Journal of Physics: Conference Series PAPER • OPEN ACCESS In-vitro Anti-Microbial Studies and GC/MS Analysis of the Leaf Extract and Fractions of Polyalthia longifolia (Engl. & Diels) Verde To cite this article: Ifedolapo O. Olanrewaju et al 2019 J. Phys.: Conf. Ser. 1299 012087 View the article online for updates and enhancements. This content was downloaded from IP address 107.173.236.38 on 09/10/2019 at 14:56 3rd International Conference on Science and Sustainable Development (ICSSD 2019) IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 1299 (2019) 012087 doi:10.1088/1742-6596/1299/1/012087 In-vitro Anti-Microbial Studies and GC/MS Analysis of the Leaf Extract and Fractions of Polyalthia longifolia (Engl. & Diels) Verde Ifedolapo O. Olanrewaju1, Raphael C. Mordi1, Johnbull O. Echeme2, Oladotun P. Bolade1, Musa Bashir3, Silas Ekwuribe3 and Joan I. Ayo-Ajayi1 1 Chemistry Department, Covenant University, Canaan Land, Km 10, Idiroko Road, Ota, Ogun State, Nigeria. 2 Department of Chemical Sciences, Michael Okpara University of Agriculture, Umudike, Abia State, Nigeria. 3 Multiuser Laboratory, Chemistry Department, Ahmadu Bello University, Zaria, Kaduna State, Nigeria E-mail: ifedolapo.olanrewaju@covenantuniversity.edu.ng Abstract. Extensive studies show that secondary metabolites in plants, used for centuries in traditional medicine, offer new sources of drugs. In the traditional setting, extracts from various parts of the plant Polyalthia longifolia (mast tree) are used in treating several ailments but the components of these extracts, which would allow for meaningful dosage, are not known. We therefore decided to examine the antimicrobial activity by testing on selected microorganisms and identify the volatile components by gas chromatography-mass spectrometry of the leaf extracts of Polyalthia longifolia (mast tree). The crude leaf extract and fractions derived from the crude exhibited anti-microbial activity against two (2) bacteria and two (2) fungi. The chloroform fraction was very active against Salmonella typhi (13.00±0.82) when compared to fractions in other solvents. The GC-MS analysis showed that the extracts were composed fatty acids and their ester along with some long chain aldehydes, like hexadecenal and tetradecenal, and Caryophyllene and Aromandendrene. These chemical constituents may be responsible for the pharmacological and therapeutic activities of this plant. 1. Introduction Historically, plant has been nature’s gift to man-kind. This is because plants and their products have always been explored as sources of drugs in the treatment of various ailments and diseases. According, to the World Health Organisation, over 80% of the world’s population use plants and their products traditionally as a primary source of health care [1]. The threat to the treatment of infectious diseases caused by bacteria and fungi has become a major health concern globally due to increased resistance to antimicrobial agents such as antibiotic drugs and other new variety of strains which are multi-drug resistant [2]. It is therefore necessary that steps be taken to tackle these concerns and to reduce the problems caused by the various strains of bacteria and fungi. Polyalthia longifolia is an ethno-medicinal plant that has been used for traditional therapeutic purposes [3]. The plant is of the genus Polyalthia and belongs to the family of Annonaceae. The genus Polyalthia has about 120 species in existence and can be found in the tropical parts of Africa, America, Asia and the Islands of Oceania. In India [4], almost all parts of the P. longifolia var pendula (Polyalthia genus) are used traditionally in the treatment of different types of diseases such Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. Published under licence by IOP Publishing Ltd 1 3rd International Conference on Science and Sustainable Development (ICSSD 2019) IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 1299 (2019) 012087 doi:10.1088/1742-6596/1299/1/012087 as diarrhoea, cough, skin infection, sore throat and cold. In