Phytochemical Analysis & Antibacterial Activity of Tobacco Plants Vol.12 (1), June 2021 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 R A D S J . B i o l . R e s . A p p l . S c i . 60 Op e n Ac c e s s F u l l L e n g t h A r t i c l e Phytochemical Analysis and Antibacterial Activity of Nicotiana tabacum and Nicotiana rustica Muhammad Tariq1, Zubair Ahmad1, Sher Ali Shah1, Zainab Gul2, Shahid Ali Khan1,* 1Department of Chemistry, University of Swabi, Khyber Pakhtunkhwa, Pakistan. 2Institute of Physical Medicine and Rehabilitation (IPMR), Khyber Medical University, Khyber Pakhtunkhwa, Pakistan. A B S T R A C T Background: All over the world, natural products containing different secondary metabolites have been used for antibacterial purposes, and as folk medicines with significant effects. Amongst many different plants, Tobacco plants are cultivated all over the world, but natively belong to America. These plants contain variety of secondary metabolites and possess significant antibacterial activity. Objectives: To conduct phytochemical analysis and measure the antibacterial potential of Tobacco plants Nicotiana tabacum and Nicotiana rustica using their extracts. Methodology: Multiple chemical tests such as Wagner’s test and Fehling’s test were used to determine the presence of different types of secondary metabolites. Both plant species were also screened for their antibacterial activity using agar well diffusion method. Results: Phytochemical analysis of the extracts from both plant species i.e. Nicotiana tabacum and Nicotiana rustica indicated the presence of secondary metabolites including tannins, alkaloids, terpenoids, saponins, steroids, and flavonoids. Significant antibacterial activity of both plant extracts was observed against Staphylococcus aureus, but not against Escherichia coli. Conclusion: It can be concluded that both plant extracts showed the presence of secondary metabolites, with significant inhibitory effect observed against Staphylococcus aureus, and no effect against Escherichia coli. Keywords Antibacterial Studies, Nicotiana tabacum, Nicotiana rustica, Phytochemical Analysis, Secondary Metabolites. *Address of Correspondence shahidsawal007@gmail.com Article info. Received: August 09, 2020 Accepted: December 31, 2020 Cite this article:Tariq M, Ahmad Z, Shah S A, Gul Z, Khan S A. Phytochemical Analysis and Antibacterial Activity of Nicotiana tobacum and Nicotiana rustica . RADS J Biol Res Appl Sci. 2021; 12(1):60-65. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. I N T R O D U C T I O N Medicinal plants have been used for treatment of various diseases in past and around 5000 species have been identified1 so far; that are used in cosmetics and pharma industries for the development of new products2-3. Various kinds of organic compounds can be produced by plants, in which some compounds do not contribute to their own functions and are referred as secondary metabolites4. Natural products contain diverse bioactive properties and can lead to the drug discovery5. Most of them are categorized as Generally recognized as safe, (GRAS) family of plants that are prominently used as folk medicines6-14 which led to the discovery of new drugs6. Medicinal plants are the source of natural products having the properties to cure the disease. The 50% available drugs; including pharmaceutical and cosmetics products in the market7 belongs to the natural products isolated from O R I G N A L A R T I C L E Phytochemical Analysis & Antibacterial Activity of Tobacco Plants Vol.12 (1), June 2021 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 R A D S J . B i o l . R e s . A p p l . S c i . 