Indian Journal of Traditional Knowledge Vol. 14(3), July 2015, pp. 370-375

Antimicrobial and antioxidant activities and total phenolic content of polycephalum Schutz.Bip. as a folkloric herb in South western Iran

Fatemeh Malekpoor1, Abdollah Ghasemi Pirbalouti2,3*, Azam Salimi4, Leila Shabani5, Majid Sharifi5 & Behzad Hamedi2 1Young Researchers Clubs, Islamic Azad University, Shahrekord Branch, Shahrekord, Iran; 2Shahrekord Branch, Islamic Azad University, Department of Medicinal , Shahrekord, Iran; 3Medicinal Plants Program, College of Natural Sciences, Massachusetts University, Amherst, 01003, MA, USA; 4Department of Biology, Faculty of Basic Science, Kharazmi University, Tehran, Iran; 5Department of Biology, Faculty of Basic Science, ShahrekordUniversity, Shahrekord, Iran E-mails: [email protected]; [email protected]; [email protected]

Received 10 November 2014, revised 22 December 2014

Medicinal plants and their essential oils are potentially useful sources of antioxidant and antibacterial compounds. Tanacetum polycephalum Schutz.-Bip belonging to the family is an important folkloric medicinal in Chaharmahal and Bakhtiari province, South western Iran. In this study, total phenolic content, antioxidant and antibacterial activities of the essential oil from the aerial parts of T. polycephalum collected from 6 different natural habitats of Chaharmahal va Bakhtiari province were investigated. The total phenolic content of the essential oilswas determined by Folin–Ciocalteu methodand antioxidant activity using α, α‐diphenyl‐β‐picrylhydrazyl (DPPH) method was tested. In addition, the antibacterial activity of the essential oils against 6 bacteria, including Staphylococcus aureus, Bacillus cereus, Proteus vulgaris, Listeria monocytogenes, Streptococcus agalactiae and Salmonella typhimurium was tested by agar disc diffusion assays. Results of this study indicated that total phenolic contents ranged between0.06 to 0.16 mg GAE/gm DWE. In addition, results indicated that the essential oils from different populations of T. polycephalum exhibited radical scavenging activity. The results of this study confirmed the essential oils from different populations of T. polycephalum exhibited marked antibacterial activity. This finding suggests that the herb can be served as a valuable source of natural antioxidants and antibacterial for further isolation and purification.

Keywords: Tanacetum polycephalum, Biological activity, Essential oil IPC Int. Cl.8: C11B 9/00, A23L 1/222, C09K 15/00, C12M, C12N

Aromatic and are known to radicals. Free radicals have been implicated in the produce certain bioactive molecules which react with etiology of several major human ailments including other organisms in the environment, inhibiting cancer, cardiovascular diseases, neural disorders, bacterial or fungal growth1,2,3,4. There is a continuous diabetes and arthritis7. Antioxidants can interfere with and urgent need to discover new antimicrobial the oxidation process by reacting with free radicals, compounds with diverse chemical structures and chelating and catalytic metals andby acting as oxygen novel mechanisms of action for new and re-emerging scavengers8. However, numerous studies have been infectious diseases5. In addition, many published on the antioxidant and antimicrobial and some common medicinal plants are good activities of plant compounds9,10,11,12,13. antioxidant compounds. Many of the biologically The genus Tanacetum (syn. Chrysanthemum), active substancesfound in plants, including phenolic belonging to the family Asteraceae, is distributed over compounds (flavonoid and phenolics) are known to and west Asia and consists of about 150- 200 6 possess potential antioxidant properties . Antioxidants species14,15. T. polycephalum Schultz-Bip. is a are micronutrients that have gained importancein taxonomically complex species, in which Rechinger recent years due to their ability to neutralize free recognized seven subspecies in Flora Iranicaarea16. —————— T. polycephalum as an important medicinal plantin *Corresponding author of Iran (Unani medicine) have MALEKPOOR et al.: ANTIOXIDANT AND ANTIBACTERIAL ACTIVITIES OF T. POLYCEPHALUM 371

