Annals of West University of Timişoara, ser. Biology, 2018, vol. 21 (2), pp.133-142 133 AN INVESTIGATION OF PHYTOCHEMICALS, ANTIOXIDANT AND GENOTOXIC POTENTIAL OF DATURA METEL LINN. Oluwole Olusoji ELEYOWO 1* , Oluwafemi Daniel AMUSA 2 , Mutiat Adetayo OMOTAYO 1 , Utomobong Udom AKPAN 1 1 Department of Science Laboratory Technology, Lagos State Polytechnic, Nigeria 2 Department of Cell Biology and Genetics, University of Lagos, Nigeria *Corresponding author e-mail: oluwoleeleyowo@gmail.com Received 6 July 2018; accepted 9 December 2018 ABSTRACT The use of plants with medicinal properties dates back decades and has been widespread. But regardless of their reported benefits, they are not completely harmless. Datura metel have been used over the years for various medicinal purposes with few reported complaints from its use. There is a need to evaluate the plant’s toxicity in a model organism other than man that could translate similar toxicity response. The phytochemicals, antioxidant and genotoxicity of crude aqueous and methanol leaf, seed and root extracts of D. metel was evaluated. The study showed no significant difference in the alkaloids, tanins and saponins in aqueous and methanol extracts. Steroids and terpenoids was not found in all plant parts evaluated regardless of extraction medium. However, flavonoid was higher in leaf and seed methanol extracts than aqueous extracts. Likewise, total flavonoid, total phenol except DPPH in aqueous leaf extract higher than methanol leaf extract. Also, total phenol in root extracts was lower than that observed in root methanol extract. Proliferation in all plant part extracts of aqueous and methanol was significantly lower when compare to the control with leaf extracts having the least mitotic index. The study observed aberrations which include adherent nucleus, c-mitosis, anaphase bridge, binucleate cells and sticky cells. Percentage aberration was highest with leaf methanol extract than other plant part extracts. Both aqueous and methanol extracts of D. metel plant parts showed high proliferation inhibition and increase aberration cells even at 2 mg/ml suggest the need for further safe dose determination of the plant before any medicinal use. Hence, still requires further safety studies to ascertain its dose limits permissive for human usage. KEY WORDS: antioxidant, Datura metel, genotoxicity, phytochemicals, toxicity INTRODUCTION Datura metel Lin., is a plant in the family of Solanaceae, indigenous to Mexico, naturalized in many other parts of the world (Satina & Blakeslee, 1941). It is usually found in dump sites in the urban northern part of Nigeria (Mann et al., 2003). Locally, it is known as zakami in Hausa, gegemu or apikan in Yoruba and Myaramuo in Igbo with common names such as nightshade plant, Thorn apple Devil’s apple, Devil’s trumpet etc. (Babalola, 2014). It is a foul-smelling, erect, annual, freely branching herb that forms a bush up to 60 to 150 cm (2-5 ft) tall with long, thick, fibrous, white root; stem is stout, erect, leafy, smooth, and pale yellow- green and leaves are about 8 to 20 cm (3-8 in) long, smooth, toothed, soft, and irregularly ELEYOWO et al: An investigation of phytochemicals, antioxidant and genotoxic potential of Datura metel Linn. 134 undulated; leaves have a bitter and nauseating taste, which is imparted to extracts of the herb, and remains even after the leaves have been dried. The egg-shaped seed capsule is 3 to 8 cm (1–3 in) in diameter and either covered with spines or bald. At maturity, it splits into four chambers, each with dozens of small, black seeds (Praseetha et al., 2009). Various parts of the plant are consumed but the seed have been reported to be use as psychoactive substance (Babalola, 2014). Local application includes local application for rheumatic swellings of the joints, lumbago, sciatica, neuralgia, painful tumours, scabies, eczema, allergy and glandular inflammations, pile (Donatus & Ephraim, 2009), swelling gum, breast pain (Rahmatullah et al., 2010), diabetes (Murthy et al., 2004) to mention a few. Scientific studies and the results on antimicrobial, antioxidant and phytochemical screening on ethanol and hydro alcoholic crude extracts of D. metel plant have been reported (Okoli et al., 2007). Despite the widespread use of herbal medicines globally and their reported benefits, they are not completely harmless (Tracy & Kingston, 2007). The presence of cytotoxic and mutagenic substances in their composition or resulting from