Phenolic contents, antimicrobial and antioxidant activity of Olea ferruginea Royle (Oleaceae)

Phenolic contents, antimicrobial and antioxidant activity of Olea ferruginea Royle (Oleaceae) Background: Olea ferruginea Royle (Oleaceae) has long been used as an important ethnomedicinal plant to cure fever and debility, toothache, hoarseness, throatache and skeleton disorders. In this study, phenolic contents, antimicrobial and antioxidant activities of leaf and bark extracts (chloroform, ethanol and methanol) of O. ferruginea were evaluated. Methods: Total phenolic contents were determined by Folin-Ciocalteu Spectrophotometric method. Antimicrobial activity was examined against Bacillus subtilis and Staphylococcus aureus (Gram positive), Escherichia coli (Gram negative), Candida albicans and Sccharomyces cerevisiae (yeas strains) by disc diffusion method. Antioxidant activity was observed through DPPH assay. Results: The higher phenolic content was found in bark extract (376 μg/mg) of O. ferruginea. Chloroform extracts was found inactive against tested microorganisms while ethanol and methanol extracts showed pronounced inhibitory activity against both gram positive and gram negative bacteria. Only methanol extract of leaves inhibited the yeast strains. None of the bark extract inhibited the growth of tested yeast strains. The zones of inhibition formed by plant extracts were compared with zones of inhibition of available reference antibiotic discs such as tetracycline, ciprofloxacin and nystatin. Higher antioxidant activity was observed with methanol extracts of leaves and bark of O. ferruginea. Conclusion: These findings show that O. ferruginea has potential antimicrobial and antioxidant activities. This study suggests a possible application of olive leaves and bark as sources of natural antimicrobial and antioxidants. Keywords: Extract, E. coli, Ethnopharmacology Background Various type of sources such as microorganisms, plant, Medicinal plants constitute a large group of econom- animals and oils have been explored to discover the new ically significant plants having raw materials for the antimicrobial agents. The systematic screening of such synthesis of medicines, flavors, perfumes and cos- sources like folk medicine result in the finding of metics. The products of these plants serve as valuable innovative effective compounds [2]. Folk medicines are foundation of income for small owners and also add great source not only for curing health of the poor in valuable foreign exchange for a country by export. developing countries but also in developed countries No doubt, anti-microbial agents are the most signifi- where conventional medicines are predominant for cant therapeutic findings of the twentiethcentury. national health care [3]. The pathogens developed resist- However, with the extensive use of antibiotics, human ance to antibiotics which opened the door to use herbal is now facing a problem of developing resistance in medicines as antimicrobial agents [4]. almost all pathogens [1]. Plant synthesized and produced different types of secondary metabolites which possess antimicrobial activ- ity [5]. Formerly it was thought that secondary metabo- * Correspondence: ansar.mehmood321@gmail.com lites, not the products of the primary metabolic pathway, Department of Botany, University of Poonch, Rawalakot, Azad Jammu and have no advantage to the plants who produced them. Kashmir 12350, Pakistan Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Mehmood and Murtaza BMC Complementary and Alternative Medicine (2018) 18:173 Page 2 of 6 However, now it is believed that they containvigorous Microorganisms functions [6]. The search for new antibiotics which can All the tested bacteria (Bacillus subtilis, Staphylococcus replace conventional antibiotics is a need of hour. To arureus and Escherichia coli) were obtained from Com- meet this need present work was carried out to scan the bined Military Hospital (CMH) Muzaffarabad while the antimicrobial and antioxidant activity of a valuable medi- tested yeasts (Candida albicans and Sccharomyces cerevi- cinal plant such as O. ferruginea. siae) were grown in Laboratory, Department of Botany, Olive tree (O. ferruginea Royle), covers 8 million hect- University of Azad Jammu and Kashmir, Muzaffarabad. ares in Mediterranean countries almost 98% of the world crop, is one of the most important fruit trees [7]. These Culture media figures show its pronounced economic and social mean- Nutrient agar medium (28 g dehydrated nutrient agar in ing and the probable aids to be derived from exploitation 1000 ml distilled water, warmed and shake) was used for of any of its byproducts [8]. The fruits and oil of Olea, culturing bacterial species. The fungal species were important constituents of daily diet in a large part of the cultured in Sabouraud’s dextrose agar (65 g Sabouraud’s world’s population, are widely studied for their alimen- dextrose agar in 1000 ml distilled water). Both the media tary use, whereas the leaves contain important secondary were autoclaved for 15 min at 121 °C. metabolites like oleuropein and oleacein, the former re- sponsible for hypoglycemic activity [9] and the latter for Antimicrobial assay hypotensive activity [10]. It was also shown by several Disc diffusion essay proposed by [14] was used to test studies that leaf extract of olive has the ability to reduce the extracts of plants for their antimicrobial activities. the blood pressure in animals [11], prevent intestinal The microorganisms were suspended in 10 ml distilled muscle spasms and relieve arrhythmia [12]. Present work water by dipping a loop of organism in sterilized labeled was aimed to investigate the phenolic contents, anti- test tube. From test tube, 1 ml dilution was transported microbial and antioxidant activity of O. ferruginea. in the corresponding sterilized petri plates. The dilution and medium were mix in petri plates by gently shaking and kept at room temperature for solidification. Methods Sterilized filter paper discs of 6 mm in diameter were Plant materials dipped in each 10 mg/ml crude extracts (chloroform, In this study, Olea ferruginea (stem bark and leaves) was ethanol and methanol) of O. ferruginea and placed on agar selected for its antimicrobial and antioxidant potential. medium in petri plates at their labeled positions. Com- The plant was collected from Kotli in 2017 and identi- mercially available antibiotics (Tetracyline, ciprolaxacine, fied by a taxonomist Dr. Sajjad Hussain, Department of and nystatin) were used as positive control and water, Botany, University of Poonch Rawalakot. The voucher chloroform, ethanol and methanol as negative control. specimen (KNV 416) was submitted in the Herbarium, The experiment was performed in aseptic environment. University of Poonch Rawalakot. The plant material was obtained in the course of flowering stage. Both bark and Incubation of plates leaves were shade dried at room temperature (25 ± 2 °C). The plates containing the bacterial and yeasts culture were incubated at 37 °C for 24 h and 25 °C for 72 h respectively. The zones of inhibition were measured in Extraction procedure millimeter by using measuring scale. After drying, a fine powder of bark and leaves was made using electric grinder. For extraction, Fifty g powder was Antioxidant activity soaked with 200 ml of chloroform, ethanol and metha- To determine antioxidant activity of selected plants, the nol solvents in three separate flasks. The maceration was DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging carried out at room temperature in each solvent for assay [15] was used to determine the antioxidant activity of 7 days with constantly shaking after every 24 h. After different extracts of leaves and bark of O. ferruginea.The maceration, the mixture was filtered using Whatmann DPPH solution fades its color when received hydrogen ions filter paper in labeled flasks. The filtrate was evaporated from antioxidant, which was initially violet. A stock of at low temperature and pressure by a rotary evaporator DPPH was prepared by adding 7 mg DPPH in 100 ml of to obtain the crude extract [13]. methanol. The DPPH solution, methanol and extracts with various concentrations (1.25 mg/ul, 2.5 mg/ul and 5 mg/ul) Dilution were added in labeled test tubes for sample and blank read- Ten mg crude extract was dissolve in 1 ml respective ing, mixed well and kept it for 30 min at room temperature. solvents (chloroform, ethanol and methanol) to make Ascorbic acid was used as control. The optical density was 10 mg/ml dilution. measured against standard at λ 517 nm by using UV max Mehmood and Murtaza BMC Complementary and Alternative Medicine (2018) 18:173 Page 3 of 6 visible spectrophotometer. The experiments were carried out in triplicate. The percentage radicals scavenging activity was calculated by using following formula Absorbance of control−Absorbance of tested sample %Inhibition ¼ Absorbance of control Where standard is the absorbance of control reaction (containing all reagents except the test compounds). 50% inhibition (IC50) of each extract concentrations against graph of inhibition was calculated by applying SSP10 software. Fig. 2 Antimicrobial activity of crude extracts of O. ferruginea (stem bark) compared to antibiotics Phenolic estimation Folin-Ciocalteu Spectrophotometric method described showed appreciable bactericidal activity against S. aureus by Kim et al. [16] was used to determine the total phe- and E. coli i.e. 11.00 ± 0.58 mm and 10.00 ± 0.58 mm. nolc content of plant extracts on a UV-vis spectropho- While C. albicans and S. cerevisiae were resistant to tometer at 650 nm. Results were expressed as catechol ethanolic leaf extract. Ethanol bark extract showed good equivalents (μg/mg). inhibitory activity against B. subtilis (12.33 ± 0.67 mm) and S. aureus (12.00 ± 0.58 mm) while it was moderately Statistical analysis active against E. coli (10.67 ± 0.33 mm). It was also un- A statistical analysis was used to interpret the antimicro- able to inhibit the growth of yeast strains. bial and antioxidant results. The experiment was con- Methanolic leaf extract induced higher antimicrobial ducted in completely randomized design with 3 activity against B. subtilis and S. aureus (14.00 ± replicates. The results are presented as means ±standard 0.58 mm, 18.33 ± 0.58 mm) respectively. It also showed a error of means using MS excel [17]. considerable amount of activity against E. coli with zone of inhibition of 11.33 ± 0.33 mm. Methanol leaf extract Results also inhibited the growth of C. albicans and S. cerevisiae Antimicrobial activity with zones of 9.00 ± 0.58 mm and 9.33 ± 0.33 mm. It was Figure 1 shows the antimicrobial activity of crude leaf also observed that Gram positive bacteria are more sus- extracts of O. ferruginea while Fig. 2 shows the anti- ceptibleto the tested extracts than gram negative bacter- microbial activity of crude bark extracts of O. ferrugnea. ium. The highest activity (18.33 ± 0.58 mm) was found The antimicrobial activity of negative controls such as of methanol extract of leaves of O. europaea against S. water, chloroform, ethanol and methanol is shown in