| Livestock Research for Rural Development 31 (10) 2019 | LRRD Misssion | Guide for preparation of papers | LRRD Newsletter | Citation of this paper |
The different geographical origins may affect on the types of plants that serve as food sources influence the honey chemical composition. The objective of this study was to evaluate an antioxidant activity, total phenolic content (TPC), total flavonoid content (TFC) and vitamin C contents of several honeys produced by the Indonesian stingless bee: Tetragonula laeviceps. Honey was obtained from three geographical origins, Sleman District was represented by the Faculty of Animal Science Universitas Gadjah Mada (UGM), Gunungkidul District, Yogyakarta was represented by Katongan Village and Klaten District, Central Java was represented by Glodogan Village. The TPC and TFC were determined by the spectrophotometric UV-Vis method, antioxidant activity by DPPH assay method, vitamin C by the 2,6-dicholorophenolindophenol titrimetric method. All data were statistically analyzed using one-way analysis of variance followed by honestly significant difference (HSD) test. The results showed that the geographical origins had a highly significant effect (p<0.01) on TFC and antioxidant activity. In addition, the geographical region had a significant effect (p<0.05) on TPC but not on vitamin C (p>0.05). It can be concluded that honey from the Sleman and Klaten District have the highest TFC, TPC, and antioxidant activity than honey from Nglipar Gunungkidul.
Keywords: gallic acid, georaphical origin, klanceng bee, nectar, quercetin
The different geographical origins affect on the types of plants that serve as food sources influence the honey chemical composition. In Indonesia, stingless bee species can be found nesting in bamboo, sugar palm stalks, tree trunk or wood and in the ground (Agussalim 2015; Agussalim et al 2015). Furthermore, In Indonesia, stingless bees species also can be called as the Klanceng bee especially in Java. For example, the product like honey is usually also called as Klanceng honey. Tetragonula laeviceps is one of stingless bee species can be found in Indonesia that nests in bamboo (Agussalim et al 2017; Agussalim et al 2019). The stingless bee Tetragonula laeviceps can produce honey, bee pollen, bee bread, and propolis (Agussalim 2015; Agussalim et al 2015, 2017).
Honey is defined as the naturally sweet substance produced by honey bees or stingless bees (Micrapis, Megapis, Meliponines) from the nectar of plant flowers and honeydew (Codex Alimentarius 2001). Honey is a natural food that is mainly composed of sugars and other constituents, such as enzymes, amino acids, organic acids, carotenoids, vitamins, minerals, and aromatic substances (Alqarni et al 2012; da Silva et al 2016). The chemical composition, color, aroma, and flavor of honey depends mainly on the flowers sources of nectar, geographical origins, climate, and honey bee species involved in its production (Agussalim et al 2019; da Silva et al 2016; Chanchao 2013; da Costa Leite et al 2000; Juan-Borrás et al 2014; Escuredo et al 2014; Tornuk et al 2013).
The chemical composition of honey from stingless bees have been studied (Ranneh et al 2018; Biluca et al 2016; Guerrini et al 2009; Oddo et al 2008; Souza et al 2006), honey from Tetragonula laeviceps in Thailand (Chuttong et al 2016; Suntiparapop et al 2012). The honey from Indonesian stingless bees has been commercialized by beekeepers but has not been studied with regard to its chemical composition; therefore, information about it is lacking. The recent study about the sugar profile of honey from Tetragonula laeviceps has been studied (Agussalim et al 2019). The objective of this study was to evaluate an antioxidant activity, total phenolic content, total flavonoid content and vitamin C contents of several honeys produced by the Indonesian stingless bee: Tetragonula laeviceps from different geographical origins.
Honey from the stingless bee, Tetragonula laeviceps, was obtained by meliponiculture from three geographical origins. Honey from the Faculty of Animal Science UGM and Klaten had a sweet flavor and honey from Nglipar has a bitter flavor. The honey was used in the study was separated from the propolis and placed in a plastic bottle before analysis. The parameters in the study were total phenolic content (TPC), total flavonoid content (TFC), vitamin C content and activity of antioxidant DPPH of honey.
