 5 Mo- ME 100 mg/kg + EMS 1000 6.52 ± 0.70 90.1 14.5 Group 6 Mo-ME 200 mg/kg + EMS 1000 5.86 ± 0.58 81.0 28.0* Group 7 Mo-ME 300 mg/kg + EMS 1000 4.90 ± 0.50 67.7 47.7** Group 8 Mo-ME 400 mg/kg + EMS 1000 3.25 ± 0.43 44.9 81.5*** NC: Negative control (distilled water), PC: Positive control [Ethyl methane sulfonate (EMS) 117.5 mg/kgbw; dose is 1/4th LD50], Mo- ME: Melissa officinalis Methanolic Extract. Values with different asterisks (*p < 0.05: significant, **p < 0.01: highly significant, ***p < 0.001: extremely significant) differ significantly from the positive control (Mann-Whitney U test). Figure 2. Graph showing number of micronucleated cells in dif- ferent groups of mice treated with EMS alone, EMS + Mo-ME and Mo-ME alone in different concentrations. Figure 3. Bar diagram showing percentage reduction in EMS treat- ed micronuclei with increase in concentration of Melissa officinalis methanolic extract (Mo-ME). 120 Hilal Ahmad Ganaie, Md. Niamat Ali, Bashir A Ganai Evans, 1998; Krings and Berger, 2001). These phenolic compounds possess biological properties such as anti- apoptosis, anti-aging, anti-carcinogen, anti-inflamma- tion, anti-atherosclerosis, cardiovascular protection and improvement of endothelial function, as well as inhibi- tion of angiogenesis and cell proliferation activities (Han et al., 2007). Tannins bind to proline rich protein and interfere with protein synthesis. Flavonoids are hydrox- ylated phenolic substances known to be synthesized by plants in response to microbial infection and they have been found to be antimicrobial substances against wide array of microorganisms in vitro. The activity is proba- bly due to their ability to complex with extracellular and soluble proteins and to complex with bacterial cell wall (Marjorie, 1999). They are also effective antioxidant and show strong anticancer activities (Salah et al., 1995; Del- Rio et al., 1997). Previous reports show that the essential oil of M. officinalis is composed of some important compounds like (E)-caryophyllene and caryophyllene oxide in addi- tion to major constituents such as citronellal, neral and geranial (Sorensen, 2000; van de Berg et al., 1997). Liter- ature reveals that the essential oil of M. officinalis subsp. officinalis contains significant amounts of citral and/or citronellal, whereas M. officinalis subsp. altissima con- tains only traces (van de Berg et al., 1997). Van de Berg et al. (1997) identified b-caryophyllene, germacrene-D, sabinene, and b-pinene as the main components in leaf oils of M. officinalis subsp. altissima. Marnewick et al. (2000) found that the aqueous extracts of fermented and unfermented rooibos tea Table 4. Frequency profile of chromosomal aberrations induced by ethyl methanesulphonate and Melissa officinalis methanolic extract sepa- rately and by their combination for different doses to evaluate the antimutagenicity in Mus musculus. Treatments Chromosomal Aberrations Concentration (mg/kgbw) No. of cells Rings Fragments Exchange Breaks Gaps Total Aberrations %age of Aberrations % Reduction Distilled water 500 1 3 - 7 - 11 2.2 - EMS 117.5 mg/kgbw 500 4 18 14 62 31 129 25.8 - Mo-ME Alone100 mg/kgbw 500 1 3 - 7 - 11 2.2 - Mo-ME Alone400 mg/kgbw 500 1 3 - 6 - 10 2.0 - Mo-ME 100 mg/kgbw + EMS 500 3 13 10 44 14 84 16.8 38.1* A-ME 200 mg/kgbw + EMS 500 2 9 7 32 8 58 11.6 60.1* Mo-ME 300 mg/kgbw + EMS 500 1 7 5 22 6 41 8.2 74.5** Mo-ME 400 mg/kgbw + EMS 500 - 4 3 10 4 21 4.2 91.5*** NC: Negative control (distilled water), PC: Positive control [Ethyl methane sulfonate (EMS) 117.5 mg/kgbw; dose is 1/4th LD50], Mo- ME: Melissa officinalis Methanolic Extract. Values with different asterisks (*p < 0.05: significant, **p < 0.01: highly significant, ***p < 0.001: extremely significant) differ significantly from the positive control (Mann-Whitney U test). Figure 5. Photomicrograph showing metaphase chromosome preparation from bone marrow. a) Normal set of chromosomes, b) Exchanges (arrow), c) Ring (arrow), d) Fragment (arrow), e) Micro- nuclei (arrow). Figure 4. Bar diagram showing percentage reduction in chromo- somal aberrations (CA) induced by EMS following post-treatment with methanolic extract of Melissa officinalis (Mo-ME). 