• Journal of Pharmacopuncture
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• v.14 no.3
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• pp.81-90
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• 2011

Objectives : Angelicae Gigantis Radix has been known traditional medicine with antimicrobial activities and it has been widely used for treatment of blood and inflammatory diseases. In the present study, some studies examined anti-inflammation effects of Angelicae Gigantis Radix but they usually were performed by ethanol extracted Angelicae Gigantis Radix pharmacopuncture. So We investigated the inhibitory effects of Angelicae Gigantis Radix pharmacopuncture by hot water and ethanol extract on Nitric oxide(NO) and Prostaglandin $E_2$($PGE_2$) production in lipopolysaccharide(LPS) induced macrophage cell. Methods : Angelicae Gigantis Radix was extracted by ethanol and hot water. Cell viability was determined by MTT assay. To evaluate anti-inflammation effects of Angelicae Gigantis Radix pharmacopuncture, we examined NO and $PGE_2$ production in LPS induced macrophages. The concentrations of NO and $PGE_2$ were measured by Griess assay and Enzyme Immuno-Assay. Results : 1) The MTT assay demonstrated that cytotoxic effect of Angelicae Gigantis Radix pharmacopuncture by hot water extract and ethanol extract in RAW 264.7 macrophage cells were not appeared. 2) Angelicae Gigantis Radix pharmacopuncture by ethanol extract and hot water extract inhibited NO production in LPS induced macrophages significantly. 3) Angelicae Gigantis Radix pharmacopuncture by ethanol extract tended to inhibiting $PGE_2$ production in LPS induced macrophages. And Angelicae Gigantis Radix pharmacopuncture by hot water extract inhibited LPS induced production of $PGE_2$ in RAW 264.7 macrophage cells significantly. Conclusions : This study suggests that Angelicae Gigantis Radix pharmacopuncture may have an anti-inflammatory property through the inhibition of NO and $PGE_2$ production in LPS induced macrophages. It may have a therapeutic potential for the treatment of various inflammatory diseases.

• Journal of Pharmacopuncture
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• v.9 no.2
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• pp.39-47
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• 2006

Objective : It has long been known about the anticancer effect of GRR-HAS, however, it has not been systemically determined the differentially regulated genes by GRR-HAS in cancer cells. The purpose of this study is to screen the GRR-HAS mediated differentially expressed genes in cancer cells such as SNU484 gastric cancer cell lines. Oligonucleotide microarray approache was employed to screen the differential expression genes. Methods : GRR-HAS was prepared by boiling and stored at $-70^{\circ}C$ until use. Cells were treated with various concentrations of GRR-HAS(0.1, 0.5, 1.5, 10, 20mg/ml) for 24 h. Cell toxicity was tested by MTT assay. To screen the differentially expressed genes in cancer cells, cells were treated with 1.5mg/ml of GRR-HAS. For oligonucleotide microarray assay, total RNA was used for gene expression analysis using oligonucleotide Genechip (Human genome Ul33 Plus 2.0., Affimatrix Co.). Results : It has no cytotoxic effects on both HepG2 and SNU484 cells in all concentrations(0.1, 0.5, 1.5, 10, 20mg/ml). In oligonucleotide microarray assay, in SNU484 cells, the number of more than twofold up-regulated genes was 346. The number of more than twofold down-regulated genes was 9. Discussion : This study showed the comprehensive gene expression analysis using oligonucleotide microarray for the screening of GRR-HAS mediated differentially regulated genes. These results will provide a better application of GRR-HAS in cancer field and drug target development.

