• Title/Summary/Keyword: ELISA assay

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Suppression of Microglial Activation by Acute Ethanol Administration through HT7 Stimulation (급성 알코올 투여 백서의 신문혈 자극이 소교세포 활성에 미치는 영향)

  • Su Yeon Seo;Se Kyun Bang;Suk Yun Kang;Seong Jin Cho;Kwang-Ho Choi;Yeonhee Ryu
    • Korean Journal of Acupuncture
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    • v.41 no.2
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    • pp.33-42
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    • 2024
  • Objectives : The sigma-1 receptor is implicated in stress, depression, psychostimulant sensitization, and addiction vulnerability. Prior studies have indicated that ethanol exposure modulates sigma-1 receptor activity within the Ventral Tegmental Area (VTA). Here, we explore the sub-mechanisms underlying sigma-1 receptor activity induced by HT7 (Shinmun) stimulation in behavioral alterations following acute ethanol (ETOH) administration. Methods : Male Wistar rats were investigated for pro- and anti-inflammatory markers after injection of ETOH (1 g/kg) using cytokine enzyme-linked immunosorbent assay (ELISA)s. After confirming that HT7 stimulation changed the total distance traveled in the open field test (OFT), protein changes in the Ventral tegmental area (VTA) were measured by Western blotting. The expression level of inducible nitric oxide synthase (iNOS) after administration of a sigma-1 receptor antagonist (dihydrobromide 1047; BD1047, 10 mg/kg i.p.) and Shenmen (HT7) stimulation was compared. Results : As a result, acute ETOH administration increased proinflammatory marker levels (TNF-𝛼 and IL-6). HT7 stimulation restored the total distance response after acute ethanol administration. In addition, in the VTA, the levels of a microglial marker (iNOS), sigma-1 receptor and protein kinase C, which are predicted to be involved in up- and downregulation, were restored by HT7 stimulation. In particular, HT7 stimulation modulates iNOS expression through effects similar to BD treatment. This study suggests that the stimulatory effect of HT7 may be driven by microglial activation. Conclusions : Microglial activity is regulated by sigma-1 receptor, and sigma-1 receptor activity is regulated by HT7 stimulation. Significantly, we demonstrate that HT7 stimulation ameliorates behavioral alterations induced by acute ETOH administration through microglial activation within the VTA.

Effects of Polysaccharide (Polycan) derived from Black Yeast in TNF-α-induced Inflammation in the Intestinal Epithelial Cells and Loperamide-induced Constipation Models (흑효모 유래 Polycan의 TNF-α 유도 장 상피세포 염증 및 Loperamide 유도 변비 모델에 미치는 효과)

  • Young Suk Kim;Bon Hwa Ku;Min Jeong Cho;Jung Hee Kwon;Seon Min Lee;Tae Woo Oh
    • Herbal Formula Science
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    • v.32 no.3
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    • pp.297-310
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    • 2024
  • Objective : This study was conducted to investigate the anti-inflammatory and laxative effects of Polycan in TNF-α-treated HT-29 intestinal epithelial cells and loperamide-induced constipation in vivo models, respectively. Methods : To evaluate the anti-inflammatory effects of Polycan, HT-29 cells were treated with TNF-α in the presence or absence of Polycan. IL-8 production was measured by enzyme-linked immunosorbent assay (ELISA). MAPK phosphorylation, nuclear translocation of NF-κB, and phosphorylation of IκB were assessed by Western blot analysis. To investigate the laxative effects of Polycan, 6-week-old SD rats (8 female rats per group) were orally administered Polycan or Chicory Fiber as a positive control for 4 weeks, and constipation was induced with loperamide treatment for 10 days before sacrifice. One day before sacrifice, a charcoal meal was administered to evaluate intestinal transit times. The periodically collected feces were used to assess the number of fecal pellets and fecal water content. Results : Polycan inhibited TNF-α-induced IL-8 expression in dose-dependent manner. Furthermore, Polycan suppressed TNF-α-induced phosphorylation of MAPKs (ERK1/2, p38 and JNK), degradation of Iκ-Bα and nuclear translocation of NF-κB. In an in vivo constipation model, the number of fecal pellets per food intake was significantly increased in rats administered with Polycan, both 1 day and 7 days after loperamide treatment. The water content of fecal pellets was restored in the Polycan groups starting 7 days after loperamide treatment. In addition, Polycan intake significantly enhanced the gastrointestinal transit ratio of a charcoal meal but reduced the number of intestinal fecal pellets. Conclusions : These results suggest that Polycan suppressed TNF-α-induced inflammation by blocking both the MAPK and NF-κB pathways in HT-29 cells. Additionally, in a loperamide-induced constipation model, Polycan showed clear laxative effects by increasing the number of fecal pellets, fecal water content, and intestinal transit ratio of a charcoal meal.