Nigeria the stem and the leaves are used traditionally in treating diseases caused by kinetoplastid protozoa such as sleeping sickness and leishmaniosis; hypertension and diabetes [11, 12]. While in other geographical regions, research has been found that the herbal drugs are used as tonic and febrifuge. The bark of this plant is the more popular as the helpful portion of this plant utilized in treating diabetes, rheumatism, pyrexia, hypertension and menorrhagia and scorpion sting. It also helps in treating constipation, urinary system, digestive system, antipyretic activity and circulatory system movements [13]. It is thought that the presence of clerodane diterpenoids and alkaloids in all parts is responsible for its use in treatment of these ailments [14]. Therefore, this study sought to investigate the antimicrobial properties against Gram-positive bacteria (Staphylococcus aureus), Gram-negative bacteria (Salmonella typhi) and fungi (Trichophyton rubrum and Candida albican) and identify the volatile chemical constituents in leaf extract and fractions of P. longifolia (Engl. & Diels) Verde. 2. Material and Methods 2.1. Plant Material and Extraction Polyalthia longifolia (Engl. & Diels) Verde plant grows wild in Covenant University campus and the required parts were taken as needed. The mature leaves of Polyalthia longifolia (Engl. & Diels) Verde were collected from the campus of Covenant University Ota, Ogun State, Nigeria (latitude 6.672065 north and longitude 3.1598830 east) in the early hours of the morning (between 6 and 7 am) of 15th July, 2015 and the temperature was about 27 ± 2oC. The plants were identified at the Department of Biological Sciences, Covenant University, Nigeria and authenticated at the Forestry Research Institute of Nigeria (FRIN) herbarium, Ibadan with the voucher number FHI: 110014 for reference purpose. The extraction of Polyalthia longifolia leaves was carried out using the procedure described by Okoronkwo et al. [5]. Air-dried leaves were pulverised and extracted in methanol by cold maceration technique. This was then fractionated in chloroform and hexane by separating funnel respectively. The fractions obtained were separated, concentrated and stored at 2oC for further use. 2.2. Antimicrobial Studies 2.2.1. Agar-well diffusion Assay. Antibacterial activities of fractions were determined using agarwell diffusion method [6, 7]. The micro-organisms were incubated at 37oC for 24 hours and the microbial cultures were adjusted by comparing them against 0.5 McFarland before transferring to the plate. A sterile cork borer was used to make 9 mm diameter wells on the agar. The extract and fractions were diluted with ethanol and screened for antibacterial activity using 100 mg/mL concentration extracts. These were then applied to each well in the culture plates previously inoculated with the test organisms. The plates were incubated at 37 oC for 24 hours for bacteria and at 28oC for 72 hours for fungi. Antimicrobial activity was determined by measuring the zone of inhibition in ‘mm’ around each well for extract and fractions. This was done in triplicate with ciprofloxacin as a positive control for bacteria and fluconazole for fungi. 2.2.2. Determination of Minimum Inhibitory Concentration (MIC’s). The MIC was determined when the’ least concentration of the extracts inhibited (stop) the growth of the test organisms after 24 hours. This was accomplished using the tube dilution method as described by the Clinical and Laboratory Standards Institute. Where, 1 mL of different concentrates (3.13 mg/mL, 6.25 mg/mL, 12.50 mg/mL, 25.00 mg/mL, 50.00 mg/mL and 100.00 mg/mL) of extract and fractions in nutrient broth was placed in different test tubes. They were further incubated at 37 oC for 24 hours after adding bacteria and observed for turbidity. The least concentration where no turbidity was observed was noted as the Minimum Inhibitory Concentration (MIC) value. The experiment was carried out in triplicates for accuracy [8]. 