• Journal of Acupuncture Research
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• v.19 no.2
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• pp.65-77
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• 2002

Objective : This study was undertaken to determine which compound of Bee Venom for herb-acupuncture has cytotoxicity on mouse mast cell line. Methods : We compared crude bee venom and its compounds such as melittin, mast cell degranulating peptide (MCD peptide), apamin with control groups on cytotoxicity by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results & Conclusion : 1. Crude bee venom showed significant cytotoxic effect(p<0.01) in 1 hour treatment with $1{\mu}g/m{\ell}$ in comparison with control group in 1 hour treatment with low concentration of $10-4{\mu}g/m{\ell}$, $10-3{\mu}g/m{\ell}$, $10-2{\mu}g/m{\ell}$, $10-1{\mu}g/m{\ell}$ and $1{\mu}g/m{\ell}$, but it showed no significant cytotoxic effect in 6 hours treatment. 2. Melittin group showed no significant cytotoxic effect in comparison with control group in 1 and 6 hours treatment with low concentration of $10-4{\mu}g/m{\ell}$, $10-3{\mu}g/m{\ell}$, $10-2{\mu}g/m{\ell}$, $10-1{\mu}g/m{\ell}$ and $1{\mu}g/m{\ell}$. 3. MCD peptide and Apamin group showed no significant cytotoxic effect in comparison with control group in 1 and 6 hours treatment with low concentration of $10-4{\mu}g/m{\ell}$ $10-3{\mu}g/m{\ell}$, $10-2{\mu}g/m{\ell}$, $10-1{\mu}g/m{\ell}$ and $1{\mu}g/m{\ell}$. 4. Crude bee venom showed significant cytotoxic effect(p<0.01) in 1 and 6 hours treatment in comparison with control group in 1 and 6 hours treatment with high concentration of $10{\mu}g/m{\ell}$, $20{\mu}g/m{\ell}$ and $102{\mu}g/m{\ell}$. 5. Melittin group showed significant cytotoxic effect(p<0.01) in 1 hour treatment in comparison with control group in 1 hour treatment with high concentration of $10{\mu}g/m{\ell}$ but it showed no significant cytotoxic effect in 6 hours treatment. 6. Crude bee venom and its compounds have more cytotoxic effect in 1 hour treatment than in 6 hours treatment. It means cytotoxicity tends to decrease according to the treatment time.

• Journal of Acupuncture Research
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• v.19 no.2
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• pp.211-220
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• 2002

Objective : This study was undertaken to determine whether Scutellaria baicalensis Georgi extract (SbG) exerts the protective effect against oxidant-induced alterations in organic cation transport in the renal proximal tubule. Methods : Organic cation transport was estimated by examining alterations in tetraethylammonium (TEA) uptake in rabbit renal cortical slices. The slices were treated with 0.2 mM tBHP for 60 min at $37^{\circ}C$. tBl-IP caused an inhibition in TEA uptake by renal cortical slices. Such an effect was accompanied by depressed Na+-K+-ATPase activity and ATP depletion. Result : SbG prevented tBHP-induced inhibition of TEA uptake in a dose-dependent manner at the concentration ranges of 0.05-0.1%. SbG also prevented H2O2-induced reduction in TEA uptake. tBHP-induced inhibition of Na+-K+-ATPase activity and ATP depletion were significantly prevented by 0.05% SbG. Oxidants increased LDH release, which was blocked by SbG. Oxidants caused a significant increase in lipid peroxidation and its effect was prevented by SbG. Conclusion : These results suggest that SbG prevents oxidant-induced alterations in organic cation transport in rabbit renal cortical slices. Such protective effects of SbG may be attributed to inhibition of peroxidation of membrane lipid.

• Journal of Pharmacopuncture
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• v.13 no.2
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• pp.5-12
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• 2010

Reactive Oxygen Species(ROS) are continuously produced at a high rate as a by-product of aerobic metabolism. Since tissue damage by free radical increases with age, the reactive oxygen species(ROS) such as hydrogen peroxide($H_2O_2$), nitric oxide(NO). Several lines of evidence provided that ROS appears to cause to develop aging-related various diseases such as cancer, arthritis, cardiovascular disease. In this study, we have conducted to investigate the biological activities of Hominis Placenta Herbal Acupuncture by measuring total polyphenol content, DPPH radical scavenging, ABTS radical scavenging, Superoxide dismutase(SOD)-like activity, Nitrite scavenging ability in vitro. The total polyphenol contents of Hominis Placenta Herbal Acupuncture was $24.6m{\ell}/m{\ell}$. Elctron donation ability on DPPH was 49.4%. The 2,2'-azinobis-3-ehtlbezothiazoline-6-sulfonic acid radical decolorization (ABTS) was 50.01%, similar 10 the DPPH free radical scavenging. The superoxide dismutase (SOD)-like activities of hominis placenta herbal acupuncture was 50.876%. The nitrite scavenging abilities at pH 1.5, pH 3.0, pH 6.0 were 52.8%, 29.4%, 15.4%, respectively; these abilities decreased as pH increased. We conclude that Hominis Placenta Herbal Acupuncture may be useful as potential sources of antioxidant.

