Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 74(1): 135-150, 2021 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1041 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: A. Okorie Asita, S. Maga- ma, T. Mahali Moahloli, S. Baholo (2021) Evaluation of extracts of wild Canna- bis sativa L. for genotoxicity and phy- tochemical composition. Caryologia 74(1): 135-150. doi: 10.36253/caryolo- gia-1041 Received: August 03, 2020 Accepted: January 23, 2021 Published: July 20, 2021 Copyright: © 2021 A. Okorie Asita, S. Magama, T. Mamoroesi Moahloli, S. Baholo. This is an open access, peer- reviewed article published by Firenze University Press (http://www.fupress. com/caryologia) 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. Evaluation of extracts of wild Cannabis sativa L. for genotoxicity and phytochemical composition Asita Okorie Asita*, Sibusisiwe Magama, Thato Mamoroesi Moahloli, Selometsi Baholo Department of Biology, National University of Lesotho, P.O. Roma 180, Maseru 100, Lesotho, Southern Africa Email address: aoasita@yahoo.co.uk, ao.asita@nul.ls; sibusisiwe.magama@gmail.com; kimanethato@gmail.com; selometsibaholo@gmail.com. *Corresponding author. Abstract. Cannabis sativa L. is used as medicine and narcotic in Lesotho. Phytochemi- cal composition and total phenolics content (TPC) for hexane, chloroform, ethyl ace- tate and methanol extracts of aerial parts of C. sativa were determined. Ethyl acetate extract (0.1875, 0.375 and 0.75 mg mL-1) and methanol extract (0.75, 1.5 and 3.0 mg mL-1) were evaluated for cytotoxicity, genotoxicity and modulation of cyclophospha- mide (CP, 1.25 mg mL-1)- and ethylmethane sulphonate (EMS, 0.25 mg mL-1)-induced genotoxicity using Allium cepa root meristem assay. CP or EMS did not reduce mitotic index (MI) of cells, hence not cytotoxic when compared with negative control using the t-test (p>0.05), but genotoxic. Both extracts were genotoxic with methanol extract also being cytotoxic. Genotoxicity was the number of aberrant cells per 100 mitotic cells. Modulatory effect (ME) was obtained by comparing mutagen-induced geno- toxicity with mixture-induced genotoxicity and expressed as the number of units of mutagen-induced genotoxicity that equalled the mixture-induced genotoxicity. ME was either positive or negative and significant only if ME = ≥ 2. Both extracts were genotoxic with methanol extract also being cytotoxic. Aberrations observed were sticky chromosomes, c-metaphase, anaphase and telophase bridges, chromosome fragments and laggards. Mixture of methanol extract with CP or EMS was more genotoxic (+ME range = 1.61-11.89) than the mutagen or extract alone which suggested synergistic interaction. Mixture of ethyl acetate extract with CP induced insignificant +ME. Mix- ture of ethylacetate extract with EMS was significantly more genotoxic (+ME = 2.20) than EMS only at high extract concentration. The methanol and ethylacetate extracts of C. sativa were not anti-genotoxic to CP- or EMS- induced genotoxicity. TPCs for hexane, chloroform, ethyl acetate and methanol extracts were 39831.46, 2544.94, 2438.20 and 56601.12 mg GAE/gram dry weight respectively. The differences in the cytotoxicity and MEs of the extracts were attributed to differences in phytochemical composition of extracts. Keywords: medicinal cannabis, phenolics, modulatory effects, cyclophosphamide, ethyl methanesulphonate, Lesotho. 136 Asita Okorie Asita, Sibusisiwe Magama, Thato Mamoroesi Moahloli, Selometsi Baholo 1. INTRODUCTION Different human civilizations have depended for many centuries on plants and plant products for their medicinal (Balandrin et al. 1985) and recreational (Siegel 1977) needs. The scientific basis for the use of plants in tradi- tional medicine, has been attributed largely, to second- ary metabolites (SMs) which have been shown to pos- sess various biological activities (Bourgaud et al. 2001); therefore much of the protective and therapeutic effects of plants have been attributed to phytochemicals such as alkaloids, terpenoids, tannins, phenolics, etc. (Harborne 1998; Hertog et al.1993; Zhang et al. 2001). The concoctions used in traditional medicine are usually crude extracts in water, alcohol, distillates or essential oils, which contain many SMs from various structural groups and their activity is often due to syn- ergistic interactions of the SMs present (Eid et al. 2012; Mulyaningsih et al. 2010). At high concentrations, SMs change membrane f luidity and increase permeability. Therefore, many lipophilic SMs exhibit antimicrobial and cytotoxic activities and are responsible for the apparent broad-spectrum activity of concoctions used in tradition- al medicine (van Wyk and Wink 2015; Wink 2015). In Lesotho, as in many other countries in the world, a system of traditional medicine based on the use of plants, birds, animals, their products and their combi- nations to treat a broad spectrum of communicable and noncommunicable diseases is still being practiced (Shale et al. 1999; Padmanabhan and Sujana, 2008). One plant species commonly used in traditional medicine in Leso- tho and Southern Africa is Cannabis sativa (C. sativa), (Ranotsi et al. 2012). Other names for cannabis are mari- juana, weed, dagga and “matekoane” in Sesotho (Ranotsi et al. 2012; Bloomer 2019). This plant has been used for multiple purposes (medicinal, recreational, seed oil and industrial fiber, etc.) for thousands of years (ElSohly and Gul, 2014). In Lesotho, C. sativa is used as medicine for all kinds of ailments such as heart burn, blood pressure and “nerves” as a recreational drug, and as part of reli- gious rites (Laniel 1999). A concoction of cannabis is a complex mixture of active compounds (phytochemicals) of which about 545 have been identified, 104 are cannabinoids or phytocan- nabinoids (as they originate from the plant) as well as 22 noncannabinoid constituents (Turner et al. 1980; ElSohly and Slade 2005; ElSohly and Gul, 2014). The cannabi- noids include Δ9-tetrahydrocanabinol (THC), canna- bidiol (CBD), cannabigerols (CBG), cannabichromenes (CBC), cannabinol (CBN) and cannabinodiol (CBDL) (El-Alfy et al. 2010) found in the flowers, to a lesser extent the leaves, and minimally in the stems, and seeds (ElSohly and Gul 2014). THC is known as the major psy- choactive component of cannabis that is responsible for causing addiction to marijuana (Ashton, 2001; National Institute on Drug Abuse (NIDA), 2018). The importance of plants as sources of medicines not withstanding, investigations have revealed that many plants which are used as food or in