2.3. Gas Chromatography-Mass spectrometry 2 3rd International Conference on Science and Sustainable Development (ICSSD 2019) IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 1299 (2019) 012087 doi:10.1088/1742-6596/1299/1/012087 GC-MS analyses were performed on Agilent 19091S-433UI system equipped with HP-5MS ultra inert capillary column (30 m × 0.25 mm × 0.25 μm). The oven temperature was programmed from 50°C to 325oC at 10oC/min for 5 min. The carrier gas was helium with a flow rate 0.73677 mL/min. The volume of sample injected was 2 μL of diluted in benzene in a spit mode of 10:1. The mass spectrometer was in the EI mode at 70 eV in m/z range 50 -550 amu. It had a run time of 47 min. The identification of components present in the extract and fractions was based on direct comparison of the retention times and mass spectral data with those for standard compounds and by computer matching with the NIST14.L GC-MS Library [15]. 3. Results and Discussion The in vitro antimicrobial activity of methanol extract, chloroform fraction and n-hexane fraction of the leaves of P. longifolia showed activity against the human pathogenic micro-organism (S. aureus, S. typhi, C. albican and T. rubrum) used in this study. Table 1. Anti-microbial Activity of Extract and Fractions P. longifolia Leaf Samples Methanol extract (mm) Chloroform fraction (mm) Hexane fraction (mm) Standards (mm) S. aureus S. typhi T. rubrum C. albican 9.67 ± 1.25 12.00 ± 0.82 9.33 ± 0.47 22.00 12.33 ± 0.47 13.00 ± 0.82 11.00 ± 0.82 23.00 7.33 ± 0.47 10.33 ± 1.24 8.33 ± 0.47 22.00 13.33 ± 0.94 10.00 ± 0.82 10.67 ± 0.47 26.00 Positive control for bacteria is ciprofloxacin; for fungi is fluconazole at 100 mg/mL; all data are reported in triplicate mean ± standard deviation The results suggested that extract and fractions from the leaves of P. longifolia possess toxic activity against Gram-positive bacteria (Staphylococcus aureus), Gram-negative bacteria (Salmonella typhi) and fungi (Trichophyton rubrum and Candida albican) as shown in Table 1. The activity index, Figure 1, shows the effectiveness of methanol extract, chloroform fraction and nhexane fractions against the micro-organisms used as compared to the standards (ciprofloxacin and fluconazole) used. This is an indication that the active component in extracts can be isolated as antimicrobial agents and can then be used in the treatment of diseases caused by such organisms as used in this study. Figure 1: Selectivity Index of Polyalthia longifolia leaf extracts and fractions 3 3rd International Conference on Science and Sustainable Development (ICSSD 2019) IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 1299 (2019) 012087 doi:10.1088/1742-6596/1299/1/012087 The minimum inhibitory concentration ranged from 12.50 mg/mL the chloroform fraction with S. typhi to 100 mg/mL as shown in Table 2 below. Some of the extracts can be used as drug boaster on already existing drug used in treatment of diseases caused by such organisms. Table 2. Minimum Inhibitory Concentration (mg/mL) of P. longifolia Extract and Fractions Samples S. aureus S. typhi C. albican T. rubrum Methanol 50.00 25.00 25.00 100.00 extract Chloroform 25.00 12.50 25.00 50.00 fraction Hexane fraction 50.00 50.00 50.00 100.00 Standards 6.25 6.25 3.13 6.25 Positive control for bacteria is ciprofloxacin; for fungi is fluconazole at 100 mg/mL The constituents of the leaf oil have been structurally identified and elucidated by GC-MS. Identification of components was based on comparison with matching library spectral using their retention time (RT). GC-MS analysis of P. longifolia revealed that active component consists of a mixture of nine compounds present in methanol extract, three compounds in the chloroform fraction and ten compounds in the n-hexane fraction; as shown on Table 3. The volatile chemical constituent in the methanol leaf extract and solvent fractions (chloroform and n-hexane) are composed of essential oils (Z)–7–hexadecenal, caryophyllene, aromandendrene, methyl palmitate, ethyl palmitate, methyl stearate, oleic acid and 9-tetradecenal. These volatile compounds are known to