In-Vitro Antioxidant, Antibacterial And Anti ...

Research Article

In-Vitro Antioxidant, Antibacterial And AntiInflammatory Characterization Of Indian Honey TOOBA NAZ SHAMSI 1# ,ROMANA PARVEEN 1# ,RIZWANA REHSAWLA 2 ,SUMBUL AFREEN 3 , MUDSSER AZAM 3 ,TASNEEM FATMA 3 ,QAZI MOHD.RIZWANUL HAQUE 3 ,SADAF FATIMA 1  1 Department of Biotechnology.; Jamia Millia Islamia, New Delhi-110025, India 2 Department of Bioscience and Biotechnology, Banasthali Vidyapeeth, Rajasthan, India 3 Department of Biosciences, Jamia Millia Islamia, New Delhi-110025, India ABSTRACT: Honey has been used since ages in medical and domestic need. It is said to possess wide range of medicinal properties including antimicrobial potential with lesser safety concerns. In this communication, we have evaluated antibacterial, anti-inflammatory and antioxidant potential of aqueous extract of Indian Honey. The extract was evaluated against eight bacterial pathogens including Klebsiella pneumonia (ATCC 700603), Staphylococcus aureus (MTCC 902), Bacillus subtilis (MTCC 736), Pseudomonas aeruginosa (MTCC 2453), Aeromonas species (A10 MDR), Klebsiella oxytoca (A13 MDR) and two strains of Escherichia coli (ATCC 25922 and MTCC 443). The antioxidant activity was evaluated using FRAP and DPPH assay. Also, anti-inflammatory property was analyzed by proteinase inhibitory and albumin denaturation assay. Results showed that honey completely inhibited K. oxytoca (100%) but could not inhibit B. subtilis. Honey showed DPPH radical scavenging activity in a concentration–dependent manner with maximum scavenging of 61% at 500 μl extract. The FRAP assay demonstrated highest value of 0.157 mM at 200μl of aqueous extract. The extract also showed anti-inflammatory activity by inhibiting the heat induced albumin denaturation with IC 50 values 540μg/ml whereas standard drug (Aspirin) showed inhibition of 71% at 100μg/ml. Also, proteinase inhibitory assay showed minimum inhibition of 7.3% at 100μg/ml and maximum inhibition of 64% at 1000μg/ml as compared to standard which showed the maximum inhibition of 87% at 1000μg/ml. The present results predict the benefits of honey in the treatment of microbial, inflammatory and free radical mediated diseases. Keywords: Antioxidant, Anti-inflammatory, Antimicrobial, Honey INTRODUCTION: The modern day lifestyle has implicated human life in the form of several adverse effects and the prior most in this context is the faulty eating habits. The deteriorating human health, sharp increase in health ailment these days are to a very large extent the result of inclination of preferences from healthy to towards unhealthy fast food. As a result with an alarming rate we need to switch to such products that are less in synthetic content and rich in natural substance. Natural product not only provides us with the wide range of medicinal properties but they are also antimicrobial, antifungal in nature and the largest benefit of them being minimal or less side effects. So the researchers even are focusing on the use of natural substitutes for curing the health ailments. For instance honey which is a natural product and has been used in various preparation since the time unknown. It not only adds to the taste but even provides us with multifold health benefits

unlike the synthetic medicine which harm us in several ways. Thus for sustainable survival and good health we need to switch on to natural products like honey. Honey is one of the most famous natural products which have been used since ages for food, health, medicine and skincare. It is mainly composed of fructose and glucose along with fructooligosaccharides [1] vitamins, minerals, amino acids, and enzymes [2]. The chemical composition of honey is very complex that varies depending on the botanical source. There occurs a variations in honey color from light to dark yellow color. Honey is viscous by nature and prepared by bees. Honey has been used since ancient times for food, the treatment of some respiratory diseases and for the healing of skin wounds, burns. It has been proposed that the healing effect of honey could be due to various physical and chemical properties [3, 4]. The Vedic civilization considered honey as one of nature's most

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Address For Correspondence: Sadaf Fatima, Assistant Professor, Lab No. 510, S. R. Block, Department of Biotechnology, Jamia Millia Islamia New Delhi-110025, India. E-mail: [email protected], Tel: +91 8447029437 Received: 20.08.15, Revised: 25.09.15, Accepted: 20.10.15 International Journal of Pharmaceutical Research | Jan – Mar 2016 | Vol 8 | Issue 1|33

