In-vitro Antibacterial and Antioxidant Activities of Sandalwood (Santalum Album)

Abstract

Santalum album commonly known as Sandalwood is used traditionally for health and wellness. It is an evergreen and hemi-parasitic tree and has a long history in Indian religious rituals and traditional Chinese medicine. Due to its wide application in cosmetics and therapeutics, we have done this study to explore the possibility of using aqueous extract of S. album as antibacterial and antioxidant agent. The S. album extract was prepared in distilled water. The activity of aqueous extract was evaluated against eight bacterial pathogens including two strains of Escherichia coli, one each of Klebsiella pneumoniae, Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Aeromonas species and Klebsiella oxytoca. The anti-oxidant activity was analyzed by two most common radical scavenging assays of FRAP (ferric reducing antioxidant power) and DPPH (1,1- diphenyl- 2-picrylhydrazyl). Results showed that S. album had strongest inhibitory activity against S. aureus (MTCC 902) i.e. 87% whereas; it showed no inhibition against E.coli (ATCC 25922) and B. subtilis (MTCC736). The S. album extract showed DPPH radical scavenging activity in a concentration-dependent manner with maximum scavenging of 64% in presence of 500μl of aqueous extract. The FRAP assay also proved antioxidant potential of S. album with the highest value of 0.628mM at 200μl of aqueous extract.

Keywords: Antibacterial activity; Antioxidant activity; Santalum album; Aqueous extract

Abbreviations

DPPH: 1,1-diphenyl-2-picrylhydrazyl; FRAP: Ferric Reducing Antioxidant Power; mg: Milligram; ml: Milliliter; CFU: Colony Forming Units; OD: Optical Density; TPTZ: 2,4,6-Tripyridyl-s- Triazine

Introduction

In present world of medical and pharmaceutical advancement, microbes have evolved with resistance against the drugs by changing their metabolism and genetic structure [1,2]. These drug resistant microorganisms are more pathogenic with high mortality rate and have become a threat against human race. To overcome microbial drug resistance, scientists are looking forward for the development of alternative and novel drugs [3]. Natural sources such as plants, algae and animals provide an array of natural medicinal compounds for the treatment of various infectious diseases [4-6]. Studies by various researchers have proved that plants are one of the major sources for drug discovery and development [7,8].

Free radicals are inevitably produced in biological systems and also encountered exogenously, and are known to cause various degenerative disorders like mutagenesis, carcinogenesis, cardiovascular disturbances and ageing [9].

Sandalwood, Santalum album, has been used since ancient times for religious purposes in incense, in fragrances, and as medicine. Various types of sandalwood trees grow in different countries of the world [10].

The present research has been conducted to study the medicinal properties like antimicrobial and antioxidant potential of aqueous extract of Santalum album so that they can be a hope in the field of phytodrugs.

Material and Methodology

Plant material

Sandalwood purchased from Local Ayurvedic Clinic.

Chemicals and reagents

All solvents and chemicals (analytical grade) used for antioxidant and antibacterial assay were purchased from Merck and Himedia. DPPH and TPTZ were purchased from Sigma-Aldrich.

Test microorganism

The following eight clinical isolates of bacteria were used for the study: Two species of Escherichia coli, one each of Klebsiella pneumoniae, Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Aeromonas species and Klebsiella oxytoca. All these cultures were maintained on nutrient agar plates at 4°C.

Methodology

Preparation of aqueous extract

he Sandalwood was ground finely and then strained through muslin cloth. 1 gram of sample was soaked for 2 hours in 20 ml of distilled water (50 mg/ml). The sample was then centrifuged and the supernatant was picked which served as aqueous extract for the further studies.

Antibacterial Assay

Antimicrobial activity of the aqueous extract was tested against three gram-positive bacteria (Bacillus subtilis, Aeromonas species. and Staphylococcus aureus) and five gram-negative bacteria (two of Escherichia coli and one each of Klebsiella pneumoniae, Klebsiella oxytoca and Pseudomonas aeruginosa). Overnight cultures were prepared in Luria broth (LB) media by inoculation with a single colony from agar plates and incubated at 37°C for 12 hrs. Overnight cultures were diluted with fresh LB media to approximately 104 colonies forming units (CFU) and incubated at 37°C for 12-14 hrs in the presence of S. album compared to the growth of the control culture where only media and bacterial inoculum was taken. The experiment was repeated twice for the confirmation. The percentage inhibition was calculated by using the formula:

Percentage Inhibition (%) = [(dc - dt)/dc] x 100,

where dc and dt represent OD₆₀₀ of control and treated sample strains respectively.

Antioxidant Activity

DPPH Assay

The antioxidant activity of S. album and the standard was checked on the basis of the free radical scavenging effect of the stable 1, 1-diphenyl-2-picrylhydrazyl (DPPH) by the method of Braca et al. with minor modifications [11]. A range of diluted working solutions of the S. album were prepared in distilled water. Ascorbic acid (1 mg/ml) in distilled water was used as standard. 0.1mM DPPH was prepared in ethanol and 500μl of this solution was mixed with 500μl of working sample solutions and standard solution separately. These solution mixtures were kept in dark for 30 min and optical density was measured at 517 nm using Spectrophotometer. 0.1mM DPPH solution was used as control. The range of diluted aqueous extracts was taken as blank. The optical density were recorded and DPPH scavenging was calculated using the formula given below:

DPPH scavenging Activity (%) = [(dc - dt)/dc] x 100,

where dc and dt represent OD₅₁₇ of control and test sample respectively.

FRAP Assay

Antioxidant activity assay was also done following the ferric-reducing antioxidant power (FRAP) method described by Benzie & Strain method with minor modifications [12]. FRAP reagents was freshly prepared by mixing 10 ml acetate buffer (300mM, pH 3.6), 1 ml 2,4,6-tris (2-pyridyl)-S-triazine (TPTZ) solution (10mM TPTZ in 40mM/L HCl) and 1 ml FeCl₃ (20mM) water solution. A range of diluted working solutions of the S. album were prepared in distilled water. Each sample (200 μl) was added in 1.5 ml of freshly prepared FRAP reagent and mixed and after 5 min, absorbance was measured at 593 nm, using FRAP working solution as blank. Ascorbic acid was used as standard. The results were expressed in mM Fe²⁺/ml of aqueous extract. Higher absorbance indicates higher reducing power.

Results

Antibacterial Assay

Antimicrobial assay of the aqueous extract was examined against various bacterial strains by accessing the percentage inhibition in presence of S. album compared to the control where only media and cultures were added. The results suggested that S. album exhibits bactericidal property in-vitro i.e. the growth of microorganisms was inhibited in its presence as shown in Table 1.