Danggwisu-san ameliorates acute inflammatory responses via NF-κB and MAPK pathway (당귀수산(當歸鬚散)의 in vitro 및 in vivo에서의 염증억제효과)

  • Chang Wook Lee;Sang Mi Park;Hyo Jeong Jin;Ye Lim Kim;Dae Hwa Jung;Sung Hui Byun;Sang Chan Kim
    • Herbal Formula Science
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    • v.32 no.2
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    • pp.155-179
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    • 2024
  • Objective : Danggwisu-san (DGSS) is an herbal formula that has been mainly used in the East Asia for the treatment of bruise, sprain and external injury. The cause of this pain is that Qi and blood become tangled and do not circulate well. DGSS can improve the tangled situation and make it well-circulated. The present study evaluated the anti-inflammatory effects of DGSS on Raw 264.7 cells and in rats with paw edema. Methods : Cell viability was measured using the MTT assay. The amount of nitric oxide (NO) production was measured the amount of nitrite content in the cultured medium using Griess reagent. The amount of tumor necrosis factor-α, monocyte chemoattractant protein 1, interleukin (IL)-1βand IL-6 in the cultured supernatant were measured by ELISA kit. Proteins expression were detected by Western blot. Furthermore, the effect of DGSS on acute inflammation was observed in rat paw edema model. Results : The DGSS ameliorates the lipopolysaccharide-activated changes in NO production, iNOS expression and pro-inflammatory cytokines. Additionally, DGSS significantly suppressed expression of p-JNK, p-ERK and nuclear NF-κB. As expected, in rat paw edema study, 1.0 g/kg of DGSS significantly reduced the carrageenan-induced paw edema and iNOS expression for 1-4 h. Moreover, administration of 1.0 g/kg (4 days) of DGSS used in this study did not show any significant change on ALT and AST. Conclusion : These results demonstrate that DGSS has anti-inflammatory effects in vitro and in vivo. Therefore, this present study can put scientific evidences up for the anti-inflammatory effect of DGSS.

Bulnesia Sarmienti Aqueous Extract Inhibits Inflammation in LPS-Stimulated RAW 264.7 Cells (RAW 264.7세포에서 lipopolysaccharide로 유발시킨 염증반응에 대한 Bulnesia sarmienti 열수추출물의 억제효과)