• The Journal of Internal Korean Medicine
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• v.31 no.2
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• pp.254-263
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• 2010

Objectives : The neuroprotective effects of bee venom (BV) have been demonstrated in many studies, but bee venom has many side effects. So we used sweet bee venom (SBV), which has high molecular elements removed to reduce the side effects. I examined the neuroprotective effect of sweet bee venom in 1-methyl-4-phenylpyridine ($MPP^+$)-induced human neuroblastoma SH-SY5Y cells. Methods : To observe the possible toxicity of SBV itself, SH-SY5Y cells were treated with SBV in various concentrations for 3 h and $MPP^+$ in concentrations (1 and 5mM) for 24h. To investigate the protective effect of SBV against $MPP^+$ toxicity, SH-SY5Y cells were pretreated with vehicle or nontoxic concentrations of SBV for 3h and the cells were not washed, followed by incubation with respective concentrations of SBV and 1 mM $MPP^+$ for 24h. To investigate the protective effect of SBV against $MPP^+$ toxicity, SH-SY5Y cells were pretreated with vehicle or nontoxic concentrations of SBV for 3h and the cells were not washed, followed by incubation with respective of SBV(0.5%), 1 mM $MPP^+$, 5uM AKT inhibitor(LY984002) and 10uM ERK inhibitor(PD98059) for 24 h. The protective effect was measured by cell viability assay. To investigate the degree of apoptosis, caspase-3 enzyme activity was measured in control, $MPP^+$, SBV+$MPP^+$. Results : SBV (0.5%) pretreatment protected the SH-SY5Y cells against $MPP^+$-induced apoptotic cell death. The cell viability was higher in the SH-SY5Y cells that were pretreated with vehicle or nontoxic concentrations of SBV than those not pretreated. The caspase-3 activity was lower in the pretreated groups than these not pretreated. ERK and AKT enzymes have a role in the neuroprotective effects of the sweet bee venom. Conclusions : The results demonstrate that SBV has a protective effect on dopaminergic neurons against $MPP^+$ toxicity. This data suggest that SBV could be a potential therapeutic tool for neurodegenerative diseases such as Parkinson's disease(PD).

• Journal of Pharmacopuncture
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• v.6 no.3
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• pp.5-14
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• 2003

Objective : Recently, Herbal-acupuncture therapeutics has been used for the treatment of inflammatory diseases such as rheumatoid arthritis. Especially, we have been interested in chemical mediators concerned with inflammation such as prostaglandin, cytokine, nitric oxide. The purpose of this study is investigated that the effect of Scutellariae Radix-acupuncture solution in lipopolysaccharide-stimulated RAW 264.7 macrophages, performed several expeimental items : those are prostaglandin E. nitric oxide and cyclooxygenase-2. Method : The cytotoxicity of Scutellariae Radix-acupuncture solution in RAW 264.7 macrophages were measured by MTT-based cytotoxicity assay. In order to observe cyclooxygenase-2 mRNA expression in lipopolysaccharide-stimulated RAW 264.7 macrophages, RT-PCR was used. Prostaglandin $E_2$ formation and nitric oxide production was measured by competitive enzyme immunoassay and Griess assay. Results : 1. The MTT assay demonstrated that cytotoxic effect of Scutellariae Radix-acupuncture solution in RAW 264.7 macrophages were not appeared before concentration of 5mg/mL 2. Scutellariae Radix-acupuncture solution inhibited cyclooxygenase-2 mRNA expression in lipopolysaccharide-treated RAW 264.7 macrophages. 3. Scutellariae Radix-acupuncture solution inhibited nitric oxide production in lipopolysaccharide-treated RAW 264.7 macrophages. 4. Scutellariae Radix-acupuncture solution inhibited prostaglandin $E_2$ formation in lipopolysaccharide-treated RAW 264.7 macrophages. Conclusion : On the basis of these results, It was shown that Scutellariae Radix-acupuncture solution is significantly able to inhibit the production of $PGE_2$ and NO, as well as COX-2 mRNA expression. Our results may provide new mechanism by which Scutellariae Radix-acupuncture solution accounts for its beneficial effect on accelerating wound healing and anti-inflammation.

