Journal List > J Nutr Health > v.52(6) > 1142007

J Nutr Health. 2019 Dec;52(6):515-528. Korean.
Published online Dec 26, 2019.  https://doi.org/10.4163/jnh.2019.52.6.515
© 2019 The Korean Nutrition Society
Splenocyte-mediated immune enhancing activity of Sargassum horneri extracts
Dong-Sub Kim,1 Nak-Yun Sung,1 In-Jun Han,1 Byung-Soo Lee,1 Sang-Yun Park,1 Eun Young Nho,1 Ji Eom,1 Geon Kim,1 and Kyung-Ah Kim2
1Division of Natural Product Research, Korea Prime Pharmacy CO., LTD., Jeonnam 58144, Korea.
2Department of Food and Nutrition, Chungnam National University, Daejeon 34134, Korea.

To whom correspondence should be addressed. tel: +82-42-821-6832, Email: kakim@cnu.ac.kr
Received Sep 23, 2019; Revised Oct 28, 2019; Accepted Oct 30, 2019.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.


Abstract

Purpose

This study examined the immunological activity and optimized the mixture conditions of Sargassum horneri (S. horneri) extracts in vitro and in vivo models.

Methods

S. horneri was extracted using three different methods: hot water extraction (HWE), 50% ethanol extraction (EE), and supercritical fluid extraction (SFE). Splenocyte proliferation and cytokine production (Interleukin-2 and Interferon-γ) were measured using a WST-1 assay and enzyme-linked immunosorbent assay, respectively. The levels of nitric oxide and T cell activation production were measured using a Griess assay and flow cytometry, respectively. The natural killer (NK) cell activity was determined using an EZ-LDH kit.

Results

Among the three different types of extracts, HWE showed the highest levels of splenocyte proliferation and cytokine production in vitro. In the animal model, three different types of extracts were administrated for 14 days (once/day) at 50 and 100 mg/kg body weight. HWE and SFE showed a high level of splenocyte proliferation and cytokine production in the with and without mitogen-treated groups, whereas EE administration did not induce the splenocyte activation. When RAW264.7 macrophage cells were treated with different mixtures (HWE with 5, 10, 15, 20% of SFE) to determine the optimal mixture ratio of HWE and SFE, the levels of nitric oxide and cytokine production increased strongly in the HWE with 5% and 10% of SFE containing group. In the animal model, HWE with 5% and 10% of SFE mixture administration increased the levels of splenocyte proliferation, cytokine production, and activated CD4+ cell population significantly, with the highest level observed in the HWE with 5% of SFE group. Moreover, the NK cell activity was increased significantly in the HWE with 5% of SFE mixture-treated group compared to the control group.

Conclusion

The optimal mixture condition of S. horneri with immune-enhancing activity is the HWE with 5% of SFE mixture. These results confirmed that the extracts of S. horneri and its mixtures are potential candidate materials for immune enhancement.

Keywords: Sargassum horneri; splenocyte proliferation; cytokine production; optimum mixture ratio; natural killer cell activity

Figures


Fig. 1
Effects of different types of Sargassum horneri extracts on the splenocyte proliferation and cytokine production in in vitro. Isolated splenocytes were treated with HWE, EE, and SFE at the concentration of 3.125, 6.25, 12.5, 25, or 50 µg/mL. CON A (1 µg/mL) was used as a positive control. After 24 h, cell proliferation and cytokine production were determined by WST-1 assay and ELISA kit. Results are expressed as the mean ± SD (n = 3). Statistical analysis was performed using Student's two tails t-test with a significant level of *p < 0.05, and **p < 0.01, ***p < 0.001 compared to control (CTRL) group. HWE, hot water extract; EE, ethanol extract; SFE, supercritical fluid extract.
Click for larger imageDownload as PowerPoint slide


Fig. 2
Effect of orally administrated different types of Sargassum horneri extracts on the splenocyte proliferation. HWE, EE, and SFE were orally treated for 14 days (once/day) at the concentration of 50 or 100 mg/kg body weight. Isolated splenocytes were stimulated with mitogen (LPS; 1 µg/mL, CON A; 1 µg/mL) for 24 h. Cell proliferation was determined by WST-1 assay. Results are expressed as the mean ± SD (n = 3). Statistical analysis was performed using Student's two tails t-test with a significant level of *p < 0.05 compared to control (CTRL) group. HWE, hot water extract; EE, ethanol extract; SFE, supercritical fluid extract.
Click for larger imageDownload as PowerPoint slide


