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1 KOREAN J. FOOD SCI. TECHNOL. Vol. 1, No. 3, pp. 313~319 (2009) q w Spirulina maxima y yáw Áw xá½ Á zá½ Á w t 1 Á 1 Á x 2 Á x 3 * w BTp yw w, 1 w tƒœ, 2w w, 3 w w The Korean Society of Food Science and Technology Enhancement of Immune Activity of Spirulina maxima by Low Temperature Ultrasonification Extraction Sung Ho Oh, Jae Gun Han, Ji Hye Ha, Young Kim, Myoung Hoon Jeong, Seong Sub Kim, Hyang Suk Jeong, Geun Pyo Choi 1, Uk Yeon Park 1, Do Hyung Kang 2, and Hyeon Yong Lee 3 * College of Bioscience and Biotechnology, Kangwon National University 1 Department of Food Processing & Bakery, Gangwon Provincial University 2 Korea Ocean Research & Development Institute 3 Research Institute of Bioscience and Biotechnology, Kangwon National University Abstract The marine microalga Spirulina maxima was extracted using water or ethanol at 100 or 80 o C and by ultrasonification in water at 60 o C. The ultrasonification technique generated the highest yield (19.8%). To be therapeutically useful, the extraction should yield a product with low cytotoxicity and high immunity against skin infections. The cytotoxicity of all extracts (1.0 mg/ml) was below 25%. Moreover, the cytotoxicity of the extract generated by ultrasonification was 5%. Extracts prepared in the described manners could inhibit hyaluronidase activity by up to 0% compared to the control. Increased growth of human B, T and NK cells and an increase in cytokine secretion were observed, confirming the interrelationship between both human immune and skin immune activity. The extract prepared by ultrasonification increased the growth of human B, T and NK cells up to cells/ml, cells/ml and cells/ml, respectively. The extract prepared by ultrasonification also greatly increased the secretion of both IL-6 and TNF-α. Moreover, it was estimated that protein, Na and leucine occupy a high ratio. Accordingly, this study has confirmed that extracts prepared as described have the potential to effectively increase skin immunity. Key words: Spirulina maxima, cytotoxicity, cytokine, NK cell, hyaluronidase Spirulina 35 ùkù y w, ¼ µm, ù 8µm, ù x Spirulina. x 35 ƒ x, v e û y w. š Spirulina Arthrospira maxima Arthrospira platensis p w ƒ s w ü y ƒ š, û w w wš, 55-70%, 6-9%, k y 15-20%», k, w wš. t y t ƒ š» mw w t *Corresponding author: Hyeon Yong Lee, College of Bioscience and Biotechnology, Division of Biomaterials Engineering, Kangwon National University, Chuncheon, Gangwon , Korea Tel: Fax: hyeonl@kangwon.ac.kr Received April 2, 2009; revised May 12, 2009; accepted May 18, ƒ j. mw v ù y w w y j vg k B12 w y w w phenoic acid, tocopherols w wš š š š (1,2). w carotinoid phycocyanin, novel sulfated polysaccharide w z w y,, w y w z ùkü š (3-6). Spirulina maxima» mw Spirulina platensis w (7-9), ƒ wš w, w 10%ƒ š, ù S. maxima œ mw» ƒ y ƒeƒ ƒw. w ¾ ƒ. w mw» w w» w ƒe. x y t t ƒ» y t y y t s š (3,9-11). v