been used to treat many disorders17,18. According to Essential oil preparation recent studies, the essential oils and extracts of Harvested flowering aerial parts (leaves and T. polycephalum have biologically activity, including flowers) were dried at room temperature for one antibacterial19,20,21, antifungal21,22, antioxidant19, week. Dried plant material were powered (100gm) antiallergic, anticancer, anti-irritant, antiseptic, and subjected to hydro-distillation (1000 ml distillated anesthetic, analgesic, and disinfectiveeffects23. water) for 3 hrs using a Clevenger- type apparatus. Some earlier works have been reported on the Samples were dried with anhydrous sodium sulphate essential oils of various Tanacetum species from all and kept in amber vials at 4°C until chromatographic over the world24,25,26.This genus is rich in essential analysis. oils, bitter substances, and sesquiterpene lactonese27. Pervious chemical investigations on different species Determination of total phenolic content (TPC) of Tanacetum have shown the presence of The total phenolic contentin each essential oil was flavonoides28 and essential oils29,30,31. determined using the Folin–Ciocalteu method. To our knowledge, there are no published reports Briefly, 0.5 ml of the sample were mixed with 2.5 ml on total phenolic content, antibacterial and antioxidant of Folin- Ciocalteu’s phenol reagent and kept for activities of T. polycephalum. The main aim of the 5 min at 37°C. Then 2 ml of saturated Na2CO3 (7.5%) present study was toevaluate content of phenolic was added, and the mixture was brought to 10 ml with compounds, antioxidant and antibacterial activities of the addition of deionized-distilled water. The mixture essential oils of wild populations of T. polycephalum was maintained at room temperature in the dark for from Chaharmahal va Bakhtiari province, Iran. 120 min and then the absorbance of the mixture was measured at 765 nm against a reagent blank using a Material and methods UV–Vis spectrophotometer (Shimadzu Corp., Japan). Gallic acid equivalent (GAE) was used as the Plant material reference standard and total phenolic contentwas The aerial parts (up to ~ 5 cm, 0.05-0.2 kg) of wild expressed as mg of GA equivalents per gram of each populations of T.polycephalum were collected from essential oil on dry basis. Chaharmahalva Bakhtiari province, South western Iranat the early flowering stage on April-June 2011. Antioxidant test The samples of the plants were identified and voucher The DPPH radical scavenging activity of essential specimens were deposited at the Herbarium of oils was determined using the method proposed I A U, Shahrekord Branch, Shahrekord, Iran (Voucher by Hung et al.32 The essential oils at concentrations specimen No. 1357). of 16 to 500 µg/ml were mixed with an equal Soil physical and chemical characteristics such as volume of 0.2 mM methanol solution of DPPH. pH, EC, texture, and OC were taken from a soil- The disappearance of the DPPH after 30 min of sampling from different locations of Chaharmahal va incubation at room temperature was determined Bakhtiari province (Table 1). The pH is determined spectrophotometrically at 515 nm. Ethanol was used using a glass and reference electrode with a pH meter to zero the spectrophotometer. The amount of sample on a 1:1 suspension (Metrohm AG, 691). The EC is necessary to decrease the absorbance of DPPH by determined using with a EC meter on a 1:1 suspension 50% (IC50) was calculated graphically and the (Jenmay 4020). Also the texture and organic carbon percentage inhibition was calculated according of soil is determined. to the equation:

Table 1—Natural habitats of six populations belonging to Tanacetum polycephalum Region pH E.C. (dS/m) O.C. (%) Clay % Silt % Sand % Biregan 7.18 1.098 0.995 28 29 43 Sarteshniz 6.82 2.010 1.677 23 49 28 Hiregan 7.35 0.980 1.131 33 43 24 Sheykh-Shaban 7.42 0.903 1.170 32 34 34 Tang-e-sayad 7.69 0.810 0.527 26 42 32 Dezak 7.42 1.016 1.287 16 50 34

372 INDIAN J TRADIT KNOWLE, VOL 14, NO. 3, JULY 2015

AAC(0)− A (t )  comparison of the means of the main constituents of % inhibition =  ×100 essential oils evaluated by Duncan’s multiple range AC (0)  test at p< 0.05 level. Where AC(0) is the absorbance of the control at t = 0 min and AA(t) is the absorbance of the Results and discussion antioxidant at t = 30 min. The food preservative Total phenolic content butylhydroxyanisole (BHA) was used as positive Total phenolic content was determined control. spectrometrically according to the Folin–Ciocalteu

Antimicrobial test method and calculated as gallic acid equivalents Four gram-positive bacteria (Staphylococcus (GAE). A significant difference (p ≤ 0.01) for total aureus, Listeria monocytogenes, Bacillus cereus and phenolic content among the essential oils of different Streptococcus agalactiae) and two Gram-negative populations of T. polycephalum was found (Fig. 1). bacteria (Proteus vulgaris and Salmonella The highest total phenolic content was obtained typhimurium) obtained from Food Microbiology from the essential oil of the Hiregan population Laboratory, Veterinary Medicine Faculty, I A U, Iran (0.16 GAE/g DWE). The lowest total phenolic and identified using conventional morphological as content was obtained from the essential oil of the well as biochemical tests. Stock cultures of bacteria Sar-teshniz population (0.063 GAE/gm DWE) were kept in 20% glycerol PBS (phosphate buffered (Fig. 2). There are no reports in assessment of saline) at 70°C. Active cultures were generated by total phenolic contentessential oil and extract of T. polycephalum inoculating 100 µl of the thawed microbial stock , but about other species of Tanacetum, et al 33 suspensions into 5ml nutrient broth (Merck, Farzam . reported that the amount T. fruticulosum Germany) followed by overnight incubation at 37°C. of total phenolic ofmethanol extract of An initial bacterial suspension containing 107CFU/ml was 0.760 mg GAE/gm. In addition, Changqing was made from the flask broth culture. Subsequent dilutions were made from the above suspension, which were then used in tests. The disc diffusion method was used with some modification to determinate rate of inhibition growth of bacteria by plant essential oils. BHI agar (Merck, Germany) was used to prepare the culture medium and autoclaved at 115°C for 10 min. Briefly, plates (8 cm diameter) were prepared with 10 ml agar inoculated with 1 ml of each bacterial suspension.

