This study investigated immunomodulatory and antimetastatic properties of polysaccharides extracted from hallabong (Citrus hybrid) peel, focusing on their effects on macrophage activation. Crude polysaccharides (HPWP) were obtained through hot water extraction and ethanol precipitation from dried hallabong peel powder. Macrophages isolated from mouse peritoneum were used to evaluate cytotoxicity of HPWP against normal cells and Colon 26-M3.1 carcinoma cells. Our findings revealed no cytotoxic effects of HPWP on either normal cells or Colon 26-M3.1 carcinoma cells. Immunoactivity was assessed by measuring cytokine production, specifically IL-6 and IL-12, using ELISA. Results indicated a significant, concentration-dependent increases of cytokine levels. Tumoricidal activity of NK cells was also evaluated, demonstrating enhanced tumor cell killing induced by HPWP, in a concentration-dependent manner. This increased activity was due to HPWP’s potent antimetastatic effects, as observed in a mouse model of Colon 26-M3.1 carcinoma. In comparison with a the negative control (NC, 100%), HPWP at concentrations of 10, 100, and 1000 μg/mouse showed antimetastatic effects of 49.4%, 61.3%, and 74.3%, respectively. These effects were attributed to only NK cell activation. These findings suggest that HPWP has potential as a functional ingredient in food products aimed at enhancing immune response and inhibiting cancer metastasis.

In a previous study, we fractionated crude polysaccharide (AME-CP) with macrophage-stimulating activity from a hot-water extract (AME) of Astragalus membranaceus. AME-CP contained glucose (Glc) as a main component sugar, suggesting that it might be rich in starch-like compounds (SLC). To enhance the immunostimulating activity of AME-CP by pruning SLC rarely known to contribute to activity, hydrolysate (AME-SH) was prepared by digesting with starch-related enzymes, including α-amylase and amyloglucosidase. AME-SH was found to contain substances with molecular weights ranging from 3.9 to 84.4 kDa. These substances were primarily composed of galactose, galacturonic acid, Glc, arabinose, rhamnose, and mannose. AME-SH significantly enhanced the production of macrophage-stimulating factors, including nitric oxide (NO), interleukin (IL)-6, and IL-12, in RAW 264.7 cells compared to AME-CP. Treatment of splenocytes isolated from C3H/HeN mice with AME-SH not only promoted IL-6 secretion, but also induced mitogenic activity. In addition, AME-SH promoted the secretion of hematopoietic growth factors including IL-6 and granulocyte-macrophage colony-stimulating factor (GM-CSF) in Peyer's patch (PP) cells and stimulated bone marrow cell proliferation through these PP cells. In conclusion, hydrolysate (AME-SH) digested from AME-CP with starch-related enzymes could be used as a potential immunostimulant.

To utilize pepper (Piper nigrum) as an immunostimulatory agent, we isolated macrophage stimulating polysaccharides from pepper and investigated their macrophage activating activities. Hot-water extracts (HW) of black pepper (BP) and white pepper (WP) were prepared, and their macrophage stimulating activities were evaluated using RAW 264.7 cells. BP-HW significantly promoted the secretion of macrophage stimulating factors such as nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-12 compared to WP-HW. When BP and WP-HW were fractionated into crude polysaccharides (CP) and low molecules (LM) by ethanol precipitation, BP-CP demonstrated significantly more potent activity. Furthermore, BP-CP not only induced mRNA gene expression of macrophage activation factors, but also promoted nuclear localization of p65 and c-Jun. In addition, component sugar analysis revealed that glucan-type polysaccharides in BP-CP played a crucial role in macrophage activation. Taken together, these findings suggest that black pepper has industrial applicability not only as a spice, but also as an immunostimulatory functional material.

For the purpose of developing new immunomodulatory agents from broccoli, ethanol extract (BCEE), hot water extract (BCHW), and crude polysaccharide (BCCP) were isolated from broccoli, and their immunomodulatory activities and chemical properties were examined. In the in vitro cytotoxicity analysis, BCHW and BCCP did not affect the growth of tumor cells and normal cells. Murine peritoneal macrophages stimulated with BCCP showed higher production of IL-6, IL-12, and TNF- α cytokines than those stimulated with BCHW. Also, BCHW and BCCP did not show proliferation of splenic lymphocytes. In the in vitro assay for intestinal immunomodulatory activities, only BCCP enhanced GM-CSF secretion and the bone marrow cell-proliferating activity via cells in Peyer’s patches at 1,000 μg/mL. Also, BCHW mainly contained 33.7% neutral sugars, such as arabinose, glucose, and galactose, and 30.7% uronic acid, and BCCP consisted of 42.6% neutral sugars, including arabinose, galactose, and glucose, and 50.5% uronic acid. The above results lead us to conclude that crude polysaccharide (BCCP) isolated from broccoli causes considerably high cytokine production in peritoneal macrophages and bone marrow cell proliferation, and the polysaccharide extraction process is indispensable for separation of new immunomodulatory agents from broccoli.