121Melissa officinalis: A potent herb against EMS induced mutagenicity in mice (Aspalathus linearis) and honey-bush tea (Cyclopia inter- media) possess antimutagenic activity against 2-acety- laminofluorine and aflatoxin B1. Vitamin C and E also significantly reduced the CA frequency in mouse bone marrow cells against rifampicin, an anti-tuberculosis drug, (Aly and Donia, 2002). According to Kaur et al. (2010), the phytoconstitu- ents from Terminalia arjuna suppressed the mutagen- ic effect of the aromatic amine, i.e., 2-aminof luorene (2-AF). The observed activity caused the inhibition of the metabolic activation of pro-mutagens. Hong et al. (2011) found that the extracts of Acanthopanax divari- catus were able to rapidly eliminate the mutagenic com- pounds from the cells before they induce the DNA dam- age. In a similar study, Nardemir et al. (2015) observed that the methanol extracts of the lichens have antimuta- genic effects against sodium azide.In another study, Prakash et al. (2014) found that the different extracts of Dioscorea pentaphylla significantly inhibited the effects of methyl methanesulphonate (MMS) induced mutagenicity. They also found that the methanolic extract was highly mutagenic in comparison to Petroleum ether and chloro- form. Entezari et al. (2014) compared the antimutagenic and anticancer activities of Echinophora platyloba DC on acute promyelocytic leukemia cancer cells and found that the methanolic extract of this plant prevented the reverted mutations and the hindrance was 93.4% in anti- mutagenic test. Akinboro et al. (2014) utilised the leaves of Myristica fragrans (Houtt.) for antimutagenic activity against benzo[a]pyrene and cyclophosphamide induced mutagenicity in Salmonella typhimurium and Mus mus- culus and found that the aqueous extract significantly suppressed more than 50 % of the mutations in all the tested concentrations. Sarac, (2015) utilised an edible wild plant, Tragopogon longirostis for the evaluation of antioxidant, mutagenic and antimutagenic properties and found that the ethanolic extract of its leaves exhibited antimutagenic properties at 2.5, 0.25, and 0.025 mg/plate concentrations. Habibi et al. (2014) found that the etha- nolic extract of Origanum vulgare reduced the frequency of MN PCR from 10.52 ± 1.07 for CP to 2.17 ± 0.6 for the synergic test of CP and the ethanolic extract. CONCLUSION Based on the above results it can be concluded that the methanolic extractof Melissa officinalis possess some important phytoconstituents which possess antimuta- genic activity. The results of the present study clearly showed that the methanolic extract of Melissa officinalis had an antimutagenic and anticlastogenic potential against the mutagenic activity of ethyl methane sulphonate in mice. Our results suggest that there may be several ways through which M. of ficinalis extract can work against EMS. Selection of M. officinalis for the present study on the basis of its folklore usage seems to be jus- tified as it is scientifically proved to have much poten- tiality. The extracts from such plants could be seen as a good source of useful drugs.However, further studies are needed in other test systems so that in the future M. officinalis can be used in reducing the occurrence of cancers or even as a coadjuvant to chemotherapy to reduce its side effects. ACKNOWLEDGEMENTS The authorsare highly thankful to the Director, Cen- tre of Research for Development, University of Kash- mir for providing the necessary facilities for the smooth research and also to Curator, Centre of Biodiversity and Plant Taxonomy, Department of Botany, University of Kashmir, Srinagar, J & K in proper identification of the plant. REFERENCES Akinboro A, Bin Mohamed K, Asmawi MZ, YekeenTA. 