2 31 w t wz 1 «3y (2009) w s k S. maxima v y ƒ y w» w w w w B s, T s Natural Killer(NK) s d IL-6 TNF-α cytokine d mw» y w, w z hyaluronidase w y z w. w S. maxima t yw S. platensis w w. S. maxima w ƒ y w v ƒ w y wš w. x w w Spirulina maxima (UTEX, LB295, USA) 105 C 3 w o, 100 C q w o 60 o C, 80 o C EtOH, y þƒ»ƒ flask w ƒƒ 10 w 3 wš, ƒ z mw ƒƒ w. x s ATCC l, s z T cell(jurkat (T lymphocytes: acute T cell leukemia), ATCC, Manassas, VA, USA), B cell (Raji (B lymphocytes: human burkitts lymphoma), ATCC), Natural Killer cell(nk-92mi cell, ATTC) w. x s, s RPMI % heating-inactivated FBS ƒ g w š, NK-cell α-mem 2 mm L- glutamine, 0.2 mm myoinositol, 20 mm folic acid, 2-mercaptoethanol, 12.5% fetal bovine serum ƒ g w. w s v s w HEPES buffer, Dulbeccos modified eagle medium(dmem) RPMI160, x fetal bovine serum(fbs) Gibco(invitrogen, Carlsbad, CA, USA) l w w. Gentamycin sulfate, sodiume chloride, sodiume bicarbonate, trypsin-edta Sigma Aldrich(St. Louis, MO, USA) l w, ethanol, z chloroform, hexane» Sigma Aldrich p w. yw w Association of Official Agricultural Chemists (AOAC)» w (12)., 10 g þƒ m k k z 60 C o dry oven r jš w. x ( cm) 5mg w 6N-HCl 5 ml ƒw k»w z w 110 C 2 o ƒ w g. 50 ml»š» 0.01 N HCl we š» 2 N NaOH 2mL š yw z 5,000 rpm 30 w d w 60 C o ƒ m j wš 0.02 N HCl 20 ml š 0.5 µm filter w z x w. yw t x» System 6300 hyperperformence analyzer(beckman Coulter, Fullerton, CA, USA) w chromatogram peak w, w Spirulina platensis xw. f Li 10 cm column No ion-exchange(Beckman Coulter) w, 150 min, buffer flow rate 20 ml/h, ninhydrin flow rate 10 ml/h, f 1380 psi, 50 µl w, ƒ 0 psi w. AOAC w w 105 o C, z w 550 C zy w o w. w micro-kjeldahl w»(foos 1035 analyzer, Tecator, Sweden), w Soxhlet w w. y w d e ww 100 w.» (Ca, P, Mg, K, Na, Fe, Zn, Cu, Mn) w AOAC w w., 0.1 mg ¾ y e w 550 C 6 zy k o, 20 o C sand bath 5mL HNO 3 ƒw 10 ƒ wš þ z, 25 ml volumetric flask š ƒw w (whatman filter paper No. 1) w. ƒ w w z Inductively Coupled Spectrometer(Perkin Elmer Instruments, Shelton, CT, USA) w w Plasma w, nabulizer pressure 3.5 bars for meinhard type C, aerosol flow rate 0.3 L/min, shealth gas flow 0.3 L/min, cooling gas 12 L/min w. s(hek293) w s d s sulforhodamine B (SRB) assay mw s HEK293(ATTC) w d w. HEK293 s 96 well plate -5Ü10 cells/ml w z 2 (37 o C, 5% CO 2 )w z, 0.2, 0., 0.6, 0.8, 1.0 mg/ ml w ƒƒ 100 µl ƒw 8 w. z wš ƒ 10% (w/v) trichloroacetic acid(tca) 100 µl ƒw C 1 o ew z -5z w TCA wš plate w ƒ well 1% (v/v) p 0.% (w/v) SRB 100 µl ƒwš 30 g. w SRB 1% p -5z, k z 10 mm Tris buffer 100 µl ƒw ü z 50 nm microplate reader (Thermo max, Molecular Devices, Sunnyvale, CA, USA) w Ÿ d w (13). w z hyaluronidase w y v z sƒw» w» hexane, chloroform, ethyl acetate, butanol water fraction z w hyaluronidase wy d w. Hyaluronidase z Rooster Comb x N-acetylglucosamine ŸŸ d w y q w. 0.1 M acetate buffer(ph 3.5) hyaluronidase(7,900 unit/ml) 50 µl ƒ 0.2, 0., 0.6, 0.8, 1.0 mg/ mlƒ 20 µl ƒwš, z y y w 12.5 mm CaCl µl yww z 37 o C 20 g. 2+ DMSO š 20 w. Ca y y hyaluronidase 0.1 M acetate buffer(ph

3 3.5) hyaluronic acid (12 mg/5 ml) 250 µl ƒw 0 w. z 0. N NaOH 100 µl 0. M potassium tetraborate 100 µl yw ƒw ò 3 k z þƒ g. þƒ k dimethyl aminobenzaldehyde (p-dimethyl aminobenzaldehyde g, 100% acetic acid 350 ml 10 N HCl 50 ml yw ) 3.28 ml yw ƒw z 37 o C 20 w z 585 nm Ÿ d w. w wš, z y 50% ww ü w wš, IC 50 w. Hyaluronidase Inhibition(%)=[(ODc-ODs)/ODc] 100» ODc OD(optical density) š, ODs 585 nm OD (1). s z cytokine d» z s T s(jurket) B s (Raji) w w. s 10% FBS w w RPMI 160 5% CO 2, 37 o C w,» z 2 well plate s cells/ml w 6 w ƒ well cell hemacytometer s d w d w