aureus. Fig. 3. The chloroform extract from both leaves and bark did not inhibit the growth of any of the tested organisms Phenolic contents and found to be inactive. Ethanol leaf extract was less Tables 1 and 2 shows the total phenolic contents in active against B. subtilis i.e. 8.33 ± 0.33 mm while it crude extracts of O. ferruginea and were reported as cat- echol equivalents (μg/mg). The higher phenolic Fig. 1 Antimicrobial activity of crude extracts of O. ferruginea (leaves) compared to antibiotics Fig. 3 Antimicrobial activity of negative controls Mehmood and Murtaza BMC Complementary and Alternative Medicine (2018) 18:173 Page 4 of 6 Table 1 Phenolic content and IC50 value in O. ferruginealeaves Solvent Stem bark used Phenolic contents (μg/mg) IC value (mg/ml) Chloroform 312 0.84 Ethanol 351 0.56 Methanol 399 0.46 compounds (376 μg/mg) were present in the methanol extract, followed by ethanol (321 μg/mg) and chloroform extract (288 μg/mg) of leaves of O. ferruginea. In stem bark extract, highest phenol was reported in methanol Fig. 4 Antioxidant activity of O. ferruginea leaves in different solvents (399 μg/mg), followed by ethanol (351 μg/mg) and chloroform (312 μg/mg). 0.37 mg/ml with chloroform, ethanol and methanol Free radical scavenging activity extracts respectively. Free radical (DPPH) scavenging activity of leaves of O. ferruginea is shown in Fig. 4 in the form of percentage Discussion inhibition. According to Fig. 4, chloroform, ethanol and Medicinal plants are the active source of both traditional methanol showed 22.22 ± 0.03, 42.48 ± 0.05 and 25.26 ± as well as modern medicines. The active compounds 0.01 free scavenging activity respectively at 1.25 mg/ml. present in herbal medicines have the compensations of the extracts of 2.50 mg/ml concentration of chloroform, being joined with several other compounds that seem to ethanol and methanol revealed 26.68 ± 0.02, 34.03 ± 0.03 be inactive. However as compared to isolated and pure and 49.71 ± 0.03 respectively. While chloroform, ethanol active compounds, the bio compounds present in plants and methanol extracts at 5.00 mg/ml concentration give them far superior security and efficiency as a whole showed 40.22 ± 0.06, 60.15 ± 0.03 and 71.24 ± 0.02 activ- [18]. Many studies have been carried out to investigate the ity respectively. Figure 5 shows the results of the free antibacterial and antifungal activities of essential oil of radical (DPPH) scavenging activity in % inhibition of olive. The presented work was conducted to observe the stem bark of O. ferruginea. The result suggested that the antimicrobial activity of medicinal plant as O. ferruginea chloroform, ethanol and methanol extract of leaves ex- against gram positive bacteria, gram negative bacterium hibited antioxidant activities of 23.56 ± 0.01, 26.05 ± 0.02 and yeast strains. From the results it was observed that and 31.41 ± 0.05 respectively at a concentration of Gram positive bacteria (B. subtilis and S. aureus)were 1.25 mg/ml. At concentration of 2.50 mg/ml, the chloro- more sensitive as compared to gram negative bacterium form, ethanol and methanol extract of leaves showed (E. coli) used. The results were well correlated to findings 29.34 ± 0.06, 35.82 ± 0.06 and 40.67 ± 0.03 antioxidant of [19] where gram negative bacterium E. coli was also activity respectively. Similarly chloroform, ethanol and more resistant to the extracts of S. xanthocarpum. It was methanol extract at 5.00 mg/ml concentration showed also observed that methanol extract showed highest antioxidant activity of 47.33 ± 0.06, 60.72 ± 0.03 and inhibitory activity because of its high polarity and it allows 73.97 ± 0.02 respectively. extracting all the phenolic compounds. In addition, as compared to isolated compounds, extracts can be more Half maximum inhibitory concentration (IC value) Tables 1 and 2 also shows the IC value of chloroform extract (0.8 mg/ml), ethanol (0.55 mg/ml) and metha- nol extract (0.45 mg/ml) of leaves of O. ferruginea. While stem bark showed IC value of 0.67, 0.46 and Table 2 Phenolic content and IC value in O. ferruginea stem bark Solvent Leaves used Phenolic contents (μg/mg) IC 50Value (μg/ml) Chloroform 288 0.67 Ethanol 321 0.47 Fig. 5 Antioxidant activity of O. ferruginea stem bark in different solvents Methanol 376 0.37 Mehmood and Murtaza BMC Complementary and Alternative Medicine (2018) 18:173 Page 5 of 6 beneficial. Since in presence of other compounds in the Many plants shows competent antioxidant properties extracts, an individual bioactive component can change its due to presence of phytochemicals including phenolic properties [20]. It was also found that n-hexane fraction compounds [30]. of leaf of O. ferruginea has higher biological activity The amount or concentration of substrate causes 50% against gram positive and gram negative bacteria as loss of the DPPH activity is known as its IC50 and it is compared to chloroform and methanol. While in our calculated by plotting a linear regression of antiradical study methanol extract was found to have higher anti- activity in percentage against the amount of compounds microbial activity [21]. tested. Methanol extracts showed being the lowest IC50 It is observed from present results that active antimicro- values which shows highest antioxidant activity. As com- bial biocompounds could be extracted in ethanol and pared to leaves extracts, stem bark extracts of O. ferrugi- methanol extracts. Except phenolics, bound to insoluble nea exhibited significant activity with low IC50 value. carbohydrates or proteins, most of the compounds extract