  • Cheon, Yong-Pil;Mollah, Mohammad Lalmoddin;Park, Chang-Ho;Hong, Joo-Heon;Lee, Gee-Dong;Song, Jae-Chan;Kim, Kil-Soo
    • Journal of Life Science
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    • v.19 no.4
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    • pp.479-485
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    • 2009
  • Bulnesia sarmienti (BS), a traditional South American herbal medicine native to Gran Chaco, has been used to treat various human ailments. We investigated the cytotoxic activities and the inhibitory effects of BS bark extract(0, 50, 100 and $200\;{\mu}g/\;mL$) on the production of nitric oxide (NO), prostaglandin $E_2$ ($PGE_2$), cyclooxygenase (COX) and proinflammatory cytokines ($IL-1{\beta}$, IL-6 and $TNF-{\alpha}$) in the lipopolysaccharide (LPS) (100 ng/ml)-stimulated murine macrophage cell line RAW264.7. The levels of NO, COX, PGE2 production and proinflammatory cytokines ($IL-1{\beta}$, IL-6 and $TNF-{\alpha}$) were measured by ELISA kit. Cell viability, as measured by the MTT assay, showed that BS extract had no significant cytotoxicity in RAW264.7 cells. BS extract significantly inhibited the LPS-induced NO, $PGE_2$ and COX production accompanied by an attenuation of $IL-1{\beta}$, IL-6 and $TNF-{\alpha}$ formation in macrophages. These results suggest that BS extract has potential as an herbal medicine for the treatment of inflammatory diseases.

Anti-inflammatory effect of Sinhyowoldo-san Extract with regard to Pro-inflammatory Mediators in PMA plus A23187-induced Human Mast Cells (인간 비만세포에서 PMA와 A23187에 의해 유도된 전염증 매개체에 대한 신효월도산 추출물의 항염증 효과)

  • Wi, Gyeong;Yang, Da-Wun;Kang, Ok-Hwa;Kim, Sung-Bae;Mun, Su-Hyun;Seo, Yun-Soo;Kang, Da-Hye;Lim, Jae-Soo;Kim, Ma-Ryong;Kwak, Nam-Won;Kong, Ryong;Kwon, Dong-Yeul
    • The Korea Journal of Herbology
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    • v.29 no.6
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    • pp.117-123
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    • 2014
  • Objectives : Sinhyowoldo-san (SHWDS) is said to be a traditional medicine used for shigellosis, abdominal pain, diarrhea. But mechanism of SHWDS mediated-modulation of immune function is not sufficiently understood. To ascertain the molecular mechanisms of SHWDS 70% EtOH extract on pharmacological and biochemical actions in inflammation, we researched the effect of pro-inflammatory mediators in phorbol-12-myristate-13-acetate (PMA)+ A23187-activated human mast cell line (HMC-1). Methods : In the present research, cell viability was measured by MTS assay. pro-inflammatory cytokine production was measured by performing enzyme-linked immunosorbent assay (ELISA), reverse transcription polymerase chain reaction (RT-PCR), and western blot analysis to analyze the activation of mitogen-activated protein kinases (MAPKs), nuclear factor kappa-light-chain-enhancer of activated B cells ($NF-{\kappa}B$). The investigation focused on whether SHWDS inhibited the expressions of interleukin-6 (IL-6), interleukin-8 (IL-8), MAPKs and $NF-{\kappa}B$ in PMA+A23187-activated HMC-1 cells. Results : SHWDS has no cytotoxicity at measured concentration (50, 100, and $250{\mu}g/ml$). SHWDS ($250{\mu}g/ml$) inhibits pro-inflammatory cytokine expression in PMA+ A23187-activated HMC-1 cells. Moreover, SHWDS inhibited cyclooxygenase (COX)-2 expression. In activated HMC-1 cells, SHWDS suppressed phosphorylation of extracellular signal-regulated kinase (ERK 1/2) and c-jun N-terminal Kinase (JNK 1/2). Then, SHWDS suppressed activation of nuclear factor $NF-{\kappa}B$ in nuclear, degradation of IkB ${\alpha}$ in cytoplasm. Conclusions : We propose that SHWDS has an anti-inflammatory therapeutic potential, which may result from inhibition of ERK 1/2, JNK 1/2 phosphorylation and $NF-{\kappa}B$ activation, thereby decreasing the expression of pro-inflammatory genes.

