Antimicrobial Activity of Frankincense of Boswellia Serrata

Int.J.Curr.Microbiol.App.Sci (2014) 3(10) 1095-1104

International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 3 Number 10 (2014) pp. 1095- 1101 http://www.ijcmas.com

Original Research Article

Antimicrobial activity of frankincense of Boswellia serrata

Shaik Ismail Mannur1, KRS Sambasivarao1 and Bhaskar Matcha2*

1Department of Biotechnology, Acharya Nagarjuna University, Guntur, India 2Division of Animal Biotechnology, Department of Zoology, S.V University, Tirupati, India *Corresponding author

ABSTRACT

This work describes the in-vitro screening of frankincense anti microbial activity of Boswellia serrata. Different concentrations (25, 50, 75 and 100 mg /ml) was K e yw or ds evaluated for the investigation of antimicrobial efficacy using Gram positive (Streptococcus pneumonia, Staphylococcus aureus and Bacillus subtilis) and Gram Boswellia serrata, negative (E. coli , Proteus vulgaris, Klebsiella pneumonia, Enterobacter aerogenes and Pseudomonas aeruginosa) microbes. Inhibition halos were evaluated and by Frankincense, Antimicrobial the use of the antibiotic Ciprofloxacin (5µg/ml) as positive control. DMSO was used as a negative control. Results demonstrated significant antimicrobial activity. Activity, Ciprofloxacin In this assay, extracts of frankincense showed antimicrobial activity comparable with standard and can be used in combating the bacterial infested diseases caused by the studied bacterial strains.

Introduction

Plant antimicrobials tender prospective Taxonomic status of Boswellia serrata agent to deal with the hazard of biological warfare (Gibbons, 2008). Consideration to Boswellia serrata Roxb. ex Colebr. the sighting of novel plant antimicrobials (Burseraceae) is a deciduous moderate to must be paid in this new era of large sized branching tree that grows chemotherapeutic healing of infection by abundantly in the seshachalam hill using plant-derived principles and on valleys.The morphological characters have discovery of new antiinfective agents crown spreading and flat, bark greenish, (Cowan, 1999). Herbal preparations can ashy grey, ex-foliating in thin flakes. Leaves supplement other systems of medicine for apically clustered, imparipinnate. Leaflets the treatment of diseases caused by bacteria 14-26, thin-coriaceous, oblong-lanceolate, (Archana and Abraham, 2011). In fact, entire or crenate, obtuse or subacute, efforts are going on to identify and isolate secondary nerves more than 16 pairs. secondary metabolites from plants as Flowers pinkish white, in little branched prospective modulators of bacterial panicles. Sepals and petals 5-7 each. resistance (Stavri et al., 2007). Stamens 10-16, inserted below disc; disc 1095

Int.J.Curr.Microbiol.App.Sci (2014) 3(10) 1095-1104 annular. Ovary 3-locular; ovules 2 per When incisions are made in the trunks of the locule, pendulous. Fruits trigonous, brown, Boswellia serrata trees to produce exuded pyrenes 3, heart shaped, each one seeded. gum (Oleo gum-resin), which appears as milk like resin. The resin hardens B. glabra Roxb.; B. serrata Roxb. var. (solidification) into orange-brown gum resin glabra (Roxb.) Benn. are the synonyms. B. known as frankincense or olibanum. There serrata vernacularly (Telugu) known as are numerous species and varieties of Guggilum, Anduga, Dhupamu, frankincense trees, including Boswellia Guggiladhpuam. Commonly known as serrata in India. Salai, White dammar, ‘Indian olibanum’, ‘Indian frankincense’, ‘dhup’ and ‘salai’ or The resins of Boswellia serrata have been ‘salai guggul’ (Siddiqui, 2011). used for the treatment of rheumatoid arthritis and other inflammatory diseases (Banno, Distributed commonly in lower hill slopes of 2006) such as Crohn's disease (Langmead, Tirumala and Talakona of Seshachalam hill 2006) in traditional medicine of many ranges. Flowering and Fruiting occurs in countries. The anti-inflammatory activity the season of March-August. Traditional has been attributing to the resin's ability in usage of Gum is diuretic, diarrhoea, regulating immune cytokines dysentery, stomachic, cardiac diseases, production (Chevrier, 2005) and leukocyte cough, haemorrhage, dyspnoea, polyuria, infiltration (Sharma et al., 1988; Singh and leucorrhoea, oligospermia, urinary troubles, Atal, 1986). Boswellia serrata extract also piles, ulcers, burns (Madhava chetty et al., exhibits anti-bacterial and anti-fungal 2013). activities (Weckesser, 2007).

