Phytochemical profiling with cytotoxic and genotoxic potential of green tobacco leaf extract

Corresponding author: Swati Sandhya Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution License 4.0. Phytochemical profiling with cytotoxic and genotoxic potential of green tobacco leaf extract Shalini Mehta 1 and Swati Sandhya 2, * 1 Assistant professor, Department of Botany, Ranchi Women’s College, Ranchi. 2 PhD Scholar, Department of Botany, Ranchi University, Ranchi World Journal of Biology Pharmacy and Health Sciences, 2025, 22(01), 395-410 Publication history: Received on 26February 2025; revised on 16 April 2025; accepted on 18 April 2025 Article DOI: https://doi.org/10.30574/wjbphs.2025.22.1.0412 Abstract Tobacco (genus Nicotiana), primarily cultivated for commercial use, poses significant occupational health risks, particularly during its green leaf stage. This study investigates the cytotoxic and genotoxic effects of green tobacco (Nicotiana tabacum L.) through phytochemical analysis using GC-MS and cytological assays on Allium cepa. GC-MS results revealed major compounds including nicotine (42.55%), naphthalene (34.08%), neophytadiene, phytol, and various methyl esters. In cytological duration dependent exposure of root tip meristematic cell and young flower buds in green leaf extracts showed reduced mitotic index as well as meiotic index and different types of chromosomal abnormalities such as stickiness, bridges, laggards, and micronuclei formation. Keywords: GC/MS;Green tobacco leaves; Genotoxicity; Cytotoxicity; Pulverization 1. Introduction Tobacco belonging to genus Nicotiana is the universally cultivated nonfood cash crop. The genus is considered to be originated in South America, several species of Australia and South pacific Islands are known as derivatives of the South America. In India tobacco was introduced by Portuguese in 1605. It belongs to family Solanaceae. There are about 70 recognized species of Nicotiana, but two species Nicotiana tabacum L.andNicotiana rustica L. are commonly cultivated for producing commercial tobacco. India, having diverse climatic condition which supports the cultivation of both the species of Nicotiana in different regions. Northern and northeastern areas of the country i.e. Uttar Pradesh, West Bengal Bihar and Assam are dominated by Nicotiana rustica as it requires cooler climate. Nicotiana tabacum is cultivated in the region of Gujarat, Andhra Pradesh, north Bihar and some area of Bengal. The N. tabacum also known as desi plants are taller than N. rustica and have broad leaves with pink flower whereas N. rustica is also known as “vilayati and calcuttia and short plants have round puckered leaves with yellow flower. Out of 70 recognized species of Nicotinana, 45 species are maintained in India. 0.45million hectare land is engaged in tobacco cultivation in India i.e.0. 27 % of the net cultivated land producing 750 M kg of tobacco leaf. Its cultivation is providing livelihood security to around 36 million people that include 6 million farmers, 20 million farm labour and 10 million people who are engaged in tobacco processing, manufacturing and export sector (ICARCTRI1). As India is the second largest producer of tobacco in the world after China and country is also second largest exporter of unprocessed tobacco. Thus tobacco exports contribute sizable foreign exchange to the Indian exchequer. There is an increase in export growth by 87% during the past 5 years i.e. it achieved a record high export value of Rs. 12,005.89 crore in the year 2023-24 as against Rs. 6,408.15 crore in 2019-20. During this period the export volume have