Mal J Nutr 22(3):Comparison 413—420, of Antioxidant2016 Contents of Thai Honeys to Manuka Honey 413

Comparison of Antioxidant Contents of Thai Honeys to Manuka Honey

1 2 3 4 5 Bundit T , Anothai T Pattaramart P , Roongpet T & Chuleeporn S

1 Department of Veterinary Clinical Medicine, Faculty of Veterinary Medicine, Khon Kaen University, 40002 Khon Kaen, Thailand 2 Department of Statistics, Faculty of Science, Khon Kaen University, 40002 Khon Kaen, Thailand 3 Department of Agricultural Extension, Ministry of Agriculture and Cooperatives, Thailand 4 Department of Biochemistry, Faculty of Medicine, Khon Kaen University, 40002 Khon Kaen, Thailand 5 Department of Veterinary Public Health, Faculty of Veterinary Medicine, Khon Kaen University, 40002 Khon Kaen, Thailand

ABSTRACT

Introduction: Natural honey has been valued in traditional medicine having demonstrated many antioxidant properties. The aim of this study was to compare the antioxidant activities of 12 types of honey from Thailand and manuka honey from New Zealand. Methods: Antioxidant contents of phenolic content, ferric reducing antioxidant power (FRAP), ascorbic acid content, 2, 2-diphenyl-1- picrylhydrazyl (DPPH), and Trolox Equivalent Antioxidant Capacity (TEAC) were determined in Thai and manuka honey samples. Results: All of the 12 types of Thai (n=54) and manuka (n=3) honey varied in range for phenolic content (210-1,519, 563-785 mg GAE/kg), FRAP value (600-9,183, 3,866-4,933 µM Fe(II)/ kg), ascorbic acid content (103-386, 913-1,212 mg/kg), DPPH radical scavenging activity (25-528, 259-310 mg Trolox/kg) and TEAC content (96-636, 328-421 mg Trolox/kg). Mangosteen honey had the highest content of phenolics (1,495±20 mg GAE/kg), FRAP value (9,083±100 µM Fe(II)/kg), DPPH radical scavenging activity (522±6 mg Trolox/kg) and TEAC content (632±3 mgTrolox/kg). Conclusion: Antioxidant activities in mangosteen and rambutan honey presented significantly higher values than manuka honey in terms of phenolic content, DPPH and TEAC. Manuka honey contained the highest vitamin C content (1,194±18 mg/ kg). However, the values for antioxidant properties were dependent on source of honey. Some honeys from Thailand were found to be a better source of antioxidant properties compared to manuka honey.

Key words: Antioxidants, free radicals, honey, Thailand

INTRODUCTION properties since it has antibacterial and Honey is a natural product obtained antioxidant activities. Antioxidants are from the nectar of flowers. It has several substances that protect the cells of the properties including those of being a body from damage caused by unstable food, supplementary diet and traditional molecules known as free radicals. Natural medicine. Honey imparts pharmaceutical antioxidants such as vitamin C and

