Extraction and characterization of polyhydroxylated-1,4- naphthoquinones (PHNQs) of the spinochrome class from Evechinus chloroticus and investigation of their bioactivities
Evechinus chloroticus is a New Zealand sea urchin species that is harvested for its edible roe and is considered by many people to be a delicacy. The shell and spines of E. chloroticus are considered as waste and are typically dumped in landfills, which leads to environmental issues. This thesis reports the investigation of the extraction of polyhydroxylated-1,4-naphthoquinone (PHNQ) pigments from E. chloroticus shell and spine waste (i) using traditional solvent extraction methods compared to the use of macroporous resin, and (ii) characterization of the PHNQs and analysis of some biological activities of these extracts. Initially, the extraction of PHNQs from E. chloroticus shell and spine was evaluated using six different macroporous resins. Four of these resins (D4006, D4020, D101 and NKA-9) were found to provide good extraction of PHNQs in terms of the overall adsorption and desorption from the macroporous resins. Extraction with organic solvents had a higher yield of PHNQs compared to the macroporous resins, as demonstrated by high-performance liquid chromatography (HPLC) analysis. The PHNQ composition was characterised by HPLC with diode‐array detection (DAD) and mass spectrometry (MS). Five PHNQ compounds (spinochromes E, B, C, A and echinochrome A), and three aminated PHNQ compounds (spinamine E and echinamines A and B) were identified. The pigments were found to be prone to degradation upon exposure to light, with the aminated PHNQ pigments being the least stable. In addition, the bioactivities of E. chloroticus shell and spine PHNQ extracts obtained by macroporous resins (NKA-9, D4020, D4006, and D101) were investigated. Spine PHNQs extracted by the D4006 resin had the highest total phenolic content (TPC) while the D101 and D4020 resin extracts had the lowest TPC (p < 0.05). The best extraction of PHNQs was obtained with the D4006 resin and spine extracts showed better antioxidant activity (DPPH, ABTS, ORAC and FRAP) than shell extracts (p < 0.05). The Minimum Inhibitory Concentration (MIC) of PHNQ extracts against Escherichia coli and Staphylococcus aureus ranged from 2.5 to 10 mg/mL, with the D4006 resin extract having the best activity. The crude PHNQs extracted by different organic solvents, and PHNQs fractionated by HPLC were all tested for antioxidant activity by the DPPH, ABTS, ORAC and FRAP assays. Ethyl acetate was the best extraction solvent and spine extracts showed better antioxidant activity than shell extracts (p < 0.05). Overall, the organic solvent extracted PHNQs showed higher levels of antioxidant activity compared to the macroporous resin extracts. The HPLC fraction containing spinochrome E showed the highest antioxidant activity (p < 0.05). The anti-microbial effect as measured by the MIC of the PHNQ crude extracts and the HPLC fractions ranged from 0.25 to 2.5 mg/mL depending on the PHNQ extract and microbial species tested. PHNQs extracted by organic solvents had overall better anti-microbial activity than those of the macroporous resin extracts. PHNQ extracts altered the morphology of the Staphylococcus aureus cell wall as observed by transmission electron microscopy. PHNQ extracts also exhibited anti-inflammatory activity in rats (ED50 = 8.26 ± 0.22 µg), which was comparable to that of celecoxib, a standard anti-inflammatory drug (6.12± 0.18 µg). A molecular docking study supported the anti-inflammatory activity of PHNQs, indicating the potential of E. chloroticus PHNQ for anti-inflammatory drug development. PHNQs were found to be non-toxic to osteoblastic cells after 21 days co-incubation with the cells, at concentrations below 62.5 µg/mL. A synergistic effect was observed when PHNQ extract at the concentration of 62.5 µg/mL and 1.5 mM CaCl2 resulted in increased mineralized nodule formation in Saos-2 cells, over that of PHNQ extract or CaCl2 alone. This indicated that PHNQs have the potential to improve the formation of the bone mineral phase and may be potentially useful for prevention or treatment of osteoporosis, but further research is required to confirm this contention.This identification and analysis of PHNQs that exhibit antioxidant, anti-microbial, anti-inflammatory activities, and mineralized nodule formation effect from shell and spine waste of E. chloroticus has made a unique contribution to current knowledge of PHNQs, demonstrating the potential for utilization of E. chloroticus sea urchin shell waste.
Advisor: Bekhit, Aladin; Carne, Alan; McConnell, Michelle
Degree Name: Doctor of Philosophy
Degree Discipline: Food Science
Publisher: University of Otago
Keywords: New Zealand sea urchin; polyhydroxylated-1,4-naphthoquinone (PHNQ) pigments; extraction and identification; antioxidant activity; anti-microbial activity; anti-inflammatory activity; mineralized nodule formation
Research Type: Thesis