Fig. 3
Effect of orally administrated different types of Sargassum horneri extracts on cytokine (IL-2 and IFN-γ) productions in splenocytes. HWE, EE, and SFE were orally treated for 14 days (once/day) at the concentration of 50 or 100 mg/kg body weight. Isolated splenocytes were stimulated with mitogen (CON A; 1 µg/mL) for 24 h. Cytokine production was determined by ELISA kit. Results are expressed as the mean ± SD (n = 3). Statistical analysis was performed using Student's two tails t-test with a significant level of *p < 0.05, and **p < 0.01, compared to control (CTRL) group. HWE, hot water extract; EE, ethanol extract; SFE, supercritical fluid extract.
Click for larger imageDownload as PowerPoint slide


Fig. 4
Determination of optimum mixing ratio of HWE and SFE extracts from Sargassum horneri in RAW264.7 macrophage cells. HWE was mixed with 5%, 10%, 15%, 20% of SFE and treated at the concentration of 6.25, 12.5, 25, and 50 µg/mL in RAW264.7 macrophage cell. After 24 h, cell proliferation and cytokine productions were determined by MTT assay and ELISA kit, respectively. NO production was detected by using Griess reagent. Results are expressed as the mean ± SD (n = 3). Statistical analysis was performed using Student's two tails t-test with a significant level of *p < 0.05, and **p < 0.01, ***p < 0.001 compared to control (CTRL) group. HWE, hot water extract; SFE, supercritical fluid extract.
Click for larger imageDownload as PowerPoint slide


Fig. 5
Effect of optimum mixing ratio of HWE and SFE extracts from Sargassum horneri on the splenocyte proliferation. HWE was mixed with 5%, 10%, or 20% of SFE and orally treated for 14 days (once/day) at the concentration of 100 mg/kg body weight. Isolated splenocytes were stimulated with mitogen (LPS; 1 µg/mL, CON A; 1 µg/mL) for 24 h. Cell proliferation was determined by WST-1 assay. Results are expressed as the mean ± SD (n = 3). Statistical analysis was performed using Student's two tails t-test with a significant level of *p < 0.05 compared to control (CTRL) group and of #p < 0.05, compared to HWE treated group. HWE, hot water extract; SFE, supercritical fluid extract.
Click for larger imageDownload as PowerPoint slide


Fig. 6
Effect of optimum mixing ratio of HWE and SFE extracts from Sargassum horneri on the splenocyte cytokine (IL-2 and IFN-γ) productions. HWE was mixed with 5%, 10%, or 20% of SFE and orally treated for 14 days (once/day) at the concentration of 100 mg/kg body weight. Isolated splenocytes were stimulated with mitogen (CON A; 1 µg/mL) for 24 h. Cytokine production was determined by ELISA kit. Results are expressed as the mean ± SD (n = 3). Statistical analysis was performed using Student's two tails t-test with a significant level of *p < 0.05 compared to control (CTRL) group and of #p < 0.05, compared to HWE treated group. HWE, hot water extract; SFE, supercritical fluid extract.
Click for larger imageDownload as PowerPoint slide


Fig. 7
Effect of optimum mixing ratio of HWE and SFE extracts from Sargassum horneri on CD4+ T cell activation. HWE was mixed with 5%, 10%, or 20% of SFE and orally treated for 14 days (once/day) at the concentration of 100 mg/kg body weight. Isolated splenocytes were treated with PerCP-Cy5.5-conjugated anti-mouse CD4, PE-Cy7-conjugated anti-CD25 antibody for 0.5 h at 4℃. T cell population was calculated by flow cytometer. Statistical analysis was performed using Student's two tails t-test with a significant level of *p < 0.05, compared to control (CTRL) group and of #p < 0.05, compared to HWE treated group. HWE, hot water extract; SFE, supercritical fluid extract.
Click for larger imageDownload as PowerPoint slide


Fig. 8
Effect of optimum mixing ratio between HWE and SFE extracts from Sargassum horneri on NK cell activity. HWE was mixed with 5%, 10%, or 20% of SFE and orally treated for 14 days (once/day) at the concentration of 100 mg/kg body weight. NK cells were isolated from spleen and purified by Magnetic activated cell sorting system. NK cells (effector cell) were mixed with YAC-1 cell (target cell) as a ratio of 1:1, 5:1, 10:1, 20:1 (effector cell : target) and NK cell activity was determined by LDH assay. Results are expressed as the mean ± SD (n = 3). Statistical analysis was performed using Student's two tails t-test with a significant level of *p < 0.05, and **p < 0.01, compared to control (CTRL) group. HWE, hot water extract; SFE, supercritical fluid extract.
Click for larger imageDownload as PowerPoint slide

Notes

This work was supported by grants from the Ministry of Trade, Industry & Energy (MOTIE), Korea Institute for Advancement of Technology (KIAT) through the Encouragement Program for The Industries of Economic Cooperation Region (P0000611).

