The present study was designed to evaluate the antibacterial and antioxidant activities of hexane, ethylacetate and ethanol extracts of Leea rubra (Leeaceae) roots and stems, which has been used as a Lanna Traditional Medicines for Màhòog. Each extract was tested for antibacterial activity by agar diffusion method and microbroth dilution method and antioxidant activity by 2,2´-azino-bis(3-ethyl- benzthiazoline-6-sulfonic acid) (ABTS) free radical scavenging assay, 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay and ferric reducing antioxidant power (FRAP) assay. The ethylacetate extract of L. rubra root showed the highest antibacterial activity against gram-positive (IZD=15.5.0±0.5 to 17.5±0.5 mm, MIC=0.098-1.562 mg/ml).  While the ethanolic extract of root showed the strongest antioxidant activity in ABTS, DPPH and FRAP method (TEAC=0.888±0.001, 0.849±0.020 and 0.733±0.037, respectively). The data obtained from this study confirms the traditional use of L. rubra for treatment Màhòog. Keywords: Leea rubra, antioxidant activity, antibacterial activit

Chansakaow, Sunee

Eakanunkul, Suntara

Kadchumsang, Sarinya

Sirisa-ard, Panee

Sookkhee, Siriwoot

English

International Institute for Science, Technology and Education (IISTE): E-Journals