본 연구는 한국의 전통발효식초가 지닌 새로운 생물학적 기능을 규명하기 위해 국내 및 국외에서 제조된 전통발효식초로부터 다당류를 분리하여 면역자극활성을 검토하였다. 국내산 현미식초 조다당(KBV-0), 일본산 현미식초 조다당(JBV-0) 및 국내산 감식초 조다당(KPV-0)을 분리하여 구성당 분석한 결과, KBV-0와 JBV-0는 주로 mannan으로 구성되어 있으며, KPV-0는 펙틴 유래 물질로 인한 조성으로 사료되었다. 3종의 다당 시료는 RAW 264.7 세포에 독성을 나타내지 않은 반면, RAW 264.7 세포를 자극하여 IL-6, IL-12 및 TNF-α와 같은 사이토카인의 생성을 농도 의존적인 경향으로 증진시켰으나, 특히 KPV-0의 활성이 KBV-0와 JBV-0보다 더 우수하였다. 또한 KPV-0는 대식세포의 포식작용과 관련있는 FcR II의 발현량을 유일하게 증가시켰다. 이상의 결과로부터 국내산 전통발효 감식초인 KPV-0는 다른 발효식초에 비해 더 우수한 면역활성을 지니는 것으로 나타났으며, 이는 기능성 소재로의 산업적 응용이 가능할 것으로 사료되었다.

본 연구에서는 일본식으로 제조된 상업용 간장과 우리나 라 전통방식으로 제조된 재래식 간장으로부터 다당을 분리 하여 RAW 264.7 대식세포주를 이용한 면역 증진활성을 비교 하였다. 간장 유래 다당, CSP-0 및 KTSP-0는 RAW 264.7 세 포주에 대해 모든 농도에서 세포 독성을 나타내지 않았다. 또 한 재래식 간장 유래 다당인 KTSP-0는 상업용 간장 유래 다 당 CSP-0보다 대식세포에 의한 NO 및 ROS의 생산을 농도의 존적으로 증가시켰으며, KTSP-0를 1,000 ㎍/㎖ 처리하였을 시 가장 높은 생산능을 나타내었다. 간장 유래 다당의 RAW 264.7 세포로부터 면역반응에 중요한 cytokine인 IL-6와 TNF-α의 cytokine mRNA의 발현량과 해당 단백질의 생산을 각각 real-time PCR과 ELISA로 확인한 결과, CSP-0는 IL-6와 TNF- α mRNA의 발현 및 생산에 유의적인 영향을 미치지 않았지 만, KTSP-0는 농도의존적으로 IL-6와 TNF-α mRNA 발현 및 생산을 증가시키는 것을 확인하였으며, 1,000 ㎍/㎖ 처리 시 최대 활성을 보였다. 한편, 대식세포의 탐식작용에서 중요한 역할을 하는 Fc 수용체의 발현 증가를 RT-PCR로 확인한 결 과, CSP-0는 FcR I, II의 발현에 모두 영향을 미치지 않았지만, KTSP-0는 FcR I의 발현을 선택적으로 증가시킴이 확인되어 항원에 결합한 IgG와 강하게 결합하여 탐식작용을 촉진시킬 것으로 예상되었다. 본 연구를 통해 상업용 간장에서 분리한 다당, CSP-0보다 우리나라 전통 재래식 간장에서 분리한 다 당, KTSP-0가 대식세포를 활성화하여 높은 면역 증진 효과를 나타내는 것으로 결론 지을 수 있었다.

