Muhammad Abdur Rehman Shah

Int. J. Biosci. 2019 International Journal of Biosciences | IJB | ISSN:- (Print),- (Online) http://www.innspub.net Vol. 15, No. 3, p. 175-182, 2019 RESEARCH PAPER OPEN ACCESS In vitro evaluation of antibacterial and antifungal activates of Iphiona aucheri leaves extracts Muhammad Abdur Rehman Shah1, Rahmat Ali Khan1*, Mushtaq Ahmed1, Fazal Dian1, Manzoor Ullah2 1 Department of Biotechnology University of Science and Technology Bannu (28100), KPK, Pakistan 2 Department of Botany University of Science and Technology Bannu (28100), KPK, Pakistan Key words: Iphiona aucheri, Antibacterial, Antifungal. http://dx.doi.org/-/ijb/- Article published on September 14, 2019 Abstract The aim of the current project was to evaluate the antibacterial and antifungal activities of Iphiona aucheri leaves methanolic extract and its n-hexane, chloroform and aqueous fractions. Fine powder of Iphiona aucheri leaves were extracted in 70% methanol and then subjected to sequential fractionation through n-hexane, chloroform and water. All the fractions were assessed for their antimicrobial characteristics using agar well diffusion assay. The results revealed that the applied extracts have significant antimicrobial characteristics at 30mg/ml concentration on gram positive bacteria (Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes) and four gram negative bacteria (Escherichia coli, Salmonella typhimurium, Klebsiella pneumoniae, Pseudomonas aeruginosa) and two fungal strains, Aspergillus flavus and Aspergillus niger. The Aspergillus fumigates was found resistant to the applied samples. The maximum inhibition of bacterial growth caused by methanolic extract and its chloroform and aqueous fractions was found (13.7±0.57), (15.7±1.52) and (9.87±1.46) respectively against S. aureus while n-hexane expressed maximum inhibition (5.2±0.57) against K. pneumonia growth. The growth of A. niger was highly inhibited by chloroform fraction (47.43±1.27%), followed by methanolic extract (42.15±1.46%), aqueous fraction (34.45±0.52%) and n-hexane fraction (21.22±1.15%). The chloroform fraction with MIC values (1.6±0.35 mg/ml) and (160.75 ±0.57 µg/ml) against S. aureus and A. niger respectively was found most effective. In conclusion, the result of our study indicated that the leaves of Iphiona aucheri possess considerable antimicrobial activities and thus will be of great use in developing plant derived antimicrobial and chemotherapeutic agents. * Corresponding Author: Rahmat Ali Khan - 175 Shah et al. Int. J. Biosci. 2019 Introduction metabolites including flavonoids, alkaloids, tannins, Currently the tendency of development of antibiotic glycosides, resistant bacteria and fungi is increasing owing to a quinines number of reasons including inappropriate utilization phytochemical are the source of natural plant-derived of antibiotics in human and animal health and their antimicrobial compounds (Srivastava et al., 2014; extended in Suresh and Nagarajan., 2009). Some natural products improving the production of livestock and poultry. are very much significant in the management of Rising antibiotic resistance and the scarcity of new bacterial infections (Fernebro., 2011). Pakistan has a antimicrobials has long been acknowledged and is number of plants varieties having many valuable now a global problem (Theuretzbacher and Mouton, bioactive compounds and its people have sufficient 2011; Walsh and Toleman, 2011). To cope with this knowledge of herbal medicines. application as growth promoters terpenoids, (Das et steroids, al., 2010). saponins These and active problem, it is required to explore and develop a novel, effective, accessible and affordable medicines for the Iphiona aucheri belongs to the family Asteraceae. It is treatment of microbial infections particularly in scattered in Pakistan, Oman, North-East Africa, the developing countries Arabian Peninsula and Iran (Anderberg., 1985). In (Awouafack et al., 2013; Srivastava et al., 2014). A number of factors such as Pakistan, its distribution in Khuzdar, Chaghi, inappropriate and prevalent use of antibiotics in the Mekran, Lasbela, and Loralai districts has been treatment of diseases, excessive use of antibiotics as a reported (Kakar et al., 2012). Antibacterial activities growth enhancer in animal feed lead to the of aerial parts methanolic extract of Iphiona aucheri development of antimicrobial-resistant microbial against S. Aureus, E. coli, S. pyogenes and K. species (Lowy., 2003). This problem in human and ponumoniae have been reported (Kakar et al., 2012). animal will persist for a long time as more species are The minerals i.e. Na, K, Ca, Fe and Ni were reported developing resistance