Separation and Characterization of Polyphenols Prevailing in NZ Herbal Leaves Kawakawa (Macropiper excelsum) and Determination of Compounds in its Essential Oil

Abstract

Kawakawa (Macropiper excelsum, G. Forst) is characteristic endemic plant prevailing throughout the coastal line of New Zealand (Aotearoa) which belongs to Piperaceae family. Kawakawa leaves have their significance as folk medicine and holistic heritage among local inhabitant of New Zealand. Kawakawa is a rich source of polyphenols. The research data is limited on polyphenols prevailing in Kawakawa leaves. Previously, work has been conducted on the major compounds prevailing in Kawakawa leaves i.e. lignans, amides, phenylpropanoids and their bioactivities have been reported. However, the research focused on phenolic compounds and free radical scavenging ability of Kawakawa leaf extract is limited. Moreover, no data has been found on purification of bioactive compounds prevailing in Kawakawa leaves. The purification of plant bioactive is an important step to gauge the actual antioxidant efficacy of plant extract. The present work is focused on phenolic extraction, antioxidant assessment and compound purification from Kawakawa leaf to fill the research gap. Furthermore, the research has contributed to the knowledge regarding the identification of major compounds prevailing in essential oil extracted from Kawakawa leaves. In the current research, extraction of Kawakawa leaf polyphenols has been carried out by conventional solvent extraction method i.e., aqueous and ethanol extraction. Based on single factor experiment, material to solvent ratio, temperature, time, pH, concentration of ethanol is selected to conduct orthogonal experiment L9 (34) (three – level – four – factor). The phenolic contents, flavonoid contents and free radical scavenging abilities of Kawakawa aqueous extract and ethanol extract were determined by TPC (total phenolic contents), TFC (total flavonoid contents), ABTS (2,2’-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid), FRAP (ferric reducing ability of plasma) and DPPH (2.2-diphenyl-1-(2,4,6-trinitrophnyl) hydrazyl. The SPSS software was used to analyze the experimental data. The orthogonal experimental design was tested by range analysis and oneway ANOVA. The verification of extraction optimization was carried out in both Kawakawa extracts. The extraction conditions in Kawakawa aqueous extraction are including 1:30g/ml (material to water ratio), pH 5, 30 min (extraction time), 60 oC (extraction temperature) which have yielded TPC and TFC as 37.68 + 0.67 mg gallic acid equivalent /g DW and 23.67 + 0.23 mg catechin equivalent /g DW, respectively. The free radical scavenging ability of Kawakawa aqueous extract was evaluated by ABTS, FRAP and DPPH as 585.02+2.72 μ M Trolox Equivalent /g, 500.14+1.41 μ M TE/g and 548.24 +9.21 μ M TE/g, respectively. In Kawakawa ethanol extraction the best extraction conditions including: 1:40 g/ml (material to solvent ratio), 80%, (ethanol concentration), 20 minutes (extraction time), 700C (extraction temperature) which have yielded phenolic contents and flavonoid contents as 87.83+0.83 mg GAE/g DW and 95.37+0.87 mg CE/g DW, respectively. The antioxidant activity of Kawakawa ethanol extract was carried out by ABTS and FRAP and evaluated as 600.72+5.57 μ M TE/g DW and 668.23+2.42 μ M TE/g DW, respectively. The purification of Kawakawa polyphenols was carried out by macroporous resins HP-20 and XAD-7HP. Batch adsorption desorption behavior of two macroporous resins by Kawakawa crude extracts were extensively studied in current research. The conditions for adsorption and desorption process were optimized. Batch adsorption, isotherm and thermodynamic parameters were studied. The adsorption of Kawakawa polyphenols on macroporous resins was described by pseudo second order kinetics. The thermodynamic parameters have shown that the adsorption of Kawakawa polyphenols on macroporous resins was an exothermic process. The column or fixed-bed adsorption desorption study was conducted on glass column. The results indicated that most phenolics eluted with acidified 30% ethanol and remining with acidified 50% ethanol. Fraction analysis was carried out by TPC, antioxidant assays (DPPH, ABTS) and compounds in purified extracts were identified and quantified by HPLC-DAD. Four major compounds were detected in purified fractions namely isorhamnetin, glycosyl vitexin, vitexin and rosmarinic acid with recovery yield 76.07+1.11%, 78.89+2.7%, 73.57+2.41 and 82.6+1.82 respectively. The quantitative analysis of compounds prevailing in essential oil of Kawakawa was carried out by GC-MS equipped with RT 5MS column. A total of 33 compounds have been identified in essential oil extracted from Kawakawa leaves. The major classes of compounds detected are including alkenyl benzene (myristicin), sesquiterpenes, terpenes (α-copene, cubene, caryophyllene, limonene, pinene, myrecene), anisols (Elemicin), acetals (Eucalyptol), ketones (bornanone) and esters (4-terpinenyl acetate, Borneol, acetate (1S, 2R, 4S) -(-)-, benzene propanoic acid, methyl esters, 2-Heptanol, acetate). The retention indices calculations have shown that essential oil extracted from kawakawa leaves mainly comprised of myristicin (2156.266+7.73 μg/μl), elemicin (110.9511+3.77 μg/μl), α-zingibirene (174.6+8.4 μg/μl), Caryophyllene (55.14+1.79 μg/μl) and 4-terpinenyl acetate (48.02+2.03μg/μl).