Antibacterial and Anti-Inflammatory Effects of Syzygium Jambos L
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Sharma et al. BMC Complementary and Alternative Medicine 2013, 13:292 http://www.biomedcentral.com/1472-6882/13/292 RESEARCH ARTICLE Open Access Antibacterial and anti-inflammatory effects of Syzygium jambos L. (Alston) and isolated compounds on acne vulgaris Richa Sharma1†, Navneet Kishore1†, Ahmed Hussein1,2† and Namrita Lall1*† Abstract Background: Acne vulgaris is a chronic skin disorder leading to inflammation as a result of the production of reactive oxygen species due to the active involvement of Propionibacterium acnes (P. acnes) in the infection site of the skin. The current study was designed to assess the potential of the leaf extract of Syzygium jambos L. (Alston) and its compounds for antibacterial and anti-inflammatory activity against the pathogenic P. acnes. Methods: The broth dilution method was used to assess the antibacterial activity. The cytotoxicity investigation on mouse melanocyte (B16-F10) and human leukemic monocyte lymphoma (U937) cells was done using sodium 3’-[1-(phenyl amino-carbonyl)-3,4-tetrazolium]-bis-[4-methoxy-6-nitrobenzene sulfonic acid hydrate (XTT) reagent. The non-toxic concentrations of the samples was investigated for the suppression of cytokines interleukin 8 (IL 8) and tumour necrosis factor (TNF α) by testing the supernatants in the co-culture of the human U937 cells and heat killed P. acnes using enzyme immunoassay kits (ELISA). The statistical analysis was done using the Graph Pad Prism 4 program. Results: Bioassay guided isolation of ethanol extract of the leaves of S. jambos led to the isolation of three known compounds namely; squalene, an anacardic acid analogue and ursolic acid which are reported for the first time from this plant. The ethanol extract of S. jambos and one of the isolated compound namely, anacardic acid analogue were able to inhibit the growth of P. acnes with a noteworthy minimum inhibitory concentration (MIC) value of 31.3 and 7.9 μg/ml, respectively. The ethanol extract and three commercially acquired compounds namely; myricetin, myricitrin, gallic acid exhibited significant antioxidant activity with fifty percent inhibitory concentration (IC50) ranging between 0.8-1.9 μg/ml which was comparable to that of vitamin C, the reference antioxidant agent. The plant extract, compounds ursolic acid and myricitrin (commercially acquired) significantly inhibited the release of inflammatory cytokines IL 8 and TNF α by suppressing them by 74 - 99%. TEM micrographs showed the lethal effects of selected samples against P. acnes. Conclusions: The interesting antibacterial, antioxidant and anti-inflammatory effects of S. jambos shown in the present study warrant its further investigation in clinical studies for a possible alternative anti-acne agent. Keywords: Syzygium jambos, Propionibacterium acnes, Antibacterial, Interleukin 8, Tumour necrosis factor, Cytotoxicity, Transmission electron microscopy * Correspondence: [email protected] †Equal contributors 1Department of Plant Science, Faculty of Agricultural and Biological Science, University of Pretoria, Pretoria 0002 South Africa Full list of author information is available at the end of the article © 2013 Sharma et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Sharma et al. BMC Complementary and Alternative Medicine 2013, 13:292 Page 2 of 10 http://www.biomedcentral.com/1472-6882/13/292 Background of pro-inflammatory cytokines like interleukins 8 (IL 8) Syzygium jambos L. (Alston) belongs to the family and tumour necrosis factors (TNF α) which attract mac- Myrtaceae and is commonly known as rose apple [1] rophages and lead to severe inflammation. Follicular wall which is widespread in sub-Saharan Africa [2], Central ruptures due to the action of hydrolytic enzymes cause America and Asia [3]. The plant is reported to be used oxidative damage with the release of free radicals [7,8]. for a variety of ailments and is known for its antipyretic Therefore, an agent that can inhibit the growth of P. and anti-inflammatory properties. All parts of the plant acnes, scavenge free radicals and suppress the inflam- are reported to have medicinal value. In Indo-China all matory response is promising. The conventional drugs parts of the plant are used for digestive and tooth available to treat acne act as antibacterial and anti- ailments. A decoction of the leaves is used as a diuretic, inflammatory agents. But these drugs have various side a remedy for sore eyes and for rheumatism. The seeds effects such as dryness, itching and hypopigmentation are used to treat diarrhoea, dysentery, diabetes and catarrh. and also have age