traditional medicine have mutagenic, cytotoxic and genotoxic effects in in vitro and in vivo assays (Higashimoto et al. 1993; Schim- mer et al. 1994; Kassie et al. 1996; Çelik and Aslantürk 2007). In a review by Marselos and Karamanakos (1999), they concluded that there was no consensus on the induction of point mutations by cannabinoids, while some experimental results suggest that cannabinoids may cause chromosomal damage (Zimmerman and Zimmerman 1990) and act as tumour promotors in ani- mals. In addition, the extracts of some plant species have been observed to induce both mutagenic and antimuta- genic effects on known mutagens in different test sys- tems (Debisri et al. 1996). The content of active compounds in plant species also vary according to their genetics, climatic factors, soil characteristics and the time of harvesting (Ramelet 2015); and when plant materials are extracted with solvents of different polarities, often the different solvent fractions contain different biomolecules (Herrera-Ruiz et al. 2008). Studies on agents that modulate carcinogen-induced genotoxic effects in experimental animals provide end points that can be used for assessing the antimutagenic or anticarcinogenic properties of putative chemopreven- tive compounds and for predicting their protective effi- cacy in humans (Khaidakov et al. 2001). In view of the foregoing therefore, the aim of this study was to evaluate hexane, methanol, ethyl acetate and chloroform extracts of wild Cannabis sativa for phy- tochemical composition, genotoxicity and the modula- tion of cyclophosphamide (CP)- and ethyl methanesul- phonate (EMS)- induced genotoxicity using the Allium cepa chromosome aberration assay system. The Allium cepa L assay is an in vivo assay and one of the established plant bioassays, validated by the inter- national programme on chemical safety (IPCS, WHO), as an efficient and standard test for chemicals screening, in situ monitoring of the genotoxicity of environmental sub- stances (Leme and Marin-Morales 2009) and to evaluate the genotoxic potential of medicinal plants (Camparoto et al. 2002; Knoll et al. 2006; Fachinetto et al. 2007; Lubi- ni et al. 2008; Fachinetto et al. 2009). The Allium cepa L assay tests genotoxicity using chromosomes and therefore detects chromosome structural and numerical altera- tions (Tedesco and Laughinghouse 2012; Bonciu et al. 137Evaluation of extracts of wild Cannabis sativa L. for genotoxicity and phytochemical composition 2018). CP is an antineoplastic indirect-acting (promuta- gen) alkylating agent (Mohn and Ellenberger 1976; Hales 1982) while EMS is a direct-acting mutagen, tetratogen, and brain carcinogen (Stubbs et al. 1997). 2. MATERIALS AND METHODS 2.1 Test organism Onion (A. cepa) seeds of the variety, Texas Grano 502 P.R.R., a product of Sakata seeds, Lanseria 1748, Republic of South Africa were purchased from Maseru Garden Centre, Lesotho in Southern Africa. 2.2 Mutagens and chemicals Cyclophosphamide (CP) and ethyl methanesulfonate (EMS) are products of Fluka (Biochemika, Germany). Methanol (absolute) is a product of Associated Chemi- cal Enterprises (Pty) Ltd (Johannesburg, South Africa); hydrochloric acid glacial and acetic acid are products of UNILAB (Krugerdorp, South Africa); acetocarmine stain was obtained from Carolina Biological Supply Company, Burlington, North Carolina, USA. 2.3 Plant material collection and preparation Aerial parts of the female plant of wild C. sativa were collected from the Thaba Bosiu area, some 12 km from the National University of Lesotho (NUL) campus, in the Maseru District of Lesotho where they grow in a location with the following geographical coordinates: Latitude: 29°22’49˝S, Longitude: 27°33’13” E and at an altitude of 1600 m. The aerial parts of the sample were dried in a fanned Labcon oven at 37°C to a constant weight and brittle, about 48 hours. Thereafter, the piec- es were segmented and ground to a fine powder using a pulveriser (Kenwood) and the powder was stored in sealed amber bottles in the dark at room temperature. 2.4 Preparation of the crude C. sativa extracts Sequential solvent extraction of the ground powder was done according to the method outlined in Razak et al. (2014); Padhi and Panda (2015); Fayera et al. (2018) with slight modifications. All crude extracts (hexane, chloroform, ethyl acetate and methanol) were stored at 4°C until further investigation for genotoxicity, modula- tory effects on mutagen-induced genotoxicity and phyto- chemical profiling. 2.5 Qualitative Phytochemical Screening of crude extracts of C sativa. The crude extracts of C. sativa prepared with hex- ane, chloroform, ethyl acetate and methanol were sub- jected to a qualitative screening for the presence of major phytochemical classes using standard phytochem- ical methods and the appropriate reagents and chemicals according to the modified methods of Trease and Evans (1984); Trease and Evans (2002); Soni and Sheetal (2013); Nwaoguikpe et al. (2014) and Uddin et al. (2014). Each reaction mixture was visually assessed as in Lu et al. (2014), for precipitate formation, foam formation, colour changes and colour intensity according to the following key: (+), Low intensity of colour and/or precipitate; (++), moderate intensity of colour and/or precipitate; (+++), strong intensity of colour and/or precipitate (-), not detected (either absent or below the detection limit). The list of screening tests that were carried out is shown in Table 1. 2.6 Determination of the total phenolic content of C. sativa extracts using the Folin-Ciocalteau assay Determination of the total phenolic content for each of the extracts was done by the method of McDonald Table 1. Phytochemical screening tests. Phytochemical Name of test Colour for positive test 1. Flavonoids Shinoda Pink 2. Alkaloids Wagner Blue-black 3. Tannins Ferric chloride Blue-black/Green 4. Terpenoids Salkowski Reddish-brown 5. Saponins Foam test Foam formation 6. Simple phenols Ferric chloride Green 7. Polyphenols Ferric chloride Blue 8. Anthocyanins NaOH Blue-green Betacyanins NaOH Yellow Quinones HCl Green Phlobatannins HCl Red precipitate Anthraquinones HCl+chloroform+ammonia Rose-pink/violet Coumarins NaOH+chlroform Yellow Phytosterols Salkowski Red Cardiac glycosides and Cardenolides Keller-Kiliani’s Brown-red ring Reducing sugars Benedict’s Red precipitate Proteins Biuret Violet Amino acids Ninhydrin purple Fatty acids Diethyl ether Transparent stain 138 Asita Okorie Asita, Sibusisiwe Magama, Thato Mamoroesi Moahloli, Selometsi Baholo et al. (2001) with slight modifications.The