In vitro Assessment of Two commercial Honey Samples for Antibacterial and Antioxidant Activities

Research Article

Austin J Trop Med & Hyg. 2015;1(1): 1002.

In vitro Assessment of Two commercial Honey Samples for Antibacterial and Antioxidant Activities

Amit Saha and Shyamapada Mandal*

Department of Zoology, University of Gour Banga, India

*Corresponding author: Shyamapada Mandal, Laboratory of Microbiology and Experimental Medicine, Department of Zoology, University of Gour Banga, Malda 732103, India

Received: November 17, 2014; Accepted: December 15, 2014; Published: January 05, 2015

Abstract

The current communication represents the antibacterial and antioxidant activities of two commercial honeys: Dabur Honey (DH) and Patanjali Honey (PH) against clinical isolates of Proteus vulgaris, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. The antibacterial activities of PH and DH (both autoclaved and non-autoclaved), were determined alone and in combination with antibiotics, Gentamicin (GM) and Kanamycin (KM) against the test isolates. The autoclaved PH and DH (at concentrations 10x103 and 15x103μg/disc) had Zone of Inhibition Diameter (ZID) 9 - 17 mm and 9 - 13 mm, respectively, against the gram-negative bacteria (P. vulgaris, E. coli, Ps. aeruginosa); S. aureus was resistant to almost all concentrations of the honeys. The non-autoclaved honeys (at concentrations 10x103 and 15x103μg/disc) showed excellent activity against both gram-positive (S. aureus) and gramnegative bacteria tested (PH honey had ZID 10-27 mm, and DH honey had ZID10-30 mm). The IC50 values of PH and DH, in 2, 2-Diphenyl-1-Picrylhydrazyl (DPPH) system, were 132.24x103 μg/ml and 66.73x103μg/ml, respectively; both the honeys contained steroids, quinones and terpenoides. In combination with KM and GM the autoclaved honey samples (PH and DH) had synergistic activity against S. aureus and E. coli ATCC 25922 standard strain, while GM-honey and KM-honey combinations had synergistic interaction against Ps. aeruginosa and P. vulgaris, respectively. Thus, PH and DH alone and in combination with GM/ KM can be used against different bacterial strains causing infection to humans.

Introduction

Honey has been in use for its healing, nutritional and therapeutic properties since ancient times, and currently it has been proved experimentally that honey possesses anti-bacterial, anti-inflammatory and anti-oxidant properties, which may be beneficial in combating multi-drug resistant bacteria as well as in preventing many chronic inflammatory processes [1]. Honey, which is a healthy food stuff and nutrition, serves as a good source of natural antioxidant, and thus it is free radical scavenger reducing the formation of free radicals, or neutralizing them that produce beneficial effects in human health [2]. The improved status of serum total anti-oxidation among young females with regular use of honey revealed it is one of the most acceptable form of food to keep balance between antioxidants and prooxidants minimizing the onset of many diseases [3]. Various studies explained the mechanism of action of different honeys against antibiotic resistant bacteria in vitro and their antibio film activity, and an important clinical advantage is that resistance to honey has not yet been detected in microorganisms causing human infection [4,5].

Mandal et al. [6] determined the antibacterial activity of honey against clinical isolates of Escherichia coli, Pseudomonas aeruginosa and Salmonella enterica serovar Typhi. Das et al. [2,7] investigated the antioxidant properties of various unifloral honeys procured from West Bengal, India. Allen et al. [3,8] have revealed that the honey is effective against Methicillin-Resistant S. Aureus (MRSA), β-haemolytic streptococci and Vancomycin Resistant Enterococci (VRE). Cooper et al. [4,9] discovered the antibacterial activity of honey against strains of S. aureus from infected wounds. Visavadia et al. [5,10] conducted research on manuka (L. scoparium) honey, and showed its activity against several human pathogens, including E. coli, Enterobacter aero genes, Salmonella typhimurium, S. aureus. Hussein et al. [6,11] reported, Gelam honey has anti-oxidative and radical scavenging activities, which are mainly attributed to its phenolic content. Khalil et al. [7,12] discovered antioxidant property of Algerian honey, as indicated by their high phenolic, flavonoid, ascorbic acid and proline contents.

The importance of honey in medical science has already been described by Mandal et al [13]. Thus, honey, both natural and commercial, has been used traditionally over the years by the people in India as food, and as traditional medicine in the treatment of various health disorders, but only a few data based on the scientific studies are available to support the medicinal claims of commonly consumed honey in our part of the globe. Therefore, the current study has been undertaken to investigate the antibacterial and Antioxidative activities of two types of commonly consumed commercial honey samples: Dabur Honey (DH) and Patanjali Honey (PH) purchased from local market (Malda, India); to the best of our knowledge, this is the first study of its kind from our part of the globe.