Sadaf Fatima et al / Biological Characterization of Indian Honey remarkable gifts to mankind. According to the Ayurveda, honey is bliss to those with weak digestion [5, 6]. The use of honey is highly beneficial in the treatment of cough. Honey is regarded, as valuable in keeping the teeth and gums healthy by Ayurvedic experts [7]. Honey is a by-product of flower nectar and the upper part of the aero-digestive tract of the honey bee, honey is concentrated through dehydration process inside the bee hive. There occur four species of honey bees in India: Apis indica, Apis florae, Apis dorsata and Apis Trigona. Among these, Apis indica can be domesticated and hived in apiaries. Its honey is therefore called as apiary honey [8].

Figure 1. DPPH free radical scavenging activity of Honey (■) was calculated and compared to ascorbic acid i.e. standard (■). Samples at different concentrations were mixed with DPPH and incubated for 30 min at room temperature. The change in color was measured spectrophotometrically at 517 nm. Nowadays, various investigations have been carried out to explore the therapeutic effects of honey. Researchers also focused their attention on the medicinal effects of honey. Particularly on its capacity to protect against cardio vascular and neurological diseases [9, 10] cancer [11-14] and microbial infections [15, 16]. The presence of antioxidant components in honey protect the health and show therapeutic impacts. Especially phenolic compounds, such as phenolic acids, flavonoids and, most of which express relevant activities such as antioxidant, anti inflammatory, antimicrobial, anti-mutagenic both in vitro and in vivo [17]. Honey shows inhibitory effect to around 60 species of bacteria which includes aerobes and anaerobes, gram-positives and gram-negatives bacteria. For some yeast and species of Aspergillus, Penicillium as well as all the common dermatophytes [18, 19]; Candidiasis caused by Candida albicans an antifungal action has also been observed. The demand of natural products in human diet, both due to the possible adverse eﬀects of synthetic and artificial food additives on human health and to the increased consumer perception of this problem in recent years. Honey is considered as a natural source of choice [20]. Unlike refined sugars, honey is safer for use by diabetic patients without any side effects and it also has more nutrition

value than artificial sweeteners [16, 21, 22]. Here, in this research, we have aimed to assess the role of aqueous extract of honey for their hole in various biological activities. As we are dealing with aqueous extract rather than methanolic one, it can stand a strong contender for phytodrug in oral administration.

Figure 2. Ferric Reducing Antioxidant Power of Honey (■) was estimated and compared to ascorbic acid (■) as standard. The samples were prepared at different concentrations, mixed with FRAP reagent and allowed to incubate for 4 min at room temperature. The absorbance was measured at 593 nm. Materials and methods: Chemicals: The chemicals used were all analytical grade. All solvents and chemicals (analytical grade) used for antioxidant, anti-inflammatory and antibacterial assay were purchased from Merck and Himedia. DPPH and TPTZ were purchased from Sigma-Aldrich. Sample: Honey was collected from gardens of IARI, New Delhi, India. Test microorganism: The following eight clinical isolates of bacteria were used for the study: Two species of Escherichia coli, one each of Staphylococcus aureus, Bacillus subtilis, Klebsiella pneumonia and Pseudomonas aeruginosa. All these cultures were maintained on nutrient agar plates at 4°C Table 1: % mean growth inhibition of various bacterial strains in the presence of honey. Bacterial strain E.coli (ATCC 25922) P.aeruginosa (MTCC 2453) S. aureus (MTCC 902) K.pneumoniae (ATCC 700603) E.coli (MTCC 443) B.subtilis (MTCC 736) S.aureus (MTCC 902) P.aeruginosa (MTCC 2453)

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%MGI 88.99 77.57 19.07 0 96.79 31.52 94.41 100

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Figure 3. Antibacterial potential of honey (• ) was calculated and it was found that maximum inhibition is shown by P. aeruginosa and K. pneumoniae show 0% inhibition. Luria broth is used for the analysis.

Figure 4. Albumin denaturation activity of Honey(■) was checked at various concentrations taking aspirin (100μg/ml) as standard. Albumin was heated it in presence of different concentration of samples at 57 °C for 20 min and cooled. The turbidity was measured at 660 nm and percent inhibition of protein denaturation was calculated.