In order to develop new physiologically active polysaccharides from persimmon leaves, two different crude polysaccharides were prepared using hot water (PLW-0) and pectinase digestion (PLE-0) and their immuno-stimulating activities were estimated. PLW-0 and PLE-0 showed similar sugar compositions with 15 different sugars, including rarely observed sugars in general polysaccharides such as 2-O-methyl-fucose, 2-O-methyl-xylose, apiose, aceric acid, 3 deoxy-D-manno-2-octulosonic acid, and 3-deoxy-D-lyxo-2-heptulosaric acid, but the uronic acid content of PLE-0 was lower than that of PLW-0 caused by pectinase treatment. Both PLW-0 and PLE-0 showed potent anti-complementary activity in a dose dependent manner which was similar to a known immuno-stimulating polysaccharide, PSK, from Coriolus versicolor. The activity of PLE-0 at a low concentration (100 ㎍/㎖) was higher than that of PLW-0. In an in vitro cytotoxicity analysis, PLW-0 and PLE-0 (up to 1, 000 ㎍/㎖) did not affect the growth of peritoneal macrophages and Colon 26-M3.1 carcinoma cells. In contrast, they enhanced lymphocyte proliferation activity. Peritoneal macrophages stimulated with PLW-0 and PLE-0 produced various cytokines, such as IL-6 and IL-12. However, PLE-0 was more effective on the cytokine production. Intravenous administration of PLW-0 and PLE-0 significantly augmented natural killer (NK) cell cytotoxicity against Yac-1 tumor cells 3 days after the treatment of polysaccharide fractions. But NK cells obtained from the PLE-treated group showed higher tumoricidal activity even at a low dose of 40 ㎍/mouse. In experimental lung metastasis of Colon 26-M3.1 carcinoma cells, prophylactic administration of PLW-0 and PLE-0 significantly inhibited lung metastasis in a dose-dependent manner and PLE-0 was more effective on the inhibition of cancer metasasis. The results lead us to conclude that the pectinase treated process is indispensable to preparing polysaccharides with higher immune-stimulating activity from persimmon leaves.

In this study, we examined immuno-modulatory activities of crude polysaccharides from wild ginseng adventitious roots (WGAR). The crude polysaccharide (WGAR-CP) was isolated from WGAR by hot water extraction, ethanol precipitation, and dialysis. The major constituents in WGAR-CP were neutral sugar (64.77%), and uronic acid (34.32%). WGAR-CP demonstrated anti-complementary activity dose-dependently. The immuno-modulatory effects of WGAR-CP were also analyzed by measuring nitric oxide and cytokines in the supernatants of mouse peritoneal macrophages. Mouse peritoneal macrophages stimulated with WGAR-CP produced nitric oxide and various cytokines such as interleukin (IL)-6 and IL-12 in a dosedependent manner. In conclusion, WGAR-CP may have immuno-modulatory activities by activating a complementary system and macrophages, which produces cytokines.

물(water, TW), 에탄올(ethanol, TE) 및 핵산(n-hexane, TH)을 이용하여 탱자씨 추출물(trifoliate orange seed extracts, TSEs)을 각각 제조한 후 그람양성 식중독균 6종(Bacillus cereus KCCM 11341, Bacillus subtilis KCTC 1022, Listeria monocytogenes ATCC 12692, Staphylococcus aureus ATCC 19111, Streptococcus mutans KCTC 3065 및 Yersinia enterocolitica KCCM 41657)에 대한 항균활성과 Lactobacillus acidophilus IFO 3025에 대한 프레바이오틱 효과(prebiotic effect)를 측정하였다. 그 결과, 탱자씨 핵산추출물(TH)은 S. aureus ATCC 19111에 대해 배양시간이 증가함에 따라 대조군에 비해 강한 증식저해활성을 보였으며, 탱자씨 에탄올 추출물(TE)은 약간의 증식저해활성을 보였다. 특히, 배양 81시간 후 대조군은 90.31%의 증식활성을 보인 것에 반해 탱자씨 핵산추출물과 에탄올추출물은 64.95%와 75.50%의 증식활성을 각각 보였다. 탱자씨 추출물의 Lb. acidophilus IFO 3025에 대한 프레바이오틱 효과는 대조군에 비해 증식활성을 보이지는 않았으나, 적어도 생육저해효과를 보이지는 않았다. 따라서 탱자씨의 핵산 및 에탄올 추출물은 S. aureus ATCC 19111에 대한 항균활성물질로서의 가능성을 제시하였다