61 the medicinal plants8-9. In Chinese and Egyptian cultures, these natural products are still used as medicine and more than 75% population believes and relies on traditional natural products as reported by WHO10-11. Plants are rich source of antioxidant, anti-fungal, anti-viral and anti-parasitic agents12-14. Among the plant family Solanaceae, Nicotiana tabacum plant is cultivated all over the world but natively belongs to America15.It has been reported to be sensitive to environmental factors including humidity, nature of land and temperature. Some species of Nicotiana tabacum have also been used for ornamental purposes16. Similarly, another plant Nicotiana rustica belongs to the family Solanaceae, contains nicotine in greater amount that is used for the production of pesticides products. These plants contain variety of secondary metabolites and possess significant antibacterial activity12-14. M A T E R I A L & M E T H O D S Distilled water and all the necessary glassware were obtained from Department of Chemistry, University of Swabi, KPK. All the chemicals used in these experiments were purchased from the Sigma Aldrich. Instrumentation The plant leaves were grinded to fine powder by using grinder. Similarly, the extract was concentrated using water bath (HH-S6-China). Filter paper discs (Oxoid USA) were used for the antibacterial assessment while, sterilization was done by using autoclave. Collection of Plants The whole plants of Nicotiana tabacum and Nicotiana rustica were collected from the district Swabi, KPK. Plants were then washed with water in order to remove dust and impurities. After washing, Nicotiana tabacum and Nicotiana rustica plants were shade dried for one week in order to avoid any phytochemical reaction. After that, the dried plants were grinded into small pieces with the help of electric grinder. The grinded powder then subjected to the extraction procedure. Extraction Procedure Grinded powder from both plants was soaked separately into a mixture of methanol and distilled water in a ratio of 1:2 in conical flask (Erlenmeyer flasks), caped with the aluminum foil. These flasks were kept in shelf for 4 days at room temperature. After 4 days, the mixture was subjected to extraction techniques; evaporated in water bath at 70°C and concentrated samples were collected. The procedure was repeated thrice and the extract was stored in clean sterile bottle for further use. S Y N T H E S I S O F R E A G E N T S Fehling’s Solution A For the preparation of Fehling solution, 7g of CuSO4.5H2O was added to 100ml of distilled water and then 2 drops of dilute H2SO4 were added to the solution, that turned the solution blue suggesting the Fehling solution A is ready.20 Fehling’s Solution B For the preparation of Fehling solution B, 35g potassium tartrate and 12g NaOH was mixed with distilled water, as a result a colorless solution was formed confirming the formation of Fehling solution B.20 Wagner’s Solution A total of 2g of iodine and 6g of KI (potassium iodide) were dissolved in 100L of distilled water to prepare Wagner solution.21 B I O L O G I C A L A C T I V I T Y Agar Well Diffusion Method The biological activity was checked by agar well diffusion method following procedure from Murray et al., well diffusion protocol17. Antibacterial activity was done using Mueller–Hinton agar (MHA). For the preparation of media, 1.9g MHA was dissolved in 50ml of deionized water, the prepared media was autoclaved for 15min. Media (15ml) each was poured into sterilized petri dishes to make MHA sensitivity testing plates. Later, the agar surface was inoculated by spreading 200µl of the microbial inoculum of S. aureus and E. coli, followed by making wells (~8mm) in agar with a sterile cork borer. 100µl Nicotiana tabacum and Nicotiana rustica extracts were transferred to their respective wells in each plate. The plates were incubated at 37°C for 24h. After 24h, the zones of inhibition were observed surrounding wells, which indicates that extract from both the species possess significant antibacterial activity. Phytochemical Analysis & Antibacterial Activity of Tobacco Plants Vol.12 (1), June 2021 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 R A D S J . B i o l . R e s . A p p l . S c i . 