total phenolic content of each extract was recorded in milligram gallic acid equivalents (GAE) per gram of dry weight of extract from the gallic acid standard curve (Wong et al., 2012; Moyo et al. 2013; Magama et al., 2018). The total phenol- ic content in each extract determined as milligram gallic acid equivalents (GAE) per gram of dry weight of extract was calculated using the following formula: (1) Where T is the total phenolic content of the extracts in mg GAE per gram of dry weight of extract, C is the con- centration of the gallic acid established from the calibra- tion curve in mgmL-1, V is the volume of extract in mL and M is the mass of the plant extract in g. 2.7 Genotoxicity Experiments The preliminary assay to select concentrations of mutagens and plant extracts to use and Genotoxicity assay (including the treatment of Allium cepa seedlings with test agents, root harvest, slide preparation and scor- ing of slides) were conducted according to the methods of Asita et al. (2017). Due to insolubility of the hexane and chloroform extracts of C. sativa only the methanol and ethyl acetate extracts were evaluated for genotoxicity using 2.5% acetone (v/v in distilled water) as the solvent. The 2.5% acetone was not toxic or genotoxic to the onion root meristem cells. From the results of the preliminary assays to select the concentrations of mutagens and plant extracts to use, the following concentrations of plant extracts (in mgmL-1); methanol extract (0.75, 1.5 and 3.00) and ethyl acetate extracts (0.750, 0.375, 0.1875 and 0.09375); CP (1.25.00) and EMS (0.250) were assessed for cytotoxicity that is, mitotic index (MI), genotoxicity (GT) and the modulatory effect (ME) of plant extracts on mutagen-induced genotoxicity. The aberrations assessed were: sticky chromosomes (S), C-metaphase (C-Mit), lagging chromosomes (L), chromosome bridges at anaphase and telophase (A.B) and chromosome fragment (F). For calculating the GT, only aberrant mitotic cells were considered. 2.8 Analysis of slide preparations 2.8.1 Cytotoxicity The mitotic index (MI) was expressed as the num- ber of dividing cells per 100 cells scored according to the formula: MI = Number of dividing cells/ Total number of cells scored x 100. (2) The MI was used as a measure of cytotoxicity (CT). The MI of each treatment group was compared with that of the negative control group using t-test at a probabil- ity level of 0.05, using the SPSS for windows, version 11.0 software. 2.8.2 Genotoxicity Genotoxicity (GT) was expressed as the number of aberrant mitotic cells (AMC) per 100 mitotic cells [i.e AMC + normal mitotic cells (NMC)] scored according to the formula: Frequency of GT = AMC/ (AMC + NMC) x 100 (3) The mean GT of each group of three slides per con- centration of test agent was compared with that of the negative control group using t-test. P values less than 0.05 (P < 0.05) were considered as indicative of signifi- cance. 2.8.3 Modulatory effect (ME) of plant extracts on muta- gen-induced genotoxicity The modulatory effect (ME) of plant extract on CP- or EMS-induced genotoxicity (GT) was calculated using the formula of Asita et al. (2017): ME = (B – C) – (A – C) / (A – C) (4) Where ‘A’ is the genotoxicity induced by the muta- gen (CP or EMS) alone, i.e. mutagen-induced genotoxic- ity; ‘B’ is the genotoxicity induced by mixture of plant extract and mutagen, i.e. mixture-induced genotoxicity and ‘C’ is the genotoxicity induced by negative control, such as tap water alone. The modulatory effect (ME) was thus obtained by comparing the mutagen-induced genotoxicity (A) with the mixture-induced genotoxicity (B). The ME value indicated the number of units of the mutagen-induced genotoxicity (A) that equaled the mixture-induced geno- toxicity (B). ME was significant only if ME was ≥ 2, i.e mixture was at least twice (200%) more (+) or less (-) genotoxic than mutagen alone. A positive ME (+ME) indicated that the mixture was more genotoxic (increased GT) than the mutagen and if mixture is also more genotoxic than the genotoxic plant extract alone then a synergistic interaction is inferred. 139Evaluation of extracts of wild Cannabis sativa L. for genotoxicity and phytochemical composition But mutagen-potentiation is inferred if mixture is less genotoxic than the non-genotoxic plant extract alone A negative ME (-ME) indicated that the mixture was less genotoxic (reduced GT) than the mutagen alone. If mixture is less genotoxic than the mutagen and the gen- otoxic plant extract then antagonism is infered. How- ever, if mixture is less genotoxic than mutagen and also, more- or less genotoxic than the non-genotoxic plant extract then it is antimutagenicity. 2.8.4 Data analysis In the determination of total phenolics content, data was expressed as means ± standard deviations of three replicate determinations using Microsoft excel 2016. Differences between controls and treatment groups were determined using Student’s t-test. P-values of less than 0.05 (p<0.05) were considered statistically sig- nificant using the IBMSPSS statistics, version 20 soft- ware. Regression equations and graphs were used for the determination of milligram gallic acid equivalents (mgGAE equivalents) per gram of dry extract and the concentration of extract needed to inhibit oxidation by 50% (IC50). For the genotoxicty assays, the mean value of each group of three slides per concentration of test agent was compared with that of the negative (solvent) control group using student’s t-test and the Chi square test. P-values less than 0.05 (p < 0.05) were considered as indicative of significance. 3. RESULTS 3.1 Qualitative biochemical profile of C. sativa solvent extracts In Table 2 is presented the qualitative phytochemical profile of different solvent extracts of C. sativa obtained from the various tests. The methanol extract con- tained the highest number of the different phytochemi- cal classes (15/19), followed by hexane and chloroform (9/15 each) and ethyl acetate (7/15). Polyphenols as a class and the polyphenols (namely- anthocyanins, beta- cyanins, coumarins, and flavonoids), quinones (aromatic ketones), simple phenols were detected in trace amounts in the hexane, chloroform and methanol extracts but not detected in the ethylacetate extract. Coumarins (also polyphenols) were present in all extracts, though in traces only in the ethyl acetate extract. Traces of amino acids were also detected in all the solvent extracts. Ter- penoids were detected only in the hexane and methanol extracts. Flavonoids (also polyphenolic), alkaloids, sapo- nins (steroid and terpenoid glycosides) and phlobatan- nins (also polyphenolic), were detected in the methanol extract only. Phytosterols (unsaturated steroid alcohols) were detected only in the hexane extract.Cardiac glyco- sides and cardenolides, proteins and fatty acids were not detected in any of the solvent extractives. 