Afr. J. Trad. CAM, Vol. 7, No. 1, January-March, 2010, pp. 59-63
Studies on the antioxidant properties of tualang honey of Malaysia
Mahaneem Mohamed1, K.N.S. Sirajudeen2, M Swamy2, Nik Soriani Yaacob2, Siti Amrah Sulaiman3
1 Department of Physiology, School of Medical Sciences, Universiti Sains Malaysia, 16150-Kubang Kerian, Kelantan, Malaysia
Code Number: tc10009
AbstractHoney has been used since ancient times for its nutritional as well as curative properties. Tualang honey is collected from wild honey bees' hives on Tualang trees found in the Malaysian rain forest. It has been used traditionally for the treatment of various diseases, where its therapeutic value has partly been related to its antioxidant properties. This study therefore assessed the colour intensity, total phenolic content, antioxidant activity and antiradical activity of gamma irradiated Tualang Honey. The colour intensity at ABS 450 was 489.5 ± 1.7 mAU, total phenolic content was 251.7±7.9 mg gallic acid /Kg honey, total antioxidant activity by FRAP assay was 322.1±9.7 (μM Fe(II)) and the antiradical activity by DPPH assay was 41.30 ± 0.78 (% inhibition). The data confirms that the antioxidant properties of gamma irradiated Tualang honey are similar to other types of honeys reported in the literature.
Keywords: Tualang honey, Malaysia, Total antioxidant activity, Gamma radiation, Phenolic content, DPPH assay
Oxidative stress has been implicated in the development of many chronic diseases (Halliwell et al., 1992). The therapeutic role of honey in the treatment of various ailments has been receiving considerable attention recently, and its therapeutic value has been partly attributed to its antioxidant properties (Aljadi and Kamaruddin 2004; The National Honey Board, 2003; Gheldof and Engeseth, 2002). In Malaysia, Tualang honey has been used in the local community for the treatment of various diseases. The content and composition of the different types of honey vary with different floral sources as well as climatic and environmental conditions (Gheldof et al., 2002; Aljadi and Kamaruddin 2004; Kuguk et al., 2007). Studies on the antioxidant activity of different types of honey from different countries and different botanical origins have been carried out (Aljadi and Kamaruddin 2004; Al-Mamary et al., 2002; Beretta et al.,2005; Estevinho et al., 2008; Socha et al., 2009), but the antioxidant properties of the local Tualang honey, have not been well documented in terms of above parameters.
Honey is frequently contaminated with various microorganisms during harvesting and packaging. In order to use honey for research in medicine, a suitable method of sterilization like gamma irradiation is highly recommended. The aim of this present study was to assess the antioxidant properties of gamma-irradiated Tualang honey of Malaysia (irradiated with 25 kGy) by using a combination of tests including colour intensity, phenolic contents, anti radical activity and total antioxidant activity (Beretta et al., 2005).
Materials and Methods
The Tualang Honey used in this study was supplied by Federal Agricultural Marketing Authority (FAMA), Malaysia. It was harvested from Apis dorsata bees′ nectar on the Tualang tree in the Rain Forest of Kedah in Peninsular Malaysia in March 2008. The honey had been previously filtered to remove solid particles, concentrated (20% w/v water) by oven drying at 40°C by FAMA, Malaysia and subjected to gamma irradiation at 25 kGy at Sterilgamma (M) Sdn. Bhd. (Selangor, Malaysia) prior to submitting to us for analysis. All the chemicals and solvents used were of analytical grade.
Assays for in vitro antioxidant properties of Tualang Honey Color intensity: Abs 450 (Beretta et al., 2005)
Tualang honey was diluted to 50% (w/v) with warm water (45-50 o C), vortex-mixed for 5 mins and then filtered (0.45μm pore size, AGILENT TECHNOLOGIES, MILAN, ITALY) to eliminate large particles. The net absorbance was defined as the difference between spectrophotometric absorbance at 450 and 720 nm.
Phenol content (PC)
The total phenol content was determined with Folin′s reagent and the result was expressed as mg gallic acid /Kg honey (Beretta et al., 2005). Tualang honey was first mixed with warm distilled water (500 mg/5mL water), and vortex-mixed for 5 mins. Then 100 microlitre of the solution, corresponding to 10 mg of honey was added to lmL of Folin-Phenol reagent (SIGMA, USA) [pre diluted with distilled water (1:10)]. The mixture was vortex-mixed for 2 mins, and was then transferred into a 1.5mL cuvette (1 cm path). The absorbance was determined against a blank on a spectrophotometer. The blank consisted of honey solution with distilled water to eliminate honey colour interference. The solutions with gallic acid (SIGMA, USA; dissolved in methanol/water: 1:1) concentrations in the range of 10-250μg/ml were used for calibration.