The first location was the Sleman District was represented by the Faculty of Animal Science UGM Yogyakarta with altitude 156 m above sea level (asl), included wet region, fertile soil, and water available all year. The second location was in Klaten District, Central Java was represented by Glodogan Village with altitude 164 m asl, included medium region (between wet and dry region), fertile soil and water available all year. The third location was in Nglipar, Gunungkidul was represented by Katongan Village with altitude 185 m asl, included dry region, soil less fertile and in the dry season was lack water because included mountain region.
Total phenolic content (TPC) was measured using the method described by Alotibi et al (2018) and Ranneh et al (2018) with some modification. Briefly, honey about 0.2 g and added 0.5 mL of Folin-Ciocalteu reagent and added 7.5 mL aquabidest. Then, the blend was allowed for 10 min at room temperature and then 1.5 mL of 20% sodium carbonate was added. Aquabidest was added up to volume 10 mL. The blended was heated in the waterbath at temperature 40oC for 20 min and immediately after that was cooled quickly using ice and allowed to react for 30 min. The absorbance was measured at 760 nm. Total phenolics were quantified as gallic acid equivalents (GAE) per 100 g of sample (% w/w) from a standard curve was prepared using a standard solution of gallic acid with concentration were 0.000; 1.563; 3.125; 6.250; 12.500; 25.000; 50.000 ppm with y = 0.00511268 x + 0.000000 and r2= 0.99941. All tests were carried out in triplicate each in duplo.
Total flavonoid content (TFC) of each honey sample was determined according to the method was reported by Khalil et al (2011) with minor modification. Briefly, honey 0.20 g was mixed with 4 mL of distilled water. At baseline, 0.3 mL of sodium nitrite (5%, w/v) were added. After 5 min, 0.6 mL aluminium chloride (10% w/v) was added, followed by the addition of 2 mL of sodium hydroxide (1 M) 5 min later. Immediately after that, the volume was increased to 10 mL by the addition of 2.4 mL distilled water. The mixture was vigorously shaken to ensure adequate mixing, and the absorbance was read at 510 nm. A calibration curve was prepared using a standard solution of quercetin with concentration were 0.000; 1.563; 3.125; 6.250; 12.500; 25.000; 50.000 ppm with y = 0.00466915 x + 0.000000 and r2= 0.99945. The results were also expressed as milligram quercetin equivalents (QE) per gram honey. All tests were carried out in triplicate each in duplo.
Vitamin C contents of the honey were determined by the 2,6-dicholorophenolindophenol titrimetric method was described by AOAC (1990). Briefly, 2 g of the honey was extracted in 5 mL of 20% metaphosphoric acid. A standard solution containing 50 mg L-ascorbic acid dissolved in 90 mL of 20% metaphosphoric acid and made up to 100 mL with water was also prepared. Two milliliters (2 mL) each of the standard and sample were titrated with the 2,6-dicholorophenolindophenol solution until a faint pink end point lasting at least 10 to 15 seconds was observed. All tests were carried out in triplicate each in duplo. The vitamin C content was calculated as follows:
Vitamin C (mg / 100 g) = [(Titer value x dye factor x 100)/weight of sample]
Dye factor (DF) = (0.5/standard titer)
The antioxidant activity can be shown by free radical scavenging activity of honey samples utilizing DPPH was measured according to the method was reported by Alotibi et al (2018), with minor modification. In the presence of an antioxidant, the purple color of DPPH decays, and the change in absorbance at 517 nm can be followed. Briefly, 100 μL of honey solution (in methanol) was mixed with 900 μL of freshly prepared DPPH methanol solution (0.1 mM). A methanol solution of DPPH (0.1 mM) was utilized as a control. After incubation for 30 min at room temperature in the dark, the absorbance was measured at 517 nm utilizing a spectrophotometer Uv-Vis. Scavenging activity (%) was figured utilizing the following equation:
DPPH (%) = [(Ac – As)/Ac] x 100
where Ac is the absorbance of the control and As is the absorbance of the sample. The mean of three measurements of each sample was calculated. All tests were carried out in triplicate each in duplo.