• Journal of Pharmacopuncture
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• v.12 no.1
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• pp.21-33
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• 2009

Objective : This experimental study was performed to investigate the safety of Soyeom Pharmacopunture solution manufactured by extraction of alcohol and water. To identify the use of it as eyedrops, the eye irritation test of rabbits and antibacterial test of Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Aspergillus niger, Fusarium oxysporum, and Candida albicans was performed. Methods : 1. The eye irritation test of this material was performed according to the Regulation of Korea Food & Drug Administration(2005. 10. 21, KFDA 2005-60). After Soyeom pharmacopuncture solution was administered in the left eye of the rabbits, eye irritation of the cornea, iris and conjunctiva was observed at 1, 2, 3, 4 & 7day. 2. After administering Soyeom Pharmacopuncture solution on bacterial species (Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Aspergillus niger, Fusarium oxysporum, Candida albicans) which cause Keratitis, MIC(Minimum Inhibition Concentration) and the size of inhibition zone were measured. Anti-bacterial potency was also measured using the size of inhibition zone. Results : 1. After Soyeom pharmacopuncture solution was administered in the left eye of the rabbits, it was found that none of nine rabbits have abnormal signs and weight changes. 2. After Soyeom pharmacopuncture solution was medicated in the left eye of the rabbits, no eye irritation of the cornea, iris and conjunctiva was observed at 1, 2, 3, 4 & 7day. 3. There was no response to MIC on bacterial species (Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Aspergillus niger, Fusarium oxysporum, Candida albicans) after Soyeom pharmacopuncture solution was medicated. Conclusions : The present study suggests that Soyeom pharmacopuncture solution is a non-toxic and non-irritant medicine, which does not cause eye irritation in rabbits, but dosen't have anti-bacterial effects on bacterial species which cause Keratitis. These study result recommends that more research on other herbal medicines of eye drop for Keratitis are required.

• Journal of Acupuncture Research
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• v.20 no.6
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• pp.128-139
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• 2003

Juglans sinensis Dode has been reported to have antioxidant activity. However, the effect of Juglans sinensis Dode aquacupuncture(JS) on reactive oxygen species(ROS)-induced alterations in membrane transport function in renal tubular cells. This study was performed to evaluate the effect of JS on the organic hydroperoxide t-butylhydroperoxide(tBHP)-induced inhibition of $Na^+$-dependent phosphate($Na^+$-Pi) uptake in opossum kidney (OK) cells, an established renal proximal epithelial cell line. tBHP inhibited $Na^+$-Pi uptake in a time-dependent manner. The inhibitory effect of tBHP was prevented by JS over concentration range of 0.05-1mg/100ml in a dose-dependent manner. Kinetic studies showed that tBHP caused an decrease in Vmax for $Na^+$-Pi uptake without any a significant change in Km. $Na^+$-dependent phosphonoformic acid binding, a irreversible inhibitor of renal $Na^+$-Pi uptake, was decreased by tBHP treatment. The reduction in Vmax and phosphonoformic acid binding by tBHP was prevented by JS. tBHP induced lipid peroxidation and its effect was completely inhibited by JS and antioxidant N,N'-diphenyl-p-phenylenediamine. These data suggest that the oxidant inhibits phosphate uptake by a reduction in the number of active carrier across the membrane. JS may prevent oxidant-induced inhibition of membrane transport function by a mechanism similar to antioxidants in renal epithelial cells. Although the precise constituents remain to be explored, JS may be employed as a useful candidate herb for drug development to prevent and treat oxidant-mediated renal failure.

• Journal of Acupuncture Research
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• v.21 no.2
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• pp.41-55
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• 2004

Objective : To investigate the possible molecular mechanism (s) of melittin as a candidate of anti-cancer drug, we examined the effects of the compound on the growth of human lung carcinoma cell line A549. Methods : Growth inhibitory study, flow cytometry analysis, SDS-polyacrylamide gel electrophoresis and Western blot analysis, RT-PCR and in vitro caspases activity assay were performed. Results : Melittin treatment declined the cell viability of A549 cells in a concentration-dependent manner, which was associated with induction of apoptotic cell death. Melittin treatment down-regulated the levels of Bcl-XS/L mRNA and protein expression of A549 cells, an anti-apoptotic gene, however, the those of Bax, a pro-apoptotic gene, were up-regulated. Melittin induced the proteolytic cleavage and activation of caspase-3 and caspase-9 protease in a dose-dependent manner without alteration of inhibitor of apoptosis proteins family and Akt expression. Western blot analysis and RT-PCR data revealed that the levels of tumor suppressor p53 and cyclin-dependent kinase inhibitor p21 were also remained unchanged. Conclusions : Taken together, these findings suggest that melittin-induced inhibition of human lung cancer cell growth is associated with the induction of apoptotic cell death via regulation of several major growth regulatory gene products, and melittin may have therapeutic potential in human lung cancer.

