Optimization of Ultrasound Extraction Conditions to Produce Antioxidant Extracts from New Zealand Bushes with Improved Inhibition of Alzheimer’s Disease Enzymes

Abstract

Alzheimer’s Disease (AD) is most common type of neurodegenerative disorder primarily affecting the elderly population. As of 2020, there are still no available therapeutic interventions to cure AD or reverse the disease progression. Therefore, the medication aims to retard the natural progression of AD and to attenuate the symptoms. The two main pathways to explain the cause of AD are inhibition of brain cholinesterase enzymes and receptor antagonists. Most of the AD drugs, whether approved or still under development, have been isolated from plant sources. New Zealand presents an abundant biodiversity of around 2000 species, which have been used by Maori people as traditional phytomedicines for different ailments. As very few species were tested for medical applications related to AD and memory disfunction, there is an opportunity to investigate the inhibition of brain enzymes related to Alzheimer’s disease treatment. In the first set of experiments extracts produced from 15 different species of New Zealand bushes or small trees were investigated based on their traditional ethnomedicinal uses for the inhibition of three enzymes related to AD treatment: acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and β-secretase (BACE1). The antioxidant activity of the plant extracts was also determined. From the first study using 30 extracts from 15 tree species, it was found that the extracts of Weinmannia racemosa bark and methanolic extract of Leptospermum scoparium leaves exhibited high AChE inhibition activity with IC₅₀ as low as 8.09 μg/ml. For BChE, strong inhibitory activity was also observed in W. racemosa bark extracts, extracts of Aristotelia serrata leaves, and ethyl acetate extract of Shefflera digitata leaves at the lowest IC₅₀ of 24.5 μg/ml. Extracts of Kunzea ericoides exhibited very strong BACE1 inhibition. The highest DPPH radical scavenging activity (RSA) was detected in the methanol extract of Aristotelia serrata, followed by both extracts of Hebe stricta and W. racemosa, and lastly the methanol extracts of K. ericoides and Pomaderris kumeraho, which presented higher RSA than the reference antioxidant (IC₅₀ <12.01 μg/mL). Then, detailed studies to optimize the extraction conditions using ultrasound assisted extraction (UAE) were conducted with 3 specific plants aiming maximum enzyme inhibition of AChE (by L. scoparium leaf extracts), BChE inhibition (by A. serrata leaf extracts), and BACE1 inhibition (by K. ericoides leaves) using response surface methodology (RSM). The extraction yield and the extract antioxidant potential expressed in terms of DPPH RSA were the two other responses investigated, together with enzyme inhibition. In the extraction of L. scoparium (manuka) leaf extracts using UAE, the effect of temperature (40-60 °C), time (1-20 min), and ethanol concentration (30-70%) was studied. The optimum extraction conditions were different for AChE inhibition, RSA and yield. Extracts produced at optimum conditions and experimentally tested, validated successfully the model predictions. The IC₅₀ concentrations of the optimized extracts were reduced from 66.0 to 28.5 μg/mL for AChE, and from 32.4 to 2.37 μg/mL for antioxidant activity. UAE of antibutyrylcholinesterase and antioxidants from Aristotelia serrata (makomako) leaves was optimized by studying the effect of acoustic power density (0.13-0.65 W/mL), time (1-20 min), and ethanol concentration (20-80%). The optimum extraction conditions were 0.53 W/mL of acoustic power density, 17.12 min of extraction time and a solvent with 74% ethanol concentration. The IC₅₀ concentrations were experimentally determined (26.2 μg/mL for BChE, 5.5 μg/mL for RSA). Lastly, optimization of UAE of β-secretase (BACE1) and antioxidants from Kunzea ericoides (kanuka) leaves was carried out. By studying three extraction parameters: acoustic power density (0.13-0.65 W/mL), time (2-20 min), and temperature (30-70 °C), the optimum extraction conditions of 0.43 W/mL, for 15.59 min, at temperature 69 °C were obtained. Experimental validation of the optimum conditions predicted by the mathematical models showed low absolute error (<6%) for the 3 responses. In addition, a comparison study between UAE, Soxhlet extraction and maceration showed that UAE method was the most efficient, producing extracts with higher BACE1 inhibition, antioxidant and extraction yield with IC₅₀ concentration of 14.25 μg/mL for BChE, and 3.17 μg/mL for RSA. This study demonstrates that UAE can be employed to enhance extraction of natural inhibitors of AChE, BChE, BACE1 enzymes, and antioxidant from plant materials.