w. Cytokine IL-6 TNF-α IL-6 TNF-α kit(chemicon, Billerica, MA, USA) w d w. Jurkat Raji s 2 well plate 1-2Ü10 cells/ml w z, 37 o C, 5% CO 2 incubator 2 w z 0.5 mg/ml w.» w d w, Sunrise(Tecan, Grodig, Austria) w 50 nm microplate reader(thermo max, Molecular Devices) w Ÿ d w t w w t š w cytokine d w (13,15,16). Natural killer(nk) cell z NK-92MI cells α-mem 2 mm L-glutamine, 0.2 mm myoinositol, 20 mm folic acid, 2-mercaptomethanol, 12.5% fetal bovine serum (FBS) 12.5% horse serum (Myelocult) cells/ml g w. B s T-25 Flask w sample n w z w 3- z s w d w. NK-92MI cell 2 well plate cells/ml 900 µl wš 2 z B s d ƒ plate 100 µl n w 8 z 6 NK-92MI cell y cell counter w s d w NK-92MI cell y d w (17). m x triplicate determinations w Mean±SD t w, ƒ s³e ANOVA test w p=0.05 w. š Spirulina maxima t Spirulina maxima» Table Spirulina maxima y 315 Table 1. Comparison of chemical composition of S. maxima and S. platensis Component S. maxima (%(w/w)) S. platensis (%(w/w)) Carbohydrate Crude protein Crude fat Water Ash Table 2. Comparison of several mineral compositions of S. maxima and S. platensis Composition S. maxima (mg/100 g) S. platensis (mg/100 g) Ca Fe K Mg Mn Na Zn P Table 2 ƒƒ ùkü. S. maxima 55.59% ƒ wš, k y z ƒƒ 1.2, 23.02% ùkü. w S. maxima S. platensis w û. w» ùp mg/100 g 60% w. Spirulina ùp e» w, p S. maxima v S. platensis w wš ùkû. w Table 3 mw S. maxima S. platensis 19 ùkü. Glutamic acid w 15.08% wš, S. platensis w v 7 wù leucine tyrosine w ƒƒ 9.83,.57% w. Leucine, v,, x w S. maxima v w v w t w» t ƒe w w y t w v» w. s(hek293) w s d S. maxima s w s sulforhodamine B assay w d w s mg/ml ƒw w ùkü š, š n 1.0 mg/ml 80 o C EtOH 2.5% ƒ, 100 o C 21.3%, 60 o C q w 18.5% z ùkü s w s û ù kû (Fig. 1). k S. platensis w š 1.0 mg/ml 26% w s ùkü w(18) û s ùkü. û s ùkü z

4 316 w t wz 1 «3y (2009) Table 3. Comparison of essential amino acids of S. maxima and S. platensis S. maxima S. platensis Composition Content (%) Content (%) Aspartic Acid Threonine Serine Glutamic Acid Proline Glycine Alanine Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Ammonia Histidine Tryptophan Arginine Fig. 1. Cytotoxicity of S. maxima extracts on human embryonic kidney (HEK293) cells. Results are expressed as mean±sd of data obtained from three independent experiments. w ƒ. S. maxima j ùkü, S. maxima 60 o C ù š, q w ƒ, w ƒ w. w z hyaluronidase w y z S. maxima v w z y w» w ƒ hyaluronidase y w z d w. Hyaluronidase š hyaluronic acid w z, z w w z» w. ƒ z Fig. 2 ùkü, ƒ z 5ƒ w ƒ w w. 60 o C q w 100 o C 1.0 mg/ml ƒƒ 62., 60.2% w y ùkü, 80 o C k û 57.2% w y ùkü. x 60 o C Fig. 2. Hyaluronidase inhibition activity of S. maxima under different extraction conditions. Results are expressed as mean±sd of data obtained from three independent experiments. q w ƒ hyaluronidase w w z» w, 0% w y ùkü S. maxima v w z» w. S. platensis hyaluronidase y w z w ƒ, w zd ƒ s y y, q w w š 20% y y ùkü (19). z w q ww y ƒ w s y ƒ w y ƒ ƒw. w, S. maxima y ùkü w wš d mw y ƒ w y w. wz mw z z, ³ mw w ƒ ƒ jš w. s z cytokine w w w s T s, B s z y w» w B s T s z cytokine d mw y w (Table, Fig. 3-). T s x d 1 l 6 ¾ ƒw w ùkü, ùkü o C, 80 o C k ƒƒ 11.3%, 11.0% ùkü, 60 o C q w cells/ml ƒ ùkü ƒ cells/ml w ƒ g y w (Table ). B s T s w. ùk ü 6 60 o C q w cells/ml ƒ cells/ml B s ƒ k ùkû. S. maxima B s T s ƒ j w y w. mw 6 ƒ z ùký» û s ƒ š w d z w. w s w cytokine(il-6, TNF-α) ùkü, T s d w ƒ ƒ mw s cytokine r,