3.2 Quantitative determination of total phenolics content In Fig. 1, is presented the gallic acid calibration curve for determination of total phenolic content of the different solvents (hexane, chloroform, ethyl acetate and methanol) extracts, where y was the mean absorbance of the sample at 760nm and x the concentration estab- lished from the gallic acid calibration curve. The regres- sion equation was y = 0.0712x – 0.1055; the total phe- nolic content of the hexane extract, x, with y = 0.0363 was found to be 39 831.46mg GAE/gram dry weight. The total phenolic content of the chloroform extract, x, with y = 0.0757 was 2544.94 mg GAE/gram dry weight. The total phenolic content of the ethyl acetate extract, x, with y = 0.0681 was 2438.20mg GAE/gram dry weight. The Table 2. Phytochemical screening test results for solvents extracts of wild C. sativa. Test C. sativa crude Plant Extracts Hexane Chloroform Ethylacetate Methanol (95%) Flavonoids - - - ++ Alkaloids - - - +++ Tannins - + - +++ Terpenoids +++ - - +++ Saponins - - - + Simple phenols + + - + Polyphenols +++ +++ +++ +++ Anthocyanins +++ +++ ++ +++ Betacyanins +++ ++ ++ ++ Quinones +++ +++ +++ +++ Phlobatannins - - - + Anthraquinones - - - +++ Coumarins ++ + + +++ Phytosterols +++ - - - Cardiac glycosides and Cardenolides - - - - Reducing sugars - + ++ +++ Proteins - - - - Amino acids + ++ ++ +++ Fatty acids - - - - Key: Low intensity = “+”; moderate intensity = “++”; strong inten- sity = “+++”; not detected or negative = “-“(Lu et al., 2014). 140 Asita Okorie Asita, Sibusisiwe Magama, Thato Mamoroesi Moahloli, Selometsi Baholo total phenolic content of the methanol extract, x, with y = 0.0960 was 56 601.12 mg GAE/gram dry weight. 3.3 Cytotoxicity and Genotoxicity 3.3.1 Figures and Tables Photographs of the most representative pictures of normal mitotic cells and cells containing the different types of chromosome aberrations that were observed and scored are presented in Figure 2. The results of the cy totoxicity and genotoxicity experiments with the methanol and ethyl acetate extracts of C. sativa are pre- sented in Tables 3 and 4 respectively. y = 0,0712x - 0,1055 R² = 0,9219 -0,1000 -0,0500 0,0000 0,0500 0,1000 0,1500 0,2000 0,2500 0,3000 0,3500 0,4000 0,4500 0 0,015625 0,03125 0,0625 0,25 0,5 1 A bs or ba nc e (7 60 nm ) Concentration (mg/ml) Gallic acid calibration curve Figure 1. Gallic acid calibration curve for determination of Total phenolic content for hexane and methanol extracts. a b c d e f g h i j k l m n o p q r s t u v Figure 2. Photomicrographs of cells of Allium cepa showing untreated cells in normal division stages and Chromosomal aberrations (arrowed) in cells treated with methanol and ethyl acetate extracts of Cannabis sativa or mixture of extracts with EMS or Cyclophosphamide. (a) Interphase (b) Normal Prophase (c) Normal metaphase (d) Normal metaphase (e) Early anaphase (f ) Late anaphase (g) Telophase (h) Pyknotic interphase nuclei with micronucleus (i) Prophase with sticky chromosomes (j) Metaphase with sticky chromosomes (k) Metaphase with sticky chromosomes (l) C-metaphase (m) Metaphase with dislocated chromosome (n) Late anaphase with dislocated chromosome (o) Anaphase with sticky and scattered chromosomes (p) Late anaphase with chromosome bridge (q) Telophase with lagging chromosome (r) Telophase with sticky chromosomes and bridge (s) telophase with chromosome bridge and lagging (t) telophase with chromosome bridge and fragment (u) Telophase with chromosome bridge (v) Telophase with chromosome fragment and lagging. Magnification is 1000 X. 141Evaluation of extracts of wild Cannabis sativa L. for genotoxicity and phytochemical composition 3.3.2 Results for methanol (95%) extract experiments in Table 3 In Table 3 are the results of cytotoxicity and geno- toxicity experiments with methanol (95%) extracts of C. sativa and the mutagens, CP and EMS. (P+M)/(A+T) Ratio: Examination of the (P+M)/(A+T) ratio in column 8 of Table 3 shows that only the treat- ment with the lowest concentration (0.75 mgmL-1) of C. sativa extract alone or in a mixture with EMS (0.75 mgmL-1) induced a significant change in (P+M)/(A+T) ratio, when compared with the solvent (2.5% acetone) treated negative control group (p < 0.05). Cytotoxicity: Examination of the MI in column 9 of Table 3 shows that CP (1.25 mg mL-1) and EMS (0.25 mg mL-1) were not toxic. All the concentrations of the meth- anol extract of C. sativa (0.75, 1.5, 3.0 mg/mL) and their mixtures with CP (1.25 mg mL-1) or EMS (0.25 mg mL-1) induced significant reduction of the MI (was toxic) when compared to the solvent (2.5% acetone) treated negative control (P<0.05). Genotoxicity (GT): Examination of induction of gen- otoxicity in column 10 of Table 3 shows that CP (1.25 mg mL-1), EMS (0.25 mg mL-1), methanol extract (0.75, 1.5, 3.0 mg/mL) and the mixtures of CP (1.25 mg mL-1) or EMS (0.25 mg mL-1) with each contration of C. sativa Table 3. Cytotoxic and genotoxic effects of methanol extracts of C. sativa, EMS and CP on meristem cells of onion root tip and the modula- tory effects (ME) of the methanol extract of C. sativa on EMS- or CP-induced genotoxicity. Treatment (TC in mg/mL) Conc. Inter. Cells Total number of cells in the stages of mitosis in 2000 cells scored Total number of cells scored (P+M)/ (A+T) MI Genotoxicity Modulatory Effect N ABN Total (N + ABN) Extract on CP Extract on EMS Acetone (2.5%) MEAN 1790.00 208.00 2.00 210.00 2000 2.60 10.50 1.05     SD 75.54 75.54 0.00 75.54 0.00 0.09 3.78 0.41     CP (1.25) MEAN 1856.00 135.33 8.67 144.00 2000 2.56 7.20 7.31#     SD 44.80 48.91 6.11 44.80 0.00 0.33 2.24 7.06     EMS (0.25) MEAN 1815.33 171.00 13.67 184.67 2000 3.86 9.23 5.50#     SD 99.36 86.63 16.50 99.36 0.00 1.60 4.97 6.42     C. sativa (0.75) MEAN 1954.33 34.33 11.33 45.67 2000 8.87 Ɉ 2.28* 24.78#     SD 1.53 0.58 1.15 1.53 0.00 0.99 0.08 1.80     C. sativa (1.5) MEAN 1984.33 11.33 4.33 15.67 2000 3.47 0.78* 27.58#     SD 1.15 0.58 0.58 1.15 0.00 0.92 0.06 1.58     C. sativa (3.0) MEAN 1942.00 39.67 18.33 58.00 2000 3.59 2.90* 31.17#     SD 16.09 9.71 6.51 16.09 0.00 2.16 0.80 2.69     C. sativa (0.75) + CP MEAN 1989.00 2.33 8.67 11.00 2000 2.50 0.55* 77.91# 11.27+†   SD 2.00 1.53 2.89 2.00 0.00 0.90 0.10 14.34     C. sativa (1.5) + CP