| Sea urchins belong to marine invertebrate Echinoidea. The edible portion of sea urchin is gonad, and the shells and spines are the major processing by-products of sea urchin. Studies indicate that the shells and spines contain polyhydroxylated 1,4-naphoquinone (PHNQ) pigments which possess antimicrobial, cardioprotective and antioxidant activities. Purple sea urchin, Strongylocentrotus nudus, is one of the most popular edible species in China. In the present study, the PHNQ pigments from Strongylocentrotus nudus shells and spines were extracted by using macroporous adsorption resin. Meanwhile, the physicochemical properties, chemical structures and bioactivities of the pigment were studied.First, the pigment mixture was prepared by using NKA-9 macroporous resin in a static adsorption mode and its physicochemical properties and stability were studied. Results showed that the pigment had relatively high solubility in polar solvents such as water and methanol but had relatively low solubility in non-polar solvents such as light petroleum and hexane. The pigment showed orange red in color when pH<6 but yellowish brown in color when pH≥6. The pigment was stable in heat, but instable in light. Na2SO3, sodium benzoate and potassium sorbate could not only cause change in color but also decrease the stability of the pigment. H2O2, NaCl, malic acid and citric acid could not cause change in color but decrease the stability of the pigment. Vitamin C could protect and enhance the color of the pigment. Ca2+ and Zn2+could not cause the change in color but decrease the stability of the pigment. Mn2+ and Mg2+ had no side-effect both on pigment stability and color, Al3+and Fe3+ formed stable and soluble metal chelates with pigments but Fe2+and Cu2+formed stable and insoluble metal chelates with pigments.Next, the pigment mixture was analysed rapidly by using an ultra-performance liquid chromatography (UPLC) coupled to hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer (Q-TOFMS). Eight PHNQ pigments were identified tentatively including 2,3,5,6,7,8-hexahydroxy-1,4-naphthoquinone, amino-pentahydroxy-1,4-naphthoquinone, 2,5,7,8-tetrahydroxy-1,4-naphthoquinone,2,3,5,7-tetrahydroxy-1,4-naphthoquinone, acetyl- amino-trihydroxy-1,4-naphthoquinone,3-acetyl-2,5,6,7,8-pentahydroxy-1,4-naphthoquinone, 3-acetyl-2,5,7,8-tetrahydroxy-1,4-naphthoquinone and 6-ethyl-2,3,5,7,8-pentahydroxy-1,4-naphthoquinone.The extract was separated and purified further on a semi-preparative HPLC. Results showed that four of them could be gained and the possible structure were 2,3,5,6,7,8-hexahydroxy-1,4-naphthoquinone,2,3,5,7-tetrahy droxy-1,4-naphthoquinone,3-acetyl-2,5,6,7, 8-pentahydroxy-1,4-naphthoquinone and 3-acetyl-2,5,7,8-tetrahydroxy-1,4-naphthoquinone.Finally, the in vitro antiproliferative,anti-inflammatory and immunoregulatory activity of the pigment extract and the in vitro antioxidant activities of the purified pigment fractions were investigated. Results showed that the 0.5 and 1 μg/mL pigment mixture could suppress the proliferation of Hela cells significantly(P<0.05), and 2 μg/mL pigment mixture could suppress the proliferation of Hela cells extremely significantly (P<0.01); the 1 μg/mL pigment mixture could suppress the proliferation of RAW264.7 cells induced by LPS significantly (P<0.05), and 5-10 μg/mL could suppress the proliferation of RAW264.7 cells induced by LPS extremely significantly(P<0.01); the 20 μg/mL pigment mixture induced a increase of spleen lymphocyte proliferation significantly(P<0.05), and 50 μg/mL pigment mixture induced a increase of spleen lymphocyte proliferation extremely significantly(P<0.01). The four pigment fractions showed strong antioxidant activity in vitro. At 0.4 mg/mL, fraction I, II, IV and V exhibited the DPPH radical scavenging rate of 60.70,86.49,70.79 and 85.14%, the reducing power of 0.491,0.860,0.652 and 0.825, and the ferrous ion chelation rate of 43.78, 31.66,53.54 and 72.57%. |
PDF Full Text Request