The data of TPC, TFC, vitamin C content, and activity of antioxidant were analyzed with one-way analysis of variance (ANOVA) using SPSS (Windows version of SPSS, release 22) (SPSS 2013). Significant differences between the means were identified with honestly significant difference (HSD) test (Steel et al 1997). Values of p<0.05 and p<0.01 were considered statistically significant.
The results showed that the different geographical origins for the meliponiculture of Klanceng bee had a significant effect (p<0.05) on the TPC of honey (Table 1). The highest TPC was in Klanceng honey from Sleman followed by honey from Klaten and the lowest was in honey from Nglipar (Table 1). The TPC of Klanceng honey from Sleman 1.69% w/w GAE and Klaten 1.21% w/w GAE did not differ, but with the TPC of honey from Nglipar 0.54% w/w GAE did differ. The different TPC content of Klanceng honey is affected by the different geographical origins that impact on the different of flowers type as the source of nectar to produce honey (Table 2). Different flowers have an impact on the chemical composition of nectar included phenolic content, therefore, it will influence the TPC of Klanceng honey. However, in our study, we did not an analyze the chemical composition of each nectar that was produced by each plant. Plants have several polyphenolic derivates with high structural diversity and complexity, and when honeybees or stingless bees collect nectar, these bioactive compounds can be transferred from plants to honey (da Silva et al 2016). The TPC in the study ranged from 0.54 to 1.69 %w/w GAE (Table 1) and was higher than those previously reported (Ranneh et al 2018; Biluca et al 2016; Guerrini et al 2009; Oddo et al 2008).
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Table 1. The TPC, TFC, vitamin C content, and antioxidant activity of several honeys produced by Klanceng bee |
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|
Parameters |
Geographical origins |
SEM |
P |
||
|
Sleman, |
Klaten, |
Nglipar, |
|||
|
TPC, %w/w GAE |
1.69b |
1.21ab |
0.54a |
0.19 |
0.02 |
|
TFC, mg/g QE |
0.90b |
0.76b |
0.21a |
0.11 |
0.00 |
|
Vitamin C, mg/100 g |
7.88 |
6.51 |
5.67 |
0.46 |
0.13 |
|
DPPH antioxidant activity, % |
90.5b |
91.2b |
47.3a |
7.23 |
0.00 |
|
a,b,c Different superscripts within rows indicate differences at p<0.05 |
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The results showed that the different geographical origins for the meliponiculture of Klanceng bee had a highly significant effect (p<0.01) on the TFC of honey (Table 1). The highest TFC was in Klanceng honey from Sleman followed by honey from Klaten and the lowest was in honey from Nglipar (Table 1). The TFC of Klanceng honey from Sleman 0.90 mg/g QE and Klaten 0.76 mg/g QE did not differ, but with the TFC of honey from Nglipar 0.21 mg/g QE did differ. The different TFC content of Klanceng honey is affected by the difference of geographical origin that impact on the different types of flowers as the source of nectar to produce honey (Table 2). Different flowers have an impact on the chemical composition of nectar included phenolic content, therefore, it will influence the TFC of Klanceng honey. However, in our study, we did not analyze the chemical composition of each nectar that was produced by each plant. Plants have several polyphenolic derivates with high structural diversity and complexity, and when honeybees or stingless bees collect nectar, these bioactive compounds can be transferred from plants to honey (da Silva et al 2016). The TFC in the study was ranged from 0.21 to 0.90 mg/g QE (Table 1) was higher to those previously reported (Ranneh et al 2018; Oddo et al 2008).