Antiradical activity: DPPH assay
The scavenging activity against 1,1-diphenyl-2-picrylhydrazil (DPPH; SIGMA, USA) radical was used in this study (Chen et al., 2000; Aljadi and Kamaruddin, 2004). Briefly, 0.75ml of the honey solution (0.1g/ml) in warm water was mixed with 1.5ml of 0.09mg/ml DPPH in methanol. The mixture was then incubated at 25°C in a water bath for 5 mins after which the absorbance was measured at 517nm against a blank sample consisting of honey solution with distilled water. The absorbance of a radical blank was also measured using 0.75ml of distilled water. The radical scavenging activity (RSA) of honey was expressed in terms of percentage inhibition of DPPH radical by honey and was calculated (Batrusaityte et al., 2007) as follows:
RSA (DPPH Inhibition, %) = [(AB-AT)/AB] x 100
Where, A B = Absorbance of radical blank (DPPH without honey)
A T = Absorbance of test sample (DPPH with honey)
Total antioxidant activity: FRAP assay
The reducing ability of honey was determined by FRAP assay (Benzie and Strain, 1999; Beretta et al., 2005) with some modifications. Briefly, working FRAP reagent was prepared by mixing 10 volumes of 300 mmol/L acetate buffer, pH 3.6 with 1 volume of 10mmol 2, 4, 6-tripyridyl-s-triazine (TPTZ; SIGMA, USA) in 40mmo1/L hydrochloric acid and with 1 volume of 20mmo1/L ferric chloride. Two hundred pl of honey solution (0.1 g/ml) was added to a test tube containing 1.5ml of freshly prepared FRAP reagent. The mixture was subsequently incubated at 37°C for 4 mins after which the absorbance value were measured at 593nm against a reagent blank (200 pl of distilled water). The difference between this absorbance and the sample blank (honey solution with distilled water), was calculated to get the final absorbance. Aqueous solutions of known Fe II concentration, in the range of 100-1000 μmol/L (FeS04.7H20) were used for calibration. The reducing ability of honey was expressed as pM of Fe II equivalent/L.
All the determinations were conducted in quadruplicate from a single honey sample. Values are expressed as mean ± standard deviation.
Results and Discussion
Honey contains many compounds that can act as antioxidants such as polyphenolics, organic acids, vitamins, catalase and glutathione peroxidase (Aljadi and Kamaruddin, 2004; Batrusaityte et al., 2007). Beretta et al. (2005) standardized the protocols to study the antioxidant properties of honey by a combination of spectrophotometric assays such as color intensity [Abs 450 ], total phenolic content, FRAP assay and DPPH assay. The results of the colour intensity of Tualang honey as well as its phenolic content and antioxidant activities were given in [Table - 1].
When compared to the other types of honey, the net absorbance of Tualang honey was in the range of the different types of Slovenian honey such as Chest nut, Fir, Spruce, Multifloral and Forest Bertoncelj et al., 2007; [Table - 2] as well as Multiflora and honeydew honey Beretta et al., 2005; [Table - 2]. The colour of the honey is usually related to the contents of the mineral, pollen and phenolic compounds (Batrusaityte et al., 2007). Honey has also been shown to have a wide range of antioxidant activities depending on the botanical source, and high correlations have been reported between the antioxidant activity and colour, and total phenolic content of the honey (Al-Mamary et al., 2002; Berrata et al., 2005; Vela et al., 2007; Al et al., 2009). The total phenolic content of Tualang honey is also within the reported range of Slovenian honey (Chestnut, Fir, Spruce, Multifloral and Forest honey), Romanian honeys like Acacia, Lime, Sunflower, Chestnut and Honeydew honey (Bertoncelj et al., 2007; Al et al., 2009; Berrata et al., 2005 ;[Table - 2]. DPPH assay reflects the activity of water soluble antioxidant (Frankel et al., 1998). The radical scavenging activity (RSA) of Tualang Honey, in terms of percentage inhibition of DPPH (-40%), is once again comparable with that reported for other types of honey such as Herb honey (pine and marigold) and Romanian honeys Socha et al., 2009;.Al et al., 2009; [Table - 2] . Unlike DPPH assay, FRAP assay directly measures the total antioxidant activity in the honey (Aljadi and Kamaruddin 2004; Beretta et al., 2005). In our study, we used this assay to measure the total antioxidant activity of gamma radiated Tualang honey. The total antioxidant activity for Tualang honey also appears to be within the range reported for some Slovenian honeys Bertoncelj et al., 2007; [Table - 2].
Many studies indicated that the colour intensity of the honey at ABS 450 reflects its total phenolic content and could be correlated to its antioxidant activity (Al-Mamary et al., 2002; Berrata et al., 2005; M.L. Al et al., 2009). Thus, in the present study, it was concluded that Tualang honey has good colour intensity and contains phenolic compounds that possess relatively good antioxidant activity, which is comparable with that reported for other types of honey. However further studies are required to identify and quantify the biologically active components present in the Tualang honey, which could serve as a source of nutraceuticals.
The financial assistance for this study was provided by the Research University Grant, Universiti Sains Malaysia (1001/PPSP/8120203). Tualang honey was provided by FAMA, Malaysia.
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