MEAN 1991.00 1.67 7.33 9.00 2000 4.67 0.45* 81.76# 11.89+†   SD 1.00 0.58 0.58 1.00 0.00 2.31 0.05 5.09     C. sativa (3.0) + CP MEAN 1981.00 10.00 9.00 19.00 2000 3.88 0.95* 47.59# 6.43+†   SD 4.00 2.65 1.73 4.00 0.00 2.51 0.20 4.65     C. sativa (0.75) + EMS MEAN 1959.00 24.00 17.00 41.00 2000 4.81 Ɉ 2.05* 41.55#   8.10+† SD 12.49 8.00 5.29 12.49 0.00 0.97 0.62 4.76     C. sativa (1.5) + EMS MEAN 1950.00 43.67 6.33 50.00 2000 2.83 2.50* 12.67#   1.61+† SD 0.00 0.58 0.58 0.00 0.00 0.54 0.00 1.15     C. sativa (3.0) + EMS MEAN 1964.33 30.33 5.33 35.67 2000 3.14 1.78* 15.00#   2.14+† SD 15.89 13.58 2.31 15.89 0.00 0.48 0.79 0.33     Key: TC = Test compound; N = Normal mitotic cells (comprising prophase, metaphase, anaphase and telophase); ABN = Aberrant mitotic cells; SD = Standard deviation; CP = Cyclophosphamide; EMS = Ethylmethane sulphonate; C.s = Cannabis sativa; MI = Mitotic index; Ɉ = P+M/A+T ratio (significant increase in ratio compared to negative control, P<0.05 in the t-test, n = 3); * = TC is Toxic (MI treatment significantly different from negative control, P<0.05 in the t-test, n = 3); # = TC is genotoxic (significant difference from negative control, P<0.05 in the t-test, n = 3); +† = C.s + Mutagen mixture more genotoxic than mutagen or C.s alone (Synergism); +‡ = C.s + Mutagen mix- ture less genotoxic than mutagen or C.s alone (antagonism); † = C.s + Mutagen mixture more genotoxic than mutagen alone but less than C.s alone; ‡ = C.s + Mutagen mixture less genotoxic than mutagen alone (antimutagenicity) but more than C.s alone. 142 Asita Okorie Asita, Sibusisiwe Magama, Thato Mamoroesi Moahloli, Selometsi Baholo extract used were all genotoxic to the root meristem cells of A. cepa when compared to the solvent (2.5% acetone) treated negative control (P<0.05). Modulatory effect (ME) of methanol (95%) extract of C. sativa on CP or EMS-induced Genotoxicity (GT): Examination of the modulatory effect (ME) in col- umn 11 of Table 3 shows that the mixture of each of the three concentrations of C. sativa (0.75, 1.5 or 3.0 mg/mL) methanol extract with CP (1.25 mgmL-1) was signifi- cantly (> twofold or 200%) more genotoxic than CP or C. sativa extract alone. The mixture of each concentra- tion of C. sativa extract with CP thus induced a positive and significant (> twofold) value of ME (11.27, 11.89 and 6.43 respectively) and synergism or synergistic interac- tion between the C. sativa extracts and CP, was inferred. Examination of the modulatory effect (ME) in col- umn 12 of Table 3 shows that the mixture of each of the three concentrations of C. sativa (0.75, 1.5 or 3.0 mg/ mL) methanol extract with EMS (0.25 mg/mL) was more genotoxic than EMS alone. The mixture of C sativa (0.75 mg/mL) was also more genotoxic than C. sativa extract alone. The mixtures of C. sativa (0.75 or 3.00 mg/mL) extract with EMS induced a positive and significant (> twofold) value of ME (8.10 and 2.14 respectively) and Table 4. Cytotoxic and Genotoxic effects of ethylacetate extracts of C. sativa, EMS and CP on meristem cells of onion root tip and the mod- ulatory effects (ME) of the ethylacetate extract of C. sativa on EMS- or CP-induced genotoxicity. Treatment (TC in mg/mL) Conc. Inter. Cells Total number of cells in the stages of mitosis in 2000 cells scored Total number of cells scored (P+M)/ (A+T) MI Genotoxicity Modulatory Effect N ABN Total (N + ABN) Extract on CP Extract on EMS Acetone (2.5%) MEAN 1790.00 208.00 2.00 210.00 2000 2.60 10.50 1.05     SD 75.54 75.54 0.00 75.54 0.00 0.09 3.78 0.41     CP (1.25) MEAN 1856.00 135.33 8.67 144.00 2000 2.56 7.20 7.31 #     SD 44.80 48.91 6.11 44.80 0.00 0.33 2.24 7.06     EMS (0.25) MEAN 1815.33 171.00 13.67 184.67 2000 3.86 9.23 5.50 #     SD 99.36 86.63 16.50 99.36 0.00 1.60 4.97 6.42     C. sativa (0.1875) MEAN 1828.67 93.67 77.67 171.33 2000 6.58 Ɉ 8.57 45.72 #     SD 21.13 22.03 11.59 21.13 0.00 0.61 1.06 7.86     C. sativa (0.375) MEAN 1805.00 180.67 14.33 195.00 2000 2.28 9.75 7.01 #     SD 21.00 11.59 11.59 21.00 0.00 0.55 1.05 5.57     C. sativa (0.75) MEAN 1824.67 171.00 4.33 175.33 2000 3.17 8.77 2.37     SD 39.63 37.59 2.08 39.63 0.00 1.55 1.98 0.76     C. sativa (0.1875) + CP MEAN 1786.33 190.67 23.00 213.67 2000 2.87 10.68 10.26 # 0.47†   SD 86.05 74.51 12.29 86.05 0.00 0.74 4.30 2.91     C. sativa (0.375) + CP MEAN 1862.33 133.33 4.33 137.67 2000 3.12 6.88 4.16 # -0.50+‡   SD 50.96 53.35 2.52 50.96 0.00 1.79 2.55 4.12     C. sativa (0.75) + CP MEAN 1817.00 178.00 5.00 183.00 2000 3.50 9.15 2.75 # -0.73‡   SD 20.66 20.42 1.00 20.66 0.00 1.18 1.03 0.55     C. sativa (0.1875) + EMS MEAN 1807.00 189.33 3.67 193.00 2000 3.09 9.65 1.88   -0.81+‡ SD 23.64 22.85 2.08 23.64 0.00 0.91 1.18 0.94     C. sativa (0.375) + EMS MEAN 1820.67 174.67 4.67 179.33 2000 2.32 8.97 2.67 #   -0.64+‡ SD 33.01 33.31 1.15 33.01 0.00 0.45 1.65 0.84     C. sativa (0.75) + EMS MEAN 1898.33 99.33 2.33 101.67 2000 3.27 5.08 15.28 #   2.20+† SD 94.50 95.00 0.58 94.50 0.00 0.67 4.73 23.89     TC = Test compound; N = Normal mitotic cells (comprising prophase, metaphase, anaphase and telophase); ABN = Aberrant mitotic cells; SD = Standard deviation; CP = Cyclophosphamide; EMS = Ethylmethane sulphonate; C.s = Cannabis sativa; MI = Mitotic index; Ɉ = P+M/ A+T ratio (significant increase in ratio compared to negative control, P<0.05 in the t-test, n = 3); * = TC is Toxic (MI treatment significantly different from negative control, P<0.05 in the t-test, n = 3); # = TC is genotoxic (significant difference from negative control, P<0.05 in the t-test, n = 3); +† = C.s + Mutagen mixture more genotoxic than mutagen or C.s alone (Synergism); +‡ = C.s + Mutagen mixture less geno- toxic than mutagen or C.s alone (antagonism); † = C.s + Mutagen mixture more genotoxic than mutagen alone but less than C.s alone; ‡ = C.s + Mutagen mixture less genotoxic than mutagen alone (antimutagenicity) but more than C.s alone. 143Evaluation of extracts of wild Cannabis sativa L. for genotoxicity and phytochemical composition synergism or synergistic interaction between the C. sativa extracts and EMS at those concentrations were inferred. 