Correspondence: Chuleeporn Saksangawong; Email: [email protected] 414 Bundit T, Anothai T, Pattaramart P, Roongpet T & Chuleeporn S phenolic compounds in many plants Agricultural Extension in the Ministry of depend on a floral source and are important Agriculture and Cooperatives of Thailand to prevent human diseases (Ferreira et al., and the Thai Beekeepers Association. 2009; Gheldof, Wang & Engeseth, 2002; The samples were Longan (n=21) from Lachman et al., 2010). In recent years, the Chiang Mai and Lamphun, sunflower identification and quantification of the (n=3) from Lop Buri and Saraburi, lychee antioxidant components of honey have (n=3) from Lop Buri and Chiang Mai, been reported worldwide (Gheldof et al., korlan (n=3) from Uttaradit, sesame (n=3) 2002; Omotayo, Siti & Mohd, 2012). from Lop Buri, rambutan (n=3) from Surat Longan honey is mainly produced Thani and Nakhon Si Thammarat, para in the northern part of Thailand, but rubber (n=3) from Nakhon Si Thammarat, other varieties (macadamia, sunflower, eucalyptus (n=3) from Khon Kaen, coconut rambutan, sesame, bitter bush, coconut, (n=3) from Nakhon Si Thammarat, korlan, bittervine, cashew nut and mangosteen (n=3) from Trat , macadamia mangosteen) are produced in different (n=3) from Chiang Rai, and bittervine geographical locations in Thailand. The (n=3) from Chiang Mai. The Manuka honey price of Thai honey range from 2.5 to 12 samples (n=3) were from New Zealand. USD/kg. In contrast, manuka honey can All honey samples were collected fresh in be sold up to 10-30 times the retail price of sterile containers and stored in darkness at Thai honey. Manuka honey is produced 5°C before the test. from the manuka bush (Leptospermum scoparium), indigenous to New Zealand Determination of phenolic compounds and Australia. It is well known for its content pharmaceutical properties and functional Thirty percent honey solution in distilled food value (Kwakman & Zaat, 2012; Majtan water (0.2 ml) was added to 0.2 ml of et al., 2012; Stephens et al., 2010). Currently, Folin-Ciocalteu reagent. The mixture was there is an increasing trend in the use of allowed to react at room temperature for 3 manuka honey in the cosmetics and food min, before adding 0.2 ml of 10% sodium industry in Thailand. Though several carbonate and 1.4 ml of distilled water. studies have reported on the composition The mixture was incubated in the dark at and quantity of antioxidant activities in room temperature for 180 min. Absorbance honey (Srisayam & Chantawannakul, 2010; was measured at a wavelength of 725 nm. Moniruzzaman et al., 2013), there is limited Gallic acid was used as a standard (20- data comparing antioxidant content 200 µg/ml; Y=15.542x-16.196; r2=0.9727) of various honeys from Thailand with to prepare a calibration curve in order to manuka honey. Therefore, the aim of this determine the total amount of phenols study is to compare antioxidant content of in each honey sample (mg of gallic acid manuka honey from New Zealand with equivalents (mg GAE) /kg of honey). various Thai honeys. Determination of ferric reducing METHODS antioxidant power (FRAP) Honey samples A quantum of 0.03 ml of diluted honey In this study, reserchers investigated in distilled water (0.1 g/ml) was mixed 12 types of 54 honey samples from with 0.9 ml of FRAP reagent and 0.09 different geographical regions and ml distilled water. The reaction mixture o provinces between January 2013 to April was then incubated at 37 C for 4 min and 2014 provided by the Department of its absorbance was read at 593 nm. Fresh Comparison of Antioxidant Contents of Thai Honeys to Manuka Honey 415

FRAP reagent was prepared by mixing 25 Determination of Trolox equivalent ml of 300 mM/L acetate buffer (pH 3.6) antioxidant capacity with 2.5 ml of 10 mM TPTZ solution in One ml of 8 mM 2,2’-azino-bis-3- HCl and 2.5 ml of 20 mM ferric chloride ethylbenzthiazoline-6-sulphonic acid

(FeCl36H2O). The resulting mixture was (ABTS) solution was added to 3 mM then pre-warmed at 37oC for 15 min. potassium persulfate. The solution was A calibration curve was prepared using stored in the dark at room temperature an aqueous solution of ferrous sulfate for 14 h and then diluted in distilled water

(FeSO47 H2O) at 25-600 µM/L (Y=0.0006x, (1:100) for a ABTS working solution. One r2=0.999). FRAP values were expressed as ml of ABTS working solution wae mixed micromoles of ferrous equivalent (mmole with 0.1 ml of 10% honey aqueous solution Fe(II)/kg of honey). in water. Absorbance was read at 734 nm and then adjusted at 0.7 with ABTS Determination of ascorbic acid content solution. Results were expressed as mg of A quantum of 20 mg honey was mixed with Trolox equivalents/kg of honey, based on 1 ml of 1% metaphosphoric acid, to which the standard calibration curve (10-60 µg/ was added 0.1 ml of DTCS reagent and ml); Y=0.749x+0.371, r2=0.999. then incubated in a water bath at 37oC for 3 h and subsequentlychilled for 10 min in Statistic analysis an ice bath. Then 0.5 ml cold sulfuric acid The significant differences represented (12 mol/L) was added slowly in solution. by Kruskal-Wallis Statistics, H test and