2014. Antimutagenic effects of aqueous fraction of Myristica fragrans (Houtt.) leaves on Salmonella typh- imurium and Mus musculus. Acta Biochim Pol. 61(4): 779-785. Aly FA, Donya SM. 2002. In vivo antimutagenic effect of vitamins C and E against rifampicin-induced chro- mosome aberrations in mouse bone-marrow cells. Mutation Research/Genetic Toxicology and Environ- mental Mutagenesis. 518(1): 1-7. Azevedo L, Gomes JC, Stringheta PC, Gontijo AMMC, Padovani CR, Ribeiro LR, Salvadori DMF. 2003. Black bean (Phaseolus vulgaris L.) as a protective agent against DNA damage in mice. Food Chem Toxicol. 41: 1671-1676. Berhow M, Wagner E, Vaughn S, Plewa M, 2000. Charac- terization and antimutagenic activity of soybean sapo- nins. Mutat Res. 448: 11-22. Bresolin S, Vargas VMF. 1993. Mutagenic potencies of medicinal plants screened in the Ames test. Phyto- ther Res. 7: 260-262. Brown JE, Rice-Evans CA (1998). Luteolin-rich artichoke extract protects low density lipoprotein from oxida- tion in vitro. Free Radical Research. 29(3): 247-255. 122 Hilal Ahmad Ganaie, Md. Niamat Ali, Bashir A Ganai Chen H, Zuo Y, Deng Y. 2001. Separation and determina- tion of flavonoids and other phenolic compounds in cranberry juice by high performance liquid chroma- tography. J. Chromatogr. A., 913: 387- 395 Dastmalchi K, Dorman HD, Oinonen PP, Darwis Y, Laakso I, Hiltunen R. 2008. Chemical composition and in vitro antioxidative activity of a lemon balm (Melissa officinalis L.) extract. LWT-Food Science and Technology, 41(3): 391-400. Del-Rio A, Obdululio BG, Casfillo J, Main FG, Ortuno A. 1997.Uses and properties of citrus flavonoids. J. Agric. Food Chem. 45: 4505-4515. Dutt HC, Bhagat N, Pandita S. 2015. Oral traditional knowledge on medicinal plants in jeopardy among Gaddi shepherds in hills of northwestern Himalaya, J&K, India. J Ethnopharmacol. 168: 337-348. Entezari M, Dabaghian FH, Hashemi M. 2014.The com- parison of antimutagenicity and anticancer activities of Echinophora platyloba DC on acute promyelocytic leukemia cancer cells. Journal of Cancer Research and Therapeutics. 10(4): 1004-1007. Fernandes JBF, Vargas VMF. 2003. Mutagenic and anti- mutagenic potential of the medicinal plants M. laevi- gata and C. xanthocarpa. Phytother Res. 17: 269-273. Florence AR, Joselin J, Brintha TSS, Sukumaran S, Jeeva S. 2014.Preliminary phytochemical studies of select members of the family Annonaceae for bioactive constituents. Bioscience Discovery. 5(1): 85-96. Ganaie HA, Ali MN, Ganai BA, Kaur J, Ahmad M. 2016. GC-MS Analysis and evaluation of mutagenic and antimutagenic activity of ethyl acetate extract of Ajuga bracteosa Wall ex. Benth: An endemic medicinal plant of Kashmir Himalaya. Indian J. Clin Toxicol. 6: 288. Ganaie HA, Ali MN, Ganai BA, Meraj M, Ahmad M. 2017. Antibacterial activity of 14, 15-dihydroajugapi- tin and 8-oacetylharpagide isolated from Ajuga brac- teosa Wall ex. Benth against human pathogenic bac- teria. Microbial Pathogenesis. 103: 114-118. Habibi E, Shokrzadeh M, Ahmadi A, Chabra A, Nagh- shvar F, Keshavarz-Maleki R. 2014. Genoprotective effects of Origanum vulgare ethanolic extract against cyclophosphamide-induced genotoxicity in mouse bone marrow cells. Pharmaceutical Biology. 