Figure 5. % inhibition of proteinase action (■) of casein protein was evaluated in the presence of aqueous extract of honey to determine the proteinase inhibitory activity. Results showed % inhibition in a concentration dependent manner. Preparation of aqueous extract: 0.1 gram of sample (honey) was taken and dissolved in 10 ml of distilled water (10mg/ml). The sample was then centrifuged and the supernatant was picked which served as aqueous extract for the further studies. Determination of Antioxidant activity

DPPH Assay: The antioxidant activity of honey and the standard were checked on the basis of the free radical scavenging effect of the stable 1, 1-diphenyl2-picrylhydrazyl (DPPH) by the method used by Fatima et al. with minor modifications [23]. A range of diluted working solutions of Honey were prepared in distilled water. Ascorbic acid (1 mg/ml) in distilled water was used as standard. 0.1mM DPPH was

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Sadaf Fatima et al / Biological Characterization of Indian Honey prepared in methanol and 500μl of this solution was mixed with 50-500μl range 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 were taken as blank. The optical density was 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 with minor modifications [24]. FRAP reagents was freshly prepared by mixing 10 ml acetate buffer (300 mM, pH 3.6), 1 ml 2,4,6-tris (2-pyridyl)-S-triazine (TPTZ) solution (10 mM TPTZ in 40 mM/L HCl) and 1 ml FeCl3 (20 mM) water solution. A range of diluted working solutions of honey 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. Assessment of Antimicrobial potential: Antimicrobial activity of the aqueous extract was tested against three gram-positive bacterial strains (Bacillus subtilis, Aeromonas species. and Staphylococcus aureus) and five gram-negative bacterial strains (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 ºC for 12 hrs. Overnight cultures were diluted with fresh LB media to approximately 104 colony forming units (CFU) and incubated at 37 ºC for 12-14 hrs in the presence of honey 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 mean growth inhibition (MGI) was calculated by using the formula: % MGI = [(dc - dt)/dc] x 100, Where dc and dt represent OD600 of control and treated sample strains respectively. Evaluating anti-inflammatory activity: Inhibition of Albumin denaturation assay: The antiinflammatory assay was done using Albumin denaturation method of Mizushima et al with minor changes [25]. A range of diluted working solutions of the Honey were prepared and mixed with 1% aqueous solution of bovine serum albumin (BSA) fraction. The samples were incubated at 37ºC for 20 min and then heated at 57 ºC for 20 min. 1% BSA

was taken as control and Tris buffer was taken as blank. Aspirin (100μg/ml) was taken as standard drug. After cooling the samples, the turbidity was measured spectrophotometrically at 660 nm. Percent inhibition of protein denaturation was calculated using formula: Percentage inhibition = (Abscontrol –Abssample) X 100/ Abscontrol Proteinase inhibitory assay: The test was performed according to the modified method of Oyedepo et al. [26]. The reaction mixture (2 ml) was containing0.06 mg trypsin, 1 ml 20 mM Tris HCl buffer (pH 7.4) and 1 ml test sample of different concentrations. The mixture was incubated at 37°C for 5 min and then 1 ml of 0.8% (w/v) BSA was added. The mixture was incubated for an additional 20 min. 2 ml of 70% acetic acid was added to terminate the reaction. Cloudy suspension was centrifuged and the absorbance of the supernatant was read at 210 nm against buffer as blank. The experiment was performed in triplicate. The percentage inhibition of proteinase inhibitory activity was calculated. Results and Discussion: Determination of Antioxidant activity: Honey is a complex structure because not only it depends on floral source but environment and season also play an important role in the composition, color and the taste of honey. The color of honey also indicates its antioxidant property. Darker the color of honey more the antioxidant activity is present. In our body, reactive oxygen species (ROS) are present, which in normal condition do not harm normal function of the body. But when free radical and ROS level increases they elevate the risk of heart disease, cancer, aging and also dysfunction of cells. In the analysis it is found that the antioxidants present in honey helps in lowering the risk of such life threatening diseases. Two very simple assays DPPH and FRAP are used for determination of antioxidant activity. They show free radical scavenging property. They are easily available and use in very less amount. Antioxidants are micronutrient and they are able to neutralize the free radicals. Antioxidant based drugs involve in treatment of Alzheimer’s disease. The antioxidant activity was analyzed by two most common radical scavenging assays of FRAP and DPPH. Honey extract showed DPPH radical scavenging activity in a concentration–dependent manner with maximum scavenging of 61% in presence of 500 μl of aqueous extract which was quite comparable to ascorbic acid (77% at 500 μl) (Fig. 1). The results showed that FRAP value of honey increase in the concentration-dependent manner. The highest absorbance of FRAP was observed in honey at 200 μl and the lowest was that in at 20μl i.e 0.628 and 0.078mM as compared to standard i.e. 0.210 and 0.965mM respectively (Fig. 2). From the