increased from 218.84 million Kg to 315.51 million Kg (Ministry of Commerce and Industry). Besides harmful usage of cured tobacco leaves in smoking and chewing green leaf was known for its medicinal values. In India and China, raw green tobacco leaves were used for treating painful piles, rheumatic swelling (Agyare 201319). World Journal of Biology Pharmacy and Health Sciences, 2025, 22(01), 395-410 396 Since ancient times these plants are used by Peruvian Amazonian community for treating mental health, parasitic illness and respiratory problems, especially Nicotiana rustica L. is described as master plant in this region. The green tobacco preparation was administered in either liquid or solid form via oral, tropical intranasal and rectal note. Although there are many reporting about the neuropsychiatric disorders caused by green tobacco leaves but still it is used in native place Peruvian Amazon because there was a notion that green tobacco having spiritual energetic property (Berlowitzet.al.202020). On small scale green tobacco extracts is used by local farmers as green pesticides to kill pests like aphids, jassids and whiteflies. The high nicotine content is also having insecticidal properties (Gudetaet. al. 202121). Tobacco farming, although economically important, has considerable health and social implications, especially for agricultural workers and labours engaged in harvesting, curing, and processing the leaves. One of the most critical health issues among tobacco workers is Green Tobacco Sickness (GTS). It is as toxic as cured form i.e. smokeless tobacco (khaini) and cigarette.Few research papers are focused on occupational anthropogenic hazard of green tobacco leaves responsible for Green tobacco sickness (GTS) prevalent in Asian and South American tobacco harvesters. The sickness occurs due to the intradermal absorption of nicotine from the wet surface of tobacco plants. GTS symptoms include nausea, vomiting, dizziness, delirium, increased perspiration, abdominal pain, diarrhea, increased salivation, weakness, breathlessness and occasional lowering of blood pressure (SHAILEE F et.al 201722). Vomiting can lead to dehydration and adds to the risk of heat illness. These symptoms are so common that GTS remains ignored and so not well documented. Tobacco is grown in more than 100 countries and tobacco processing is all done manually by the labourer so there is extended exposure of GTS which may lead to disease like cardiovascular or may have mutagenic effect which manifested as cancer (Mc Bride et.al.199823). The prevalence of GTS among workers varies between 8% to 47%, with increased susceptibility among women, adolescents, and individuals working without protective gear. In this study phytochemicals analysis of fresh green tobacco leaf has been done through GC/MS and genotoxicity and cytotoxicity were assessed by analyzing chromosomal aberrations in meiotic and mitotic cells of Allium cepa flower bud as well as meristematic cells of root tip respectively. 2. Material and methods 2.1. Sample collection Fresh green tobacco leaves were grown from seeds procured from Indian Agricultural Research Institute Pusa, Bihar. 