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Additionally, extracts from Boswellia Microorganisms species gum resins might possess anticancer activities, based on their anti- Clinical isolates of Gram positive proliferative and pro-apoptotic activities in (Streptococcus pneumonia, Staphylococcus rat astrocytoma cell lines and Clinically, aureus and Bacillus subtilis) and Gram extract from the resin reduces the negative (E. coli, Proteus vulgaris, peritumoral edema in glioblastoma Klebsiella pneumonia, Enterobacter patients (Winking, 2000) and in human aerogenes and Pseudomonas aeruginosa) leukemia cell lines (Hostanska, 2002), as authentic bacterial strains were used in the well as their anti-carcinogenic activity in study which are procured from Institute of chemically induced mouse skin cancer Microbial Technology (IMTECH), models (Huang, 2000). Chandigarh. The stock culture maintained at Division of Animal Biotechnology, Sri The pharmacological characteristics and Venkateswara University. All the clinical efficacy of Boswellia serrata have microorganisms were maintained at 4°C on been studied, with research published and nutrient agar slants. systematically reviewed in the medical literature (Ernst, 2008). These results Preparation of the Bacterial Suspension suggest that frankincense resin contains active ingredients that modulate important The turbidity of each of the bacterial biological activities. B. serrata flowers and suspension was prepared to match to a 0.5 leafs showed significant antibacterial McFarland standard (1.5x108 CFU/ml). activity (Mohammed Aman et al., 2010). In Measure the turbidity with the aid of a addition B. serrata has versatile spectrophotometer at an optical density pharmacological activities (Arshiya Sultana 0.08-0.13 and turbid suspension at 625 nm et al., 2013). as per Bauer-Kirby Method (1966).

However, there are no enough scientific Determination of antimicrobial activity reports to support these supposed antimicrobial activity. The present Culture of the bacterial organism was investigation was undertaken which deals aseptically introduced and evenly spread with the evaluation of antimicrobial activity using sterile ‘L’ rod on the surface of sterile of aqueous crude extraction of frankincense Mueller Hinton agar (M173/M1084, resin of Boswellia serrata. HiMedia) plates. The agar disc diffusion test is the most convenient and widely used Materials and Methods method for routine antimicrobial susceptibility testing according to CLSI The crude gum is collected and processed. (Clinical Laboratory Standards Institute; The collected material is dried under shade formerly NCCLS). 25 µL of different and made into powder and subjected to hot concentrations (25, 50,75,100 mg/ml) of the percolation by using soxhlet apparatus with resin coarse powder extract were added on water. The extract was filtered using whatman No. 1 filter paper disc (6mm) and Whatsman-No. 1 filter paper and the was inoculated with a loopful of the test extraction procedure were repeated three organism previously diluted to 0.5 times. The filtrate was used for the McFarland turbidity standards seeded on the biological assay. medium. The experiment was run in

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Int.J.Curr.Microbiol.App.Sci (2014) 3(10) 1095-1104 triplicate for each extract and each organism known ethnobotanical value and has shown tested. The plates were incubated at 37°C potent activity against the tested pathogens. for 24 hours and the zones of inhibition were Investigation in the potential discovery of measured in millimeters using a calibrated new natural bioactive compounds (Graph-1). instrument like zone scale (HiMedia). DMSO was used as a negative control. The highest antimicrobial activity was These inoculated tubes were then incubated observed on E. coli with zone of inhibition Control experiments comprising inoculums as 21.87±0.98 and the lowest effect was on without the extract were set up. E. aerogenes with a zone of inhibition of 11.67±1.00 mm. Whereas, Standard showed Determination of MIC highest activity against E.aerogenes (30.27±1) and lowest activity against S. The determination of the MIC was done pneumoniae (20.07±0.74). The order of with different dose levels of (25, 50, 75 inhibitory activity of extract against and100 mg/ml) of extract. The procedure different organisms is: E. coli>S. aureus>B. was repeated on all the test organisms using subtilis>S. typhi>K. pneumoniae>S. the standard antibiotic Ciprofloxacin (5 pneumoniae> E. aerogenes > P. vulgaris. µg/mL Disc, Himedia, Mumbai, India). The order of inhibitory activity of standard The statistical analysis was undertaken using against different organisms are summarized t-test in SPSS statistics software (Version as P. vulgaris > E.coli >S. aureus> E. 20, IBM Corporation, New York, USA), aerogenes> P. aureginosa> B. subtilis> K. considered significant when p≤0.05. pneumonia. This shows that the studied Boswellia Serrata frankincense extract Results and Discussion mostly exhibited bacteriostatic effects.