본 연구는 bisphenol A의 실제 식품을 통한 사람의 노출농도에서 에스트로젠 효과를 알아보기 위하여 사람의 유방암 세포인 MCF-7와 난소를 적출한 무흥선 마우스를 이용하여 살펴보았다. MCF-7세포를 이용한 genistein과 bisphenol A가 용매대조군과 무처치대조군에 비하여 genistein(10nM, 100nM, 1 uM, l0 uM)과 bisphenol A(2 ng/㎖, 4 ng/㎖, 8 ng/㎖, 16ng/㎖)의 세포성장촉진 효과가 용량의 증가에 따라 증가하는 경향을 보였다. genistein의 경우 1 uM을 정점으로 세포성장촉진이 극에 달하다가 l0 uM 투여군에서 1 uM 투여군에 비하여 약 40% 정도 세포성장이 감소하였으나 대조군에 비하여 여전히 세포의 성장을 촉진하였다. 이러한 결과는 bisphenol A와 genistein이 에스트로젠과 같이 에스트로젠 수용체와 결합하는 경로로 MCF-7세포의 성장을 촉진한 것으로 생각된다. 따라서 이러한 결과를 확인하기 위하여, 에스트로젠 의존적으로 유전자가 발현되는 유전자 중의 하나인 pS2 유전자의 발현을 살펴보았다. Genistein과 bisphenol A가 pS2유전자의 발현을 유도하는 것이 northern blot분석에 의하여 관찰되었다. 또한, 21일령의 무흉선 마우스를 이용한 in vivo연구에서, 무처치 대조군에 비하여 genistein과 bisphenol A를 투여한 마우스 군에서 유선의 발달과 end-bud의 형성이 유의하게 증가됨이 관찰되었다(p<0.05). 결론적으로, bisphenol A와 genistein이 MCF-7 세포에서 에스트로젠 효과가 매우 낮은 농도에서도 나타날 수 있다는 것은 식품위생상 큰 의미를 갖고 있으며, 이러한 물질들의 실제 사람에서의 위해성 특히 성장기 유아의 위해성에 대하여 좀 더 면밀히 연구되어야 할 것이다.

The growth inhibition effect of orally administrated yogurt ACE and Metchnikoff upon E. coli O157:H7 and S. typhimurium inoculated into gastric lumen of rabbits was investigated. The rabbits challenged with each 1 ml of suspension containing 10^8 CFU/ml of the pathogens were divided into 4 groups by the interval of yogurt administration: A group; preadministrated 7 days before inoculation of the pathogens and fed daily; B group; administrated daily after inoculation of the pathogens, C group; administrated every 3 days after inoculation of the pathogens; Control group, not fed after inoculation of the pathogens. Each 3 ml of yogurt containing 10^9 CFU/ml was orally administrated into rabbits. All yogurt administrated groups (A, B, C) showed growth inhibition effect on E. coli O157:H7 in one day after inoculation of the pathogen by the level of 0.8-1.0 log CFU/g, compared with the result differences between the control group and the yogurt administrated groups. In the control group after 5 days of inoculation, the number of colonized pathogens was 10^5-10^6 CFU/g, whereas 10³-10⁴ CFU/g was detected in the yogurt administrated groups. After 10 days of inoculation, the viable pathogen number per gram (g) of the rabbit feces was 10³ CFU/g in the control group, whereas the number below 10¹ CFU/g was detected in the group A, and 10² CFU/g in the group B and C. The growth inhibition effect of yogurt administration on E. coli O157:H7 was highly increased in the order of A, B, and C group. The same effect on S. typhimurium was observed at the level of 2 log CFU/g in the Metchnikoff yogurt administrated groups, compared with the control group result in one day after inoculation of the pathogen. In 7 days after inoculation of the pathogen, the viable number was increasingly decreased, and finally after 15 days no viable cell of S. typhimurium was discharged into the fecal samples in the group A, and the mean level of 10¹ CFU/g was detected in the group B, but there was no growth inhibition effect in the group C. The growth inhibition effect on S. typhimurium was observed at the same level of viable cell number between the yogurt ACE administrated groups and the control group in 5 days after inoculation. But, after 10 days of inoculation the viable cell number was started to decrease, and the viable cell of S. typhimurium was not discharged from rabbit intestinal contents after 15 days of inoculation in the yogurt ACE administrated groups. In such a case that yogurt was administrated in order to prevent the pathogens, pre-administration on a daily basis one week before inoculation of the pathogens exerted considerable effect in growth inhibition. In comparison with two kinds of yogurt tested in this study, the growth inhibition effect on two kinds of pathogens was observed more highly in the Metchnikoff administrated group than the ACE administrated group.