5 Spirulina maxima y 317 Table. The growth of human immune T, B cell in adding 0.5 mg/ml of S. maxima extracted from various extraction conditions Extraction condition of sample Cultivation time (day) T cell B cell Viable cell density ( 10 cells/ml) Viable cell density ( 10 cells/ml) Extracts from water ultrasonification at 60 o C 3.3±0.02.3±0.0 a 6.2±0.05 b 8.3±0.06 a 10.8±0.07 a 12.8±0.08 b Extracts from water at 100 o C 3.1±0.08.0±0.07 a 5.5±0.11 a 7.3±0.13 a 9.5±0.16 b 11.3±0.18 c Extracts from EtOH 80 o C 2.8± ±0.09 a 5.8±0.07 a 7.3±0.08 a 9.1±0.10 c 0.11±0.09 b Control 2.8± ±0.13.8±0.09 a 6.3±0.07 c 8.5±0.11 c 10.3±0.16 Extracts from water ultrasonification at 60 o C 3.1±0.06.3±0.03 a 6.3±0.08 c 8.±0.10 a 9.8±0.11 b 11.3±0.09 a Extracts from water at 100 o C 2.8± ±0.10 c 6.2±0.11 a 7.8±0.13 a 9.3±0.10 c 10.8±0.7 c Extracts from EtOH 80 o C 2.8± ±0.09 b.8±0.07 a 6.3±0.06 c 8.±0.08 c 09.3±0.09 a Control 2.5± ±0.08 b.5±0.12 a 5.8±0.08 a 7.5±0.09 c 09.2±0.08 Values are expressed as mean±sm of data obtained from three independent experiments. Fig. 3. Secretion of IL-6 and TNF-α from human immune T cells in adding 0.5 mg/ml of the extracts. Results are expressed as mean±sd of data obtained from three independent experiments. (A) IL-6 (B) TNF-α Fig.. Secretion of IL-6 and TNF-α from human immune B cells in adding 0.5 mg/ml of the extracts. Results are expressed as mean±sd of data obtained from three independent experiments. (A) IL-6 (B) TNF-α ùkü 6 IL-6 TNF-α ƒ w 60 o C q w ƒƒ pg/cell, pg/ cell w, ƒƒ pg/cell,.6 10 pg/cell w w ƒ ùkü y w (Fig. 3). ƒ B s mw cytokine d w ƒ w s IL-6 TNFα 60 o C q w w IL-6 TNF-α ƒƒ pg/cell, pg/cell ƒƒ pg/cell, pg/cell w w ƒ (Fig. ). mw S. maxima ƒƒ s w ƒ š, y, cytokine. S. maxima ƒ mw s cytokine ƒ mw y ƒ y w, k w x wù hyaluronidase y w mw v z k y t y ƒ». w, S. platensis n w T cell d z» y k ƒ, w z n w H9 š 8.2% ƒ sw y w» y k. x mw y ww, w. x S.

6 318 w t wz 1 «3y (2009) Fig. 5 Effects of S. maxima extracts on the enhancement of NK cell growth by adding the supernant of human B cells. Results are expressed as mean±sd of data obtained from three independent experiments maxima w swwš ƒ ƒ mw w x w z». Natural killer(nk) s z NK s z x x B s z cytokine d mw y w ƒ y ùkü 60 o C q w w d w. NK s y d B s ƒw z NK s ƒw y ƒw w y d w. Fig. 5 B s ƒ ƒw z NK s ƒw, NK s y ùk ü. 6 w w ƒ ƒw ùkü š, ƒ ùkü cells/ml ùkü cells/ml ùkü w y ùkü, y swwš. m NK s y d ƒw B cell NK s ƒw xw, 6 w 6 m tg cells/ ml ùkü, S. maxima w y ù kü. w cytokine w ƒ, S. maxima w z ù kü, S. maximaƒ ƒ š w y ùkü. x w Spirulina maxima 100 o C q w 60 o C, 80 o C EtOH w x ww. AOAC w»,» ùp ƒƒ 56, 60% w, w ³ v swwš. v leucine w 9.83% t w S. maxima v y z ƒ y w. v w T s B s cytokine d NK s y w z hyaluronidase w y z d w. s(hek293) w s j ù kü ùkù S. maxima z w 60 o C q w B s T s ƒƒ cells/ml, cells/ml 6 ƒ, w s w cytokine(il-6, TNF-α) ƒ 60 o C q w ƒw B s, T s IL- 6 TNF-α ƒ,» y ù ùkû. B s ƒ ƒw z NK s ƒw, 6 w w ƒ ƒw, cells/ml ùkü cells/ml ùkü w y ùkü, y swwš. w w z hyaluronidase w y z 60 o C q w 1.0 mg/ml 62.% w y 100 o C š» ù y q ƒ y š q ƒ y y x w mw v y y ƒ w z yƒ ƒ w y w., S. maxima v y mw k wz, mw v y w š w w w w w w tw (PM570). 