their metabolism can cause damage to human health (Tedesco & Laughinghouse, 2012). However, the ability for recognition for toxic plants has been by trial and error in recent times. This has made information about their safety and efficacy generally sparse, D. metel is no exception. Therefore, there is a need to evaluate the plant’s toxicity in a model organism other than man that could translate similar toxicity response. The use of Allium cepa L. (onion) is an excellent model in vivo suitable for genotoxic studies and has become well established for the determination of the genotoxic substances in various environments. This is because the root growth dynamics is very sensitive to the pollutants; the mitotic phases are very clear in the onion; it has a stable chromosome number; diversity in the chromosome morphology; stable karyotype; clear and fast response to the genotoxic substances and spontaneous chromosomal damages occur rarely (Firbas & Amon, 2013). Hence the use of A. cepa test for such purpose. The aim of the study was therefore to evaluate phytochemical, antioxidant and genotoxicity potential of aqueous and methanol extracts of D. metel. MATERIALS AND METHODS COLLECTION AND PREPARATION OF PLANT MATERIALS Fresh leaves, roots and seeds of D. metel were collected from Alakia area in Ibadan, Oyo state. The collected plant materials were identified at the herbarium, Lagos State Polytechnic, Ikorodu, Lagos state, Nigeria. The plant materials were washed with distilled water, dried under shade for 30 days and then ground into fine powder using electrical grinder. The powdered materials were stored in airtight containers at 4 o C until needed. 100 grams each of leaf, root and seed powder of D. metel were soaked in 500 ml of distilled water and 350 ml of methanol separately, and kept for 24 hours at room temperature. The mixtures were filtered through a clean muslin cloth and the filtrate again filtered by using Whatmann No.1 filter paper. The above procedure was repeated for another 24 hours before the residuals were discarded. Then, the extracts were concentrated and dried in a rotary evaporator at 37 °C till a sticky mass was obtained. After evaporation, the dried extracts were stored at 4°C until needed. Annals of West University of Timişoara, ser. Biology, 2018, vol. 21 (2), pp.133-142 135 QUALITATIVE PHYTOCHEMICAL ANALYSIS Stock solutions were prepared from each of the crude extracts dissolving 100 mg each of the methanol and water extracts in 10 ml of its own mother solvents. The obtained stock solutions were subjected to phytochemical screening using the method of Sangeetha et al. (2014). Phytochemicals assayed for include alkaloids, steroids, flavonoids, terpenoids, tannins and saponins. ANTIOXIDANTS EVALUATION 4 mg of both aqueous and methanol extracts were taken and dissolved in 40 ml of methanol separately. The concentrations of the solution were diluted to 100 µg/ml each. Evaluation of total phenol, total falvonoids and 2,2-diphenyl-1-pycrilhydrazyl (DPPH) was done according to Sangeetha et al. (2014). GENOTOXIC EVALUATION TEST Solutions from both methanol and aqueous extract were prepared into various concentrations and subjected to cytotoxic evaluation using the A. cepa test. Onion bulbs of average size (15-22 mm) in diameter were purchased locally in Mile-12 market, Lagos State Nigeria. They were sun dried and the dried roots present at the base were carefully removed. Bulbs were rooted for 24 hours in distilled water. The rooted bulbs were then transferred into the treatments. Three replicates were set up for each treatment and a control setup, and then left for 48 hours. Root tips of each bulb were harvested, fixed in ethanol/acetic acid (3:1 v/v), fixed on a clean glass slide and stained with Aceto-orcein stain. Prepared slides were then viewed under a microscope. ANALYSIS OF DATA Mitotic activity and any aberration kind were recorded for treatments. Mitotic index was estimated according to Sehgal et al. (2006) where MI, P, M, A, T are mitotic index, prophase, metaphase, anaphase and telophase respectively. where PAB is percentage number of aberrant observed RESULTS AND DISCUSSIONS Analysis of phytochemicals in leaf, seed and root extracts of D. metel The study evaluated the presence of six phytochemicals in both the aqueous and methanol extract of D. metel. The results obtained from phytochemical screening revealed the absence of steroids and terpenoids in all extracts. The level of alkaloids, flavonoids