Extraction of bioactive compounds from New Zealand Manuka leaves (Leptospermum scoparium) using emerging technologies

Abstract

The impact of high UVB levels on the natural flora of New Zealand represents a unique case study for those unusual plants. Its pharmacological properties consider the most promising field in which the research area can expand uses of bioactive compounds. Manuka (Leptospermum scoparium) is one of the shrubs that used medicinally by Māori. The high level of bioactive compounds in Manuka leaves is responsible for the powerful antibacterial, antifungal, antioxidant/anti-ageing, and anti-inflammatory activities. In light of this, the potential of green technologies in improving the extractability of bioactive compounds and enhancing the value of Manuka leaves was undertaken in this present study. Deep eutectic solvents (DESs) and pulsed electric field (PEF) are two emerging technologies that have great potential for extracting target compounds from Manuka leaves. With respect to the particle size study, two types of milling were used for particle size reduction. Mild milling (around 1 min) to get particle size between 1400-250μm and severe milling (approximately 5 mins) to achieve the finest particle size possible, between 200-68μm. It was observed that by decreasing the particle size to 200-68 μm, most of the extraction achieved as soon as the powder was exposed to the solvent (at zero time) with no further extraction at an extended period. The results of using different hydrophobic and hydrophilic DESs in the extraction of β-caryophyllene revealed that the hydrophobic menthol: lactic acid DES at 1:2 molar ratio was more efficient than n-hexane, methanol, and ethanol as reference solvents and conventional steam distillation and Soxhlet extraction. β-caryophyllene showed good stability in menthol: lactic acid DES over nine days of storage. Moreover, results also approved good reusability of the potential DES after four cycles of extraction. Further, the Manuka extract was tested for its antioxidant, total phenolic content (TPC) and, antibacterial. Results were compared with ethanol and n-hexane extracts. The ethanolic extract possessed the highest amount of TPC (86.08±6.22 mg GAE/g DW) among tested extracts. Moreover, the extraction of TPC using hydrophobic deep eutectic solvent (HDES) was at a sufficient level (79.82±0.75 mg GAE/g DW), while n-hexane presents poor yield (2.74±0.70 mg GAE/g DW). Higher radical scavenging antioxidant activity (DPPH) and ferric reducing antioxidant power (FRAP) values were found to be in ethanolic extract. Disc diffusion method was used to assess the antibacterial activity against Staphylococcus epidermidis, E. coli, and Pseudomonas aeruginosa strains. The results were also compared with HDES, lactic acid (as an individual component), steam distilled Manuka oil, commercial Manuka essential oil from the East Cape (CMEO) and β-caryophyllene pure oil. The results revealed that higher toxicity was found in HDES, HDES extract, and lactic acid against all tested bacteria. However, the commercial Manuka essential oil has a superior toxic effect against Staphylococcus epidermidis. The effect of ammonium-based DESs on the extraction of total phenolic content and antioxidant capacity were also studied and compared with ethanol. Results revealed that three DESs were more efficient as compared to the ethanol as a benchmark solvent. ChCl: lactic acid DES was preferred and studied further for optimization. The highest amount of TPC was obtained when the extraction temperature, biomass content and extraction time were 49.96 ℃, 5.07wt%, and 1.07 h, respectively. Furthermore, ChCl: ethylene glycol DES extract showed a better DPPH value (12.78473896 μg TE/ml) while ChCl: lactic acid DES extracts gave higher FRAP value (199.7823129 μg TE/ml) with both being higher than the extract obtained by ethanol, indicating a potent antioxidant activity of the DESs extracts. PEF treatment studied as a direct extraction of total phenolic content from a suspension of ungrounded Manuka leaves using a binary mixture of ethanol and water 75% (v/v) in a batch treatment chamber. The maximum improvement was achieved when pulses frequency (Hz), time, and extraction temperature of 788 Hz, 59 min, and 60 ℃, respectively. Results were found to be 1.58 times higher than thermal extraction. The electric field intensity was limited to 2.27 kV/cm due to cell design limitation and higher electric field would have given even higher extraction. In addition, higher DPPH and FRAP values were found in samples treated with PEF. The obtained results demonstrate the advantages of both deep eutectic solvents and pulsed electric field in enhancing the bioactive extraction from Manuka leaves and enhancing the bioactivity of the extract.