The development of transgenic maize expressing Actinobacillus pleuropneumoniae ApxIIA gene using Agrobacterium (아그로박테리움을 이용한 Actinobacillus pleuropneumoniae ApxIIA (ApxII toxin) 유전자 발현 옥수수 형질전환체 개발)

  • Kim, Hyun-A;Yoo, Han-Sang;Yang, Moon-Sik;Kwon, Suk-Yoon;Kim, Jin-Seog;Choi, Pil-Son
    • Journal of Plant Biotechnology
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    • v.37 no.3
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    • pp.313-318
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    • 2010
  • To develop edible vaccines for swine, the embryogenic calli (type II) derived from HiII genotype were inoculated with A. tumefaciens strain C58C1 containing the binary vector pMYV611, 613, 616, and V621, 622 and 623 respectively. Six of those vectors carry nptII gene which confers resistance to paromomycin and apxIIA gene producing ApxII toxin which is generated in various serum types of A. pleuropneumoniae as a target gene. The 4,120 callus clones for pMYV611, 5,959 callus clones for pMYV613, 7,581 callus clones for pMYV616, 52,329 callus clones for V621, 48,948 callus clones for V622, and 56,188 callus clones for V623 were inoculated. The frequency of positive response clone was confirmed into range of 2.3% - 4.4% for each vectors by NPTII ELISA kit assay, and the selected callus clones of them were finally 3 callus clones from pMYV611 (0.07%), 4 callus clones from pMYV613 (0.07%), 2 callus clones from pMYV616 (0.03%), 51 callus clones from V621 (0.1%), 72 callus clones from V622 (0.15%), and 102 callus clones from V623 (0.18%) respectively. From the selected callus clones of each binary vector, the integration of the apxIIA gene into maize genome was detected from 2 plants of pMYV613 and 2 plants of V623 by Southern blot analysis.

The Seroprevalence and Related Factors of Helicobacter pylori Infection (Helicobacter pylori 감염의 유병률과 관련요인에 관한 연구)

  • Kim, Yeung-Wook;Lee, Su-Ill;Cho, Byung-Mann;Koh, Kwang-Wook;Kim, Young-Sil;Kang, Su-Yong;Cha, Oae-Ri;Kim, Don-Kyoun
    • Journal of Preventive Medicine and Public Health
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    • v.29 no.3 s.54
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    • pp.669-678
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    • 1996
  • Helicobacter pylori is now recognized as causative agent of chronic gastritis and peptic ulcer, and strongly associated with development of gastric carcinoma. With development of sensitive and specific serologic tests to identify individuals infected with Helicobacter pylori, the epidemiologic study of this diseases has been investigated. But it's transmission route is not established, yet. The purpose of this study is to measure the prevalence of Helicobacter pylori infection in healthy children and young adults and to evaluate related factors for Helicobacter pylori infection in Korea. The seroprevalence of Ig G antibodies to Helicobacter pylori was determined using a Enzyme Linked Immunosorbent Assay and we obtained the information, such as demographic characteristics, monthly household income, numbers of family members in the house, numbers of persons using same room, type of house, and type of drinking water through the questionnaire survey. The observed overall seropositivity rate was 25.7%. The rate is increased progressively from 5.8% in the age group $1\sim3$ years to 44.4% in the age group $20\sim29$years($\chi^2$ for trend, p<0.001). Especially, the rate increased steeply from 6.5% in the age group $4\sim6$ years to 20.8% in the age group $7\sim9$ years, and this suggested that elementary school age was the major acquisition time of Helicobacter pylori infection. In multivariate logistic regression model, age, numbers of family members in the house, and type of house was statistically significant variables for Helicobacter pylori infection. Each odds ratio(93% CI) were as follows; base to age group $1\sim9$ years, age group $10\sim19$ years $3.6(2.0\sim6.4)$, age group $20\sim29$ years $7.3(4.1\sim13.1)$ and base to group of $1\sim3$ family members, group of $4\sim5$ family members $2.1(1.1\sim4.0)$, group of 6 or more family members $2.7(1.3\sim5.4)$ and base to apartment, single and multihouse $1.9(1.1\sim3.5)$. Sex, monthly household income, numbers of persons using same room, and type of drinking water was not statistically significant for Helicobacter pylori infection.