and saponins were higher in both aqueous and methanol extracts of leaf and seeds of the plant than the root. The level of tannins showed no difference in all plants evaluated regardless of the medium of extraction (Table 1). Also more phytochemical concentrations were observed with methanol extracts than water extracts. Generally, some of the secondary metabolites studied in leaf, seed and root parts of D. metel were present in higher amount in methanol extracts than the aqueous solvent. This may be as a result of the polarity level of the medium of extraction playing major role in extracting the secondary metabolites as suggested by Ghasemzadeh et al. ELEYOWO et al: An investigation of phytochemicals, antioxidant and genotoxic potential of Datura metel Linn. 136 (2011). The presence of alkaloids in high quantity did not corroborate with the report of Akharaiyi (2011) who reported a moderate quantity of alkaloids in the leaves of D. metel. The absence of terpernoids was also not in corroboration with Akharaiyi (2011) who reported moderate quantity of terpernoids in the seeds and leaves of D. metel. More phytochemicals were observed with methanol extracts than the aqueous extracts contrary to the reports of Akharaiyi (2011) who reported more phytochemicals with aqueous extracts than ethanol extracts samples. Methanol extacts showed more flavonoids than the aqueous extracts of both leaf and seeds. Tannins was found more in the leaves than the roots in the work of Jamdhade et al. (2010) which did not agree with this present work which showed similar tannin concentration in this study. Prasanna & Yuwvaranni (2014) reported the absence of alkaloid and the presence of steroids with aqueous extract which did not corroborate with the present study. Sangeetha et al. (2014) reported similar result with phytochemicals evaluated in this study. Phytochemical constituents which are present in plant samples are known to be biologically active compounds and they are responsible for different activities such as antimicrobial, antioxidant, antifungal, anticancer and antidiabetic (Hossain & Nagooru, 2011) Phytochemical constituents which are present in plant samples are known to be biologically active compounds and they are responsible for different activities such as antimicrobial, antioxidant, antifungal, anticancer and antidiabetic (Hassain & Nagooru, 2011). Different phytochemicals have been found to possess a wide variety of pharmacological activities, which may help in protection against chronic diseases (Sangeetha et al., 2014). Tannins, glycosides, saponins, flavonoids, and aminoacids have hypoglycemic and anti- inflammatory activities. Terpenoids, and steroids shows analgestic properties and central nervous system (CNS) activities. Saponins are involved in plant defense system because of their antimicrobial activity (Ayoola et al., 2008) and also possess hypocholesterolemic and antidiabetic properties. The most effective bio active compounds are alkaloids, aminoacids and saponins these were found in all four types of crude extracts. Flavonoids were found in methanol, chloroform and ethylacetate except hexane extracts. Chloroform and methanol extracts shows the presence of majority phytoconstituents. Many reports are available on flavanoid groups which exhibiting high potential biological activities such as antioxidant, anti- inflammatory, antiallergic reactions (Anyasor et al., 2010; Chao et al., 2002; Igbinosa et al., 2009; Thitilertdecha et al., 2008) TABLE 1: Phytochemical screening for Aqueous and Methanol leaf, seed and root extracts of D. metel Extract Sample Alkaloids Flavonoids Tannins Saponins Steroids Terpenoids Aqueous DLF ++ + + ++ ND ND DSD ++ + + ++ ND ND DRT + + + + ND ND Methanol DLF ++ ++ + ++ ND ND DSD ++ ++ + ++ ND ND DRT + + + + ND ND ++: Highly present; +: moderately present; ND: not detected; DLF: sample leaf; DSD: sample seed; DRT: sample root Annals of West University of Timişoara, ser. Biology, 2018, vol. 21 (2), pp.133-142 137 Antioxidant evaluation The contents of total flavonoid, total phenol and DPPH were evaluated in plant parts of D. metel are shown in Table 2. Sangeetha et al. (2014) in their work reported that total phenolic content ranges from 0.53-3.99mg/ml which did not corroborate with this present study which observed total phenol range between 42-116 mg/g. The study showed that antioxidant contents evaluated were highest in the leaf part of D. metel for both aqueous and methanol extracts