• Journal of Acupuncture Research
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• v.27 no.3
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• pp.105-116
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• 2010

목적 : 이 연구는 MPTP 유발 파킨슨병 동물 모델에서 봉독약침의 신경보호 효과 및 항염증 효과를 확인하기 위해 시행되었다. 방법 : C57BL/6 mice에 신경독소인 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)를 하루에 2시간 간격으로 MPTP-HCl(20mg/kg per dose)을 4번 복강 내 주입하여 중뇌 흑질의 도파민 신경세포를 파괴한 파킨슨병 동물 모델을 유발하였다. 실험군은 MPTP군, MPTP 현종 BVA군, MPTP 곡지 BVA군, MPTP 신수 BVA군의 4군으로 하였다. 마지막 MPTP 투여 2시간 후에 1차로 봉독약침을 시술하고, 그 후 48시간 간격으로 총 5차 연속 시술하였다. 봉독약침액의 농도는 0.2mg/Kg으로 하였고, 경혈은 양측 현종($GB_{39}$), 곡지($LI_{11}$), 신수($BL_{23}$)를 사용했고, 주입량은 각 경혈당 양측으로 각 $20{\mu\ell}$씩 주입하였다. 항염증작용을 알아보기 위해 TH, MAC-1, iNOS HSP70을, 세포사멸에 대한 신경세포의 보호효과를 알아보기 위해 caspase-3을 면역조직화학법을 사용하여 실시하였다. 결과 : 실험 결과 MPTP 유발 파킨슨병 동물 모델에서 현종 곡지 신수혈에 대한 봉독약침은 TH-Immunoreactivity neuron의 감소와 microglial activation을 억제하였다. 봉독약침군 모두 효과를 보였으나 그 중 현종과 신수혈에서 특히 억제작용이 컸다. MAC-1에서는 현종혈이 억제작용이 컸다. HSP70-IR neuron은 곡지에서 유의한 억제작용을 보였으나, iNOS neuron은 모든 군에서 유의한 차이를 보이지 않았다. 또한 세포사멸억제여부 실험에서 봉독약침은 모두 억제작용을 보였으나 특히 곡지자침군에서 caspase-3 발현을 유의하게 억제하였다. 결론 : 이러한 결과는 봉독약침이 MPTP 투여로 인한 중뇌 흑질의 염증에 의한 도파민 신경세포 손상을, 염증을 억제함으로써 항염 효과를 나타냄을 알 수 있으며, 신경세포를 보호하는 활성이 있음을 보여줌과 동시에 세포사멸을 억제하는 활성이 있다고 사료된다.

• Journal of Acupuncture Research
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• v.23 no.3
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• pp.177-190
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• 2006

Objectives : It has long been known about the anticancer effect of GRR-HAS, however, it has not been systemically determined the differentially regulated genes by GRR-HAS in cancer cells. The purpose of this study is to screen the GRR-HAS mediated differentially expressed genes in cancer cells such as HepG2 hepatoma cell lines. Oligonucleotide microarray and proteomic approaches were employed to screen the differential expression genes. Methods : GRR~HAS was prepared by boiling and stored at $-70^{\circ}C$ until use. Cells were treated with various concentrations of GRR-HAS (0.1, 0.5, 1.5, 10, $20mg/m{\ell}$) for 24 h. Cell toxicity was tested by MTT assay. To screen the differentially expressed genes in cancer cells, cells were treated with $1.5mg/m{\ell}$ of GRR-HAS. For oligonucleotide microarray assay, total RNA was used for gene expression analysis using oligonucleotide genechip (Human genome Ul33 Plus 2.0., Affimatrix Co.). For proteomic analysis, total protein was analyzed by 2D gel electrophoresis and Q-TOF mass spectrometer. Results : It has no cytotoxic effects on both HepG2 cells in all concentrations(0.1, 0.5, 1.5, 10,$20mg/m{\ell}$). In oligonucleotide microarray assay, the number of more than twofold differentially regulated known genes was 320 with 6 up-regulated and 314 down-regulated genes in HepG2 cells. In proteomic analysis, three spots were identified by 2D-gel electrophoresis and Q-TOF analysis. One down -regulated protein was protein disulfide isomerase and up-regulated proteins were fatty acid binding protein 1 and 14-3-3 gan1lTIa protein by $1.5mg/m{\ell}$ of CRR-HAS. Discussion : This study showed the comprehensive gene expression analysis using oligonucleotide microarray for the screening of GRR-HAS mediated differentially regulated genes. These results will provide a better application of GRR-HAS in cancer field and drug target development.