2.2. Extract Preparation for GC/MS Analysis Fresh green tobacco leaves were dried in shade and powdered.For extract preparation 100 gm of dried leaf powder was extracted with 500ml of methanol using an orbital shaker for 72 hours. The extraction process was repeated with the same solvent until the solvent became clear and colourless. The resulting extract was then evaporated for drying and stored in an airtight container at 4 °C for future use. 2.3. GC/MS Analysis Methanol extract of green tobacco leaves was sent to AIRF, JNU, New Delhi for GC-MS analysis. GC-MS analysis was carried out in Shimadzu QP-2010 Plus with Thermal Desorption System TD 20. The carrier gas used was Helium at the flow rate of 16.3 ml/min and column flow rate of 1.21 ml/min. The amount of sample used was 6µl, and the mode of injection was split with a temperature of 260 ºC. The column oven temperature was set at 100 ºC. The total running time of GC-MS was 40 minutes. For determining the phytochemicals, the obtained retention time and mass weight were compared with the GC-MS spectra database of online Wiley library and NIST (National Institute of Standard and Technology). 2.4. Extract preparation for Cytological study For extract preparation 100grams of green leaves were thoroughly crushed with 1000ml double distilled water and left for 10 hours and then filtered through whatman filter paper 5. 2.5. Allium cepa assay Allium cepa seeds were soaked in double distilled water for 9 to 10 hr. Soaked seeds were kept on moist filter paper for germination in flat dishes. Immediately after germination germinated seeds were exposed to green tobacco leaves extract for 15, 30, 45and 60 minutes. After extract exposure root tips were transferred in carnoy’s fixative (1:3 acetic acid: alcohol) for 24 h and were preserved in 70% ethanol. Some unexposed germinated seeds were directly fixed by transferring in carnoy’s fixative and preserved in 70% alcohol which serves as negative control. The exposed tips as World Journal of Biology Pharmacy and Health Sciences, 2025, 22(01), 395-410 397 well as negative control tips were separately heated in acetocarmine for staining and slides were prepared by squash and smear technique and were observed under Magnus s/n: C197050239 microscope. Photographs were taken at 40X. To determine the effect of the leaves extract, mitotic index (MI) were calculated for different time period as: - MI =Total number of dividing cells Total number of cells observed in one focus ×100 Abnormality percentage (AB %) was calculated by following formula. AB % = Total number of abnormal cells Total number of dividing cells in one focus ×100 3. Results The GC-MS analysis of green tobacco leaves revealed the presence of several major compounds (Table 1 and Figure 1) such as nicotine, naphthalene, 1,2,3,6-tetrahydro-2,3′-bipyridine, neophytadiene, phytol, stigmasterol, β-sitosterol, hexadecanoic acid methyl ester, methyl stearate, and 9,12-octadecadienoic acid methyl ester includingsclareolide, palmitic acid derivatives, and various long-chain hydrocarbons and esters. Microscopic examination of exposed meristematic root tips cells and young flower buds of Allium cepa showed gradually decreasing number of dividing mitotic cells as well as dividing meiocytes of bud as compared to control. Different types of chromosomal abnormalities were observed in different phases of cell divisions. The increase in abnormalities was dependent on the exposure time period as mentioned in Tables 2 and 3. The entire experiment was conducted in five replicates. A broad spectrum of clastogenic, aneugenic and non-clastogenic aberrations were observed in mitotic cells of root tips as well as flower bud exposed to green tobacco leaves extract. The clastogenic abnormalities like stickiness (Fig.7,8,9,16,17), laggards (Fig. 8, 12, 19) and non-clastogenic like disorientation at metaphase, anaphase and telophase (Fig.11, 18)were observed at 15 min, 30 min, 45 min and 60 min of exposure time. While micronuclei (Fig. 20), nuclear budding (Fig. 13), abnormal Sporades like triads (Fig 22), disoriented