Moreover, a linear relationship was found between the in methanol or acetone [22]. In present study, ethanolic total polyphenol and the reciprocal of IC50 value, indi- and methanolic extractions of O. ferruginea were found to cating that polyphenols are directly proportional to anti- have acceptable antimicrobial activities with respect to ref- oxidant activity. Similar results were recorded by [31]. erence antibiotic discs (Fig. 1). It was observed in present study that plant extracts showed high inhibitory activity Conclusion against bacteria in contrast to fungi. Previously extracts of O. ferruginea is known for treating many important infec- Bellis perennis have also shown more activity towards bac- tious diseases including to kill cancerous cells. Different teria [23]. Moreover, methanolic extract was found more crude extracts of tested plants were prepared by using vari- active in our studies which is correlated to results of [24], ous solvents with the aim of screening better antimicrobial methanol extract of H. afficinalis was more active against activities in comparison with some standard antibiotics. P. aeruginosa and B. subtillus. The antimicrobial activity Among these, the extract obtained with methanol was of plants may be the consequence of presence of wide found to have a better effectiveness against the tested range of bioactive compounds. Plant extracts comprise bacteria and also has highest antioxidant activities. The re- many polyphenols, flavonoids and alkaloids which could sults of present study support the use of this valuable plant be antimicrobial representatives. Many studies show the in traditional medicines for treating infectious diseases. association between antimicrobial activities of plants and Further phytochemicals studies are needed to find the the phytochemicals present in them. Flavonoids are well major bioactive compounds responsible for antimicrobial known for their antiviral [25], antimicrobial [26] and spas- and antioxidant effect of this plant. molytic [27] activities. Likewise, alkaloids extracted from plants have also shown antimicrobial activity [28]. The in- Acknowledgments hibition of microbial growth may result from the binding We are thankful to Combined Military Hospital for providing clinical pathogens. of biocompounds to the cell wall. The antioxidant activity of plants and their natural prod- Availability of data and materials ucts is widely evaluated by using scavenging activity for The datasets used and/or analysed during the current study available from free radicals of 1.1-diphenyl-2-picrylhydrazyl (DPPH).The the corresponding author on reasonable request. methanol extract of stem bark of O. ferruginea showed Authors’ contributions higher radical scavenging activity (73.97 ± 0.02) followed by AM carried out the experimental work and wrote the manuscript. GM ethanol and chloroform extracts. Overall, methanol ex- designed experiment and helped out in data analysis. Both authors have tracts have shown higher antioxidant activity. It has already read and approved the manuscript, and ensure that this is the case. been proven that phenolic compounds are best extracted in ethanol and methanol solvents [29]. Total phenolic con- Ethics approval and consent to participate Not applicable. tent was found higher in methanol extract as compared to ethanol and chloroform extracts. Therefore, it can be easily Competing interests assume that DPPH free radical scavenging activity is corre- The authors declare that they have no competing interests. sponded to present of bioactive compounds like phenols, as methanol extract showed highest antioxidant activity. A Publisher’sNote significant variation was found in total phenolic content Springer Nature remains neutral with regard to jurisdictional claims in among the different extracts of stem bark and leaf extract published maps and institutional affiliations. of O. ferruginea. Free radicals such as superoxide, hydroxyl, Author details peroxyl and singly oxygen play a vital role in many disease Department of Botany, University of Poonch, Rawalakot, Azad Jammu and conditions. Herbal drugs, comprising free radical scaven- 2 Kashmir 12350, Pakistan. Department of Botany, University of Azad Jammu gers, are getting prominence in treating such diseases. and Kashmir, Muzaffarabad 13100, Pakistan. Mehmood and Murtaza BMC Complementary and Alternative Medicine (2018) 18:173 Page 6 of 6 Received: 24 January 2018 Accepted: 23 May 2018 27. Julianeli T, Jackson R, Kelly S, Sílvia S. Selective spasmolytic effect of a new furanoflavoquinone derivative from diplotropin on Guinea-pig trachea. J Chem Pharm Res. 2011;3:249–58. 28. Ahmed EHM, Nour BY, Mohammed YG, Khalid HS. Antiplasmodial activity of some medicinal plants used in Sudanese folk-medicine. Environ Health References Insights. 2010;4:1. 1. Peterson LR, Dalhoff A. Towards targeted prescribing: will the cure for 29. Siddhuraju P, Becker K. Antioxidant properties of various solvent antimicrobial resistance be specific, directed therapy through improved extracts of total phenolic constituents from three different agroclimatic diagnostic testing? 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Zarzuelo A, Duarte J, Jimenez J, Gonzalez M, Utrilla M. Vasodilator effect of olive leaf. Planta Med. 1991;57:417–9. 12. Benavente-Garcıa O, Castillo J, Lorente J, Ortuno A, Del-Rio J. Antioxidant activity of phenolics extracted from Olea europaea L. leaves. Food Chem. 2000;68:457–62. 13. Hussain A, Murtaza G, Mehmood A, Qureshi RA. Conservation of indigenous knowledge of medicinal plants of Western Himalayan region Rawalakot, Azad Kashmir, Pakistan. Pak J Pharm Sci. 2017;30:773–82. 