restrictions. Moreover, bacterial resist- A decoction of bark is administered to relieve asthma and ance is an ongoing problem. The use of medicinal plants bronchitis [1]. dates back thousands of years. In light of previous Previous researchers who have investigated the plant reports regarding S. jambos as an antimicrobial agent on have documented its potential pharmacological value. a variety of micro-organisms, the present study was con- Acetone and aqueous bark extracts of this plant have ducted to explore its efficiency as an anti-acne agent. been reported to be active against Staphylococcus aureus, Thus, the antibacterial activity against P. acnes, the anti- Yersinia enterocolitica, Staphylococcus hominis, Staphylo- oxidant and anti-inflammatory properties of S. jambos coccus cohnii and Staphylococcus warneri [4]. In another and its compounds were investigated. study, the scientists have investigated the antimicrobial activity of both acetone and aqueous extracts of the leaves, Methods bark and seeds against eight microorganisms namely, S. Chemicals, microbial strain and culture media aureus, Bacillus subtilis, Escherichia coli, Klebsiella pneu- Silica gel 60 (70–230 mesh); sephadex LH-20 and all the moniae, Proteus vulgaris, Pseudomonas aeruginosa, Sal- analytical grade chemicals were purchased from Sigma- monella typhi and Vibrio cholera. The acetone bark Aldrich and Merck SA Pty Ltd. Three chemical com- extract showed growth inhibitory activity against all the pounds namely, myricetin, myricitrin and gallic acid microorganisms tested whereas, the leaf extract inhibited were acquired from Sigma-Aldrich.The ELISA kits were only S. aureus and the seed extract did not show any bought from BD Biosciences, Johannesburg, South Africa. inhibitory activity. The aqueous bark extract exhibited Cell proliferation Kit II (XTT) was supplied by Roche growth inhibitory effect against S. aureus, E. coli and S. diagnostics Pty Ltd., Johannesburg, South Africa. Propioni- typhi, whereas, the seed extract inhibited the growth of P. bacterium acnes (ATCC 11827) was purchased from aeruginosa and V. cholerae, and leaf extract exhibited an Anatech, Johannesburg, South Africa. The cell lines and inhibitory effect only against S. typhi [5]. In a study done medium were purchased from Highveld Biological Pty by Kuiate et al. [6], it was found that the ethanol bark Ltd., Johannesburg, South Africa. extract of S. jambos and its isolated triterpenoids such as friedelin, β -amyrin acetate, betulinic acid and lupeol Plant material exhibited antidermatophytic activity against Microsporum The leaves of Syzygium jambos L. (Alston) were collected audouinii, Trichophyton mentagrophytes and Trichophyton in August 2010 from the botanical garden of the Univer- soudanense. sity of Pretoria. The plant was identified at the H.G.W.J Acne vulgaris is a chronic inflammatory disorder of Schweicherdt Herbarium (University of Pretoria, Pretoria) the pilosebaceous unit with multifactorial etiology. It where a voucher, specimen number (PRU 119053), has affects almost everybody during the course of their life. been deposited for future reference. There are four key processes in the pathogenesis of acne: 1. Increased sebum production, 2. Follicular hyperke- Extraction and purification ratinization which leads to follicular obstruction, 3. The air-dried and powdered leaves (1.9 kg) of S. jambos Colonization by the causative agent, Propionibacterium were soaked in 7.5 L ethanol for three days at room acnes and 4. Host inflammatory responses triggered as a temperature. The crude ethanol extract (70.5 g) was results of bacterial infection [7,8]. obtained by concentrating the filtrate under reduced pres- As a result of the increased sebum production due to sure. About 60 g of this ethanol extract was applied to a high androgen levels, P. acnes, a gram positive anaerobic silica gel column (70 cm × 120 cm) using hexane fractions commensal, produces various hydrolytic enzymes that (Hex): ethyl acetate (EtOAc) of increasing polarity (100:0 act on the sebum to release free fatty acids. These free to 0:100) followed by 100% methanol (MeOH) as eluents. fatty acids acts as chemokines and increase the release In total forty three (43) fractions (500 ml) were collected. Sharma et al. BMC Complementary and Alternative Medicine 2013, 13:292 Page 3 of 10 http://www.biomedcentral.com/1472-6882/13/292 Similar fractions were combined, according to the thin- the bacteria was cultured from a Kwik-Stick on nutrient layer chromatography (TLC) profiles, which resulted in agar and incubated at 37°C for 72 h under anaerobic con- twelve (12) major fractions (MF). All the twelve (12) major ditions before the assay. The 72