1. Kay RA. Microalgae as food and supplement. Crit. Rev. Food Sci. Nutr. 30: (1991) 2. Yang HN, Lee EH, Kim HM. Spirulina platensis inhibits anaphylactic reaction. Life Sci. 61: (1997) 3. Hernandez AC, Nieves I, Meckes M, Chamorro G, Barron BL. Antiviral activity of Spirulina maxima against Herpes simplex virus type 2. Antivir. Res. 56: (2002). Jeong SW, Lee NK, Kim SJ, Han DS. Screening of tyrosinase inhibitor from plants. Korean J. Food. Sci. Technol. 27: (1995) 5. Kim IS, Cho ZS. Modulation of human fibroblast proliferation and collagen production by prostaglandin E2. Korean Biochem. J. 26: 0-52 (1993) 6. Cho JH, Lee KM, Kim NS, Kang WH. The effects of whitening components on human melanocytes in vitro. Korean Cosmet. Sci. 23: (1997) 7. Grinstead GS, Tokach SS, Goodband RD, Nelssen JL. Effects of Spirulina platensis on growth performance of weanling pigs. Anim. Feed Sci. Technol. 83: (2000) 8. Nandeesha MC, Gangadhara B, Manissery KK, Venkataraman LV. Growth performance of two Indian Major carps, catla (catla catla) fed diets containing different levels of Spirulina platensis. x

7 Spirulina maxima y 319 Bioresour. Technol. 80: (2001) 9. Diego JM, Gomez C, Ibanez E, Ruperez FJ, Barbas C. Tocopherol measurement in edible products of vegetable origin. J. Chromatogr. A 105: (200) 10. Kaji T, Fujiwara Y, Inomata Y, Hamada C, Yamamoto C, Shimada S, Lee JB, Hayashi T. Repair of wounded monolayers of cultured bovine aortic endothelial cells is inhibited by calcium spirulan, a novel sulfated polysaccharide isolated form Spirulina platensis. Life Sci. 70: (2002) 11. Lee HS, Lee SH, Mun HC, Lee HY. Screening of the Immunostimulatory activity of the marine alga Chlorella capsulate. Korean J. Biotechnol. Bioeng. 18: 19-2 (2003) 12. Thompson RH, Merola GV. A simplified alternative to the AOAC official method for cholesterol in multicomponent foods. AOAC Int. 76: (1993) 13. Lee MK, Choi GP, Ryu LH, Lee GY, Yu CY, Lee HY. Enhanced immune activity and cytotoxicity of Artemisia capillaris Thunb. extracts against human cell lines. Korean J. Med. Crop Sci. 12: 36-2 (200) 1. Kim YS, Noh YK, Lee GI, Kim YK. Inhibitory effects of herbal medicines on hyaluronidase activity. Korean J. Pharm. 26: (1995) 15. Han BH, Park MH, Cho JY, Park JS, Yoo ES, Baiik KU. Effect of ginsenosides from Panax ginseng on TNF-α production and T cell proliperation. Yakhak Hoeji 2: (1988) 16. Kwon SY, Lee HS, Lee SH, Im GI, Kim SN, Kim HS, Hwang SW, Hwang SY. Analgesic effect and inhibition of prostaglandin E2 Activity and pro-inflammatory cytokines production by ethyl alcohol extract from new herbal formula. Korean J. Pharmacogn. 37: (2006) 17. Limdbolum CK. IL-2 receptor signaling through the Shb adapter protein in T and NK cells. Biochem. Biophys. Res. Commun. 296: (2002) 18. Kim HS, Kim CH, Kim JH, Kwon MC, Cho JH, Gwak HG, Hwang BY, Kim JC, Lee HY, Comparison of anticancer activities from the culture and extraction conditions of the Spirulina platensis. Korean J. Microbiol. Biotechnol. 3: (2006) 19. Choi SI, Lee YM, Heo TR. Screening of hyaluronidase inhibitory and free radical scavenging activity in vitro of traditional herbal medicine extracts. Korean J. Biotechnol. Bioeng. 18: (2003)