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African Journal of Traditional, Complementary and Alternative Medicines
African Ethnomedicines Network
ISSN: 0189-6016
Vol. 7, Num. 1, 2010, pp. 59-63

Afr. J. Trad. CAM, Vol. 7, No. 1, January-March, 2010, pp. 59-63

Research Paper

Studies on the antioxidant properties of tualang honey of Malaysia

1 Department of Physiology, School of Medical Sciences, Universiti Sains Malaysia, 16150-Kubang Kerian, Kelantan, Malaysia
2 Department of Chemical Pathology, School of Medical Sciences, Universiti Sains Malaysia, 16150-Kubang Kerian, Kelantan, Malaysia
3 Department of Pharmacology, School of Medical Sciences, Universiti Sains Malaysia, 16150-Kubang Kerian, Kelantan, Malaysia

Correspondence Address:
K.N.S. Sirajudeen
Department of Chemical Pathology, School of Medical Sciences, Universiti Sains Malaysia, 16150-Kubang Kerian, Kelantan
Malaysia
knssiraj@yahoo.com


Code Number: tc10009

Abstract

Honey 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

Introduction

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.

Data presentation

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.

Acknowledgement

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.[17]

References

1.Aljadi, A. M. and Kamaruddin, M. Y. (2004). Evaluation of the phenolic contents and antioxidant capacities of two Malaysian floral honeys. Food Chemistry. 85 : 513-518.  Back to cited text no. 1    
2.Al-Mamary, M., Al-Meeri, A. and Al-Habori, M. (2002). Antioxidant activities and total phenolics of different types of honey. Nutr. Res. 22 : 1041-1047.  Back to cited text no. 2    
3.Al, M.L., Daniel, D., Moise, A., Bobis, 0., Laslo, L. and Bogdanov, S. (2009). Physico-chemical and bioactive properties of different floral origin honeys from Romania. Food Chemistry, 112 (4) : 863-867.  Back to cited text no. 3    
4.Baltrusaityte, V., Venskutonis, P.R. and Ceksteryte, V. (2007) Radical scavenging activity of different floral origin honey and beebread phenolic extracts, Food Chemistry. 101 : 502-514.  Back to cited text no. 4    
5.Benzie, I. F. and Strain, J. J. (1999). Ferric reducing/antioxidant power assay: direct measure of total antioxidant activity of biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration. Methods in Enzymology. 299 : 15-27.  Back to cited text no. 5    
6.Beretta, G., Granata, P., Ferrero, M., Orioli, M. and Facino, R. M. (2005). Standardization of antioxidant properties of honey by a combination of spectrophotometric/fluorimetric assays and chemometrics. Anal. Chim. Acta. 533 : 185-191.  Back to cited text no. 6    
7.Bertoncelj, J., Dobersek, U.,Jamnik, M. and Golob, T. (2007). Evaluation of the phenolic content, antioxidant activity and colour of Slovenian honey. Food Chemistry. 105 (2): 822-828.  Back to cited text no. 7    
8.Chen, L., Mehta, A., Berenbaum, M., Zangerl, A. R. and Engeseth, N. J. (2000). Honeys from different floral sources as inhibitors of enzymatic browning in fruit and vegetable homogenates. J. Agric. Food Chemistry. 48 : 4997-5000.  Back to cited text no. 8    
9.Estevinho, L., Paula Pereira, A., Moreira, L., Dias, L. G. and Pereira, E. (2008). Antioxidant and antimi­crobial effects of phenolic compounds extracts of Northeast Portugal honey. Food and Chemical Toxicology. 46 (12): 3774-3779.  Back to cited text no. 9    
10.Frankel, S., Robinson, G.E. and Berenbaum, M.RL. (1998). Antioxidant content and correlated characteristics of 14 monofloral honeys. J. Apic. Res. 37 : 27-31.  Back to cited text no. 10    
11.Gheldof, N. and Engeseth, N. J. (2002). Antioxidant capacity of honeys from various floral sources based on the determination of oxygen radical absorbance capacity and inhibition of in vitro lipoprotein oxidation in human serum samples. J. Agric. Food Chemistry. 50 : 3050-3055.  Back to cited text no. 11    
12.Gheldof, N., Wang, X.-H. and Engeseth, N. J. (2002). Identification and quantification of antioxidant components of honeys from various floral sources. J. Agric. Food Chemistry. 50 : 5870-5877.  Back to cited text no. 12    
13.Halliwell, B., Gutteridge, J. M. and Cross, C. E. (1992). Free radicals, antioxidants, and human disease: where are we now. J. Lab. Clin. Med. 119 : 598-620.  Back to cited text no. 13    
14.Kiigiik, M., Kolayh, S., Karaoglu, $., Ulusoy, E., Baltaci , C. and F. Candan, F. (2007). Biological activities and chemical composition of three honeys of different types from Anatolia. Food Chemistry. 100 : 526-534.  Back to cited text no. 14    
15.Socha, R., Juszczak, L., Pietrzyk, S. and Fortuna, T. (2009). Antioxidant activity and phenolic composition of herbhoneys. Food Chemistry. 113 ( 2) : 568-574.  Back to cited text no. 15    
16.The National Honey Board (2003). Honey-Health and therapeutic qualities, 390 Lashley street Longmont. www. nhb. org.  Back to cited text no. 16    
17.Vela, L, De lorenzo, C. and Perez, RA. (2007). Antioxidant capacity of Spanish honeys and its correlation with some physico-chemical parameters and polyphenolic content. J. Sci. Food Agric. 87 : 1069-1075.  Back to cited text no. 17    

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