possess enormous potential to exhibit microbial pathogens. For instance, caryophyllene and aromandendrene are known for their anti-microbial potential as reported in other studies [3]. Other compounds identified such as Hexadecanoic acid have antioxidant, hypocholesterolemic and haemolytic properties [16]. Octadecanoic acid is used in cosmetic, flavour, lubricant and perfumery [17]. It has been reported that the compound, phytol contains phytochemicals such as terpenoids, flavonoids, alkaloids, tannins etc. In killing bacteria, anti-cancer agents and treatment of diseases. Phytol is also known as an acrylic diterpene alcohol which is used to produce synthetic Vitamin K (for improving bone health and treatment of gastrointestinal illness) and Vitamin E (for stronger immune system, healthy skin, and reduced cell aging) which have essential roles of the human system. It is essential in reducing blood cholesterol and effective in enzymes activation helping in the production of insulin. When phytol in plants are digested in the intestine, they are released, converted to phytanic acid which is stored in the body’s plant tissue [18]. Studies from literatures suggest that P. longifolia contains diterpenoids, alkaloids and sesquiterpene derivatives [9]. Hydro-distillation by Clevenger type apparatus, also led to identification of some similar compounds in leaf extract of Polyalthia longifolia sonnerat (Thwait) [10]. The difference in the composition of the plant extracts could be as a result of different mode of extraction and environmental factors. 4 S/N 1 2 3 4 5 6 7 8 9 10 Methanol extract tR (min) Name of Compound 1.1957 (Z)-7-Hexadecenal 18.1957 Caryophyllene 18.7390 18.9723 24.2210 26.1721 26.3896 26.4354 36.0139 Aromandendrene α-Curcumene Methyl 14-methyl Pentadecanoate Methyl trans-13-Octadecenoate Phytol Methyl stearate 9-Octadecenoic acid (Z)-, 2-hydroxy-1(hydroxymethyl)ethyl ester tR (min) 24.9018 31.6079 35.6476 Chloroform fraction Name of Compound Ethyl palmitate 2,2'-methylenebis[6-(1,1dimethylethyl)-4-methyl-Phenol Oleic Acid N-Hexane fraction tR (min) Name of Compound 1.1955 cis-9-Hexadecenal 18.7391 Aromandendrene 18.9222 20.4957 24.1578 25.1935 26.0976 26.3608 31.3103 α-Curcumene Caryophyllene oxide Methyl palmitate 2-Methyl-Z,Z-3,13-octadecadienol 9-Tetradecenal, (Z)Methyl stearate 2-Methyl-Z,Z-3,13-octadecadienol 5 36.0141 9-Octadecenoic acid (Z)-, 2-hydroxy1-(hydroxymethyl)ethyl ester 3rd International Conference on Science and Sustainable Development (ICSSD 2019) IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 1299 (2019) 012087 doi:10.1088/1742-6596/1299/1/012087 Table 3. GC-MS Results of Identified Compounds in P. longifolia Leaf Extract and Fractions 3rd International Conference on Science and Sustainable Development (ICSSD 2019) IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 1299 (2019) 012087 doi:10.1088/1742-6596/1299/1/012087 4. Conclusion The bio-activity of leaf extract in methanol and the fractions in chloroform and n-hexane of P. longifolia has been established at least for the microorganisms used in this work. We have also been able to identify some components in the extract that may be contributing to the bioactivity of the extracts. The presence of these compounds in the extracts confirms a potential role for the use of P. longifolia (Engl. & Diels) Verde in pharmaceuticals. 5. Acknowledgement We would like to acknowledge the Covenant University management for providing us the fund to carry out this work. Also, we acknowledge the staff of the Multi-User laboratory Ahmadu Bello University, Zaria, Kaduna State, Nigeria. 