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The specific antibody response of olive flounder Paralichthys olivaceus to route of antigen administration and change in environmental conditions (항원의 투여방법 및 사육환경 변화에 따른 넙치의 특이항체 반응)

  • Kim, Wi-Sik;Han, Jong-Seok;Jang, Min-Seok;Seo, Han-Gil;Jung, Sung-Ju;Park, Jung-Jun;Kim, Seok-Ryel;Oh, Myung-Joo
    • Journal of fish pathology
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    • v.24 no.2
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    • pp.153-160
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    • 2011
  • The specific antibody response of olive flounder, Paralichthys olivaceus to change in rearing-environmental conditions post immunization with antigen (bovine serum albumin, BSA) and different routes of antigen administration were investigated by enzyme-linked immunosorbent assay (ELISA). To test the effect of routes of antigen administration, flounder were injected intraperioneally or intramuscularlly with 1 mg of BSA. In addition, to test the effect of change in environmental condition post immunization, flounder were injected intraperioneally with 1 mg of antigen, and then were exposed to acute thermal change (the water temperature (WT) was decreased from $21^{\circ}C$ to $15^{\circ}C$ within 30 min and maintained at $15^{\circ}C$ for 3 h), handling (fish were caught and subsequently held out of water for 1 min) or heavy oil (76 g/200 L for 2 days). Consequently, there was no significant difference between intraperioneal (IP) and intramuscular (IM) injections except at 10 days post-immunization. With these results, it suggests that both 1M and IP injections may be used as route of vaccination. Furthermore, no significant difference was observed in the antibody response among the groups exposed to heavy oil, handling, sudden drop of WT and positive control except at 10 days post-immunization. From these results, it was confirmed that specific antibody response was not affected by the above mentioned rearing-environmental conditions, suggesting that vaccination can be employed at changing rearing-environmental conditions.

EFFECTS OF HYDROQUINONE ON NEOPLASTIC TRANSFORMATION OF HUMAN EPITHELIAL CELLS IN CULTURE (Hydroquinone이 인체 상피세포의 발암화에 미치는 영향)

  • Sohn, Jung-Hee;Kim, Chin-Soo
    • Maxillofacial Plastic and Reconstructive Surgery
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    • v.32 no.3
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    • pp.218-228
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    • 2010
  • Components of dental resin-based restorative materials are reported to leach from the filling materials even after polymerization. Hydroquinone (HQ) is one of the major monomers used in the dental resin and is known as a carcinogen. Thus, carcinogenic risk of HQ leaching from the dental resin becomes a public health concern. The present study attempted to examine the carcinogenic potentials of HQ on the human epithelial cell, which is the target cell origin of the most of oral cancers. Cytotoxicity of HQ was observed above 50${\mu}M$ as measured by LDH assay, indicating a relatively low toxicity of this substance in human epithelial cells. The parameters of neoplastic cellular transformation such as cell saturation density, soft agar colony formation and cell aggregation were analyzed to examine the carcinogenic potential of HQ. The study showed that 2-week exposure of HQ showed the tendency of increase in the saturation density and the significant enhancement of soft agar colony formation at the highest dose, 50 ${\mu}M$ only. It is suggested that HQ has a weak potential of carcinogenicity. When cells were treated with HQ and TPA, a well-known tumor promoter, the parameters of neoplastic cellular transformation was significantly increased. This result indicates that the potential risk of carcinogenicity from HQ is largely dependent upon the presence of promoter. Exposure of 50 ${\mu}M$ HQ increased the time-dependent apoptosis as measured by the ELISA kit. This concentration coincides with a dose of neoplastic transformation, indicating a possible link between apoptosis and HQ-induced cellular transformation. Hydroquinone generated Reactive Oxygen Species (ROS) which was evidenced by the treatment of antioxidants such as trolox and N-acetyl cysteine and the GSH depleting agent, BSO. Antioxidants blocked the generation of ROS and the GSH depleting agent, BSO dramatically increased the ROS production. Since HQ is known to increase ROS production thru activation of transcriptional factor such as c-Myb and Pim-1, it is speculated that ROS generation by HQ plays a role in the activation of oncogene, which may lead to neoplastic transformation. In addition, ROS is involved in the alteration of signal transduction, which regulates the apoptosis in many cellular systems. Thus, ROS-mediated apoptosis may be involved in the HQ-induced carcinogenic processes. Protein kinase C (PKC) is known to play pivotal roles in neoplastic transformation of cells and its high expression is often found in a variety of types of tumors including oral cancer. PKC translocation of PKC-${\alpha}$ was observed following HQ exposure. Altered signaling system may also play a role in the transformation process. Taken together, HQ leached from the dental resin does not pose a significant threat as a cancer causing agent, but its carcinogenic potential can be significantly elevated in the presence of promoter. The mechanism of HQ-induced carcinogenesis involved ROS generation, apoptosis and altered signaling pathway. The present study will provide a valuable data to estimate the potential risk of HQ as a carcinogen and understand mechanism of HQ-induced carcinogenesis in human epithelial cells.