• Journal of Acupuncture Research
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• v.17 no.3
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• pp.208-218
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• 2000

This study was undertaken to determine if Salviae Radix (SR) exerts protective effect against oxidant-induced inhibition of phosphate uptake in renal proximal tubular cells. Membrane transport function and cell death were evaluated by measuring phosphate uptake and trypan blue exclusion, respectively, in opossum kidney (OK) cells, an established proximal tubular cell line. $H_2O_2$ was used as a model oxidant. $H_2O_2$ inhibited the phosphate uptake in a dose-dependent manner over the concentration range of 0.1-0.5 mM. Similar fashion was observed in cell death. However, the phosphate uptake was more vulnerable to $H_2O_2$ than cell death, suggesting that $H_2O_2$-induced inhibition of phosphate uptake is not totally attributed to cell death. Decreasedphosphate uptake was associated with ATP depletion and inhibition of $Na^+$-pump activity as determined by direct inhibition of $N^+-K^+$-ATPase activity. When cells were treated with $H_2O_2$ in the presence of 0.05% SR, the inhibition of phosphate uptake and cell death induced by $H_2O_2$ was significantly attenuated. SR restored ATP depletion and decreased $Na^+-K^+$-ATPase activity, and this is likely responsible for the protective effect of SR on decreased phosphate uptake. The protective effect of SR was similar to the $H_2O_2$ scavenger catalase. SR reacts directly with $H_2O_2$ to reduce the effective concentration of the oxidant. The iron chelator deferoxamine prevented the inhibition of phosphate uptake and cell death induced by $H_2O_2$, suggesting that $H_2O_2$-induced cell injury is resulted from an iron-dependent mechanism. These results indicate that SR exerts the protective effect against $H_2O_2$-induced inhibition of phosphate uptake by reacting directly with $H_2O_2$ like the $H_2O_2$scavenger enzyme catalase, in OK cells. However, the underlying mechanism remains to be explored.

• Korean Journal of Acupuncture
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• v.23 no.3
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• pp.133-160
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• 2006

Objectives : Scolopendrae Corpus has a broad array of clinical applications in Korean medicine, including treatment of inflammatory conditions such as arthritis. To explore the global gene expression profiles in human Raw cell lines treated with Scolopendrae Corpus herbal-acupuncture solution (SCHAS), cDNA microarray analysis was performed. Methods : The Raw 264.7 cells were treated with lipopolysaccharide (LPS), SCHAS, or both. The primary data was normalized by the total spots of intensity between two groups, and then normalized by the intensity ratio of reference genes such as housekeeping genes in both groups. The expression ratio was converted to log2 ratio. Normalized spot intensities were calculated into gene expression ratios between the control and treatment groups. Greater than 2 fold changes between two groups were considered to be of significance. Results : Of the 8 K genes profiled in this study, with a cut-off level of two-fold change in the expression, 20 genes (BCL2-related protein A1, MARCKS-like 1, etc.) were upregulated and 5 genes (activated RNA polymerase II transcription cofactor 4, calcium binding atopy-related autoantigen 1, etc.) downregulated following LPS treatment. 139 genes (kell blood group precursor (McLeod phenotype), ribosomal protein S7, etc.) were upregulated and 42 genes (anterior gradient 2 homolog (xenopus laevis), phosphodiesterase 8B, etc.) were downregulated following SCHAS treatment. And 10 genes (yeast saccharomyces cerevisiae intergeneic sequence 4-1, mitogen-activated protein kinase 1, etc.) were upregulated and 8 genes (spermatid perinuclear RNA binding protein, nuclear receptor binding protein 2, etc.) were downregulated following co-stimulation of SCHAS and LPS. Discussions : It is thought that microarrays will play an ever-growing role in the advance of our understanding of the pharmacological actions of SCHAS in the treatment of arthritis. But further studies are required to concretely prove the effectiveness of SCHAS.

• Korean Journal of Acupuncture
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• v.27 no.2
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• pp.95-105
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• 2010