The ascorbic acid content was calculated Tamhane’s T2 method was used for multiple based on a calibration curve of ascorbic comparison tests when equal variances acid (0.1-3.0 mg/ml; Y=0.2610x, r2=0.9982) were not assumed. Values were presented while absorbance was measured at 520 as mean±SD, maximum and represented nm. The results were expressed as mg of a minimum of three determinations. Data ascorbic acid/kg of honey. were analysed using IBM SPSS software Version 16. Determination of DPPH radical-scavenging activity RESULTS AND DISCUSSION One part of methanol solution was mixed Phenolic compounds are some of the most with 1 part of methanol solution containing important groups of antioxidant substances DPPH (α, α-diphenyl-β-picrylhydrazyl) present in honey. The assay measured the radicals (0.2mM/L) called DPPH solution. phenol and polyphenol derivatives, and Then 10% honey solution in water (0.1 other electron-donating antioxidants. All ml) was added to 0.2 ml of methanol and of the different types of Thai and manuka 1 ml of DPPH solution. The mixture was honey had a varied range of phenolic allowed to react at dark room temperature content of 210-1,519 and 563-785 mg GAE/ for 15 min. The reduction of DPPH kg, respectively. radical was determined by measuring The highest phenolic content was the absorption at 517 nm. The radical- found in mangosteen honey followed by scavenging activity (RSA) was calculated rambutan (Table 1), both of which were as a percentage of DPPH discoloration higher than the phenolic content in manuka using the equation (% RSA) then calibrated honey. However, the range and average using a standard curve of Trolox (10-60 µg/ 2 values of total phenolic content observed ml; Y=1.162x-5.546; r =0.998). Results were for the Thai honey samples used in this expressed as mg of Trolox equivalents per study (lychee, longan, korlan, sunflower, 1 kg honey. 416 Bundit T, Anothai T, Pattaramart P, Roongpet T & Chuleeporn S

Table 1. Antioxidant properties of Thai and manuka honey Type Phenolic FRAP Vit C DPPH TEAC (mg GAE/kg) (uM Fe(II)/kg) (mg/kg) (mg Trolox/kg) (mg Trolox /kg) Korlan 465.49b 2,755.56d 200.19a 203.49e 363.79e s = 14.90 s = 141.75 s = 0.96 s = 0.01 s = 1.05 Macadamia 485.10b 2,150.00c 323.75b 144.59d 277.94d s = 7.65 s = 33.33 s = 1.92 s =2.81 s = 2.09 Sunflower 432.55b 2,755.56d 219.35a 178.72e 360.30e s = 8.91 s = 91.79 s = 0.96 s = 4.93 s = 15.18 Coconut 576.47c 3,305.55e 165.71a 194.61e 343.55e s = 8.15 s = 125.09 s = 2.87 s = 2.14 s =6.81 Bittervine 253.28a 938.89a 106.32a 57.16b 124.73a s = 6.95 s = 108.44 s = 2.88 s = 0.81 s = 1.60 Rambutan 1,361.68e 4,016.67f 164.75a 306.35g 467.44f s = 1.32 s = 83.34 s = 1.92 s = 4.92 s = 1.05 Lychee 216.79a 605.55a 120.69a 26.90a 107.98a s = 8.04 s = 9.62 s = 7.66 s = 0.81 s = 10.53 Pararubber 478.43b 2,622.22d 185.82a 182.31e 293.29d s = 8.26 s = 58.53 s = 3.84 s = 2.16 s = 5.77 Mangosteen 1,495.79f 9,083.33g 379.31b 522.79h 632.51g s = 20.60 s = 100.00 s = 7.66 s = 6.37 s = 3.68 Eucalytus 354.60a 2,050.00c 147.51a 91.89c 168.01b s = 25.13 s = 83.33 s = 1.92 s = 9.08 s = 2.10 Sesame 314.18a 1,372.22b 134.10a 91.35c 170.10b s = 5.79 s = 108.44 s = 1.92 s =2.15 s = 3.14 Longan 425.67b 2,588.89d 190.61a 157.51d 235.01c s = 14.64 s = 175.07 s = 0.96 s = 3.54 s = 8.38 Manuka 657.91d 4,342.59f 1,067.37c 284.07f 374.72e s = 83.16 s = 397.05 s = 121.52 s = 17.17 s = 38.30 Test of Levene Statistic Levene Statistic Levene Statistic Levene Statistic Levene Statistic homogeneneity = 4.87 = 3.74 = 6.45 = 2.83 = 4.49 of variances p = 0.000*** p =0.001** p =0.000*** p = 0.009** p = 0.000*** Test statistic H = 42.55 H = 42.45 H = 43.74 H = 43.67 H = 41.42 p-value 0.000 *** 0.000 *** 0.000 *** 0.000 *** 0.000 *** Note : ** for p <0.01, *** for p < 0.001 H is Kruskal- Wallis test ‘s statistic value Tamhane’s T2 method was used for multiple comparison tests when equal variances were not assumed. plaunoi, sesame and sabsue) are lower However, the phenolic content range (587-1,652 mg GAE/kg; Limpawattana, in Thai honey(wild flowers, sunflowers, 2013) than the level of the Venezuelan longan and lychee) was higher (100-144 honey (3811,821 mg GAE/kg; Vit et mg GAE/kg; Sangsrichan & Wanson, 2008) al., 2009), Nigeria honey (362-1,028 mg than that of honey from south-west Nigeria GAE/kg; Buba, Gidado & Shugaba, 2013.) (7-28 mg GAE/kg; Adetuyi et al., 2009), Comparison of Antioxidant Contents of Thai Honeys to Manuka Honey 417

