Classification of Propolis Extract of Trigona spp Bee from Four Regions in Indonesia Using FTIR based Metabolomic Approach

Abstract

Propolis is a resinous substance collected by honeybees from various plant sources. Propolis obviously possesses antioxidant activity, as reported by many researchers. Although numerous researchers have reported the biological activities of propolis collected worldwide, information about Indonesian propolis, particularly from Trigona spp., is only a few. Some studies reported that propolis has various bioactivities including antimicrobial, anticancer and antioxidant. These properties are influenced by phenols and flavonoids compounds.. The complex constituents and properties of propolis is different from each other depends on location and plant of origin. This variation will have an effect on its bioactivity. Indonesia has many local honey bees; one of them that produce propolis is Trigona spp. The location of propolis source used in this research may relate to its physical and chemical characteristics. Some researches have examined the correlation between phenol and flavonoid content with antioxidant activity of propolis. It was found that for some cases there is no correlation which implies that other compounds may have influence to the antioxidant activity. Therefore metabolomic is further used to observe this possibility. Metabolomic approach is capable of determination which regions have a potential propolis as a source of antioxidant. Metabolomic is defined as metabolite process analysis of the organism comprehensively quantitatively or qualitatively. Metabolomic approach in this study is focussed on classification of propolis as a result the difference of regions and its correlation to antioxidant activity. The objective of this research is to apply metabolomic in classification of propolis from four regions based on its FTIR metabolite profile and its antioxidant activity. Those four regions are West Nusa Tenggara, West Java, West Kalimantan and South Sulawesi. Since the composition of propolis varies with its origin, the intensity of antioxidant activity should be different as well. Metabolomic approach was used to identify which region has propolis with better antioxidant activity. FTIR based metabolomic was applied to measure the spectra of propolis in order to examine the differences among propolis samples then to classify them. For this purpose, the FTIR spectra were analyzed by PCA and PLS. In addition, the correlation between propolis phenol and flavonoid content to its antioxidant capacity was also determined. Interpretation of FTIR spectra showed the presence of O-H, C-O and C-H aromatic bond for phenol and its ester. Meanwhile, the presence of flavonoid compound is designated to C=O, C-H, C-H aromatic and supported by O-H bond. In addition, C=C, C-O bonds were also found which may relate to the presence of terpenes. The samples also confirm the presence of N-O symmetric, C-N and N-H which point to amino acid and aromatic amine. There were also C-O and O-H bonds which may relate to fatty acid. Nevertheless the existence of these compounds according to the functional groups is not confirmed yet. Then further confirmation by targeted analysis of propolis total phenol and flavonoid content and its correlation to propolis antioxidant activity are required. Antioxidant potential of propolis from four regions in Indonesia was accessed by analyze propolis scavenging effect on DPPH radicals which IC50 were in the range 0,54-2,90 μg/mL. Low values of IC50 on DPPH scavenging assay were obtained for West Kalimantan and West Java propolis are 0,54 μg/mL and 0,86 μg/mL, respectively. The IC50 of propolis from four regions of Indonesian were found to be lower as compared to commercial propolis. All IC50 of propolis from four regions were also lower than reference IC50 of trolox and vitamin C. According to PCA score plot, the propolis samples were classified into two groups. Propolis from West Kalimantan, West Java, South Sulawesi and commercial tend to group itself which imply chemical composition similarities, meanwhile West Nusa Tenggara can be remarked as being totally different. The three members of the first group were recognized by the presence of C=O, C-O, C-H aromatic bond which are interpreted as phenol and flavonoid compound. By contrast, the second group was designated by O-H, C-O, C-C aliphatic which are interpreted as fatty acid, then C-N, N-H and N-O that refers to amino acid. Based on PLS, the propolis samples were classified in the same clusters as in PCA. Propolis from West Nusa Tenggara as group 2 demonstrates lower antioxidant activity than group 1 which consist of propolis from West Kalimantan, West Jaca, South Sulawesi and commercial. The major compounds in group 2 were attributed by N-H, C-N, C=C, C=O, C-H aliphatic bonds which shows the existence of terpene, fatty acid and amino acid. Then, the three members of the group 1 which have higher antioxidant activity have predominant O-H, C-H, C=C, C=O bonds which indicate the presence of phenol and flavonoid compounds. From this, the assumption that the composition of complex chemical constituent and antioxidant activity do vary according to its geographical origin was proven. Total flavonoid and phenols contents of extracts of propolis samples were determined by using aluminium nitrate and Folin–Ciocalteu colorimetric methods were in the range of 39.9 – 97.4 mg/mL of GAE and 3.12 – 20.2 mg/mL of QE, respectively. Both phenol and flavonoid contents of commercial propolis was higher as compared to propolis from four regions of Indonesian. All the samples exhibited strong positive correlation between phenolic, flavonoid content and its antioxidant activity. Therefore, it is concluded that the result from PLS analysis which mentioned functional groups of phenol and flavonoids as a major difference between propolis with good antioxidant activity and those with lower antioxidant activity was proven.