Effects of Plant Water Extract Mixture Ixeris Sonchifolia Hance, Oenanthevjavanica, Fagopyrum Esculentum Moench, Hizikia Fusiforme, Zingiber Officinale Roscoe on Mouse Immune Cell Activation Ex vivo (5가지 (고들빼기, 돌미나리, 메밀, 톳, 생강)혼합식품 물 추출물의 마우스 면역세포 활성화 효과)

  • Ryu, Hye-Sook;Kim, Jung-Hee;Kim, Hyun-Sook
    • Journal of Nutrition and Health
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    • v.41 no.2
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    • pp.141-146
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    • 2008
  • Ixeris sonchifolia Hance (Godulbaegi), Oenanthe javanica (Dolminari), Fagopyrum esculentum Moench (Buckwheat>, Hizikia fusiforme (Seaweed Fusiforme) and Zingiber ojficinale Roscoe (Ginger) have been used respectively as one of folk remedies as well as food materials. However, reportedly few studies on their immunomodulating effects have been made, although it has been known from other preceding studies that the ex vivo supplementation of each Ish, OJ, Fem, Hf, Zor water extracts tends to enhance the proliferation of splenocyte in comparison to the control group. This study on the combined immunomodulative effect of water extract mixture of these five food materials (Ish + Oj + Fem +Hf+Zor) lasted covering seven or eight weeks. The old mice (balb/c) was fed ad libitum on chow diet, and the water extract of plant mixture was orally administrated every other day for four weeks at two different concentrations (50 and 500 mg/kg B.W) . After preparing the single cell suspension, the proliferation of splenocyte was determined by MTT (3-[4,5-dimethylthiazol-2-y]-2,5-diphenyl terazolium bromide) assay. The production of cytokine ($IL-{\beta}$, IL-6, and $TNF-{\alpha}$) which was secreted by macrophages stimulated with LPS or not was detected by ELISA assay using the cytokine kit. After the 48 hours of incubation with the mitogen (ConA or LPS) stimulation, the proliferation of the mice splenocyt in the experimental group statisticaly increased at both of two different concentrations in comparison to the control group. The cytokines production was more significantly enhanced at the lower supplementation (50 mg/kg B.W.) group than at the higher concentration (500 mg/kg B.W.). The result of this study may suggest that the supplementation of water extract of plant mixture can regulate and enhance the immune function by increasing the splenocyte proliferation and regulating the cytokine production capacity by the activated macrophages in mice.