References
1. Tomasi TB Jr, Tan EM, Solomon A, Prendergast RA. Characteristics of an immune system common to certain external secretions. J Exp Med 1965;121(1):101–124.
2. Byun EH. Immunomodulatory activities of crude polysaccharide fraction separated from Perilla frutescens Britton var. acuta Kudo. Korean J Food Sci Technol 2017;49(5):559–566.
3. Cho EJ, Lee JH, Sung NY, Byun EH. Anti-inflammatory effects of Annona muricata leaf ethanol extracts. J Korean Soc Food Sci Nutr 2017;46(6):681–687.
4. Kim YE, Lee JH, Sung NY, Ahn DH, Byun EH. A comparative study of the immuno-modulatory activities of ethanol extracts and crude polysaccharide fractions from Annona muricata L. Korean J Food Sci Technol 2017;49(4):453–458.
5. Klimp AH, de Vries EG, Scherphof GL, Daemen T. A potential role of macrophage activation in the treatment of cancer. Crit Rev Oncol Hematol 2002;44(2):143–161.
6. Schepetkin IA, Quinn MT. Botanical polysaccharides: macrophage immunomodulation and therapeutic potential. Int Immunopharmacol 2006;6(3):317–333.
7. Sung NY, Park WY, Kim YE, Cho EJ, Song H, Jun HK, et al. Increase in antioxidant components and reduction of off-flavors on radish leaf extracts by extrusion process. J Korean Soc Food Sci Nutr 2016;45(12):1769–1775.
8. Kim JS, Oh CH, Jeon H, Lee KS, Ma SY. Immuno-regulatory property of fruit-extracts of Cornus kousa Burg. Korean J Med Crop Sci 2002;10(5):327–332.
9. Lee JY, Hwang WI, Lim ST. Effect of Platycodon grandiflorum DC extract on the growth of cancer cell lines. Korean J Food Sci Technol 1998;30(1):13–21.
10. Byrd JC, Park JH, Schaffer BS, Garmroudi F, MacDonald RG. Dimerization of the insulin-like growth factor II/mannose 6-phosphate receptor. J Biol Chem 2000;275(25):18647–18656.
11. Brown ES, Allsopp PJ, Magee PJ, Gill CI, Nitecki S, Strain CR, et al. Seaweed and human health. Nutr Rev 2014;72(3):205–216.
12. Kim NG. Effects of temperature, photon irradiance, and photoperiod on the growth of embryos of Sargassum horneri in laboratory culture. Korean J Fish Aquat Sci 2015;48(1):76–81.
13. Liu L, Heinrich M, Myers S, Dworjanyn SA. Towards a better understanding of medicinal uses of the brown seaweed Sargassum in Traditional Chinese Medicine: a phytochemical and pharmacological review. J Ethnopharmacol 2012;142(3):591–619.
14. Preeprame S, Hayashi K, Lee JB, Sankawa U, Hayashi T. A novel antivirally active fucan sulfate derived from an edible brown alga, Sargassum horneri. Chem Pharm Bull (Tokyo) 2001;49(4):484–485.
15. Matsumura Y. Nutrition trends in Japan. Asia Pac J Clin Nutr 2001;10 Suppl:S40–S47.
16. McHugh DJ. In: A guide to the seaweed industry. Rome: Food and Agriculture Organization of the United Nations; 2003.
17. Cardoso SM, Pereira OR, Seca AM, Pinto DC, Silva AM. Seaweeds as preventive agents for cardiovascular diseases: from nutrients to functional foods. Mar Drugs 2015;13(11):6838–6865.
18. Yamaguchi M. Regulatory mechanism of food factors in bone metabolism and prevention of osteoporosis. Yakugaku Zasshi 2006;126(11):1117–1137.
19. Shao P, Chen X, Sun P. Chemical characterization, antioxidant and antitumor activity of sulfated polysaccharide from Sargassum horneri. Carbohydr Polym 2014;105:260–269.
20. Shao P, Liu J, Chen X, Fang Z, Sun P. Structural features and antitumor activity of a purified polysaccharide extracted from Sargassum horneri. Int J Biol Macromol 2015;73:124–130.
21. Kim DS, Sung NY, Park SY, Kim G, Eom J, Yoo JG, et al. Immunomodulating activity of Sargassum horneri extracts in RAW264.7 macrophages. J Nutr Health 2018;51(6):507–514.