Sporades (Fig 23) were observed only at high exposure time i.e. 60 minutes. Multiple bridges were observed at 30 and 45 minute exposure time (Fig. 10). The study also showed that mitotic and meiotic abnormalities were directly proportional to the exposure time (Figure 4 and 6). Table 1 Compounds identified in methanolic extract of green tobacco leaves (GL) by GC/MS PeakReportTIC Peak# R.Time Area Area% Name 1 7.096 9608896 34.00 NAPHTHALENE 2 8.270 143907 0.51 Sulfurousacid,2-ethylhexylisohexylester 3 9.153 218939 0.77 4-(DIMETHOXYMETHYL)-1,2-DIMETHYLBENZENE 4 9.351 12024327 42.55 PYRIDINE,3-(1-METHYL-2-PYRROLIDINYL)-,(S)- 5 11.063 106355 0.38 2-Buten-1-ol,2-ethyl-4-(2,2,3-trimethyl-3-cyclopenten-1-yl 6 11.144 126991 0.45 Sulfurousacid,2-ethylhexylhexylester 7 11.590 227002 0.80 1,2,3,6-Tetrahydro-2,3'-bipyridine 8 13.667 135320 0.48 Heneicosane 9 15.148 520184 1.84 Neophytadiene 10 15.601 137873 0.49 2,6,10-TRIMETHYL,14-ETHYLENE-14-PENTADECNE 11 16.044 1537843 5.44 Hexadecanoicacid,methylester 12 17.677 102526 0.36 9,12-Octadecadienoicacid,methylester 13 17.738 472748 1.67 8,11,14-Docosatrienoicacid, methylester 14 17.842 620504 2.20 2-HEXADECEN-1-OL,3,7,11,15-TETRAMETHYL-,[R-[ World Journal of Biology Pharmacy and Health Sciences, 2025, 22(01), 395-410 398 15 17.979 917719 3.25 Methylstearate 16 18.378 118181 0.42 4,8,13-Cyclotetradecatriene-1,3-diol,1,5,9-trimethyl-12-(117 18.752 286025 1.01 Cholest-22-ene-21-ol,3,5-dehydro-6-methoxy-,pivalate 18 20.663 40928 0.14 2-Methoxydecanoicacid 19 21.090 130936 0.46 Glycerol,2-TMS20 21.894 63432 0.22 1,2-Cyclohexanedicarboxylicacid,bis(2-ethylhexyl)ester 21 22.913 51424 0.18 Per-O-trimethylsilyl-(3-O-.beta.-d-mannopyranosyl-d-gluci 22 26.065 97789 0.35 7-(2-HYDROXY-1-METHYLETHYL)-1,4A-DIMETHYL 23 27.153 83569 0.30 Stigmasterol 24 27.448 330176 1.17 Stigmasterol 25 28.195 157161 0.56 .beta.-Sitosterol 28260755 100.00 Figure 1 GC-MS Chromatogram of methanol extract of green tobacco leaves World Journal of Biology Pharmacy and Health Sciences, 2025, 22(01), 395-410 399 Table 2 Effect of green tobacco leaf extract on mitotic cells of root tip of Allium cepa Duration control 15 minutes 30 minutes 45 minutes 60 minutes Total number of cell observed 2500 2500 2500 2500 2500 Total number of dividing cell 566 500 453 380 300 Total number of abnormal cells 0 38 47 62 70 Mitotic index (%)± S.E 22.64 ±2.86 20.00± 2.07 18.1 ±2.19 15.2±2.0 12.0 ±1.7 Sticky metaphase - 1.7 ±0.7 1.8 ±0.75 2.8 ±1.01 3.68 ±1.1 Bridge - 0.9 ±0.63 1.05 ±0.62 1.64 ±0.49 2.36 ±0.4 Laggard - 0.96 ±0.64 1.2 ±0.41 1.88 ±0.47 2.63 ±0.62 Micronuclei - 0.7 ±0.41 0.84 ±0.43 1.64 ±0.44 2.36 ±0.44 Pulverized cell - 0.7 ±0.45 1.05 ±0.1 1.4 ±0.74 1.31 ±0.61 Disorientation - 0.5 ±0.49 0.84 ±0.44 1.64 ±0.43 1.81 ±0.60 Blebs - 0.5 ±045 1.05 ±0.1 1.1 ±0.63 1.81 ±0.44 Other abnormalities - 0.7 ±0.42 1.05 ±0.1 1.1 ±0.61 1.31 ±0.65 Abnormality (%)± S.E - 7.6 ± 1.8 10.37 ±1.07 16.31± 4.2 23.33± 1.17 Table 3 Effect of green tobacco leaf extract on meiocytes of bud of Allium cepa Duration Control 15 minutes 30 minutes 45 minutes 60 minutes Total number of cell observed 1750 1750 1750 1750 1750 Total number of dividing cell 1415 1370 1335 1310 1255 Meiotic Index% ± S.E. 80.85± 5.07 78.2 ±5.8 76.2 ±4.5 74.8 ±5.2 71.7 ±3.4 Diakinesis/ Diplotene - - 0.15 ±0.52 0.23 ±0.4 0.3 ±0.43 Metaphase-IStickiness, Disorientation - - 0.35 ± 0.41 0.5 ±0.48 0.6 ±0.37 Anaphase IBridge, Laggard - - 0.33 ± 0.45 0.38 ±0.45 0.55 ± 0.32 Telophase IBridge, Laggard - 0.29 ±0.58 0.39 ±0.41 0.5 ±0.3 Anaphase I, Telophase –IMicronuclei, Disorientation - - 0.22 ± 0.47 0.52±0.37 0.6 ±0.32 Metaphase IIStickiness, Disorientation -- - 0.29 ±0.41 0.44 ±0.51 0.56 ±0.36 Anaphase IIBridge, Laggard - - 0.30 ±0.51 0.35 ±0.44 0.3 ±0.52 Telophase IIBridge, Laggard - - 0.3 ±0.64 0.15 ±0.69 0.15 ±0.51 Anaphase II, Telophase IIMicronuclei, Disorientation - - 0.14 ±0.54 0.35 ±0.56 0.45 ±0.34 Meiotic Products (triad, Pentad Hexad) - - 0.15 ±0.66 0.22 ±0.41 0.35 ±0.64 Sterile Pollen - - - 0.07 ±0.69 0.23±0.58 Other Abnormality Abnormality % ± S.E - - 3.5±0.78 4.2 ±0.49 5.09 ±1.1 World Journal of Biology Pharmacy and Health Sciences, 2025, 22(01), 395-410 400 Figure 2 Effect of green tobacco (Nicotiana tabaccum) leaves extract : Mitotic Index % of Allium cepa L Figure 3 Effect of green tobacco (Nicotiana tabaccum) leaves extract on mitotic cells of Allium cepa L Figure 4 Effect of green tobacco (Nicotiana tabaccum) leaves extract : Abnormality % of Allium cepaL.mitotic cells World Journal of Biology Pharmacy and Health Sciences, 2025, 22(01), 395-410 401 Figure 5 Effect of green tobacco (Nicotiana tabaccum) leaves extract on bud cells of Allium cepa Figure 6 Effect of green tobacco (Nicotiana tabaccum) leaves extract on bud cells of Allium cepa L. (Column) : Abnormality 3.1. Photograph showing effect of green tobacco leaf extract on mitotic cells of root tip of Allium cepa Figure 7 Ring at sticky Prophase World Journal of Biology Pharmacy and Health Sciences, 2025, 22(01), 395-410 402 Figure 8 Laggard and Stickiness at Metaphase Figure 9 Sticky Metaphase Figure 10 Multiple Bridges at Anaphase World Journal of Biology Pharmacy and Health Sciences, 2025, 22(01), 395-410 403 Figure 11 Diagonal Anaphase Figure 12 Laggards at Telophase Figure 13 Nuclear budding World Journal of Biology Pharmacy and Health Sciences, 2025, 22(01), 395-410 410 [14] Kahl VFS, Dhillon VS, Simon D, et al. Chronic occupational exposure endured by tobacco farmers from Brazil and association with DNA damage. Mutagenesis. 2018;33(2):119–128. doi:10.1093/mutage/gex045. [15] Khatab HA, Elhaddad NS, Salih HA. Naphthalene induced “Cross-Talk” between pollen mother cells in Vicia faba L. plants. World J Biol Pharm Health Sci. 2024;19(2):294–310. doi:10.30574/wjbphs.2024.19.2.0506. [16] Klasterska, I., Natrajan, A. T., and Ramel, C., An interception of the origin of chromatid aberrations and chromosome stickiness as a category of chromatid aberrations. Hereditas, 1976, 83, 153. [17] Krupina K, Goginashvili A, Cleveland DW. Causes and consequences of micronuclei. CurrOpin Cell Biol. 2021 Jun; 70: 9199. Doi: 10.1016/j. ceb. 2021. 01. 004. Epub 2021 Feb 18. PMID: 33610905; PMCID: PMC8119331. [18] Kumar, V., and Nagpal, A. K., Genotoxic effects of lead acetate employing Allium sativum root chromosomal aberration assay. Int. J. Adv. Res. Biol. Sci., 2015, 2(1), 193-199. [19] MCBRIDE JEFFERY, D G ALTMAN, M KLEIN AND W WHITE 1998 Green tobacco sickness Tobacco control 7:294298. [20] McGill, M., Pathak, S., and Hsu, T. C., Effects of ethidium bromide on mitosis and chromosomes: A possible material basis for chromosome stickiness. Chromosoma, 1974, 47, 157-167 [21] Pessim, C., Pagliarini, M. S., Silva, N., & Jank, L. (2015). Chromosome stickiness impairs meiosis and influences reproductive success in Panicum maximum (Poaceae) hybrid plants. Genetics and Molecular Research, 14(2), 4195–4202. https://doi.org/10.4238/2015.April.28.2. [22] SHAILEE F AND FOTEDAR V 2017 “Green Tobacco Sickness: A Brief Review” IJOEM vol 21 3 110-104. [23] Yilmaz S, Ünal F. Evaluation of the genotoxic potential of 1-naphthaleneacetamide (NAAm) in human peripheral lymphocytes. ToxicolInd Health. 2016;32(8):1372–1380. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4960144/