3.3.3 Results for ethylacetate extract experiments in Table 4 In Table 4 are the results of cytotoxicity and geno- toxicity experiments with ethylacetate extracts of C. sati- va and the mutagens, CP and EMS. (P+M)/ (A+T) Ratio: Examination of the (P+M)/ (A+T) ratio in column 8 of Table 4 shows that only the treatment with the lowest concentration (0.1875 mg/ mL) of C.sativa extract induced a significant change in (P+M)/(A+T) ratio, when compared with the solvent (2.5% acetone) treated negative control group (p < 0.05). Cytotoxicity: Examination of the MI in column 9 of Table 4 shows that none of the treatments induced a reduction of the MI when compared to the solvent (2.5% acetone) treated negative control (P<0.05) and were adjudged none toxic to the root meristem cells of A. cepa i.e. CP (1.25 mg mL-1), EMS (0.25 mg mL-1 and all con- centrations of the ethylacetate extract of C. sativa (0.1875, 0.375, 0.75 mg/mL) and the mixtures of the individual concentrations of the C. sativa extract with CP (1.25 mg mL-1) or EMS (0.25 mg mL-1) were all none toxic. Genotoxicity (GT): Examination of induction of gen- otoxicity in column 10 of Table 4 shows that CP (1.25 mg mL-1) and EMS (0.25 mg mL-1) were genotoxic to the root meristem cells of A. cepa, when compared to the solvent (2.5% acetone) treated negative control (P<0.05). The two lowest concentrations of ethylacetate extracts of C. sativa (0.1875 and 0.375 mg mL-1) were also genotox- ic. The mixture of each concentration (0.18750, 0.375 or 0.75 mg/mL) of C. sativa extract with CP (1.25 mg mL-1) was also genotoxic. The mixture of each concentration of the two higher concentrations (0.375 or 0.75 mg/mL) of C. sativa extract with EMS was also genotoxic, but not the mixture of the lowest concentration (0.1875 mg/mL) with EMS. Modulatory effect (ME) of ethylacetate extract of C. sativa on CP or EMS-induced Genotoxicity (GT): Examination of the modulatory effect (ME) in col- umn 11 of Table 4 shows that the mixture of the low- est concentration of the ethylacetate extract of C. sativa (0.1875 mg/mL) with CP (0.25 mg/mL) was none signifi- cantly (< twofold, ME = 0.47) more genotoxic than CP alone but not the ethylacetate extract (0.1875 mg/mL) of C. sativa alone. Each mixture of C. sativa extract (0.375 or 0.75 mg/mL) with CP was none significantly (< two- fold, ME = -0.50 and -0.73 respectively) less genotoxic than CP or C.s extract alone. Each mixture of C. sativa extract (0.1875 or 0.375 mg/mL) with EMS was none sig- nificantly (< twofold, ME = -0.81 and -0.64 respectively) less genotoxic than EMS or C.s extract alone. However the mixture of the highest concentration of C.s extract (0.75 mg/mL) with EMS was significantly (> twofold (200%), ME = 2.20) more genotoxic than EMS or C.s extract alone, and synergism or synergistic interaction between the EMS and C. sativa extract, at that concen- tration, was inferred. 4. DISCUSSION In this study, hexane, chloroform, ethyl acetate and methanol extracts of the aerial parts of Cannabis sativa (C. sativa) growing in Lesotho and used in traditional medicine to treat some diseases and for recreational pur- poses were evaluated for phytochemical composition, genotoxicity and modulatory effects on EMS- and CP – induced genotoxicity using the onion (Allium cepa L.) chromosome aberration assay system. The results of the phytochemical screening tests are presented in Table 2 while the results of the genotoxicity tests are presented in Tables 3 and 4. In the present qualitative study presented in Table 2, based on the intensity of the colour in the colorimetric tests and (or) the appearance of precipitates, during the identification reactions, methanol extract contained the highest number of the different phytochemical classes (15/19) followed by hexane and chloroform (9/19 each) and ethylacetate (7/19). Such tests allow a semi-quanti- tative evaluation for the presence of secondary metabo- lites in extract solutions (Chukwudi and Yusha’u 2016). The phytochemicals detected in the extracts of C. sativa in the present study which have been detected in extract of C. sativa previously include flavonoids, sterols and alkaloids (Pollastro et al. 2018) and flavonoids, alkaloids, sterols, saponins, tannins and terpenoids in extracts of Cannabis indica (Pollastro et al. 2018). Cardiac glyco- sides and cardenolides, proteins and fatty acids were not detected in any of the solvent extractives. A study by Audu et al. (2014) using C. sativa L. procured from the National Drug Law Enforcement Agency (NDLEA) in Nigeria revealed a high presence of cardiac glycosides in petroleum ether (a non polar solvent) crude extract of C. sativa leaves. The absence of these biomolecules from the extracts of the solvents used in the present study to dif- ferences in the strain of C. sativa used and the diffenc- es in the climate, soil and topography between Lesotho (temperate climate with two-thirds of the terrain being mountainous and over 80% of soils in the lowlands being acidic) and Nigeria (tropics) where the plants were grown, and the different extraction methods used, petro- 144 Asita Okorie Asita, Sibusisiwe Magama, Thato Mamoroesi Moahloli, Selometsi Baholo leum ether (Audu et al. 2014) and methanol, hexane, chloroform and ethylacetate in the present study (Rame- let 2015).The most ubiquitous classes were the polyphe- nols, anthocyanins, betacyanins, quinones, coumarins and amino acids which were detected in all the solvent extracts. These compounds were also detected in petro- leum ether extracts of leaves of C. sativa in the study by Audu et al. (2014). The flavonoids, alkaloids, saponins, phlobatannins and anthraquinones were detected at different colour intencies only in the methanol extract while phytosterols were present at high intensity only in the hexane extract, since hexane is a non polar sol- vent. Terpenoids were detected only in the hexane and methanol extracts. Constituents such as carotenoids, terpenoids, ascorbates, reducing sugars and tocopherols are known to contribute to the antioxidant, antiviral, anticancer and anti-inflammatory properties of phenolic compounds (Bang et al. 2015; ElSohly et al. 2017; Andre et al. 2016). In another study by ElSohly et al. (2017) they identified the quinone 2-geranyl-5-hydroxy-3-n- pentyl-1,4-benzoquinone in extracts of leaves and buds of C. sativa using several chromatographic techniques. The differences in biological activities of different solvent fractions have been demonstrated by other research- ers. Mihailović et al. (2013) studied the antioxidant and antigenotoxic activities of chloroform, ethyl acetate and n-butanol fractions obtained from the methanolic extract of Gentiana asclepiadea L. roots. Among all frac- tions, the ethyl acetate fraction exhibited the highest antioxidant activity, as well as total phenolics (146.64 GAE/g), flavonoids, flavonols and gallotannins contents. Only the total phenolics content of crude hexane, chloroform, ethylacetate and methanol extracts of C. sativa were determined in this