J Nutr Health.  2019 Dec;52(6):515-528. 10.4163/jnh.2019.52.6.515.

Splenocyte-mediated immune enhancing activity of Sargassum horneri extracts

• Kim DS
• Sung NY
• Han IJ
• Lee BS
• Park SY
• Nho EY
• Eom J
• Kim G
• Kim KA

• KMID: 2466666
• DOI: http://doi.org/10.4163/jnh.2019.52.6.515

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.

Keyword

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

MeSH Terms

Body Weight
Chromatography, Supercritical Fluid
Enzyme-Linked Immunosorbent Assay
Ethanol
Flow Cytometry
In Vitro Techniques
Killer Cells, Natural
Macrophages
Models, Animal
Nitric Oxide
Sargassum*
Water
Ethanol
Nitric Oxide
Water

Figure

• 

  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.

• 

  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.

• 

  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.

• 

  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.

• 

  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.

• 

  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.

• 

  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.

• 

  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.

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