Objectives : The purpose of this study is to investigate the effect of Bacillus-fermented Scutellariae Radix acupuncture solution (SB) on interleukin(IL) production in mouse macrophage stimulatedby lipopolysaccaride(LPS). Methods : Productions of interleukins were measured y High-throughput Multiplex Bead based Assay with Bio-plex Suspension Array System based on $xMAP^{(R)}$(multi-analyte profiling beads) technology. To begin with, cell culture supernatant was obtained after treatment with LPS(1 ${\mu}g/mL$) and SB for 24 hour. Then, it was incubated with the antibody-conj${\mu}g$ated beads for 30 minutes. And detection antibody was added and incubated for 30 minutes. After incubating for 30 minutes, Strepavidin-conjugated Phycoerythrin(SAPE) was then added. Incubating for another 30 minutes, the level of SAPE fluorescence was analyzed on Bio-plex Suspension Array System. Results : The results of the experiment are as follows. SB significantly inhibited the LPS-induced production of IL-3($9.15{\pm}0.35$ pg/mL) by $6.92{\pm}0.05,\;7.21{\pm}0.11,\;6.96{\pm}0.33,\;and\;7.45{\pm}0.74$ pg/mL at the concentration of 25, 50, 100, and 200 ${\mu}g/mL$ in mouse macrophage RAW 264.7 cells (p<0.05). SB significantly inhibited the LPS-induced production of IL-5($7.30{\pm}0.48$ pg/mL) by $6.50{\pm}0.29,\;6.30{\pm}0.25,\;6.30{\pm}0.25,\;and\;5.80{\pm}0.25$ pg/mL at the concentration of 25, 50 100, and 200 ${\mg}g/mL$ in RAW 264.7 cells (p<0.05). SB significantly inhibited the LPS-induced productiion of IL-9($17.26{\pm}0.19$ pg/mL) by $15.01{\pm}0.43$ pg/mL at the concentration of 25 ${\mu}g/mL$ in RAW 264.7 cells(p<0.05). SB significantly inhibited the LPS-induced productioh of IL-13($187.80{\pm}2.90$ pg/mL) by $152.80{\pm}4.25,\;172.80{\pm}3.97,\;162.10{\pm}6.67,\;and\;165.30{\pm}11.80$ pg/mL at the concentration fo 25, 50, 100, and 200 ${\mu}g/mL$ in RAW 264.7 cells(p<0.05). SB significantly inhibited the LPS-induced production of IL-17($18.30{\pm}0.95$ pg/mL) by $13.30{\pm}1.25,\;13.80{\pm}1.11,\;13.30{\pm}0.75,\;and\;14.00{\pm}1.08$ pg/mL at the concentration of 25, 50 100, and 200 ${\mu}g/mL$ in RAW 264.7 cells(p<0.05). SB significantly inhibited the LPS-induced production of IL-23($43.90{\pm}0.83$ pg/mL by $39.50{\pm}1.26,\;38.00{\pm}1.78,\;and\;39.60{\pm}2.49$ pg/mL at the concentration of 25, 100, and 200 ${\mu}g/mL$ in RAW 264.7 cells(p<0.05). Conclusions : These results suggest that SB has anti-inflammatory activity related with its inhibition of IL-3, IL-5, IL-13, IL-17, and IL-23 production in macrophages.

• Journal of Pharmacopuncture
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• v.9 no.2
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• pp.5-16
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• 2006

Objective : It has long been known about the osteogenic effect of CPC-HAS on bone tissues. However, it has not been determined the effect of CPC-HAS on cancer cells. The purpose of this study is to screen the CPC-HAS mediated differentially expressed genes in cancer cells such as HepG2 hepatoma cells. Oligonucleotide microarray and proteomics approaches were employed to screen the differential expression genes. Methods : CPC-HAS was prepared by boiling and stored at $-70^{\circ}C$ until use. Cells were treated with various concentrations of CPC-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 CPC-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 cell in all concentrations(0.l, 0.5, 1.5, 10, 20mg/ml). In oligonucleotide microarray assay, the number of more than twofold differentially regulated known genes was 23 with 5 up-regulated and 18 down-regulated genes in HepG2 cells. In proteomic analysis, three spots were identified by 2D-gel electrophoresis and Q-TOF analysis. Two down-regulated proteins were aldehyde dehydrogenase 1 and enolase 1, and up-regulated protein was fatty acid binding protein 1 by 1.5mg/ml of CPC-HAS. Discussion : This study showed the screening of CPC-HAS mediated differentially regulated genes using combined approaches of oligonucleotide microarray and proteomic analysis. The screened genes will be used for the better understanding of the therapeutic effects of CPC-HAS on cancer fields.

• Journal of Pharmacopuncture
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• v.9 no.2
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• pp.49-65
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• 2006

Objective : The purpose of this study is to investigate nucleotide sequence and extract cytotoxicity of Scolopendrae corpus. The nature and taste of Scolopendrae corpus is hot, Warm and toxic, and the effect of this is dispelling wind, anti-spasmodic action and detoxication so it has been used for C.V.A, facial palsy, sensory disorder at extremities, wounds and arthritis. Methods : Scolopendrae corpus were collected by locality on the market. They were morphologically classified. Their nucleotide sequence was investigated and compared among them. In addition, the water-alcohol extract cytotoxicity of them was studied by MTT-based cytotoxicity assay. Results : It was shown that the each Scolopendrae corpus by locality is almost identical at genetic result and is identified as Scolopendra subspinipes mutilans L. Koch. Nucleotide sequence of Scolopendra subspinipes mutilans L. Koch in this study will help to discriminate other species of Scolopendrae corpus. The water-alcohol extract of Scolopendra subspinipes mutilans L. Koch did not induce cytotoxicity on Hep G2, L929 cell and peritoneal macrophages. Besides, it did not influence nitrite production of peritoneal macrophages. These results can be used as basic data for genetic discrimination with another species of scolopendrae corpus.