except for DPPH where aqueous seed extract gave the highest value for DPPH than both leaf and root aqueous extracts. Usually in many plants, the leaf shows higher antioxidant activities than other parts (Pyo et al., 2004; Wong & Kitts, 2006). Aqueous root extract showed the least DPPH while methanol root extract showed least for both total flavonoids and DPPH. Aqueous seed extract gave the least total flavonoids while methanol seed extract gave the least total phenol among evaluated sample parts. This is similar to Akharaiyi (2011) who also reported higher antioxidant activity in the leaves of D. metel. More antioxidant activities were observed with aqueous extracts than ethanol extracts of the plant. More phytochemicals were also reported for aqueous extracts than ethanol extract (Akharaiyi, 2011). TABLE 2: Antioxidant screening for aqueous and methanol leaf, seed and root extracts of D. metel Extract Plant Part Total Flavonoid (mg of QE/g) Total Phenol (mg of QE/g) DPPH (IC50) Aqueous DLF 7.64 116.75 49.85 DSD 1.19 78.63 65.99 DRT 2.00 42.00 3.45 Methanol DLF 4.07 106.80 81.01 DSD 3.64 46.95 56.34 DRT 3.36 64.70 12.06 DLF: sample leaf; DSD: sample seed; DRT: sample root; IC50: Inhibition concentration at 50%; DPPH: 2,2-diphenyl- 1-pycrilhydrazyl Genotoxic evaluation of D. metel extracts Genotoxicity and cytotoxicity tests using A. cepa test in vivo have been validated by several researchers, who jointly performed animal testing in vitro and the results obtained are similar providing valuable information for human health (Vicentini et al., 2001; Teixeira et al., 2003). Cell proliferation evaluation of water and methanol extracts treatments is presented in Table 4. There was a significant difference between the varied aqueous extract concentrations and the control. Although, the different stages of mitosis observed among the various plant parts varied, there was significant difference between the evaluated plant parts regardless of concentration. Methanol extracts were observed to be significantly reduced when compare to both the control and aqueous extracts evaluated in the study although similar result was observed in terms of plant part comparison and concentration differences (Table 3). In this study, all concentrations evaluated showed reduction in mitotic index when compared to control for both aqueous and methanol extracts in all plant parts used. Mitotic index in the methanol extract was significantly lower than that observe with aqueous plant part extracts. The leaf extracts in both medium of extraction was observed to have lower proliferation index when compared to the other plant parts. ELEYOWO et al: An investigation of phytochemicals, antioxidant and genotoxic potential of Datura metel Linn. 138 TABLE 3: Proliferation evaluation of aqueous and methanol extracts of D. metel plant parts Sample Conc. (mg/ml) Total cells P M A T DC MI Aqueous Extract CTRL 0 1000 339 5 35 160 539 0.54a DLF 2 1001 66 22 8 18 114 0.19b 4 1006 61 19 12 13 105 0.15b 6 980 50 18 17 15 100 0.10b 8 1000 52 21 13 7 93 0.09bc DRT 2 1000 78 23 21 10 132 0.23b 4 1001 54 20 21 11 106 0.21b 6 1006 60 25 19 9 113 0.21b 8 995 72 15 10 12 109 0.20b DSD 2 992 79 25 11 13 128 0.20b 4 997 78 8 14 9 109 0.18b 6 991 60 13 17 10 100 0.15b 8 1002 59 15 10 13 97 0.12b Methanol Extract DLF 2 1000 23 17 22 10 72 0.03c 4 988 24 14 21 12 71 0.03c 6 890 21 16 20 8 65 0.02c 8 1032 20 12 19 21 72 0.02c DRT 2 987 23 12 12 12 59 0.10c 4 993 22 20 10 11 63 0.08c 6 978 21 18 15 8 62 0.08c 8 987 22 11 16 10 59 0.07c DSD 2 996 19 17 16 19 71 0.07c 4 1055 27 12 20 13 72 0.07c 6 1018 21 11 18 15 65 0.06c 8 1014 21 17 16 8 60 0.05c DLF: sample leaf; DSD: sample seed; DRT: sample root; P: prophase, M: metaphase, A: anaphase, T: telophase, MI: mitotic index, DC: dividing cells, MI: mitotic index. Percentage aberration (PAB) was higher with methanol extract than aqueous extracts of D. metel evaluated in the study. PAB was not significantly different between the plant parts so also between concentrations (Table 4). The cytotoxic effect of D. metel extract treatments was evident as observed by a significant decrease in the mitotic index. Our observations support the antiproliferative effects of D. metel extracts similar to that reported by Danhof and McAnally (1983). The production of chromosome abnormalities in all the plant parts’ extracts can be regarded as a reliable evidence of the genotoxicity of the plant’s part. The results indicated that all the treatments induced different