study. The determined value of total phenolics (Figure 1) of the hexane, chloro- form, ethylacetate and methanol extracts were, 39831.46, 2544.94, 2438.20 and 56601.12 mg GAE/gram dry weight respectively. The methanol extract, being the most polar, had the highest content of phenolics. In a study con- ducted by Mkpenie et al. (2012), the polyphenol content of the acetone and methanolic extracts of C. sativa was found to be in the range of 0.090 – 0.556 mg GAE/g dry weight. We therefore attribute the higher content of total phenolics observed in the present study to the differ- ent extraction times; 2, 8 and 18 hours in the study by Mkpenie et al. (2012) compared to the extraction time of 48 hours in the current study. As shown in Tables 3 and 4, the concentrations of CP (1.25 mg mL-1) and EMS (0.25 mg mL-1) used in the present study did not reduce the mitotic index (MI) of meristem cells of the treated roots compared with the negative control, and were adjudged not cytotoxic. The concentrations of CP and EMS used however induced genotoxicity in the root meristem cells of A. cepa. In a study by Çelik and Aslantürk (2010), EMS at a concen- tration of 2x10-2 M (0.2484 mg mL-1) was both toxic and mutagenic to root meristem cells of A. cepa. CP at a concentration of 1% (1 mg mL-1) was also both toxic and clastogenic to onion root meristem cells (Akeem et al. 2011). The results of the assessments of the cytotoxic and genotoxic effects of the methanol and ethyl ace- tate extracts of C. sativa are presented in Tables 3 and 4. Only the lowest concentration (0.75 mg mL-1) of the methanol extract and its mixture with EMS (0.25 mg mL-1) (Table 3) and the lowest concentration (0.1875) of the ethyl acetate extract (Table 4), significantly reduced the (P+M)/(A+T) ratio. A decrease in the proportion of dividing cells in A+T is an indication of metaphase arrest due to the poisoning of the spindle fibers, akin to the action of the well documented spindle poison, col- cemid (Parry et al. 1999). The chemotherapeutic agents taxol, vincristine, vinblastine and nocodozole act in a similar manner (Alberts et al. 2008). These compounds act by binding to and stabilizing microtubules, inhibit- ing their dynamic instability and cuasing various genetic disruption, including the induction of cell cycle arrest (Alberts et al. 2008; Zhang et al. 2015). In the present study, it seems that cell cycle arrest at the metaphase/ anaphase junction by these extracts depended on con- centration as only the lowest concentration (0.75 mgmL- 1) of the methanol extract and the lowest concentra- tion (0.1875 mg/mL) of the ethylacetate extract induced mitotic cell arrest. All three concentrations (0.75, 1.5 and 3.0 mg mL-1) of the methanol extract of C. sativa and their individual mixtures with CP or EMS (Table 3) tested were cyto- toxic to the onion root meristem cells. None of the three concentrations (0.1875, 0.375 and 0.75 mg mL-1) of the ethylacetate extract of C. sativa alone or in mixtures with CP or EMS (Table 4) was cytotoxic to the onion root meristem cells. Plant secondary metabolites, such as the ones detected in the extracts of C. sativa in the present study, are the key drivers of the pharmacologi- cal actions of medicinal plants and have been shown to possess various biological effects including antibiotic, antifungal, antiviral and cytotoxic effects and therefore are able to protect plants from pathogens (Asche 2005; Hussein and El-Anssary 2018, Priyanka et al. 2019). The toxicity of the extracts observed in the present study was therefore attributed to the presence of cytoxic sec- ondary plant metabolites in the solvent extracts. In the Ames assay with extracts of C. sativa diluted with olive oil as well as the extracts produced with an isopropanol 145Evaluation of extracts of wild Cannabis sativa L. for genotoxicity and phytochemical composition and supercritical CO2 extraction method, toxicity was evident for strains TA 98, TA 1535, TA 1537 and E. coli WP2 uvrA at≥50 μg/plate, with and without S9, in the plate incorporation and/or pre-incubation tests (Dzi- wenka et al. 2020). These results are similar to results of other researches that demonstrated cytotoxicity of plant extracts including betel and tobacco leaf extracts and some Nigerian folk medicines to root-tip cells of A. cepa (Sopova et al. 1983; Abraham and Cherian 1978). Regarding the genotoxicity of the extracts, all three concentrations (0.75, 1.5 and 3.0 mg mL-1) of the meth- anol extract of C. sativa and their individual mixtures with CP or EMS (Table 3) tested were genotoxic to the onion root meristem cells. The 0.1875 and 0.375 mg mL-1 dilutions of the ethyl acetate extract and their individual mixtures with CP and the mixture of 0.75 mg/mL ethyl acetate extract with CP were also genotoxic. In addition, the mixture of each concentration (0.375 or 0.75 mgmL-1) of the ethyl acetate extract with EMS was also genotoxic. The chromosomal abnormalities observed following treatment of the root tip cells of A. cepa with methanol and ethyl acetate extracts of C. sativa alone or in mix- ture with CP or EMS included sticky chromosomes, c-mitosis, chromosome largards, Chromosome frag- ments, and anaphase and telophase bridges. Genotoxic effects of different medicinal herbs in A. cepa have been demonstrated (Soliman 2001; Bidau et al. 2004; Çelik and Aslantürk 2007; Akinboro and Bakare 2007; Akintonwa et al. 2009; Oyedare et al. 2009). Mari- juana smoke condensates were mutagenic to the TA98 strain in the Ames Salmonella/microsome bioassay but only in the presence of liver homogenates (Busch et al. 1979). However a supercritical CO2 extract of the aerial parts of the C. sativa, was not mutagenic in the Ames bacterial reverse mutation test, in vitro mammalian chromosomal aberration test, or in an in vivo mouse micronucleus study (Marx et al. 2018). In another assess- ment of extracts of hemp (C. sativa) using the Ames reverse mutation assay, the extracts produced with an isopropanol and supercritical CO2 extraction methods were diluted with olive oil and the undiluted extract for- mulated as a solution in DMSO; no mutagenic effect was observed in the four strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and one strain of E. coli (WP2 uvrA) that were used (Dziwenka et al. 2020). In the present study, methanol and ethyl acetate extracts of the areal parts of C. sativa dissolved in 2.5% acetone as solvent, induced genotoxiciy in the A. cepa root mer- istem cells. The modulatory effects (ME) of the extracts on mutagen-induced genotoxicty are presented