to the available antimicrobial in Iphiona aucheri while Al was found absent medicines (Andersson and Hughes., 2011). To (Lanjwani et al., 2016). The aim of this project was to overcome these circumstances, it is urgently required explore the antibacterial and antifungal activities of to develop alternative drugs to treat such infectious Iphiona aucheri leaves methanolic extract and its n- diseases (Srivastava et al., 2014). hexane, chloroform and aqueous fractions. Local information of herbal medicine is a principal Material and methods source of modern knowledge. Today, thousands of Plant material plants species, conventionally used as medicines, are The plants of Iphiona aucheri were collected in being searched for their antimicrobial characteristics March 2017 from District, Bannu, Pakistan. Its and chemical constituents (Sofowora et al., 2013). taxonomic recognition was carried out by Prof. Abdur Plants possess numerous chemical compounds which Rehman, Govt. Post Graduate College Bannu, and may be therapeutically active or inactive. In addition Khyber Pakhtunkhwa (KP) Pakistan. to the carbohydrates, proteins and lipids, plants synthesis an array of secondary metabolites such as Preparation of crude extract alkaloids, flavonoids, The freshly collected plants were washed with the tap tannins and essential oils which exert physiological water, separated its leaves, shade dried and was activities (Kokate et al., 2002). Most of the secondary pulverized into a fine powder with the help of pestle metabolite synthesized by the plants are extracted in and mortar. 500g powder of leaves was put into 70% different solvents and their therapeutic characteristics methanol (1.5 L) and kept at room temperature for 72 are identified (Gonzalez-Guevara et al., 2004). hours with frequent agitation and then filtered glycosides, triterpenoids, (Whatman No. 3 filter paper, Whatman Ltd., Plants generate a large number of secondary 176 Shah et al. England). The filtrate was placed at room Int. J. Biosci. 2019 temperature to evaporate the liquid content. The physiological saline (0.9% NaCl w/v.) and its resulting gummy methanolic extract of leaves (29.63 turbidity was adjusted with standard turbidity of g) was stored in falcon tube for further use. McFarland 0.5 BaSO4 (106 CFU) by the addition of further sterile physiological saline. These inoculums Preparation of fractions were used for seeding the nutrient agar. The stored gummy methanolic extract of leaves was subjected to fractionation. 20 g of methanolic crude Antibacterial assay extract of Iphiona aucheri leaves was sequentially The antibacterial activities of crude methanolic extracted with 300 ml n-hexane, chloroform and extract and its hexane, chloroform and aqueous water using separating funnel to avoid any sort of fractions were determined by using agar well damages to the filtrate. The respective solvents were diffusion method (Bagamboula et al., 2003). 2 g evaporated completely at room temperature. The nutrient agar media and 0.8 nutrient broth media was resulting fractions of leaves (n-Hexane 2.73 g, dissolved in 100 ml water, autoclaved at 121C for 15 chloroform 5.19 g and water 8.89 g) were stored for minutes and poured in autoclaved petri dishes up to further designed assays. 4cm depth. Following the solidification of media, the bacterial strains were swabbed on petri dishes by Samples preparation using aseptic aluminum borer in the laminar flow The crude methanolic extract of Iphiona aucheri cabinet. Aseptic tips were used to cut five wells in the leaves and its n-hexane, chloroform and aqueous medium (growth medium) layer of each petri dish. An fractions were dissolved in DMSO (30mg/ml) to equal volume of Levaquin (positive control), DMSO prepare the stock solutions which were further (negative control) tested sample with different diluted to the desired working solutions. Similarly the concentrations were added into separate wells in the solutions of Levaquin (levofloxacin; positive control petri dishes and incubated at 37°C for 24 hours. for bacteria) and terbinafine (positive control for Following the incubation, the zone of inhibitions was fungi) were prepared. The DMSO was used as a measured in millimeters and recorded. negative control. Antifungal assay Test microorganism To investigate the antifungal characteristics of the Streptococcus pneumoniae, Streptococcus pyogenes, crude methanolic crude extract of Iphiona aucheri Staphylococcus aureus (Gram positive bacteria) leaves, the standard protocol of Duraipandiyan and Klebsiella Lgnacimuthu pneumoniae, Escherichia coli, (Duraipandiyan and Ignacimuthu., Pseudomonas aeruginosa, Salmonella typhimurium 2009) were used. 6.5 gm Sabouraud Dextrose Agar (Gram negative bacteria) and Aspergillus fumigates, (SDA) media was dissolved in 100ml water and Aspergillus flavus and Aspergillus niger (fungi) were autoclaved at 121 C° for 20 minutes. In laminar flow, used in the current experiment. 