홍조류 Kappaphycus alvarezii의 신경세포 성장활성 평가

Abstract

Neurodegenerative disorders are associated to the brain aging. This condition in conjunction with life style, such as alcohol drinking, cigarette smoking, and incorrect diets will affect brain health. Neurodegenerative disorders are causing neuronal death and synaptic loss in brain which affect memory, thinking, and social abilities in a human. The most prevalent cause of progressive neurodegenerative diseases is Alzheimer’s disease (AD). People with AD are showing impaired-brain especially in the hippocampus area, where the area for memory-processing and learning. One alternative to treating AD is by using neurotrophic factors (NF) that play a role in development and maintenance of neuron cells. The Rhodophyta Kappaphycus alvarezii has been known to support neurite growth and maturation of neuron cell. Thus, the investigations of further research related to the axodendritic maturation, spinogenesis and synaptogenesis of neurons and as well as the ideal cultivation condition and post-harvest treatment of K. alvarezii might increase the possibility of this seaweed as a candidate for neurotrophic agent.

The first part of this study, we evaluated the optimal cultivation condition of K. alvarezii for neurotrophic activities. The difference between 3 depths, 4 growing periods, and 14 areas of cultivation were evaluated. At the same cultivation condition (at a depth of 0 m, in the 45-day growth period), K. alvarezi exhibited higher neurotrophic activities than the other two carrageenan producers, K. striatum and Eucheuma denticulatum. The neurotrophic activities of K. alvarezii based on the number of primary neurites, the total length of primary neurites, and the length of the longest neurites (1.4-, 1.8-, and 1.7-fold, respectively, compared with controls). Thalli harvested from deep (2 m) and early (15 d) cultures represented significantly lower activities than those from 0-1 m and 30-45 d (P < 0.05). Thalli harvested from Ternate and Garut areas, Indonesia showed high activities. Commercially, the cultivation conditions at the surface for 45 days were optimal for both neurotrophic compounds and carrageenan production.

Based on the above results, we next compared K. alvarezii for its neurotrophic activities by five different postharvest treatments (oven drying, sun drying, freeze drying, shade drying, and salting following shade drying). All postharvest treatments showed significant neurotrophic activities (P<0.05) compared to control. In addition, we extracted carrageenan waste using ethanol from carrageenan processing at 90 oC for 2h, pH 9, and added the extract to the hippocampal neuron cultures. The ethanol extract of carrageenan waste significantly promoted the neurotrophic activity (P<0.05), with an optimum dose at 1 µg mL-1. From carrageenan processing, we concluded that neurotrophic compounds of K. alvarezii showed heat- and alkaline-stable. We also observed the neurotrophic activities of main and branch thalli of K. alvarezii with insignificant differences. These results exhibit that K. alvarezii exposed to sunlight, heat, and/or alkaline conditions is still retaining its neurotrophic activity.

The development and the neuronal cytoarchitecture of K. alvarezii ethanolic extract (EKA) on hippocampal neuron cultures were investigated in the third part. The extract at optimal concentration of 1 µg mL-1 significantly enhanced the axonal length, the number of secondary axonal collateral branches, the length of primary dendrites, the number of secondary dendritic branches by 58%, 8-fold, 68%, and 2.6-fold, compared with control (p < 0.05). The number of axodendritic intersections, branching points, and branching tips were also increased in treated neurons. Several kinds of lipophilic compounds are assumed to have neurotrophic activities to increase neurites in a robust form.

Having found the lipophilic group was active for enhancing neurite growth, we isolated the neurotrophic activity compound. From high-performance liquid chromatography (HPLC) and NMR analysis, we found that the most abundant phytosterol contained in K. alvarezii was cholesterol. Thus, we investigated the effect of EKA and its isolated cholesterol (PTS) on spinogenesis and synaptogenesis of hippocampal neuron cultures. EKA and PTS significantly increased the dendritic filopodia and spine formations (P<0.05) and the pre- and post-synaptic which results in synapse enhancement (P<0.05). The functional presynaptic plasticity was significantly improved by the addition of EKA or PTS on neuron cultures (P<0.05). These results support the use of K. alverezii as a diet supplement and/or source of neurotrophic compounds for the people who are prone to neurodegenerative diseases.