The results showed that the different geographical origins for the meliponiculture of Klanceng bee had no significant effect (p>0.05) on the vitamin C of honey (Table 1). The vitamin C content in Klanceng honey in the study ranged from 5.67 to 7.88 mg/100 g was similar for all geographical origins. The similar of vitamin C content of honey in the study might be attributed with the flowers type (Table 2) that have a similar nectar chemical composition especially vitamin C content, which causes no difference on vitamin C of Klanceng honey. However, in our study, we did not analyze the chemical composition of each nectar that was produced by each plant. The vitamin C of Klanceng honey in the study was lower to those previously reported (Ranneh et al 2018; Syam et al 2016).
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Table 2. Predominant plants type for nectar source to produce honey in each geographical origin |
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|
Geographical origins |
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|
Sleman, Yogyakarta |
Klaten, Central Java |
Nglipar, Yogyakarta |
|
Banana (Musa paradisiaca L.) |
Coconut (Cocos nucifera) |
Calliandra (Calliandra calothyrsus) |
|
Rambutan (Nephelium lappaceum) |
Mango (Mangifera indica L.) |
Mexican creeper (Antigonon leptopus) |
|
Canarium (Canarium indicum L.) |
Rambutan (Nephelium lappaceum) |
Banana (Musa paradisiaca L.) |
|
Tamarind (Tamarindus indica) |
Banana (Musa paradisiaca L.) |
Mango (Mangifera indica L.) |
|
Matoa (Pometia pinnata) |
- |
White albizia (Paraserianthes falcataria L.) |
|
Cattapa (Terminalia catappa) |
- |
- |
|
Caimito (Chrysophyllum cainito) |
- |
- |
The results showed that the different geographical origins for the meliponiculture of Klanceng bee had a highly significant effect (p<0.01) on the antioxidant activity DPPH of honey (Table 1). The Klanceng honey from Sleman 90.5% and Klaten 91.2% was the highest radical scavenger than honey from Nglipar 47.3% (Table 1). The highest radical scavenger of Klanceng honey from Sleman and Klaten was caused by the highest of TPC and TFC than honey from Nglipar. In addition, based on the Pearson correlation coefficients (Table 3) showed that TPC had a significant effect on antioxidant activity DPPH and TFC had a highly significant effect on the antioxidant activity DPPH, but not on vitamin C. The TPC and TFC have a positive correlate on the antioxidant activity DPPH was shown in Table 3. Thus, the higher of TPC and TFC in honey could be increase the radical scavenger DPPH of Klanceng honey. da Silva et al (2016) explained that phenolic acids and flavonoids present in honey have been used as floral markers and they are compounds that act as the antioxidants, eliminating or reduce the formation of free radicals, and inhibiting lipid oxidation. The DPPH antioxidant activity of Klanceng honey from Sleman and Klaten was similar reported by Ranneh et al (2018) for Kelulut honey and was higher to those previously reported (Guerrini et al 2009; Oddo et al 2008).
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Table 3.
Correlation matrix (Pearson correlation coefficients) between
total |
||||
|
TPC |
TFC |
Vitamin C |
DPPH |
|
|
TPC |
1 |
|||
|
TFC |
0.84a |
1 |
||
|
Vitamin C |
0.45 |
0.73b |
1 |
|
|
DPPH |
0.79b |
0.96a |
0.56 |
1 |
|
a
Correlation is significant at the 0.01 level (2-tailed) |
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The authors would like to thank the Directorate of Research and Community Service, Ministry of Research, Technology, and Higher Education of the Republic of Indonesia for financial support of the research through Penelitian Terapan Unggulan Perguruan Tinggi (PTUPT), Penelitian Disertasi Doktor (PDD), and Directorate of Research Universitas Gadjah Mada (UGM) through Rekognisi Tugas Akhir (RTA) 2019.
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Received 1 August 2019; Accepted 26 August 2019; Published 2 October 2019