4ml media was poured in each autoclaved test tube marked up to 10cm and then added 55 µl working Preparation of 0.5 McFarland standard solution of terbinafine (final concentration 200 The requisite standard was prepared by adding µg/ml; positive control), samples (final concentration together 0.5 ml. of 0.048 M BaCl2 (1.17% w/v 400 µg/ml; experimental) and DMSO (negative BaCl2·2H2O) with 99.5 ml. of 0.18 M H2SO4 (1% w/v) control) to non-solidified SDA. All the test tubes were whilst stirring continuously (Andrews, 2004). arranged in the laminar flow in slanting position for the solidification of the media in the test tubes at the Preparation of inoculums room temperature. Following the solidification, each The 24 hours old culture of the selected bacterial tube was inoculated with a piece of inoculums strains in nutrient broth (MERCK) was mixed with approximately 4 mm in diameter from the 7days old 177 Shah et al. Int. J. Biosci. 2019 culture. Packed all the test tubes air tightly and placed the slanted surface in the test tubes. them for 1 week in the incubator at 28°C. After incubation, their growth was measured, compared The petri dishes were incubated at 37°C for 24 hours with the (bacteria) and the test tubes (fungal) were placed for 7 percentage inhibition with reference to the negative days in the incubator at 28°C. Plates/test tubes were control (Umadevi et al., 2003). arranged in triplicate. % Mycelia inhibition Gn-Gt/Gn ×100 Statistical analysis Where Gn = Mycelial growth in normal; Gt = Mycelial Microsoft Excel was to calculate the mean and growth in test standard deviation. the negative control and calculated MIC determination by agar well diffusion method The samples which revealed the antibacterial and Results and discussion antifungal their Antibacterial and antifungal activities of crude minimum inhibitory concentration (MIC) using agar activities methanol extracts (CME) of Iphiona aucheri and its well diffusion method (Owuama., 2017). Bacterial/ n-hexane, chloroform and aqueous fractions were fungal suspension (1 ml) was mixed with 20 ml explored growth Streptococcus media, were compared evaluated with 0.5 for McFarland against Streptococcus pyogenes, pneumoniae, Staphylococcus aureus standards, poured into petri dishes/test tubes, (Gram positive bacteria) Klebsiella pneumoniae, allowed to solidify and made wells in agar layer of Escherichia petri dishes. Two fold serial dilutions of 30mg/ml Salmonella typhimurium (Gram negative bacteria) were prepared in DMSO (w/v) and introduced in the and Aspergillus fumigates, Aspergillus flavus and wells and approximately 10-12 fungal spores put on Aspergillus niger (fungi). coli, Pseudomonas aeruginosa, Table 1. Antibacterial activities of crude methanolic extract (CME) of Iphiona aucheri leaves and its n-hexane, chloroform and aqueous fractions. Samples Crude methanolic Inhibition zones (mm) Spn Spy Sa Kp Ec Pa St 11.67±1.52 12.3±1.52 13.7±0.57 8.3±0.57 13.3±1.2 12.7±0.57 7.3±1.16 extract (CME) n-hexane fraction 6.42±0.52 7.9±1.62 7.45±1.52 5.2±0.57 5.7±1.57 7.25±1.57 4.7±1.52 Chloroform fraction 13.67±1.57 14.8±1.52 15.7±1.52 9.6±1.52 14.7±1.2 14.0±1.73 8.6±1.15 Aqueous fraction 8.57±0.52 7.97±1.57 9.87±1.46 4.7±1.27 7.7±1.52 8.0±0.57 4.7±0.57 Levaquin® 18.75±0.52 19.83±1.57 20.8±0.50 17.7±0.57 18.25±0.75 22.4±0.57 13.3±0.52 Spn; Streptococcus pneumoniae, Spy; Streptococcus pyogenes, Sa; Staphlococcus aureus, Kp; Klebsiella pneumoniae, Ec; Escherichia coli, Pa; Pseudomonas aeruginosa, St; Salmonella typhi, NA ; Nil activity. The results of antibacterial activities of crude extract fraction expressed maximum antibacterial activities and fractions of Iphiona aucheri, Levaquin® and followed by the crude methanolic extract, aqueous DMSO are articulated in Table 1. fraction and n-hexane fraction. Among the tested samples, the chloroform fraction expressed highest The results indicated that all the samples were active antibacterial activities. Similar results were reported against all the tested bacterial strains to a different during the investigation of antibacterial activities of extent. The antibiotic, Levaquin® (levofloxacin) was Vernonia found active against tested bacterial strains while Vernonia oocephala (Aliyu et al., 2011) and Aspilia DMSO did not show any activity. The chloroform latissima (Souza et al., 2015). The effectiveness of 178 Shah et al. ambigua, Vernonia blumeoides and Int. J. Biosci. chloroform