• Title/Summary/Keyword: collagenase

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Screening of Anti-wrinkle Resource from Herbal Medicinal Extracts and Stability Test of Its Cosmetic Products (생약재 추출물로부터 주름 개선 소재의 발굴 및 이를 이용한 화장품의 안정성 시험)

  • Cho, Eun-Ah;Cho, Eun-Hye;Choi, Sun-Ju;Park, Keun-Hyoung;Kim, So-Young;Jeong, Yoon-Joo;Ku, Chang-Sub;Ha, Byung-Jhip;Jang, Dong-Il;Chae, Hee-Jeong
    • Korean Journal of Medicinal Crop Science
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    • v.19 no.2
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    • pp.126-135
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    • 2011
  • Various herbal medicinal extracts were examined for the development of cosmetic products with anti-oxidative and anti-wrinkle activity. First, total polyphenol contents and DPPH radical scavenging activities of herbal medicinal extracts were measured. Most herbal samples, except for DW extracts of Portulaca oleracea, Caesalpinia sappan, Taraxacum platycarpum, Carthamus tinctorius, and 70% EtOH extracts of Taraxacum platycarpum and Carthamus tinctorius, showed DPPH radical scavenging activity over 80% at a concentration of $400{\mu}g/m{\ell}$. SOD-like antioxidant activity of DW extracts of Syzygium aromaticum, DW extracts of Eriobtrya japonica and 70% EtOH extracts of Sophora japonica was measured as 40%, 35% and 80%, respectively at a dry matter concentration of $50{\mu}g/m{\ell}$. In elastase inhibition assay, DW extracts of Lycium chinense ($50{\mu}g/m{\ell}$) and 70% EtOH extracts of Areca catechu ($50{\mu}g/m{\ell}$) showed 50% and 40% of inhibition, respectively. At a concentration of $1.250{\mu}g/m{\ell}$, DW extracts of Lycium chinense and 70% EtOH extracts of Areca catechu showed 10% and 30% of collagenase inhibition, respectively. Skin and lotion samples were prepared using the two herbal extracts of high anti-wrinkle activity: Lycium chinense extract and Areca catechu extract. The storage stability of skin and lotion containing each of the selected herbal extracts was evaluated. pH and viscosity were used as stability indicators for the stability test under different storage temperatures and freeze-thaw cycle conditions. The skin and lotion containing each of DW extract of Lycium chinense and 70% EtOH extract of Areca catechu was showed high pH and viscosity stability. The skin and lotion containing DW extracts of Lycium chinense showed relatively higher stability than the skin and lotion containing 70% EtOH extract of Areca catechu, at cycle chamber and freeze-thaw conditions. In summary, these results indicated that cosmetics containing DW extract of Lycium chinense were relatively stable, and this herbal extract could be used as a stable functional cosmetic material.

Calcium Current and Background Current Activation in L-triiodothyronine Loaded Ventricular Myocytes of the Rabbit

  • Han, Jin;Kim, Eui-Yong;Han, Jae-Hee;Park, Choon-Ok;Hong, Seong-Geun;Leem, Chae-Hun;So, In-Suk;Ho, Won-Kyung;Earm, Yung-E;Sung, Ho-Kyung
    • The Korean Journal of Physiology
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    • v.26 no.2
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    • pp.99-111
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    • 1992
  • Permissive action of thyroid hormone at the level of Ca channel and responsible mechanisms underlying thyroid hormone-induced change in myocardial contractile state and $T_3-induced$ arrhythmias were investigated in rabbit ventricular or atrial myocytes using whole cell patch clamp technique. Single cells were isolated by Langendorff perfusion with collagenase. Cardiac myocytes were incubated in $low-Cl^-,$, $high-K^+$ medium containing $1_{\mu}M\;L-triiodothyronine\;(T_3)$ at $4^{\circ}C$ for 2.10 hours. The calcium currrent $(I_{Ca})$ was increased in $T_3$ loaded cells, however, the shape of current voltage curve and reverse potential did not altered. Cyclic AMP, cyclic GMP, isoprenaline and 3-isobutyl-1-methyl-xanthine increased $I_{Ca}$ in euthyroid and hyperthyroid conditions, and acetylcholine blocked the increase of $I_{Ca}\;in\;T_3$ loaded cells. The amplitude of $I_{Ca}$ was much larger after perfusing cGMP than cGMP in both conditions, whereas the degree of increase of $I_{Ca}$ was greater after perfusing cAMP than cGMP in $T_3$ loaded cells. The degree of increase of $I_{Ca}$ after perfusing isoprenaline or IBMX also was greater in $T_3$ loaded cells than in control cells. Background current induced by isoprenaline also increased in $T_3$ loaded cells. The Ca release dependent inward current was increased in amplitude but its activation and inactivation time course was not changed in $T_3$ loaded cells. Activation of Na pump current was not changed in $T_3$ loaded cells. From the above results it is suggested that thyroid hormone induced increase in the contractile state of cardiac myocytes are accompanied by augmented $I_{Ca}$ and the increase of Ca release from sarcoplasmic reticulum and the permissive action of thyroid hormone to catecholamines could induce arrhythmias through the increase of $I_{Ca}$ and background current.