53(1): 92-97. Han X, Shen T, Lou H. 2007. Dietry polyphenols and their biological significance. Int. J. Mol. Sci. 950-988. Haraguchi FK, Pedrosa ML, de Paula H, dos Santos RC, Silva ME. 2009. Influência das proteínas do soroso- breenzimashapáticas, perfillipídico e formaçãoóssea de ratoshipercolesterolêmicos. Rev Nutr. 22: 517–525. Hernandez-Ceruelos A, Madrigal-Bujaidadar E, De La Cruz C. 2002. Inhibitory effect of chamomile essen- tial oil on the sister chromatid exchanges by dauno- rubicin and methyl methanesulfonate in mouse bone marrow. Toxicol Lett. 135: 103-110. Herodez SS, Hadolin M, Skerget M, Knez Z. 2003. Sol- vent extraction study of antioxidants from Balm (Melissa officinalis L.) leaves. Food Chem. 80: 275- 282. Hong CE, Cho MC, Jang HA, Lyu SY. 2011. Mutagenicity and anti-mutagenicity of Acanthopanax divaricatus var. albeofructus. The Journal of Toxicological Sci- ences 36(5): 661-668. Jeeva S, Johnson M, Aparna JS, Irudayaraj V. 2011. Pre- liminary phytochemical and antibacterial studies on flowers of selected medicinal plants.International Journal of Medicinal and Aromatic Plants 1(2): 107- 114. Kaur H, Kalotra R, Walia GK, Handa D. 2013. Genotox- ic effects of dyeing industry effluent on a freshwater fish, Cirrhinus mrigala by chromosomal aberration test. International Journal of Pharmacy and Biologi- cal Sciences 3: 423-431. Krings U, Berger RG. 2001. Antioxidant activity of roast- ed foods. Food Chem. 72: 23-229. Kwak Y, Ju J. 2015. Inhibitory activities of Perilla frutes- cens britton leaf extract against the growth, migra- tion, and adhesion of human cancer cells. Nutr. Res. Pract. 9: 11–16. Liu HX, He MT, Tan HB, Gu W, Yang SX, Wang YH, Li L, Long CL. 2015. Xanthine oxidase inhibitors iso- lated from Piper nudibaccatum. Phytochem. Lett. 12: 133–137. Marjorie MC. 1999. Plant products as antimicrobial agents. Clinical Microbiology Review 12(4): 564-582. Marnewick JL, Gelderblom WC, Joubert E. 2000. An investigation on the antimutagenic properties of South African herbal teas. Mutation Research/ Genetic Toxicology and Environmental Mutagenesis 471(1): 157-166. Nardemir G, Yanmis D, Alpsoy L, Gulluce M, Agar G, Aslan A. 2015. Genotoxic, antigenotoxic and antioxi- dant properties of methanol extracts obtained from Peltigera horizontalis and Peltigera praetextata. Toxi- cology and Industrial Health 31(7): 602-613. Pereira RP, Fachineto R, Prestes AS, Puntel RL, Boschetti TK, Athayde ML, Büerguer ME, Morel AF, Morsch VM, Rocha JBT. 2009. Antioxidant effects of differ- ent extracts from Melissa officinalis, Matricaria recu- tita and Cymbopogon citratus. Neurochem Res. 34: 973-983. Prakash G, Hosetti BB, Dhananjaya BL. 2014. Antimuta- genic effect of Dioscorea pentaphylla on genotoxic effect induced by methyl methanesulfonate in the 123Melissa officinalis: A potent herb against EMS induced mutagenicity in mice drosophila wing spot test. Toxicology International. 21(3): 258-263. Sadraei H, Ghannadi A, Malekshahi K. 2003. Relaxant effect of essential oil of Melissa officinalis and citral on rat ileum contractions. Fitoterapia 74: 445- 52. Salah N, Miller NJ, Pagange G, Tijburg L, Bolwell GP, Rice E, Evans C. 1995. Polyphenolic flavonoids as scavenger of aqueous phase radicals as chai breaking antioxidant. Arc. Biochem. Broph. 2: 339-346. Sarac N. 2015. Antioxidant, mutagenic, and antimuta- genic activities of Tragopogon longirostis var. longiro- stis, an edible wild plant in Turkey. Indian Journal of Pharmacology 47(4): 414-418. Schnitzler P, Schuhmacher A, Astani A, Reichling J. 2008. Melissa officinalis oil affects infectivity of enveloped herpesviruses. Phytomed. 15(9): 734-740. Shrikumar S, Ravi TK. 2007.Approaches towards devel- opment and promotion of herbal drugs. Pharmacog- nosy Reviews 1(1): 180-184. Sorensen JM. 2000. Melissa officinalis, essential oil authenticity, production and pharmacological activ- ity. International Journal of Aromatherapy 10(1): 7-15. Souza AC, Alviano DS, Alves PB, Alviano CS, Gattass CR. 2004. Melissa officinalis L. essential oil: Antitu- moral and antioxidant activities. J Pharm Pharmacol. 