Journal of Natural Sciences Research                                                                                                                                               www.iiste.org ISSN 2224-3186 (Paper)   ISSN 2225-0921 (Online) Vol.4, No.11, 2014  1 Antibacterial and Antioxidant Activities of Various Fraction of Leea rubra (Leeaceae)  Sarinya Kadchumsang1 Panee Sirisa-ard1 Siriwoot Sookkhee2 Suntara Eakanunkul1 Sunee Chansakaow1* 1. Department of Pharmaceutical Sciences, Faculty of Pharmacy, Chiang Mai University, Chiang Mai 50200 Thailand 2. Department of Microbiology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand *E-mail of the corresponding author: chsunee@gmail.com  Abstract The present study was designed to evaluate the antibacterial and antioxidant activities of hexane, ethylacetate and ethanol extracts of Leea rubra (Leeaceae) roots and stems, which has been used as a Lanna Traditional Medicines for Màhòog.  Each extract was tested for antibacterial activity by agar diffusion method and microbroth dilution method and antioxidant activity by 2,2´-azino-bis(3-ethyl- benzthiazoline-6-sulfonic acid) (ABTS) free radical scavenging assay, 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay and ferric reducing antioxidant power (FRAP) assay.  The ethylacetate extract of L. rubra root showed the highest antibacterial activity against gram-positive (IZD=15.5.0±0.5 to 17.5±0.5 mm, MIC=0.098-1.562 mg/ml).  While the ethanolic extract of root showed the strongest antioxidant activity in ABTS, DPPH and FRAP method (TEAC=0.888±0.001, 0.849±0.020 and 0.733±0.037, respectively).  The data obtained from this study confirms the traditional use of L. rubra for treatment Màhòog. Keywords: Leea rubra, antioxidant activity, antibacterial activity  1. Introduction Leea rubra Blume ex Spreng is and erect or suberect soft-wooded shrub of family Leeaceae.  Root and stem of this plant has been used as a Lanna Traditional Medicines for Màhòog.   Màhòog is a group of intestinal diseases (Brun et al., 1987).  The exact cause of Màhòog disease is not well established, however there are many factors that provoke this disease including half-cooked and spicy food consumptions, drinking less water and hard work.  Although the disease is not severe and occurs much less frequently, this disease is a chronic disease if left untreated, and it may result in death.  The characteristic symptoms of Màhòog are pain, inflammation and wound infection.  Especially, when wound is occurred, it is accompanied with pain, reddening and edema within a short time, which are the classical symptoms of inflammation.  These symptoms are caused by releasing of eicosanoids, prostaglandins, leukotrienes, and reactive oxygen species (ROS).  Not only is ROS produced in large amount at the site of wound as a defense mechanism against invading bacteria, but also at the same time, the presence of free radicals may be hampered the process of wound healing, resulting in wound damage or microbial infection (Houghton et al., 2005; Srinivas et al., 2008).  Therefore, the objectives of the present study were to investigate the antibacterial and antioxidant activities of various fraction of Leea rubra.  The data obtained from the study will be used as a scientific evidence to support the pharmacological properties of Lanna medicinal plants.  2. Experimental 2.1 Plant materials The plants used in the work were collected from Chiang Mai provinces, Thailand.  The identity of the Lanna medicinal plants were verified by a taxonomist at Faculty of Pharmacy and the voucher specimens were deposited in the Herbarium of Faculty of Pharmacy, Chiang Mai University. 2.2 Plant extracts The stem and root samples of Leea rubra were cut into small pieces, dried at 50°C for 24 hours and then ground into powder.  Each powder was successively extracted with hexane, ethylacetate and ethanol by soxhlet apparatus, respectively.  The extract solutions were filtered through Whatman filter paper No. 1 and then the solvents were removed by using rotary evaporator. 2.3 Antibacterial activity 2.3.1 Microorganisms Four bacteria; 2 Gram-positive and 2 Gram-negative were used for the antibacterial assays.  The following strains of microorganisms were used: Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 9027. 2.3.2 Agar diffusion method Diameter of zone of inhibition was determined using agar well diffusion technique modified from Kirby-Bauer method (Bauer et al., 1966).  A swab of the bacteria suspension containing 1×10⁸ cfu/ml was spread on to sterile Journal of Natural Sciences Research                                                                                                                                               www.iiste.org ISSN 2224-3186 (Paper)   ISSN 2225-0921 (Online) Vol.4, No.11, 2014  2 Petri dish (Greiner bio-one, Austria) containing Tryptic Soy Agar (TSA; Difco, MI) media.  Each extract was dissolved in propylene glycol to concentration 50 mg/ml and autoclaved at 121°C, 15 psi for 15 min.  Wells were cut with sterile borer (6 mm) and 50 µl of the extracts were added into the wells.  The plates were incubated at 37°C for 24 hours.  The propylene glycol used as negative control while the standard chloramphenicol 10 mg/ml and gentamicin 1 mg/ml were used as positive controls.  Antibacterial activity was indicated by the presence of clear inhibition zone around the wells. Tests were performed in triplicate. 2.3.3 Minimum inhibitory concentration (MIC)  The MIC was determined using microbroth dilution method.  The extracts were dissolved in 50% DMSO (Sigma-Aldrich, Germany).  The extracts were diluted by two-fold to obtain a concentration range 100–0.05 mg/ml with Tryptic Soy Broth (TSB; Difco, MI) in the 96-well microplates (Greiner bio-one, Austria).  The microorganism suspension (1x105 cfu/ml) of 50 µl was added to the broth dilutions.  These were incubated for 24 hours at 37ºC. MIC of each extract was taken as the lowest concentration that did not permit any turbidity of the tested microorganism. 2.4 Antioxidant Activity 2.4.1 ABTS free radical scavenging assay The ABTS assay was performed using a modified method (Re et al., 1999).  ABTS•+ stock solution was generated by oxidation of 7.0 mM ABTS (Sigma-Aldrich, Germany) with 2.45 mM potassium persulfate (UNILAB, Austria).  The solution was protected from light and stored at room temperature for 12-16 hrs. The 2.0 ml of ABTS•+ working solution was mixed with 100 µl dilute extract, comparing it to Trolox (Sigma-Aldrich, Germany).  After 3 min incubation at room temperature, the color reaction was measured at 734 nm using a UV/VIS spectrophotometer (Varian Cary 1E UV/Visible Spectrophotometer, USA).   The results of ABTS assay were expressed as Trolox equivalent antioxidant capacity (TEAC).  This index is defined as gram of standard is equivalent to 1.0 gram of the extract. 