• Journal of Acupuncture Research
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• v.20 no.2
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• pp.98-111
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• 2003

Objective : This study was designed to analyze the effects of bee venom and melittin on cell death in neuroblastoma cell line. Methods : MTT assay, morphologic method, DNA fragmentation, flow cytometry, immunocytochemistry analysis, RT-PCR and Western blot were performed. Results : The obtained results are summarized as follows: 1. The MTT assay demonstrated that neuroblastoma cell viability was significantly inhibitted dose-dependently by treatment with bee venom and melittin in comparison with control. 2. Cell culture demonstrated that control group proliferated highestly at he 5th day in comparison with the 4th day in bee venom and melittin group. And in bee venom and melitti group cell proliferation decreased 2.5 times than control group. 3. The morphologic study demonstrated that neuroblastoma cell showed apoptosis after treatment with bee venom and melittin for 6 hours using microscope. 4. The Flow cytometry demonstrated that apoptosis of neuroblastoma cell treated with bee venom and melittin was related with stop of cell cycle in stage of $G_0/G_1$. 5 .DNA fragmenation demonstrated that neuroblastoma cell treated with bee venom and melittin showed DNA ladder below 1 Kbp. 6. Immunocytochemistry assay demonstrated that Fos and MAPK which are related with cancer were down-regulated by treatment with bee venom and melittin. 7. RT-PCR analysis demonstrated that Fos and MAPK mRNA were transcripted. Fos was down-regulated form treatment with $5{\mu}g/ml$ bee venom and MAPK was down-regulated form $1{\mu}g/ml$ bee venom. 8. Western blot demonstrated that Fos was down-regulated from $1{\mu}g/ml$ bee venom whereas MAPK was expressed by $1{\mu}g/ml$ bee venom but down-regulated by $10{\mu}g/ml$ bee venom. Conclusions : We found that some cancer related genes ware down-regulated by treatment with bee venom and melittin. Further study is needed for investigating the anti-cancer effect of bee venom and melittin.

• Journal of Acupuncture Research
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• v.21 no.5
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• pp.179-190
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• 2004