14. Bauer A, Kirby W, Sherris JC, Turck M. Antibiotic susceptibility testing by a standardized single disk method. Amer J Clin Pathol. 1996;45:493. 15. Amarowicz R, Estrella I, Hernández T, Robredo S, Troszyńska A, Kosińska A, Pegg RB. Free radical-scavenging capacity, antioxidant activity, and phenolic composition of green lentil (Lens culinaris). Food Chem. 2010;121:705–11. 16. Kim KT, Yoo KM, Lee JW, Eom SH, Hwang IK, Lee CY. 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Antimicrobial activity of polyacetylenes from Bellis perennis and their synthetic derivatives. Planta Med. 1997;63:503–7. 24. Jankovsky M, Landa T. Genus Hyssopus L. Recent knowledge. Horticult Sci. 2002;29:119–23. 25. Karamoddini MK. Antiviral activities of aerial subsets of Artemisia species against herpes simplex virus type 1 (HSV1) in vitro. Asian Biomed. 2011;5:63. 26. Maria LA, Maria RF. Studies on the antimicrobial activity and brine shrimp toxicity of Z. tuberculosa extracts and their main constituents. Ann Clin Microbiol Antimicrob. 2009;8:16. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png BMC Complementary and Alternative Medicine Springer Journals

Phenolic contents, antimicrobial and antioxidant activity of Olea ferruginea Royle (Oleaceae)

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Abstract

Background: Olea ferruginea Royle (Oleaceae) has long been used as an important ethnomedicinal plant to cure fever and debility, toothache, hoarseness, throatache and skeleton disorders. In this study, phenolic contents, antimicrobial and antioxidant activities of leaf and bark extracts (chloroform, ethanol and methanol) of O. ferruginea were evaluated. Methods: Total phenolic contents were determined by Folin-Ciocalteu Spectrophotometric method. Antimicrobial activity was examined against Bacillus subtilis and Staphylococcus aureus (Gram positive), Escherichia coli (Gram negative), Candida albicans and Sccharomyces cerevisiae (yeas strains) by disc diffusion method. Antioxidant activity was observed through DPPH assay. Results: The higher phenolic content was found in bark extract (376 μg/mg) of O. ferruginea. Chloroform extracts was found inactive against tested microorganisms while ethanol and methanol extracts showed pronounced inhibitory activity against both gram positive and gram negative bacteria. Only methanol extract of leaves inhibited the yeast strains. None of the bark extract inhibited the growth of tested yeast strains. The zones of inhibition formed by plant extracts were compared with zones of inhibition of available reference antibiotic discs such as tetracycline, ciprofloxacin and nystatin. Higher antioxidant activity was observed with methanol extracts of leaves and bark of O. ferruginea. Conclusion: These findings show that O. ferruginea has potential antimicrobial and antioxidant activities. This study suggests a possible application of olive leaves and bark as sources of natural antimicrobial and antioxidants. Keywords: Extract, E. coli, Ethnopharmacology Background Various type of sources such as microorganisms, plant, Medicinal plants constitute a large group of econom- animals and oils have been explored to discover the new ically significant plants having raw materials for the antimicrobial agents. The systematic screening of such synthesis of medicines, flavors, perfumes and cos- sources like folk medicine result in the finding of metics. The products of these plants serve as valuable innovative effective compounds [2]. Folk medicines are foundation of income for small owners and also add great source not only for curing health of the poor in valuable foreign exchange for a country by export. developing countries but also in developed countries No doubt, anti-microbial agents are the most signifi- where conventional medicines are predominant for cant therapeutic findings of the twentiethcentury. national health care [3]. The pathogens developed resist- However, with the extensive use of antibiotics, human ance to antibiotics which opened the door to use herbal is now facing a problem of developing resistance in medicines as antimicrobial agents [4]. almost all pathogens [1]. Plant synthesized and produced different types of secondary metabolites which possess antimicrobial activ- ity [5]. Formerly it was thought that secondary metabo- * Correspondence: ansar.mehmood321@gmail.com lites, not the products of the primary metabolic pathway, Department of Botany, University of Poonch, Rawalakot, Azad Jammu and have no advantage to the plants who produced them. Kashmir 12350, Pakistan Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Mehmood and Murtaza BMC Complementary and Alternative Medicine (2018) 18:173 Page 2 of 6 However, now it is believed that they containvigorous Microorganisms functions [6]. The search for new antibiotics which can All the tested bacteria (Bacillus subtilis, Staphylococcus replace conventional antibiotics is a need of hour. To arureus and Escherichia coli) were obtained from Com- meet this need present work was carried out to scan the bined Military Hospital (CMH) Muzaffarabad while the antimicrobial and antioxidant activity of a valuable medi- tested yeasts (Candida albicans and Sccharomyces cerevi- cinal plant such as O. ferruginea. siae) were grown in Laboratory, Department of Botany, Olive tree (O. ferruginea Royle), covers 8 million hect- University of Azad Jammu and Kashmir, Muzaffarabad. ares in Mediterranean countries almost 98% of the world crop, is one of the most