types of chromosomal abnormalities in non- dividing cells as well as different mitotic stages. These abnormalities were observed to affect almost all the stages of mitosis. Plants and toxins such as Calotropis procera and podophylatoxins have been described to impact negatively on these processes to cause cell proliferation inhibition or arrest (Sehgal et al., 2006). Annals of West University of Timişoara, ser. Biology, 2018, vol. 21 (2), pp.133-142 139 TABLE 4: Proliferation evaluation of aqueous and methanol extracts of D. metel plant parts Sample Conc. (mg/mL) Total cells AN CMIT BRG BNC STK PAB Aqueous Extract CTRL Neg. 1000 0 0 0 0 0 0 DLF 2 1001 37 16 1 2 0 5.59 4 1006 38 15 5 1 2 6.06 6 980 42 12 3 3 5 6.63 8 1000 33 19 9 5 2 6.80 DRT 2 1000 15 15 3 1 1 3.50 4 1001 21 12 3 0 0 3.60 6 1006 20 10 4 3 2 3.88 8 995 19 18 4 2 1 4.42 DSD 2 992 16 20 1 1 0 3.83 4 997 27 18 2 1 1 4.91 6 991 26 17 8 3 1 5.55 8 1002 29 22 2 7 0 5.99 Methanol Extract DLF 2 1000 41 18 7 3 6 7.50 4 988 51 20 6 5 8 9.10 6 890 55 21 11 6 2 10.67 8 1032 53 36 13 8 3 10.94 DRT 2 987 25 10 11 8 5 5.98 4 993 24 16 5 12 3 6.06 6 978 36 18 4 3 3 6.54 8 987 30 30 2 2 2 6.69 DSD 2 996 39 13 9 5 0 6.62 4 1005 38 25 3 7 1 7.36 6 1018 35 27 10 2 2 7.47 8 1014 45 18 5 9 3 7.89 DLF: sample leaf; DSD: sample seed; DRT: sample root; TOT: total cells counted; AN: adherent nucleus; CMT: c- mitosis; BRG: bridge; BNC: bi-nucleate; STK: sticky cells; PAB: percentage aberration (%) Aberrations observed in the study include adherent nucleus, c-mitosis, anaphase bridge, binucleate cells and sticky cells. Adherent nucleus was observed to be the highest aberration among both the aqueous and methanol extracts while the least aberration was sticky cells (Table 4; Figure 1). In aetiology terms, c-mitosis has been explained to occur due to inhibition of microtubule formation during mitosis and this may lead to aneuploidy and cell death, while stickiness is due to interchromosomal linkages of sub-chromatid strands coupled with excessive formation of nucleoproteins and inappropriate protein-protein interaction (Chattopadhyay et al., 2004; Turkoglu, 2007). The latter is also believed to have resulted from altered physico-chemical properties of DNA due to interactions with other chemicals viz-aviz: mutagens, carcinogens and clastogenic agents (Badr & Ibrahim, 1987). Adherent nucleus was observed as the highest aberration in all test plant part extracts regardless of extraction medium. This is followed by C-mitosis. This indicates a relatively as suggested in the works of Badr & ELEYOWO et al: An investigation of phytochemicals, antioxidant and genotoxic potential of Datura metel Linn. 140 Ibrahim (1987). The observation of binucleated cells formation signifies inhibition of cytokinesis following telophase (Majewska et al., 2003). In A. cepa, such inhibition arrest cell plate formation and this has been attributed to phlamogram inhibition at the early stage of telophase (Badr & Ibrahim, 1987; Majewska et al., 2003). Generally, these aberrations were observed to be mostly caused by root extract of D. metel concentrations, suggesting their greater genotoxic effects on A. cepa when compared with extracts from other plant parts regardless of the concentrations of the extract. Furthermore, some of the chromosomal aberrations educe by D. metel were also comparable to effects due to NaN3, a known mutagenic and clastogenic agent (Iwalokun et al., 2011). FIG. 1. Cell aberrations observed during the study (A) Prophase (B) Metaphase (C) Anaphase (D) Telophase (E) Anaphase bridge (F) Adherent nucleus (G) C-mitosis (H) Sticky cell (I) Bi-nucleate cell A B C D E F G H I Annals of West University of Timişoara, ser. Biology, 2018, vol. 21 (2), pp.133-142 141 Studies using bioindicators of toxicity and mutagenicity, such as the in vivo test of A. cepa are necessary for contributing to their safe and efficient use (Ianovici et al, 2009; Tedesco & Laughinghouse, 2012). CONCLUSIONS Extracts of D. metel plant parts elucidates that methanol root extract was the most toxic from this study with aqueous leaf extract being the least toxic in the A. cepa test. This suggest the need for safe dose determination of the plat before any medicinal use. Hence, still requires further safety studies to ascertain its dose limits permissive for human usage. REFERENCES • Akharaiyi F.C. 2011. Antibacterial, Phytochemical and Antioxidant activities of Datura metel. 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