6. Disclosure Statement: The authors wish to declare there is no potential conflict of interest. References [1] WHO, 2004. WHO Guidelines on Safety Monitoring of Herbal Medicines in Pharmacovigilance Systems. Geneva, Switzerland: World Health Organization. [2] Tchinda, C.F; Voukeng, I.K; Beng, V.P; Kuete, V. 2017. Antibacterial activities of the methanol extracts of Albizia adianthifolia, Alchornea laxiflora, Laportea ovalifolia and three other Cameroonian plants against multi-drug resistant Gram-negative bacteria SJBS, ,24, 950 – 955. [3] Katkar, K.V.; Suthar, A.C.; Chauhan, V.S. 2010. The Chemistry, Pharmacologic, and Therapeutic applications of Polyalthia longifolia. Pharma Rev, 4, 62–68. [4] Jothy, S. L., Choong, Y. S., Saravanan, D., Deivanai, S., Latha, L. Y., Vijayarathna, S., & Sasidharan, S. (2013). Polyalthia longifolia Sonn: An ancient remedy to explore for novel therapeutic agents. Research Journal of Pharmaceutical, Biological and Chemical Sciences, 4(1), 714–730. [5] Okoronkwo, N.E.; Echeme J.O.; Onwuchekwa, E.C. 2012. Cholinesterase and bacterial inhibitory activities of Stachytarpheta Cayennensis Acad. Res. Int. 2, 209 – 217. [6] Parekh, J.; Chanda, S. 2007. Antibacterial and phytochemical studies on twelve species of Indian medicinal plants. Afr. J. Biomed. Res., 10, 175 – 181. [7] Aida, P.; Rosa, V.; Blamea, F.; Tomas, A.; Salvador, C. 2001. Paraguyan plants used in traditional medicine. J. Ethnopharm., 16, 93 – 98. [8] CLSI, 2017. Performance Standards for Antimicrobial Susceptibility Testing, 27th ed., Informational Supplement CLSI Document M100-S27, Clinical and Laboratory Standards Institute, Wayne, PA. [9] Ogunbinu, A.O.; Ogunwande, I.A.; Essien, E.; Cioni, PL.; Flamini, G. 2007. Sesquiterpenes-Rich Essential Oils of Polyalthia longifolia Thw. (Annonaceae) from Nigeria. J. Essent. Oil Res., 19, 419–421. [10] Ouattara, Z.A.; Yapi T.A.; Boti, J.B.; Ahibo, C.A.; Békro, Y.; Mamyrbékova-Békro, A.J.; Casanova, J.; Tomi, F.; Bighelli, A. 2018. Chemical Composition of Leaf Oil from Polyalthia longifolia (Sonnerat) Thwait. Grown in Côte d’Ivoire. J. Essent. Oil Res., 30(3), 153-158 [11] Ebiloma, G.U.; Igoli, J.O.; Katsoulis, E.; Donachie, A.-M.; Eze, A.; Gray, A.I.; Koning, H.P. de. 2017. Bioassay-Guided Isolation of Active Principles from Nigerian Medicinal Plants Identifies New Trypanocides with Low Toxicity and No Cross-Resistance to Diamidines and Arsenicals. Journal of Ethnopharmacology, 202, 256–264. [12] Bankole, A.E.; Adekunle, A.A.; Sowemimo, A.A.; Umebese, C.E.; Abiodun, O.; Gbotosho, G.O. 2016. Phytochemical Screening and in Vivo Antimalarial Activity of 6 3rd International Conference on Science and Sustainable Development (ICSSD 2019) IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 1299 (2019) 012087 doi:10.1088/1742-6596/1299/1/012087 [13] [14] [15] [16] [17] [18] Extracts from Three Medicinal Plants used in Malaria Treatment in Nigeria. Parasitology Research, 115(1), 299–305. Sampath, M.M.; Vasanthi, M. 2012. Isolation, Structural Elucidation of Flavonoid from Polythia longifolia (sonn.) thawaites and Evaluation of Antibacterial, Antioxidant and Anticancer Potential. International Journal of Pharmacy and Pharmaceutical Science, 5(1), 336-341. Khan, A.S. 2017. Medicinally Important Trees. In Khan, A.S. (ed.), Trees with Antimicrobial Activities, pp. 85–108. Switzerland: Springer. http://doi.org/10.1007/978-3319-56777-8 Osuntokun, O.; Olanbiwonnu, A.A.; Orimolade, G.F.; 2017. Assessment of Antibacterial, Phytochemical Properties and GCMS Profiling of Crude Polyalthia Longifolia Extract International Journal of Medical, Pharmacy and Drug Research, 1(1), Mujeeb, F.; Bajpai, P.; Pathak, N. 2014. Phytochemical Evaluation, Antimicrobial Activity, and Determination of Bioactive Components from Leaves of Aegle marmelos. BioMed. Research International. http://dx.doi.org/10.1155/2014/497606 Sudharsan, S.; Saravanan, R.; Shanmugam, A.; Vairamani, S.; Mohan kumar, R.; Menaga, S.; Ramesh, N. 2011. Isolation and Characterization of Octadecanoic Acid from The Ethyl Acetate Root Extract of Trigonella foneum graecum L. by Using Hydroponics Method. Journal of Bioterrorism & Biodefense, 2(1). http://doi.org/10.4172/2157-2526.1000105 Phatangare, N.D.; Deshmukh, K.K.; Murade, V.D.; Hase, G.J; Gaje, T.R. 2017. Isolation and Characterization of Phytol from Justicia gendarussa Burm. f.-An Anti-Inflammatory Compound. International Journal of Pharmacognosy and Phytochemical Research 9(6), 864-872. http://doi.org/10.25258/phyto.v9i6.8192 7