important fruit trees [7]. These Culture media figures show its pronounced economic and social mean- Nutrient agar medium (28 g dehydrated nutrient agar in ing and the probable aids to be derived from exploitation 1000 ml distilled water, warmed and shake) was used for of any of its byproducts [8]. The fruits and oil of Olea, culturing bacterial species. The fungal species were important constituents of daily diet in a large part of the cultured in Sabouraud’s dextrose agar (65 g Sabouraud’s world’s population, are widely studied for their alimen- dextrose agar in 1000 ml distilled water). Both the media tary use, whereas the leaves contain important secondary were autoclaved for 15 min at 121 °C. metabolites like oleuropein and oleacein, the former re- sponsible for hypoglycemic activity [9] and the latter for Antimicrobial assay hypotensive activity [10]. It was also shown by several Disc diffusion essay proposed by [14] was used to test studies that leaf extract of olive has the ability to reduce the extracts of plants for their antimicrobial activities. the blood pressure in animals [11], prevent intestinal The microorganisms were suspended in 10 ml distilled muscle spasms and relieve arrhythmia [12]. Present work water by dipping a loop of organism in sterilized labeled was aimed to investigate the phenolic contents, anti- test tube. From test tube, 1 ml dilution was transported microbial and antioxidant activity of O. ferruginea. in the corresponding sterilized petri plates. The dilution and medium were mix in petri plates by gently shaking and kept at room temperature for solidification. Methods Sterilized filter paper discs of 6 mm in diameter were Plant materials dipped in each 10 mg/ml crude extracts (chloroform, In this study, Olea ferruginea (stem bark and leaves) was ethanol and methanol) of O. ferruginea and placed on agar selected for its antimicrobial and antioxidant potential. medium in petri plates at their labeled positions. Com- The plant was collected from Kotli in 2017 and identi- mercially available antibiotics (Tetracyline, ciprolaxacine, fied by a taxonomist Dr. Sajjad Hussain, Department of and nystatin) were used as positive control and water, Botany, University of Poonch Rawalakot. The voucher chloroform, ethanol and methanol as negative control. specimen (KNV 416) was submitted in the Herbarium, The experiment was performed in aseptic environment. University of Poonch Rawalakot. The plant material was obtained in the course of flowering stage. Both bark and Incubation of plates leaves were shade dried at room temperature (25 ± 2 °C). The plates containing the bacterial and yeasts culture were incubated at 37 °C for 24 h and 25 °C for 72 h respectively. The zones of inhibition were measured in Extraction procedure millimeter by using measuring scale. After drying, a fine powder of bark and leaves was made using electric grinder. For extraction, Fifty g powder was Antioxidant activity soaked with 200 ml of chloroform, ethanol and metha- To determine antioxidant activity of selected plants, the nol solvents in three separate flasks. The maceration was DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging carried out at room temperature in each solvent for assay [15] was used to determine the antioxidant activity of 7 days with constantly shaking after every 24 h. After different extracts of leaves and bark of O. ferruginea.The maceration, the mixture was filtered using Whatmann DPPH solution fades its color when received hydrogen ions filter paper in labeled flasks. The filtrate was evaporated from antioxidant, which was initially violet. A stock of at low temperature and pressure by a rotary evaporator DPPH was prepared by adding 7 mg DPPH in 100 ml of to obtain the crude extract [13]. methanol. The DPPH solution, methanol and extracts with various concentrations (1.25 mg/ul, 2.5 mg/ul and 5 mg/ul) Dilution were added in labeled test tubes for sample and blank read- Ten mg crude extract was dissolve in 1 ml respective ing, mixed well and kept it for 30 min at room temperature. solvents (chloroform, ethanol and methanol) to make Ascorbic acid was used as control. The optical density was 10 mg/ml dilution. measured against standard at λ 517 nm by using UV max Mehmood and Murtaza BMC Complementary and Alternative Medicine (2018) 18:173 Page 3 of 6 visible spectrophotometer. The experiments were carried out in triplicate. The percentage radicals scavenging activity was calculated by using following formula Absorbance of control−Absorbance of tested sample %Inhibition ¼ Absorbance of control Where standard is the absorbance of control reaction (containing all reagents except the test compounds). 50% inhibition (IC50) of each extract concentrations against graph of inhibition was calculated by applying SSP10 software. Fig. 2 Antimicrobial activity of crude extracts of O. ferruginea (stem bark) compared to antibiotics Phenolic estimation Folin-Ciocalteu Spectrophotometric method described showed appreciable bactericidal activity against S. aureus by Kim et al. [16] was used to determine the total phe- and E. coli i.e. 11.00 ± 0.58 mm and 10.00 ± 0.58 mm. nolc content of plant extracts on a UV-vis spectropho- While C. albicans and S. cerevisiae were resistant to tometer at 650 nm. Results were expressed as catechol ethanolic leaf extract. Ethanol bark extract showed good equivalents (μg/mg). inhibitory activity against B. subtilis (12.33 ± 0.67 mm) and S. aureus (12.00 ± 0.58 mm) while it was moderately Statistical analysis active against E. coli (10.67 ± 0.33 mm). It was also un- A