62 Scheme-1. Extraction procedure, phytochemical analysis and anti-bacterial activity of Nicotiana tabacumand Nicotiana rustica. R E S U L T S A N D D I S C U S S I O N Phytochemical analysis of the Nicotiana tabacum (Fig. 1a) and Nicotiana rustica (Fig. 1b) extracts was performed using different reagents. The following test has been performed for the identification of different secondary metabolites in the extract of Nicotiana tabacum and Nicotiana rustica species. The entire procedure is depicted in scheme-1. Tannins Identification For the identification of tannins in the extract of both plant species, specific amount of the selected plant extract was taken and mixed with distilled water. The filtrate was then collected from the reaction mixture after heating. After this, the filtrate was poured in the test tube and ferric chloride drops were added to that filtrate resulting in the formation of new color i.e. dark greenish. The appearance of new color confirms the presence of tannins in both plants as shown in the inset of Fig. 1(c)17. Alkaloid Identification For the identification of alkaloids, 0.3g of each extract was dissolved in 2ml of distilled water separately and warmed up with 2% sulphuric acid for 3min, and then filtered. After that, few drops of Mayer’s solution was individually added to each plant extract in the test tube. The creamy white precipitates were observed in both extracts which indicates the presence of alkaloids in Fig. 1(d) 18. Amidine Identification Both extracts (0.5g) were separately mixed in 3ml of ammonium hydroxide. Benzene was added to the reaction medium in test tubes. Lack of reddish color indicated the absence of amidine in the both extracts.19 Phlobatannins Identification For the detection of phlobatannins, 0.5g of both extracts was mixed with distilled water in the separate test tubes, followed by filtration. After this, 3% HCl was added and boiled. Absence of reddish color suggests the absence of phlobutannins in both plants.19 Terpenoid Identification For this purpose, 0.3g from both extracts was added to 2ml chloroform and filtered into the test tube. After that, 3ml of sulphuric acid was introduced resulting in the layer formation followed by the appearance of reddish brown color, which shows the positive result for terpenoids. We have observed that each plant extract possesses terpenoids see Fig. 1(c).19 Phytochemical Analysis & Antibacterial Activity of Tobacco Plants Vol.12 (1), June 2021 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 R A D S J . B i o l . R e s . A p p l . S c i . 63 Fig 1. Raw extracts of Nicotiana tabacum(a) and Nicotiana rustica (b). After the addition of specific reagents, change in color indicates the presence of tannins (c), alkaloides (d), terpenoides (e), saponins (f), reducing sugars (g), steroids (h) and flavonoids (i). Saponins Identification For the identification of saponins, 0.4g of both extracts was mixed with distilled water separately. After 10min, the formation of froth and change in color in test tube B indicated the presence of saponins as manifested in Fig. 1(f).19 Reducing Sugar Identification Each plant extract (0.4g) was mixed with 2ml of distilled water in test tube and then filtered. After that, Fehling solution A & B were introduced and boiled for 8min. According to the literature, the presence of precipitates in orange-red color is the indication of reducing sugars which was absent in these species see Fig. 1(g).19 Steroid Identification For the confirmation of steroids in the selected plant species, the acetic anhydride (3ml) was mixed with both plant extracts separately and then filtered into the test tubes, followed by the addition of 2ml of conc. sulphuric acid. The blue green color was observed in both species, which confirmed the presence of steroids; see Fig. 1(h). 19 Flavonoids Identification About 0.3g from each extract was mixed with dilute NaOH, and then HCl was added in test tubes. Appearance of yellow color confirmed the presence of flavonoids in the plant extracts. It was observed that both plant species have flavonoids; see Fig. 1(i).19 Antibacterial Activity Both the species were subjected to antibacterial activity using Agar Well diffusion method against Staphylococcus aureus and Escherichia coli. Results showed that the inhibitory effect against Staphylococcus aureus was found in the extract of both Nicotiana tabacum and Nicotiana rustica see Fig. 2(b), while no inhibitory effect was observed against E. coli species; see Fig. 2(a).19 Fig 2. Antibacterial activity of Nicotiana tabacum and Nicotiana rustica against Escherichiacoli (a) and Staphylococcus aureus (b). C O N C L U S I O N Keeping in view the findings of present research work, it can be concluded that like many different plants, quantitative assessment of Nicotiana tabacum and Nicotiana Phytochemical Analysis & Antibacterial Activity of Tobacco Plants Vol.12 (1), June 2021 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 R A D S J . B i o l . R e s . A p p l . S c i . 