Czech (39-167.1 mg GAE/kg; Lachman et from Romania (2,260 to 2,960 mg/kg; al., 2010), Poland (71.7- 201 mg GAE/kg; Matei et al., 2004). Kaskoniene et al., 2009), Malaysia (144-580 The DPPH radical scavenging analysis mg GAE/kg; Moniruzzaman et al., 2013) was used to investigate the overall hydrogen and monofloral Cuban honey (213- 595 mg or electron donating activity of single GAE/kg of honey; Alvarez-suarez et al., antioxidants. The values for all of the 2010). different types of Thai and manuka honey Determination of antioxidant activity of ranged from 25-528 and 259-310 mgTrolox/ honey by FRAP assay is based on the ability kg in DPPH radical scavenging activity with of the sample to reduce the ferric ions to the average values being 179±127 and 284±17 ferrous ions. The FRAP activities for the mg Trolox/kg, respectively. The DPPH different types of Thai and manuka honey levels in Thai and Manuka honey were lower ranged from 600-9,083 and 3866-4,933 µM than those obtained in buckwheat honey Fe(II)/kg, respectively. Similar to phenolic (304 mg Trolox/kg, Cheng et al., 2015), but content, the highest FRAP values were higher than Mexican honey (81 to 255 μmol found in mangosteen honey followed by Trolox/kg; Beatriz et al., 2012 ) rambutan and manuka honey (Table 1). The TEAC content from Thai and manuka FRAP values of Thai honey(lychee, longan, honey were 96-636 and 328-421 mg Trolox/ korlan, sunflower, plaunoi, sesame and kg, respectively. The TEAC contents sabsue) in the present study were higher obtained for the honey samples used in (1,475-5,500 µM Fe(II)/kg; Limpawattana, this study were within the range reported 2013) than those of Croatian monofloral by Buenocosta et al., (2016) from Brazil (82 honey (250-1160 µM Fe(II)/kg; Piljac- and 1,114 mg trolox/kg), but higher than Zegarac, Stipcevic & Belscak, 2009) and the values reported for Mexican honey Algerian honey (3377± 10 µM Fe (II)/kg; (910 to 2,927 μmol Trolox/kg; Beatriz et al., Khalil et al., 2012). 2012). Ascorbic acid content from Thai and In this study, lychee and bittervine manuka honey ranged from 103-379 and honey from the north of Thailand 913-1,212 mg/kg, respectively.The highest showed the weakest antioxidant properties vitamin C content was found in manuka (Table 1) as in the study by Srisayam and honey followed by mangosteen honey and Chantawannakul (2010) who found lychee macadamia honey (Table 1). Similar results had the weakest antioxidant properties. were obtained from Nigeria honey which Whereas mangosteen from the East and the ranged from 138 to 273 mg/kg (Buba et rambutan from the southern of Thailand al., 2013) and the Venezuelan honey (128- showed high antioxidant properties (Table 370 mg/kg ; Vit et al., 2009). Thai honey 1) similar to that of the report of Srisayam showed a higher content of ascorbic acid and Chantawannakul (2010) who found than Malaysian honey (129-132 mg/kg; rambutan to have the highest antioxidant Moniruzzaman et al., 2013), Bangladesh property. honey (129-154 mg/kg; Islam et al., 2012), Each of the antioxidant assays of DPPH, Portuguese honey (140–145 mg/kg; Ferreira FRAP, ascorbic acid antioxidant content, et al., 2009) and Malaysian pineapple honey and TEAC have their own advantages and (146 mg/kg; Kishore et al., 2011). disadvantages. This study found at least The range values for Vitamin C content one method to detect the difference in observed in the honey samples used in this amount of antioxidant activity in honey (p study are lower than the level of a various < 0.05). Mangosteen honey was found to types of honey from Bosnia Herzegovina have the highest amount of antioxidants (372 to 3783 mg/kg; Kesic et al., 2009) and using FRAP, phenolic content, DPPH and 418 Bundit T, Anothai T, Pattaramart P, Roongpet T & Chuleeporn S

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