statistical analysis was used to interpret the antimicro- able to inhibit the growth of yeast strains. bial and antioxidant results. The experiment was con- Methanolic leaf extract induced higher antimicrobial ducted in completely randomized design with 3 activity against B. subtilis and S. aureus (14.00 ± replicates. The results are presented as means ±standard 0.58 mm, 18.33 ± 0.58 mm) respectively. It also showed a error of means using MS excel [17]. considerable amount of activity against E. coli with zone of inhibition of 11.33 ± 0.33 mm. Methanol leaf extract Results also inhibited the growth of C. albicans and S. cerevisiae Antimicrobial activity with zones of 9.00 ± 0.58 mm and 9.33 ± 0.33 mm. It was Figure 1 shows the antimicrobial activity of crude leaf also observed that Gram positive bacteria are more sus- extracts of O. ferruginea while Fig. 2 shows the anti- ceptibleto the tested extracts than gram negative bacter- microbial activity of crude bark extracts of O. ferrugnea. ium. The highest activity (18.33 ± 0.58 mm) was found The antimicrobial activity of negative controls such as of methanol extract of leaves of O. europaea against S. water, chloroform, ethanol and methanol is shown in aureus. Fig. 3. The chloroform extract from both leaves and bark did not inhibit the growth of any of the tested organisms Phenolic contents and found to be inactive. Ethanol leaf extract was less Tables 1 and 2 shows the total phenolic contents in active against B. subtilis i.e. 8.33 ± 0.33 mm while it crude extracts of O. ferruginea and were reported as cat- echol equivalents (μg/mg). The higher phenolic Fig. 1 Antimicrobial activity of crude extracts of O. ferruginea (leaves) compared to antibiotics Fig. 3 Antimicrobial activity of negative controls Mehmood and Murtaza BMC Complementary and Alternative Medicine (2018) 18:173 Page 4 of 6 Table 1 Phenolic content and IC50 value in O. ferruginealeaves Solvent Stem bark used Phenolic contents (μg/mg) IC value (mg/ml) Chloroform 312 0.84 Ethanol 351 0.56 Methanol 399 0.46 compounds (376 μg/mg) were present in the methanol extract, followed by ethanol (321 μg/mg) and chloroform extract (288 μg/mg) of leaves of O. ferruginea. In stem bark extract, highest phenol was reported in methanol Fig. 4 Antioxidant activity of O. ferruginea leaves in different solvents (399 μg/mg), followed by ethanol (351 μg/mg) and chloroform (312 μg/mg). 0.37 mg/ml with chloroform, ethanol and methanol Free radical scavenging activity extracts respectively. Free radical (DPPH) scavenging activity of leaves of O. ferruginea is shown in Fig. 4 in the form of percentage Discussion inhibition. According to Fig. 4, chloroform, ethanol and Medicinal plants are the active source of both traditional methanol showed 22.22 ± 0.03, 42.48 ± 0.05 and 25.26 ± as well as modern medicines. The active compounds 0.01 free scavenging activity respectively at 1.25 mg/ml. present in herbal medicines have the compensations of the extracts of 2.50 mg/ml concentration of chloroform, being joined with several other compounds that seem to ethanol and methanol revealed 26.68 ± 0.02, 34.03 ± 0.03 be inactive. However as compared to isolated and pure and 49.71 ± 0.03 respectively. While chloroform, ethanol active compounds, the bio compounds present in plants and methanol extracts at 5.00 mg/ml concentration give them far superior security and efficiency as a whole showed 40.22 ± 0.06, 60.15 ± 0.03 and 71.24 ± 0.02 activ- [18]. Many studies have been carried out to investigate the ity respectively. Figure 5 shows the results of the free antibacterial and antifungal activities of essential oil of radical (DPPH) scavenging activity in % inhibition of olive. The presented work was conducted to observe the stem bark of O. ferruginea. The result suggested that the antimicrobial activity of medicinal plant as O. ferruginea chloroform, ethanol and methanol extract of leaves ex- against gram positive bacteria, gram negative bacterium hibited antioxidant activities of 23.56 ± 0.01, 26.05 ± 0.02 and yeast strains. From the results it was observed that and 31.41 ± 0.05 respectively at a concentration of Gram positive bacteria (B. subtilis and S. aureus)were 1.25 mg/ml. At concentration of 2.50 mg/ml, the chloro- more sensitive as compared to gram negative bacterium form, ethanol and methanol extract of leaves showed (E. coli) used. The results were well correlated to findings 29.34 ± 0.06, 35.82 ± 0.06 and 40.67 ± 0.03 antioxidant of [19] where gram negative bacterium E. coli was also activity respectively. Similarly chloroform, ethanol and more resistant to the extracts of S. xanthocarpum. It was methanol extract at 5.00 mg/ml concentration showed also observed that methanol extract showed highest antioxidant activity of 47.33 ± 0.06, 60.72 ± 0.03 and inhibitory activity because of its high polarity and it allows 73.97 ± 0.02 respectively. extracting all the phenolic compounds. In addition, as compared to isolated compounds, extracts can be more Half maximum inhibitory concentration (IC value) Tables 1 and 2 also shows the IC value of chloroform extract (0.8 mg/ml), ethanol (0.55 mg/ml) and metha- nol extract (0.45 mg/ml) of leaves of O. ferruginea. While stem bark showed IC value of 0.67, 0.46 and Table 2 Phenolic content and IC value in O. ferruginea stem bark Solvent Leaves used Phenolic contents (μg/mg) IC 50Value (μg/ml) Chloroform 288 0.67 Ethanol 321 0.47 Fig. 5 Antioxidant activity of O. ferruginea stem bark in different solvents Methanol 376 0.37 Mehmood and Murtaza BMC Complementary and Alternative Medicine (2018) 18:173 