22. Kang BK, Kim KBWR, Ahn NK, Choi YU, Kim MJ, Bark SW, et al. Immuno-stimulating activities of skipjack tuna Katsuwonus pelamis cooking juice concentrates on mouse macrophages and spleen cells. Korean J Fish Aquat Sci 2014;47(6):776–784.
23. Shan BE, Yoshida Y, Kuroda E, Yamashita U. Immunomodulating activity of seaweed extract on human lymphocytes in vitro. Int J Immunopharmacol 1999;21(1):59–70.
24. Mischell BB, Shiigi SM. Normal peritoneal cells. In: Mischell BB, Shiigi SM, editors. Selected Methods in Cellular Immunology. San Fransisco (CA): W. H. Freman and Company; 1980. pp. 156.
25. Kim P, Ko SK, Pyo MY. Effects of hot water extract of chaga mushroom on the proliferation and cytokines production of mouse splenocytes in vitro. Yakhak Hoeji 2010;54(3):187–191.
26. Lee JK, Lee MK, Yun YP, Kim Y, Kim JS, Kim YS, et al. Acemannan purified from Aloe vera induces phenotypic and functional maturation of immature dendritic cells. Int Immunopharmacol 2001;1(7):1275–1284.
27. Kim BH, Cho DH, Cho JY. Modulatory effect of kaempferitrin, a 3,7-diglycosylflavone, on the LPS-mediated up-regulation of surface co-stimulatory molecules and CD29-mediated cell-cell adhesion in monocytic- and macrophage-like cells. Yakhak Hoeji 2007;51(6):482–489.
28. Virchow JC Jr, Oehling A, Boer L, Hansel TT, Werner P, Matthys H, et al. Pulmonary function, activated T cells, peripheral blood eosinophilia, and serum activity for eosinophil survival in vitro: a longitudinal study in bronchial asthma. J Allergy Clin Immunol 1994;94(2 Pt 1):240–249.
29. Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol 1995;155(3):1151–1164.
30. Byun MW, Byun EH. Immunological synergistic effects of combined treatment with herbal preparation (HemoHIM) and red ginseng extracts. J Korean Soc Food Sci Nutr 2015;44(2):182–190.
31. Chiou WF, Chou CJ, Chen CF. Camptothecin suppresses nitric oxide biosynthesis in RAW 264.7 macrophages. Life Sci 2001;69(6):625–635.
32. Hibbs JB Jr, Taintor RR, Vavrin Z, Rachlin EM. Nitric oxide: a cytotoxic activated macrophage effector molecule. Biochem Biophys Res Commun 1988;157(1):87–94.
33. Pyo SN, Son EH. In: Introduction to immunology. Seoul: Shinil Books; 2008.
34. Lee GN, Kwon OH. In: Clinical laboratory file. 3rd edition. Seoul: Medical Publisher; 2003.
35. Pisa P, Halapi E, Pisa EK, Gerdin E, Hising C, Bucht A, et al. Selective expression of interleukin 10, interferon gamma, and granulocyte-macrophage colony-stimulating factor in ovarian cancer biopsies. Proc Natl Acad Sci U S A 1992;89(16):7708–7712.
36. Sypek JP, Chung CL, Mayor SE, Subramanyam JM, Goldman SJ, Sieburth DS, et al. Resolution of cutaneous leishmaniasis: interleukin 12 initiates a protective T helper type 1 immune response. J Exp Med 1993;177(6):1797–1802.
37. Lee JW, Jang MH, Choi JS, Ahn TW. The effect of Yongyukjowitang distillate on the immune activity of spleen cells of aged rats. J Sasang Const Med 2013;25(3):218–232.
38. Biron CA, Nguyen KB, Pien GC, Cousens LP, Salazar-Mather TP. Natural killer cells in antiviral defense: function and regulation by innate cytokines. Annu Rev Immunol 1999;17(1):189–220.
39. Wang R, Jaw JJ, Stutzman NC, Zou Z, Sun PD. Natural killer cell-produced IFN-γ and TNF-α induce target cell cytolysis through up-regulation of ICAM-1. J Leukoc Biol 2012;91(2):299–309.
40. Frese-Schaper M, Keil A, Yagita H, Steiner SK, Falk W, Schmid RA, et al. Influence of natural killer cells and perforinmediated cytolysis on the development of chemically induced lung cancer in A/J mice. Cancer Immunol Immunother 2014;63(6):571–580.
41. Li Q, Morimoto K, Nakadai A, Inagaki H, Katsumata M, Shimizu T, et al. Forest bathing enhances human natural killer activity and expression of anti-cancer proteins. Int J Immunopathol Pharmacol 2007;20 2 suppl 2:3–8.