2.4.2 DPPH free radical scavenging assay The DPPH free radical scavenging assay was done according to the method of Brand-Williams et al., (1995) with some modifications.  The 2.1 milliliters of reaction mixture containing 2.0 ml ethanolic DPPH (Sigma-Aldrich, Germany) and 100 µl diluted extract.  The mixture was incubated in the dark for 30 min at room temperature.  The absorbance was measured at 517 nm using a UV/VIS spectrophotometer.  The results were calculated in terms of TEAC.  This index is defined as gram of standard is equivalent to 1.0 gram of the extract. 2.4.3 FRAP assay The FRAP assay was done according to Benzie and Strain, (1996) with some modifications.  The 3.1 milliliters of reaction mixture containing 3.0 ml FRAP reagent and 100 µl diluted extract.  The mixture was incubated in the dark for 4 min at 37°C.  The absorbance was measured at 593 nm.  The results were calculated in terms of TEAC.  This index is defined as gram of standard is equivalent to 1.0 gram of the extract.  3. Results and discussions 3.1 Antibacterial Activity In this study, the antibacterial activities of hexane, ethylacetate and ethanol extracts of L. rubra root and stem were evaluated against 2 gram-positive and 2 gram-negative bacterial.  Chloramphenicol and gentamicin were used as standards for bacteria at concentration 0.5 and 0.05 mg/well, respectively.  The results showed that the ethylacetate extracts of root and stem, ethanol and hexane extracts of root were active against the gram-positive bacteria: B. subtilis and S. aureus (IZD=9.0±0.5 to 17.5±0.5 mm).  None of extracts showed activity against the gram-negative bacteria: E. coli and P. aeruginosa.  The zone of inhibition values are summarized in Table 1. Journal of Natural Sciences Research                                                                                                                                               www.iiste.org ISSN 2224-3186 (Paper)   ISSN 2225-0921 (Online) Vol.4, No.11, 2014  3 Table 1: Antibacterial activity of L. rubra extracts in agar well diffusion method  The MIC values of different microbes, tested in a concentration range 100–0.05 mg/ml, are given in Table 2. All of root extracts showed activity against gram-positive bacteria, B. subtilis and S. aureus, with MIC ranging from 0.098-12.5 mg/ml.  The ethylacetate extract of stem showed MIC against B. subtilis and S. aureus were 50 mg/ml while the MIC of hexane and ethanol extracts of stem could not be observed even at a concentration of 100 mg/ml. Table 2: The minimum inhibitory concentration (MIC)  3.2 Antioxidant Activity The antioxidant activity of hexane, ethylacetate and ethanol extracts of L. rubra root and stem were evaluated by using ABTS, DPPH and FRAP method.  The results of antioxidant activity were expressed as Trolox equivalent antioxidant capacity (TEAC) (Table 3).  It was found that the antioxidant activity of ethanol extract (TEAC=0.790±0.001 to 0.888±0.001) and ethylacetate extract (TEAC=0.733±0.037 to 0.884±0.001) of L. rubra root were higher than ehanol and ethylacetate extracts of L. rubra stem 7-8 times.  While the hexane extracts showed low antioxidant activity in all method. Table 3: The antioxidant activity of hexane, ethylacetate and ethanol extracts of L. rubra root and stem  4. Conclusions In conclusion, the ethylacetate extract of L. rubra root showed the highest antibacterial activity against gram-positive (IZD=15.5.0±0.5 to 17.5±0.5 mm, MIC=0.098-1.562 mg/ml).  While the ethanolic extract of root showed the strongest antioxidant activity in ABTS, DPPH and FRAP method (TEAC=0.888±0.001, 0.849±0.020 and 0.733±0.037, respectively).  The results of this study indicated L. rubra to have potential antibacterial and antioxidant activities that could be used as a scientific evidence to support the pharmacological properties of Lanna medicinal plants.  In this study, the hexane extracts of this plant indicated less active than the ethylacetate and ethanol extracts.  This trial for these activities guided isolation led us knows that, the active principle(s) belongs to moderately polar fraction.  References Bauer AW, Kirby WM, Sherris JC, Turck M. (1966), “Antibiotic susceptibility testing by a standardized single Journal of Natural Sciences Research                                                                                                                                               www.iiste.org ISSN 2224-3186 (Paper)   ISSN 2225-0921 (Online) Vol.4, No.11, 2014  4 disk method”,  American Journal of Clinical Pathology 45, 493-496. Benzie IFF, Strain JJ. (1996), “The ferric reducing ability of plasma (FRAP) as a measure of ‘‘antioxidant power’’: the FRAP assay”,  Analytical Biochemistry 239, 70–76. Brand-Williams W, Cuvelier ME, Berset C. (1995), “Use of free radical method to evaluate antioxidant activity”, Lebensmittel Wissenschaft und Technologie 28, 25–30. Brun V, Schumacher T. (1987), “Traditional herbal medicine in Northern Thailand”, University of California Press, Berkeley. Houghton PJ, Hylands PJ, Mensah AY, Hensel A, Deters AM. (2005), “In vitro tests and ethnopharmacological investigations: wound healing as an example”, Journal of ethnopharmacology, 100, 100-107.  Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C. (1999), “Antioxidant activity applying an improved ABTS radical cation decolorisation assay”, Free Radical Biology and Medicine 26, 1231-1237. Srinivas RB, Reddy RKK, Naidu VG, Madhusudhana K, Agwane SB, Ramakrishna S, et al. (2008), “Evaluation of antimicrobial, antioxidant and wound-healing potentials of Holoptelea integrifolia”, Journal of ethnopharmacology 115, 249-256. The IISTE is a pioneer in the Open-Access hosting service and academic event management.  The aim of the firm is Accelerating Global Knowledge Sharing.  More information about the firm can be found on the homepage:  http://www.iiste.org  CALL FOR JOURNAL PAPERS There are more than 30 peer-reviewed academic journals hosted under the hosting platform.   Prospective authors of journals can find the submission instruction on the following page: http://www.iiste.org/journals/  All the journals articles are available online to the readers all over the world without financial, legal, or technical barriers other than those inseparable from gaining access to the internet itself.  Paper version of the journals is also available upon request of readers and authors.   MORE RESOURCES Book publication information: http://www.iiste.org/book/  IISTE Knowledge Sharing Partners EBSCO, Index Copernicus, Ulrich's Periodicals Directory, JournalTOCS, PKP Open Archives Harvester, Bielefeld Academic Search Engine, Elektronische Zeitschriftenbibliothek EZB, Open J-Gate, OCLC WorldCat, Universe Digtial Library , NewJour, Google Scholar   

Antibacterial and Antioxidant Activities of Various Fraction of Leea rubra (Leeaceae)

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Antibacterial and Antioxidant Activities of Various Fraction of Leea rubra (Leeaceae)

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