Objectives : The purpose of this study was to investigate the effect of Bee Venom and Melittin acupuncture solution on the lipopolysaccharide and sodium nitroprusside- induced expression of cytosolic phospholipase $A_2$, tumor necrosis factor-${\alpha}$ and calcium concentration in RAW 264.7 cells, a murine macrophage cell line. Methods : The expression of cytosolic phospholipase $A_2$ and tumor necrosis factor-${\alpha}$ was determined by western blotting with corresponding antibodies, and the generation of intracellular calcium concentration was investigated by delta scan system in RAW 264.7 cells. Results : 1. Compared with control, expressions of lipopolysaccharide-induced cytosolic phospholipase $A_2$ were decreased significantly by $5{\mu}g/mL$ of bee venom and 5, $10{\mu}g/mL$ of melittin acupuncture solution and decreased by 0.5, $1{\mu}g/mL$ of bee venom. 2. Compared with control, expressions of sodium nitroprusside-induced phospholipase $A_2$ were decreased significantly by 0.5, 1, $5{\mu}g/mL$ of bee venom and by 5, $10{\mu}g/mL$ of melittin acupuncture solution. 3. Compared with control, expressions of lipopolysaccharide-induced tumor necrosis factor-${\alpha}$ were decreased significantly by $10{\mu}g/mL$ of melittin acupuncture solution and were not changed significantly by 0.5, 1, $5{\mu}g/mL$ of bee venom and $5{\mu}g/mL$ of melittin acupuncture solution. 4. Compared with control, expressions of sodium nitroprusside-induced tumor necrosis factor-${\alpha}$ were decreased significantly by 1, $5{\mu}g/mL$ of bee venom and 5, $10{\mu}g/mL$ of melittin acupuncture solution and decreased by $0.5{\mu}g/mL$ of bee venom 5. Compared with control, lipopolysaccharide-induced intracellular calcium concentrations were decreased by 0.5, 1, $5{\mu}g/mL$ of bee venom and $10{\mu}g/mL$ of melittin acupuncture solution and increased by $5{\mu}g/mL$ of melittin acupuncture solution. 6. Compared with control, sodium nitroprusside-induced intracellular calcium concentrations were decreased by 0.5, 1, $5{\mu}g/mL$ of bee venom and 5, $10{\mu}g/mL$ of melittin acupuncture solution.

• Journal of Acupuncture Research
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• v.29 no.1
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• pp.15-24
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• 2012

Objectives : In recent years, many studies have been widely researching anti-inflammation effect of various medicinal plants. $Cinnamomi$ $Cortex$ was not enough in researching of the anti-inflammation. Moreover, there is no comparative study about extraction methods. Therefore, we investigated the inhibitory effects of $Cinnamomi$ $Cortex$ pharmacopuncture by EtOH and Hot water extraction on Nitric oxide(NO), Prostaglandin E2(PGE2) production, Cyclooxygenase(COX)-2, inducible NOS(iNOS) expression and extracellular signal regulate kinase(ERK)1/2 phosphorylation in lipopolysaccharide(LPS) induced RAW 264.7 macrophage cell. Methods : $Cinnamomi$ $Cortex$ was extracted by EtOH and Hot water. RAW 264.7 macrophage cell viability was measured by MTT assay. Effect of $Cinnamomi$ $Cortex$ pharmacopuncture on NO and PGE2 production in LPS induced macrophages was accessed by Griess assay and enzyme-linked immunospecific assay(ELISA), respectively. Inhibition effect on COX-2, iNOS expression and ERK1/2 phosphorylation was examined by Immunoblotting assay. Results : 1. Cytotoxic effect of $Cinnamomi$ $Cortex$ pharmacopuncture by Hot water extraction in RAW 264.7 macrophages was not appeared, except $3125{\mu}g/m{\ell}$. And cytotoxic effect was not appeared in EtOH extraction method. 2. $Cinnamomi$ $Cortex$ pharmacopuncture by EtOH and Hot water extraction inhibited NO production in LPS induced macrophages significantly. 3. $Cinnamomi$ $Cortex$ pharmacopuncture by EtOH and Hot water extraction inhibited PGE2 production in LPS induced macrophages significantly. 4. $Cinnamomi$ $Cortex$ pharmacopuncture by EtOH and Hot water extraction inhibited COX-2, iNOS expression in LPS induced macrophages. Especially, it has been confirmed that COX-2, iNOS expression were effectively inhibited in Hot water extraction. 5. $Cinnamomi$ $Cortex$ pharmacopuncture by EtOH and Hot water extraction inhibited ERK1/2 phosphorylation in LPS induced macrophages. Especially, it has been confirmed that ERK1/2 phosphorylation was effectively inhibited in Hot water extraction. Conclusions : According to the results, $Cinnamomi$ $Cortex$ pharmacopuncture suppresses NO, PGE2 production, COX-2, iNOS expression and ERK1/2 phosphorylation in LPS induced macrophages. It has a potential for treating various inflammatory diseases, and Hot water extraction method could be used more extensively than EtOH extraction method.