Materials and Methods

Bacterial strains

The bacterial strains used in the study included E. coli, Ps. aeruginosa, P. vulgaris, and S. aureus; the E. coli ATCC 25922 strain was used as control. The identified bacterial isolates were kindly provided by Dr. N. K. Pal, Professor and Head, Department of Microbiology, Malda Medical College, Malda (India).

Honey samples and disc preparation

Two commercial honeys: Dabur Honey (DH) and Patanjali Honey (PH) were purchased from market (Malda, India), and were utilized in the study. One gram of honey diluted in 5ml of double distilled water (200μg/μl) was autoclaved at 121°C for 15 min. Similarly, non-autoclaved aqueous honey sample (200μg/μl) was also prepared for the study. The PH and DH were subjected to screening tests for bioactive compounds following standard protocol.

Both autoclaved and non-autoclaved honey samples (PH and DH) were utilized in disc preparation. The autoclaved blank paper discs (6 mm diameter; punched from what man No. 1 filter paper) were soaked either with diluted PH or DH, to prepare honey discs of different concentrations: 2.5x103, 5x103, 10x103 and 15x103μg/disc.

Antibacterial activity

The antibacterial activity of PH and DH were determined for the E. coli ATCC 25922, E. coli, Ps. aeruginosa, P. vulgaris, and S. aureus; by disc diffusion method using sterile Nutrient Agar (NA) plates, each of which were inoculated with 108 CFU from young broth culture of the test bacteria. The discs containing different concentrations of honey, as mentioned above, were placed aseptically on the inoculated NA plates, and incubated at 350C for 16-18 h. The sensitivity of the test bacterial isolates to PH and DH (autoclaved and non-autoclaved) were considered with Zone of Inhibition Diameter (ZID) =7 mm.

Antibiotic susceptibility testing

Antibiotic sensitivity testing was performed by disc diffusion method following the NCCLS (National Committee for Clinical Laboratory Standards) guidelines for the bacterial isolates the E. coli ATCC 25922, E. coli, Ps. aeruginosa, P. vulgaris, and S. aureus:, tested against Gentamicin (GM) and Kanamycin (KM). The antibiotic discs, GM (10 μg/disc) and KM (10 μg/disc), were purchased from Hi-Media, India.

In order to determine the combined effect of honey and antibiotic (GM/KM) against the test isolates, PH and DH (both non-autoclaved), two different amount of each of the honey samples:1x103 μg (5 μl) and 10x103μg (50 μl), were dropped on GM and KM discs on the NA plates inoculated with 108 CFU. The ZIDs were recorded after 16- 18 h incubation at 35°C. The combined antibiotic (GM/KM)-honey (PH/DH) activity was considered synergistic when the ZID from the combined action for a given bacterial strain was increased compared to the ZIDs obtained from the single action of both antibiotics and honey samples.

DPPH-free radical-scavenging assay for antioxidant activity

The free radical-scavenging activity for DH and PH was studied following the protocol of Habib et al. [14], through the evaluation of free radical-scavenging effect on 2, 2-Dipheny-1-Picrylhydrazyl (DPPH) radical. To 3.8 ml of methanolic DPPH solution (0.25 mM) aliquots (200 μl) of PH and DH (aqueous solution) at different concentration (25 x 103, 50 x 103, 75 x 103, 100 x 103, 125 x 103 and 150 x 103μg/ml) were mixed, and incubated in the dark for 30 min, following which the absorbance was measured colorimetric ally at 520 nm against methanol without DPPH as blank. The results were expressed as % inhibition of DPPH radical, which was calculated according to the equation: % inhibition of DPPH = Absorption (control) - (Absorption honey)/Absorption (control) x 100; where Absorption (control) is the absorbance of DPPH solution without the test sample (PH and DH). The IC50 values of PH and DH (honey concentration, mg/ml, which scavenges the DPPH radicals by 50 %) were calculated using linear regression of plots where the x-axisrepresented the concentration of honey and the y-axis represented the % inhibition (antioxidant activity).

Results

The ZIDs obtained due to the action of DH (both non-autoclaved and autoclaved), at different concentrations, against clinical isolates of E. coli, P.vulgaris, Ps. aeruginosa and S. aureus are presented in Table 1. The bacterial isolates were resistant to the autoclaved honey at concentrations 2.5x103 -5x103 μg/disc, while the ZIDs ranged 9-16 mm for clinical isolates of E. coli, P. vulgaris, Ps. aeruginosa and S. aureusat concentrations 10x103 -15x103 μg/disc; the S. aureus was resistant to the honey at concentration 15x103 μg/disc. The bacterial isolates of P. vulgaris and S. aureus were resistant to the non-autoclaved honey at concentrations 2.5x103-5x103 μg/disc; Ps. aeruginosa was resistant to the honey (2.5x103 μg/disc) and E. coli was resistant to the honey at concentration 5x103 μg/disc; the ZIDs ranged 10-24 mm for the clinical isolates of E. coli, P. vulgaris, Ps. aeruginosa and S. aureus at concentrations 10x103-15x103μg/disc.