56: 677-681. Sumathi BM, Uthayakumari F. 2014. GC MS analysis of Leaves of Jatropha maheswarii Subram & Nayar. Sci- ence Research Reporter 4(1): 24-30. Tovart RT. 2009. Clinical approach to clinical herbal tox- icity. Sem Diagn Pathol. 26: 28-37. Van den Berg T, Freundl E, Czygan FC. 1997. Melissa officinalis subsp. altissima: characteristics of a pos- sible adulteration of lemon balm. Pharmazie 52(10): 802-808. Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Volume 73, Issue 1 - 2020 Firenze University Press Karyotypic investigation concerning five Bromus Species from several populations in Iran Sara Sadeghian, Ahmad Hatami, Mehrnaz Riasat High genetic diversity and presence of genetic structure characterise the endemics Ruta corsica and Ruta lamarmorae (Rutaceae) Marilena Meloni1, Caterina Angela Dettori2, Andrea Reid3, Gianluigi Bacchetta2,4,*, Laetitia Hugot5, Elena Conti1 Cytogenetic effects of C6H4 (CH3)2 (xylene) on meristematic cells of root tips of Vicia faba L. and mathematical analysis Cihangir Alaca1, Ali Özdemir1, Bahattın Bozdağ2, Canan Özdemir2,* Clethodim induced pollen sterility and meiotic abnormalities in vegetable crop Pisum sativum L. Sazada Siddiqui*, Sulaiman Al-Rumman Temporal Analysis of Al-Induced Programmed Cell Death in Barley (Hordeum vulgare L.) Roots Büşra Huri Gölge, Filiz Vardar* Genetic diversity, population structure and chromosome numbers in medicinal plant species Stellaria media L. VILL. Shahram Mehri*, Hassan Shirafkanajirlou, Iman Kolbadi A new diploid cytotype of Agrimonia pilosa (Rosaceae) Elizaveta Mitrenina1, Mikhail Skaptsov2, Maksim Kutsev2, Alexander Kuznetsov1, Hiroshi Ikeda3, Andrey Erst1,4,* Study regarding the cytotoxic potential of cadmium and zinc in meristematic tissues of basil (Ocimum basilicum L.) Irina Petrescu1, Ioan Sarac1, Elena Bonciu2, Emilian Madosa1, Catalin Aurelian Rosculete2,*, Monica Butnariu1 Chemical composition, antioxidant and cytogenotoxic effects of Ligularia sibirica (L.) Cass. roots and rhizomes extracts Nicoleta Anca Şuţan1,*, Andreea Natalia Matei1, Eliza Oprea2, Victorița Tecuceanu3, Lavinia Diana Tătaru1, Sorin Georgian Moga1, Denisa Ştefania Manolescu1, Carmen Mihaela Topală1 Phagocytic events, associated lipid peroxidation and peroxidase activity in hemocytes of silkworm Bombyx mori induced by microsporidian infection Hungund P. Shambhavi1, Pooja Makwana2, Basavaraju Surendranath3, Kangayam M Ponnuvel1, Rakesh K Mishra1, Appukuttan Nair R Pradeep1,* Electrophoretic study of seed storage proteins in the genus Hypericum L. in North of Iran Parisa Mahditabar Bahnamiri1, Arman Mahmoudi Otaghvari1,*, Najme Ahmadian chashmi1, Pirouz Azizi2 Melissa officinalis: A potent herb against EMS induced mutagenicity in mice Hilal Ahmad Ganaie1,2,*, Md. Niamat Ali1, Bashir A Ganai2 Population genetic studies in wild olive (Olea cuspidata) by molecular barcodes and SRAP molecular markers Rayan Partovi1, Alireza Iaranbakhsh1,*, Masoud Sheidai2, Mostafa Ebadi3 In Vitro Polyploidy Induction in Persian Poppy (Papaver bracteatum Lindl.) Saeed Tarkesh Esfahani1, Ghasem Karimzadeh1,*, Mohammad Reza Naghavi2 Long-term Effect Different Concentrations of Zn (NO3)2 on the Development of Male and Female Gametophytes of Capsicum annuum L. var California Wonder Helal Nemat Farahzadi, Sedigheh Arbabian*, Ahamd Majd, Golnaz Tajadod A karyological study of some endemic Trigonella species (Fabaceae) in Iran Hamidreza Sharghi1,2, Majid Azizi1,*, Hamid Moazzeni2 Karyological studies in thirteen species of Zingiberacaeae from Tripura, North East India Kishan Saha*, Rabindra Kumar Sinha, Sangram Sinha