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 10.3 10 cells/ml, 11.3 10 cells/ml and 19.1 10 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 300-500 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 200-701, Korea Tel: 82-33-250-655 Fax: 82-33-253-6560 E-mail: hyeonl@kangwon.ac.kr Received April 2, 2009; revised May 12, 2009; accepted May 18, 2009 313 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
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 160 10% 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 (1.6 1.6 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. 338051ion-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 2 200 µl yww z 37 o C 20 g. 2+ DMSO š 20 w. Ca y y hyaluronidase 0.1 M acetate buffer(ph
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 1.0 10 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) 2 10 7 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 -5 10 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 1.2 16.21 Crude protein 56.59 56.1 Crude fat 0.9 0.75 Water 5.03.92 Ash 23.02 20.98 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 2.38 1.16 Fe 5.3 3.02 K 2.26 25.32 Mg 3.52 2.03 Mn 0.03 1.23 Na 65.80 56.32 Zn 0.01 1.32 P 13.31 10.62 1 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 65.80 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 0.2-1.0 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
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 9.83 10.20 Threonine 5.26 5.92 Serine 5.03.79 Glutamic Acid 15.080 1.930 Proline 3.3.51 Glycine 5.8 5.07 Alanine 8.23 7.61 Cystine 0.27 0.56 Valine 6.63 6.76 Methionine 1.37 3.10 Isoleucine 5.9 6.76 Leucine 9.83 0.85 Tyrosine.57 0.56 Phenylalanine 5.03 6.76 Lysine 5.26 7.0 Ammonia 0.27 1.1 Histidine 1.60 2.5 Tryptophan 0.07 0.00 Arginine 6.86 10.20 100.0000 100.0000 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 6 100 o C, 80 o C k ƒƒ 11.3%, 11.0% ùkü, 60 o C q w 12.8 10 cells/ml ƒ ùkü ƒ 10.3 10 cells/ml w ƒ g y w (Table ). B s T s w. ùk ü 6 60 o C q w 11.3 10 cells/ml ƒ 9.0 10 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,
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) 1 2 3 5 6 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±0.06 3.5±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±0.10 3.5±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±0.09 3.8±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±0.07 3.3±0.09 b.8±0.07 a 6.3±0.06 c 8.±0.08 c 09.3±0.09 a Control 2.5±0.07 3.1±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 5.08 10 pg/cell,.90 10 pg/ cell w, ƒƒ 3.93 10 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 5.38 10 pg/cell, 5.89 10 pg/cell ƒƒ 3.87 10 pg/cell,.67 10 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.
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 6 12.0 10 cells/ml ùkü 11.9 10 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 1.5 10 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 11.3 10 cells/ml, 12.8 10 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, 6 12.0 10 cells/ml ùkü 11.9 10 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. 2009 w w w w w tw (PM570). 1. Kay RA. Microalgae as food and supplement. Crit. Rev. Food Sci. Nutr. 30: 555-573 (1991) 2. Yang HN, Lee EH, Kim HM. Spirulina platensis inhibits anaphylactic reaction. Life Sci. 61: 1237-12 (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: 297-285 (2002). Jeong SW, Lee NK, Kim SJ, Han DS. Screening of tyrosinase inhibitor from plants. Korean J. Food. Sci. Technol. 27: 891-896 (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: 115-121 (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: 237-27 (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
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