fraction 2019 was crude methanolic extract and aqueous fraction Streptococcus exhibited significant antibacterial activities. The pyogenes (14.8±1.52), Escherichia coli (14.7±1.2), methanolic extract of aerial parts of Iphiona aucheri Streptococcus pneumonia (13.67±1.57), Klebsiella has also indicated antibacterial activities against Staphlococcus in aureus descending (15.7±1.52), order pneumonia (9.6±1.52), Salmonella typhi (8.6±1.15). Staphlococcus Escherichia aureus, coli, Streptococcus Streptococcus pyogenes, pneumonia, Klebsiella pneumonia and Salmonella typhi (Kakar et The n-hexane fraction exhibited the lowest al., 2012). In the previous study, the methanolic and antibacterial activities. Harmonious effects were chloroform extracts also exhibited antibacterial determined during the antibacterial study of Enantia activities chlorantha Oliver (Ebelle Etame et al., 2018). The (Ahameethunisa and Hopper., 2010). against several bacterial strains Table 2. MIC of methanolic extract (CME) of Iphiona aucheri leaves and its n-hexane, chloroform and aqueous fractions. Samples Minimum inhibitory concentration (MIC) values (mg/ml) Crude methanolic extract (CME) Spn 2.6 ±0.25 Spy 2.8±0.52 Sa 2.3±0.35 Kp 5.6±0.57 Ec 2.5±0.25 Pa 3.1 ±0.15 St 2.9±1.1 n-hexane fraction 4.2 ±0.45 4.3±0.25 4.6±0.15 6.2±0.25 5.3±0.45 4.8±0.0 3.6±0.25 Chloroform fraction Aqueous fraction 1.8±0.35 3.1 ±1.15 2.1±0.57 4.4±1.2 1.6±0.35 2.5±1.15 4.6±0.52 5.7±0.45 2.3±0.15 3.6±0.57 2.7±0.0 4.6±0.0 2.7±0.52 3.7±0.45 In the current study, bioassays were employed to crude extracts and its fractions are illustrated in Table characterize the plant fractions. A quantitative 2. The lowest MIC value of chloroform fraction was method, of found (1.6±0.35) against Staphlococcus aureus while antimicrobial agent which is capable to inhibit the n-hexane fraction against indicate the highest measurable microbial growth. According to Lambert MIC value (3.6±0.25) against Salmonella typhi. and Pearson (Singh et al., 2001), in biological assays, Congruent MIC values were reported for the it is a standard measure documented for the methanolic extract of aerial parts of Iphiona aucheri susceptibility of organisms to inhibitors. The MIC of (Kakar et al., 2012). MIC is the lowest concentration Table 3. Antifungal activities of methanolic extract (CME) of Iphiona aucheri leaves and its n-hexane, chloroform and aqueous fractions. Samples Crude methanolic extract (CME) n-hexane fraction Chloroform fraction Aqueous fraction Terbinafine Percentage inhibition of fungal growth A. flavus 32.4±-±-±-±-±1.52 A. niger 42.15±-±-±-±-±0.57 A; Aspergillus. The results of antifungal characteristics of crude fungal strains. The highest inhibition of fungal growth extract and fractions of Iphiona aucheri, terbinafine was caused by chloroform fraction (47.43±1.27%) and DMSO are presented in Table 3. The crude and followed by crude methanolic extract (42.15±1.46%). fraction were active to various extents against A. The n-hexane fraction showed lowest (17.35±1.27%) niger and A. flavus and did not any activity against A. antifungal activities against A. flavus (Omezzine et fumigates. Terbinafine was active against all tested al., 2011). 179 Shah et al. Int. J. Biosci. 2019 The MIC values for antifungal activities of crude and the highest (270.25 ±0.25µg/ml) one which indicated fractions were determined and articulated in Table 4. that chloroform fraction is more effective. Synergistic The chloroform fraction has the lowest MIC value results have been reported in previous studies (Habbu (160.75 ±0.57µg/ml) while the n-hexane fraction has et al., 2009). Table 4. The MIC of methanolic extract (CME) of Iphiona aucheri leaves and its n-hexane, chloroform and aqueous fractions. Samples Minimum inhibitory concentration (MIC) values (µg/ml) A. fumigates A. niger Crude methanolic extract (CME) 270.25 ±0.25 190.15±0.52 n-hexane fraction 540.25 ±0.57 49.55±0.45 Chloroform fraction 210.57 ±0.25 160.75 ±0.57 Aqueous fraction 310.45 ±0.35 230.25 ±0.75 Antimicrobial activities of Iphiona species have not Srivastava J, Chandra H, Nautiyal AR, Kalra been explored yet. However, the previous reports SJ. 2014. Antimicrobial resistance (AMR) and plant- show that it has. Moreover, a number of substances derived antimicrobials (PDA m s) as an alternative including non-toxic pyrrolizidine alkaloid and two drug line to control infections. 3 Biotech 4, 451-460. diterpene glycosides, atractyloside and carboxyatractyloside have been isolated from the Lowy FD. 2003. Antimicrobial resistance: the aerial parts of Iphiona aucheri in which atractyloside example of Staphylococcus aureus. 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