64 rustica plant extracts also indicates the presence of bioactive phytochemicals like flavonoids, alkaloids, steroids, saponins, terpenoids, alkaloids and tannins. Interestingly, both plant extracts also confirmed significant antibacterial effect toward Staphylococcus aureus, while no significant effect have been observed against Escherichia coli. C O N F L I C T S O F I N T E R E S T None. F U N D I N G S O U R C E None. A C K N O W L E D G E M E N T S The authors greatly acknowledge the management of Department of Chemistry, University of Swabi, Pakistan for providing research facilities. L I S T O F A B B R E V I A T I O N S GRAS KI CuSo4 MHA HCl Generally Recognized as Safe Potassium Iodide Copper Sulphate Mueller–Hinton agar Hydrochloric Acid H2SO4 Sulphuric Acid NaOH Sodium Hydroxide WHO World Health Organization R E F E R E N C E S 1. Chen SL, Yu H, Luo HM, Wu Q, Li CF, Steinmetz A. Conservation and sustainable use of medicinal plants: Problems, progress, and prospects. Chinese Med. 2016; 11(1):37-45. 2. Huang H. Plant diversity and conservation in China: Planning a strategic bioresource for a sustainable future. Bot J Linnean Soc. 2011; 166(3):282-300. 3. Halberstein RA. Medicinal plants: Historical and cross- cultural usage patterns. Ann Epidemiol. 2005; 15(9):686-99. 4. Croteau R, Kutchan TM, Lewis NG. Natural products (secondary metabolites). Biochem Mol Biol Plants. 2000; 24:1250-319. 5. Sarker SD, Nahar L. An introduction to natural products isolation. In Natural products isolation, Springer: 2012; 1-25. 6. Koehn FE, Carter GT. The evolving role of natural products in drug discovery. Nature Rev Drug Dis. 2005; 4(3):206-20. 7. Gordaliza M. Natural products as leads to anticancer drugs. Clin Transl Oncol. 2007; 9(12):767-76. 8. Yarnell E, Abascal K. Dilemmas of traditional botanical research. HerbalGram. 2002; 55:46-54. 9. Jain SK. Medicinal plants. National Book Trust, India.1968; 18-65. 10. Samuelson G, Bohlin L. Drugs of Natural origin. A texboot of Pharmacognosy. Stockholm: Swedish Pharmaceutical Pres. 1992; 1-25. 11. Gibbons S. Anti-staphylococcal plant natural products. Nat Prod Rep. 2004; 21(2):263-77. 12. Rajaei A, Barzegar M, Mobarez AM, Sahari MA, Esfahani ZH. Antioxidant, anti-microbial and antimutagenicity activities of pistachio (Pistachia vera) green hull extract. Food Chem Toxicol. 2010; 48(1):107-12. 13. Wink M. Medicinal plants: A source of anti-parasitic secondary metabolites. Mol. 2012; 17(11):12771-91. 14. Ruma K, Sunil K, Prakash H. Antioxidant, anti- inflammatory, antimicrobial and cytotoxic properties of fungal endophytes from Garcinia species. Int J Pharm Pharm Sci. 2013; 5(3):889-97. 15. Li L, Shen QP, Liu CB, Wang Y, Yao JJ, Zhang T, et al. Isoflavones from the leaves of Nicotiana tabacum and their anti-tobacco mosaic virus activities. Phytochem Let. 2015; 13:156-9. 16. Kenton A, Parokonny AS, Gleba YY, Bennett MD. Characterization of the Nicotiana tabacum L. genome by molecular cytogenetics. Mol Gen Genetics. 1993; 240(2):159-69. 17. Roux DG. Some recent advances in the identification of leuco-anthocyanins and the chemistry of condensed tannins. Nature. 1957; 180(4593):973-5. 18. Preininger V, Antavý F. Isolation and chemistry of the alkaloids of the genus Papaver. 51st Report: Isolation of the alkaloids from P. bracteatum, P. fugax and P. triniaefolium, and the identification of several earlier isolated alkaloids from plants of the sections Orthorhoeades, Mecones and Pilosa. Pharmazie. 1970: 25:356-60. Phytochemical Analysis & Antibacterial Activity of Tobacco Plants Vol.12 (1), June 2021 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 R A D S J . B i o l . R e s . A p p l . S c i . 65 19. Sokunvary Oeung, Voleak Nov, Huykhim Ung, Koemlin Roum and et.al Phytochemical analysis of different extracts of leaves of Nicotiana tabacum L. of Cambodia,2017, 1(3):18-26. 20. Prof. Robert J. Lancashire, 2015, http://wwwchem.uwimona.edu.jm/courses/Fehling.ht ml 21. Fatima Tuz Zohra,Extraction of secondary metabolites, phytochemical screening and the analysis of antibacterial activity in Stevia rebaudiana, 2015,pg 18.