in col- umns 11 and 12 of Tables 3 and 4 for the methanol and ethyl acetate extract respectively. The ME indicated the type of interaction between the extract and the muta- gen. The mixture of each concentration (0.75, 1.5 and 3.0 mg mL-1) of the methanol extract with CP, Table 3, was significantly (>two-fold) more genotoxic than the mutagen (CP) alone with +ME values of (11.27, 11.89 and 6.43) respectively. The mixtures were also more genotoxic than the C. sativa extract alone at each con- centration. The mixtures of the different concentrations (0.75, 1.5 and 3.0 mg mL-1) of the methanol extract of C. sativa with EMS were also more genotoxic than the mutagen (EMS) alone with positive ME (+ME) values of (8.10, 1.61 and 2.14) respectively. Only the +ME val- ues of the mixture of EMS with the lowest (0.75 mgmL-1) or highest (3.0 mgmL-1) concentration were significant, i.e. >two-fold the genotoxicity induced by the muta- gen alone. Theses results indicated a synergistic inter- action between each of the three concentrations of the methanol extract of C. sativa with CP and between two concentrations of the methanol extract C. sativa with EMS. The mixture of 0.1875 mgmL-1 of the ethylyac- etate extract of C. sativa and CP (Table 4) was insig- nificantly more genotoxic (ME = 0.47) than the muta- gen (CP) alone; the mixture of 0.375 or 0.75 mgmL-1 of the ethylacetate extract with CP was insignificantly less genotoxic (ME = -0.50 and -0.73 respectively) than CP alone and therefore no interaction between CP and ethy- lacetate extract was inferred. Each mixture of 0.1875 or 0.375 mgmL-1 of the ethylacetate extract with EMS was insignificantly less genotoxic than EMS alone with nega- tive ME (-ME) values of -0.81 and 0.64 respectively. The mixtures were also less genotoxic than the ethylacetate extract alone. The mixture of 0.75 mgmL-1 of the ethyl acetate extract with EMS was significantly more geno- toxic (+ME value of 2.20) than EMS or C. sativa extract alone. The significant +ME value indicated a synergistic interaction between the ethyl acetate extract of C. sati- va with EMS at the highest concentration only. These results demonstrated that the methanol and ethylacetate extracts of C. sativa did not exert any anti-genotoxic effects on CP- or EMS- induced genotoxicity. Lack of or differential anti-genotoxic activity of different sol- vent extracts have been demonstrated in other test sys- tems. Mihailović et al. (2013) studied the antioxidant and antigenotoxic activities of chloroform, ethyl acetate and n-butanol fractions obtained from the methanolic extract of Gentiana asclepiadea L. roots and found no significant difference in the antigenotoxic effect of the different fractions against EMS-induced DNA damage in the in vivo sex linked recessive lethal mutations assay in Drosophila melanogaster males (Mihailović et al., 2013). The differential effects of different concentrations of 146 Asita Okorie Asita, Sibusisiwe Magama, Thato Mamoroesi Moahloli, Selometsi Baholo plant extracts or plant derivatives on mutagens-induced genotoxicity have been demonstrated in many test sys- tems. In mice, an increase in the anticlastogenic activity of CP-induced clastogenicity by β-carotene at lower dos- es and an absence of a protective effect at higher concen- trations were observed (Salvadori et al. 1992). Salvadori et al. (1992) interpreted the observations to mean differ- ent mechanisms of β-carotene modulation and a possible alteration of the balance of CP activation/detoxification mechanism of the promutagen. While no study on the antimutagenic activity of C. sativa extract was found, however, two pure terpenes that are found in canna- bis (Bedini et al. 2016), D-linalool (LNL) and myrcene (MYR), were efficient against t-butyl hydroperoxide (t-BOOH) induced genotoxicity in the reverse mutation assay with E. coli and oxyR mutants and in the com- et assay using cultured human hepatoma HepG2 and human B lymphoid NC-NC cells, which was predomi- nately mediated by direct radical scavenging activity (Mitić-Ćulafić et al. 2009). Another pure terpene, found in cannabis, bisabolol (BISA), caused a reduction in the levels of Aβ-induced chromosomal damage and apop- tosis in PC12 cells (Shanmuganathan et al. 2018). It is now well accepted that the health benefits of fruits, veg- etables and other plant foods are due to the synergy or interactions between the different bioactive compounds or other nutrients present in the whole foods, and not to the action of a sole compound (Liu, 2013). Similarly, we attribute the differences in the actions between the ethyl acetate and methanol extracts in the induction of cytotoxicity, genotoxicity and modulatory effects at dif- ferent extract concentrations observed in this study to the synergistic or antagonistic interactions between vari- ous phytochemicals present in the extracts. According to Efferth and Koch (2011) cannabis-based therapeutics in humans, exert their pharmacological effects via syn- ergistic or antagonistic interactions between the various phytochemicals it contains. 5. CONCLUSION The order of effectiveness at extracting, from the aerial parts of A. sativa, the 19 different phytochemi- cals investigated in the present study was 95% metha- nol (15/19), followed by hexane and chloroform (9/19 each) and ethylacetate (7/19). Total phenolics content, in mg GAE/gram dry weight of extract was 95% methanol (56601.12) > ethylacetate (2438.20) > chloroform (2544.94) > hexane (39831.46). The methanol extract was both cyto- toxic and genotoxic to the A. cepa root meristem cells, but the ethyl acetate extract was not cytotoxic but genotoxic. Mixtures of methanol extract (0.75, 1.5 and 3.0 mg mL-1) with either CP or EMS were more genotoxic than the CP, EMS or extract alone which demonstrated a synergis- tic interaction between the methanol extract of C. sativa with CP and between two concentrations of the metha- nol extract with EMS. Mixtures of ethyl acetate extract of C. sativa (0.1875, 0.375, 0.75 mg/mL) with either CP or EMS were generally insignificantly (p<0.05) less genotoxic than CP, EMS or extract alone. Thus no interaction was observed between all three concentrations and the two lower concentrations of ethylacetate extract with CP or EMS. There was however a synergistic interaction between the highest concentration of the ethylacetate extract with EMS (+ ME of 2.2). The methanol and ethylacetate extracts of C. sativa did not exert any anti-genotoxic effects on CP- or EMS- induced genotoxicity. 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