Sadaf Fatima et al / Biological Characterization of Indian Honey observations, it is clear that honey showed fair antioxidant activity comparable to ascorbic acid. Assessment of Antimicrobial potential: Honey as a therapeutic agent has been used from the ancient time. At that time people don’t know about its antibacterial property. In the recent time they come to know about its antibacterial property. It was found very effective in clearing up of infection and quick healing. Honey is also very effective in treating bacterial gastroenteritis in children especially infants. Many bacterial species cause various infections. Honey shows inhibitory effect against the bacteria. It was found that many species of bacteria which are responsible for causing many types of diseases like skin disease, respiratory disease, intestinal infection and urinary tract infection are treated by using honey as a medicine. The antibacterial property of honey is of great importance for mankind. During the experiment we found many species such as Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Aeromonas species and Klebsiella oxytoca are inhibited by honey sample. Due to the inhibitory effect the risk of diseases like diarrhea, Septicaemia, urinary infection and wound infection caused by Escherichia coli, Pneumoniae caused by Klebsiella pneumoniae , abscesses, boils, carbuncles, impetigo, wound infection caused by Staphylococcus aureus is lower down. Antimicrobial assay of the aqueous extract was examined against various bacterial strains by accessing the % MGI in the presence of honey compared to the control where only media and cultures were added. The results suggested that honey exhibits bactericidal property in-vitro i.e. the growth of microorganisms was inhibited in its presence. It was found that honey had strongest inhibitory activity against K.oxytoca (MTCC 2453) i.e. 100% whereas, it showed no inhibition against K.pneumoniae (ATCC 700603) as shown in Fig. 3. Evaluating Anti-inflammatory activity: If there occur any infection or damage to the body or tissue then body show response against the infection through inflammation. But sometimes inflammation is so severe that it leads to the discomfort or extraordinary pain. Allopathic anti-inflammatory drugs are very commonly used but there excessive used may harm the body or our immune system. Also, we cannot use pharmaceutical drugs on the sensitive part of body like eyes also these drugs are cannot used in wounds because they slow down the healing process of wounds. On the other hand honey can be used on the sensitive part of body and wound healing. Honey lowers down the activity of cyclooxygenase1 and cyclooxygenase2 and this leads to the anti-inflammatory activity of honey [27]. The results showed that the extract inhibited albumin denaturation in dose-dependent manner. The IC50 value of honey was found out to be 540μg/ml .The

Aspirin which was used as standard drug showed the inhibition of 71% at 100μg/ml (Fig. 4). Also, proteinase inhibitory assay showed that honey exhibits anti-inflammatory property in concentrationdependent manner. Results exhibited significant antiproteinase activity at different concentrations of aqueous extract as shown in Fig. 5. It showed minimum inhibition of 7.3% at 100μg/ml and maximum inhibition of 64% at 1000μg/ml. Aspirin which was used as standard showed the maximum inhibition of 87% at 1000μg/ml. Conclusion: The present study provides the evidence that the aqueous extract of honey possess high antioxidant potential which may help in the prevention of free radical mediated diseases by scavenging the reactive oxygen species and can reduce the deleterious effects caused by free radical in the cell system. Also, the extract showed the fair anti-inflammatory and antibacterial activity which might help in the reduction of various side effects caused due to the drugs consumed for the treatment of inflammation (anti-inflammatory drugs) and bacterial infections (antibiotics). As honey is known to have various phytochemicals such as flavonoids, phenolic acids etc and possess antibacterial, anti-inflammatory and antioxidant activity, the ingestion of extract may be beneficial in reducing the inflammation and oxidative damage and also can hamper the growth of bacteria. Acknowledgments: The authors acknowledge financial support from DST-SERB (SR/FT/LS-194/2009). The authors are thankful to Central Instrumentation Facility, Department of Biotechnology, JMI and Cyanobacterial Biotechnology Lab and Microbiology Research Lab, Department of Biosciences, JMI, New Delhi to allow the access to instruments for the research work. References: 1. 2.

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