Page 5 of 6 beneficial. Since in presence of other compounds in the Many plants shows competent antioxidant properties extracts, an individual bioactive component can change its due to presence of phytochemicals including phenolic properties [20]. It was also found that n-hexane fraction compounds [30]. of leaf of O. ferruginea has higher biological activity The amount or concentration of substrate causes 50% against gram positive and gram negative bacteria as loss of the DPPH activity is known as its IC50 and it is compared to chloroform and methanol. While in our calculated by plotting a linear regression of antiradical study methanol extract was found to have higher anti- activity in percentage against the amount of compounds microbial activity [21]. tested. Methanol extracts showed being the lowest IC50 It is observed from present results that active antimicro- values which shows highest antioxidant activity. As com- bial biocompounds could be extracted in ethanol and pared to leaves extracts, stem bark extracts of O. ferrugi- methanol extracts. Except phenolics, bound to insoluble nea exhibited significant activity with low IC50 value. carbohydrates or proteins, most of the compounds extract Moreover, a linear relationship was found between the in methanol or acetone [22]. In present study, ethanolic total polyphenol and the reciprocal of IC50 value, indi- and methanolic extractions of O. ferruginea were found to cating that polyphenols are directly proportional to anti- have acceptable antimicrobial activities with respect to ref- oxidant activity. Similar results were recorded by [31]. erence antibiotic discs (Fig. 1). It was observed in present study that plant extracts showed high inhibitory activity Conclusion against bacteria in contrast to fungi. Previously extracts of O. ferruginea is known for treating many important infec- Bellis perennis have also shown more activity towards bac- tious diseases including to kill cancerous cells. Different teria [23]. Moreover, methanolic extract was found more crude extracts of tested plants were prepared by using vari- active in our studies which is correlated to results of [24], ous solvents with the aim of screening better antimicrobial methanol extract of H. afficinalis was more active against activities in comparison with some standard antibiotics. P. aeruginosa and B. subtillus. The antimicrobial activity Among these, the extract obtained with methanol was of plants may be the consequence of presence of wide found to have a better effectiveness against the tested range of bioactive compounds. Plant extracts comprise bacteria and also has highest antioxidant activities. The re- many polyphenols, flavonoids and alkaloids which could sults of present study support the use of this valuable plant be antimicrobial representatives. Many studies show the in traditional medicines for treating infectious diseases. association between antimicrobial activities of plants and Further phytochemicals studies are needed to find the the phytochemicals present in them. Flavonoids are well major bioactive compounds responsible for antimicrobial known for their antiviral [25], antimicrobial [26] and spas- and antioxidant effect of this plant. molytic [27] activities. Likewise, alkaloids extracted from plants have also shown antimicrobial activity [28]. The in- Acknowledgments hibition of microbial growth may result from the binding We are thankful to Combined Military Hospital for providing clinical pathogens. of biocompounds to the cell wall. The antioxidant activity of plants and their natural prod- Availability of data and materials ucts is widely evaluated by using scavenging activity for The datasets used and/or analysed during the current study available from free radicals of 1.1-diphenyl-2-picrylhydrazyl (DPPH).The the corresponding author on reasonable request. methanol extract of stem bark of O. ferruginea showed Authors’ contributions higher radical scavenging activity (73.97 ± 0.02) followed by AM carried out the experimental work and wrote the manuscript. GM ethanol and chloroform extracts. Overall, methanol ex- designed experiment and helped out in data analysis. Both authors have tracts have shown higher antioxidant activity. It has already read and approved the manuscript, and ensure that this is the case. been proven that phenolic compounds are best extracted in ethanol and methanol solvents [29]. Total phenolic con- Ethics approval and consent to participate Not applicable. tent was found higher in methanol extract as compared to ethanol and chloroform extracts. Therefore, it can be easily Competing interests assume that DPPH free radical scavenging activity is corre- The authors declare that they have no competing interests. sponded to present of bioactive compounds like phenols, as methanol extract showed highest antioxidant activity. A Publisher’sNote significant variation was found in total phenolic content Springer Nature remains neutral with regard to jurisdictional claims in among the different extracts of stem bark and leaf extract published maps and institutional affiliations. of O. ferruginea. Free radicals such as superoxide, hydroxyl, Author details peroxyl and singly oxygen play a vital role in many disease Department of Botany, University of Poonch, Rawalakot, Azad Jammu and conditions. Herbal drugs, comprising free radical scaven- 2 Kashmir 12350, Pakistan. 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BMC Complementary and Alternative MedicineSpringer Journals

Published: Jun 5, 2018

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