In this study the aqueous extract of Extract inhibitory activity was statistically commercially available Boswellia serrata compared to the inhibitory activity of powder was evaluated for their antimicrobial standard and observed that extract inhibitory properties. Preliminary antimicrobial activity on the microbes has significantly screening assay of commercially available lower activity than the standard (P≤0.01, Boswellia serrata gave relatively wide P≤0.001). inhibition zone against the test strains. The MIC values of both revealed similar results. Minimum inhibitory concentration (MIC) is Showed different activities in vitro against defined as the lowest concentration that will the nine tested bacteria at the concentrations inhibit the growth of a test organism over a 10, 25, 50, 100 mg/ml. To determine the defined interval related to the organism’s antibacterial sensitivity of B.S growth rate, most commonly 18-24 h Frankincense resin aqueous extracts. All (Turnidge et al., 2003). The zone size is the bacterial strains were compared with inversely proportional to the minimum Ciprofloxacin under the same experimental inhibitory concentration (MIC). Disc conditions. In this study both the extract diffusion test is a qualitative test method. and commercial product have shown greater The recommended medium for disc antimicrobial activity which may explain diffusion testing is Mueller-Hinton agar anonymous claim on the topical use of (MH; Himedia, Mumbai, India) (CLSI, Boswellia serrata for microbial infections 2012). (Table-1). Boswellia serrata has much

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Graph.1 Antimicrobial efficacy of Boswellia Serrata frankincense resin extracts against some bacterial pathogens vs Standard (Ciprofloxacin -5 µg/ml)

Values are the Mean ± SD; Astringent indicate level of significance (** = p≤0.01, *** = p≤0.001)

Table.1 Minimum Inhibitory Concentrations (MIC)

S. No Test Organism G+ / G- I II III IV 1. B. subtilis G+ ++ + + * 2. S. aureus G+ + + * - 3. S. pneumoniae G+ + + + * 4. E. coli G- + * - - 5. K. pneumoniae G- ++ + + * 6. P. aeruginosa G- ++ ++ + + 7. E. aerogenes G- ++ ++ + * 8. P. vulgaris G- ++ ++ ++ +

I = 25mg/ml; II =50 mg/ml; III =75 mg/ml; IV =100 mg /ml,*=MIC, - = No growth, + = Moderate growth, ++ =Dense growth

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This medium demonstrates good batch-to- (2013). Boswellia serrata Roxb. A batch reproducibility, and supports the Traditional Herb with Versatile growth of most non fastidious bacterial Pharmacological Activity: A Review. pathogens (Jorgensen and Turnidge, 2003). IJPSR 4(6): 2106-2117. Well-variant of the diffusion method was Banno N, Akihisa T, Yasukawa K, Tokuda more sensitive and best conditions for the H, Tabata K, Nakamura Y, Nishimura determination of minimal inhibitory R, Kimura Y, Suzuki T (2006): Anti- concentration (Valgas et al., 2007). inflammatory activities of the Dimethyl sulfoxide (DMSO) used as solvent triterpene acids from the resin of for natural as well as synthetic antibacterial Boswellia carteri. J compounds (Wadhwani et al., 2009; Ethnopharmacol. 107:249-253. Houghton and Raman, 1998). Bauer, Kirby, Sherris and Turck, (1966), Antibiotic susceptibility testing by However, exclusive focus on individual standard single disc diffusion method biochemical targets neglects the fact that Am. J. Clin. Path., 45: 493. strong synergy of multiple constituents in a Chevrier MR, Ryan AE, Lee DY, Zhongze crude drug may prove more potent and M, Wu-Yan Z, Via CS effective than any single purified compound, (2005). Boswellia carterii extract or that interactions of co-occurring inhibits TH1 cytokines and promotes phytochemicals may help nullify the toxic TH2 cytokines in vitro. Clin Diag Lab effects of individual constituents. So while it Immunol, 12:575-580. is important to understand the active agents CLSI (Clinical and Laboratory Standards within medicinal plants, it should also be Institute). (2012). Performance with caution that we extract and use standards for antimicrobial constituents in isolation. susceptibility testing. National Committee for Clinical Laboratory In Conclusion, the screening of Standards, Wayne, Pa. antimicrobial activity performed on aqueous Cowan M. (1999). Plant products as fractions of 25, 50, 75, 100 mg/ml of antimicrobial agents. Clinical Boswellia serrata, traditionally important Microbiology Reviews, 12(4):564- medicinal plant proved to be a bacteriostatic 582. agent. The current work will provide new Ernst E. (2008). Frankincense: systematic reference data for the drug development and review. BMJ. ;337:a2813. possesses the ability to inhibit pathogenic Gibbons S. (2008). Phytochemicals for bacteria. Further studies should be done on bacterial resistance - Strengths, fractionation and identification of bioactive weaknesses and opportunities. Planta constituents which are responsible for Medica 74, 594–602. antibacterial activity. Hostanska K, Daum G, Saller R (2002). Cytostatic and apoptosis- References inducing activity of boswellic acids toward malignant cell lines in vitro. Archana S. and Abraham J. (2011). Journal Anticancer Res 22:2853-2862. of Applied Pharmaceutical Science. Houghton P and Raman A. (1998). 1(8): 149-152 Laboratory handbook for the Arshiya Sultana, Khaleeq Ur Rahman, A.R. fractionation of natural extracts. Padmaja and Shafeeq Ur Rahman Chapman and Hall. 1-199.

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