Sterile paper discs (6 mm in diameter) were impregnated with 60 µl of dilutions of known Fig. 1—Total phenolic content of the essential oils of essential oils concentrations (100µg/disc) and Tanacetum polycephalum incubated at 35°C for 18 hrs. The essential oilswere dissolved in dimethyl sulfoxid (DMSO, 15 µl) before the test for antimicrobial activity. Standard antibiotic discs (6 mm diameter) of tetracycline and streptomycin (10 µg) were used as positive controls. Bacterial growth inhibition was determined as the diameter of the inhibition zones around the discs (mm). The growth inhibition diameter was an average of three measurements, taken at 3 different directions. All tests were performed in triplicate.

Statistical analyses

The data was statistically analyzed using Fig. 2—Antioxidant activity of the essential oils of one-way ANOVA by the program SPSS (17.0), and Tanacetum polycephalum MALEKPOOR et al.: ANTIOXIDANT AND ANTIBACTERIAL ACTIVITIES OF T. POLYCEPHALUM 373

et al.34 reported that the amount total phenolic activity of phenolics is mainly due to their redox content of the extract of T. was measured properties which make them act as reducing as agents, in 21.21 µg GAE/mg DWE. hydrogen donors, and singlet oxygen quenchers. They 43,44 The composition and quantity of the phenolics also may have a metallic chelating potential . 45 vary significantly according to different intrinsic and Atoui et al. mention that the antioxidant activity of extrinsic factors, such as species, plant genetics and phenolic is mainly due to their redox properties, cultivar, soil and growing conditions, maturity state which allow them to act as reducing agents, hydrogen and harvest conditions35. donors, and singlet oxygen quenchers.

Antioxidant test Antibacterial test Antioxidant activity is very important in The antibacterial activity of essential oil from the counteracting the deleterious role of free radicals in various populations of T. polycephalum was tested foods and biological systems. DPPH method is widely against the 6 pathogenic bacteriaby using the disc reported for screening of antioxidants and for diffusion method. Results indicate that the essential determining comparative antioxidant effectiveness36. oils used in this study inhibited development of The reduction capability of DPPH was determined by bacteria at different ratios.Generally,the antimicrobial the decrease in its absorbance at 517 nm, which is activity of essential oils of T .polycephalum against induced by antioxidants. The effect of antioxidants the studied bacteria was found as moderate to good on DPPH is based on their ability to donate a (Table 2). hydrogen atom to DPPH, thus converting the radical Previous investigations on different species of 37 Tanacetum have shown the antibacterial effects into a stable molecule .The lower IC50 value 46,47 indicates a stronger ability of the essential oils to act of this genus . The results indicated a broad spectrum activity against both Gram-positive and as a DPPH scavenger while the higher IC50 value indicates a lower scavenging activity of the Gram-negative bacteria. Gram-negative bacteria were scavengers as more scavengers were required to more resistant to the essential oils. Presence of an achieve 50% scavenging reaction38. outer membrane in Gram-negative bacteria may explain this resistance effect48. In our study, the antioxidant activity of essential oils of the various populations of T. polycephalum Table 2—Zones of growth inhabitation (mm) of different population of Tanacetum polycephalum (500µg/ml) was expressed as IC50 with value between 1.18 to 7.6 mg/ml that indicating the essential oils acts as population L. m S.a St. Ag B.C S.T.M P.v moderate DPPH scavenger (Fig. 3). Significant Biregan 18 26 25 15 22 15 differences (p< 0.01) in DPPH values were found Sarteshniz 31 42 54 32 31 32 in antioxidant activity of different populations of Hiregan 31 26 28 26 23 28 T. polycephalum. The antioxidant activity of the Sheykh-Shaban 22 19 25 30 23 21 Tanacetum species were reported by numerous Tang-e-sayad 26 30 27 34 25 24 17,39,40,41,42 investigators . These results suggest that the Dezak 27 24 30 37 23 30 higher levels of antioxidant activity were due to the Estereptomycin 19 - - 21 22 16 presence of phenolic components.Therefore, the Tetracycelin - 13 31 21 27 13 phenolic compounds are the major group contributing L. m: Listeria monocytogenes; S.a: Staphylococcus aureus; to the antioxidant activity of vegetables, fruit, cereals St. Ag: Staphylococcus agalactie; B. c: Bacilluscereus; and other plant-based materials.The antioxidant S.T.M: Salmonella tiphimorium and P.v: Proteus vulgaris.

Fig. 3—Antibacterial activity of the essential oils from Tanacetum polycephalum against six bacteria by disc diffusion method 374 INDIAN J TRADIT KNOWLE, VOL 14, NO. 3, JULY 2015

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