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Induction of Heat Shock Protein 70 Inhibits Tumor Necrosis $Factor{\alpha}-induced$ Lipid Peroxidation in Rat Mesangial Cells (Heat Shock Protein 70이 흰쥐 배양 혈관간 세포에서 관찰되는 $TNF{\alpha}$에 의한 지질과산화에 미치는 보호 효과)

  • Ha, Hun-Joo;Park, Young-Mee;Ahn, Young-Soo;Kim, Kyung-Hwan
    • The Korean Journal of Pharmacology
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    • v.31 no.3
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    • pp.323-331
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    • 1995
  • Monocyte/macrophage infiltration is the well known initial features associated with the development of glomerular disease including non-immune mediated nephropathy. Tumor necrosis factor ${\alpha}(TNF{\alpha})$, a cytokine produced primarily by monocyte/macrophage, exhibits similar effects as observed at the initial stages and during the progression of glomerular injury. Because the mesangial cells are target cells for glomerular injury, the present study examined the effect of $TNF{\alpha}$ on glomerular mesangial cell membrane lipid peroxidation as an index of cytotoxicity attributing to $TNF{\alpha}$. Primary culture of rat mesangial cell was established by incubation of glomeruli isolated from male Sprague-Dawley rat kidneys utilizing a standard sieving method. The levels of lipid peroxides in the mesangial cells were quantitated by malondialdehyde- thiobarbituric acid adduct formation. During an 8 hour incubation at $37^{\circ}C$, $TNF{\alpha}$ at 10 to 10,000 units/ml increased the levels of lipid peroxides dose dependently. Western blot analysis demonstrated that a short thermal stress induced heat shock response and the synthesis of heat shock protein 70(hsp70) in this mesangial cells. Further, this induction of hsp 70 prevented increase of lipid peroxides in the mesangial cells exposed to $TNF{\alpha}$. These data suggest that $TNF{\alpha}-induced$ lipid peroxidation in the mesangial cells may have pathophysiological relevance to glomerular injury and prior induction of heat shock response may play a role in the cellular resistance against $TNF{\alpha}-induced$ glomerular injury.

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옻나무 유래 Flavonoid처리가 흰쥐 Leydig 세포의 체외배양에서 Testosterone분비에 미치는 영향

  • 성환후;최선호;민관식;장유민;나천수;정일정
    • Proceedings of the KSAR Conference
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    • 2001.03a
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    • pp.80-80
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    • 2001
  • 옻나무 유래 Flavonoid(F)는 항암 효과, 숙취해소 등의 효능이 있으며, 신장의 기능을 촉진하고 생식에도 영향을 준다고 알려져 있다. 그 주성분으로는 fustin, fisetin, sulfuretin, butein으로 추정되고 있다. 본 연구팀의 예비실험에서 성숙 흰쥐에게 F를 30일간 5mg씩 구강투여 하여 정소 중량 및 혈액 내 Testosterone 농도를 분석한 결과, 정소 중량이 증가되고 혈중 Testosterone의 농도도 대조구에 비해 유의적으로 높은 결과를 얻었다. 이에 본 연구의 목적은 옻나무 유래 F가 성숙 수컷 횐쥐의 생식 기능에 직접 미치는 영향을 알아보고자, 흰쥐 Leydig 세포를 분리, 회수하여 체외배양에서 Testosterone농도를 조사하였다. SD계 흰쥐(약 12주령, 체중 250g 전후)의 정소를 적출 한 후, Leydig 세포를 회수하기 위해 0.25% collagenase용액에 넣어 34$^{\circ}C$에서 15분간 진탕 수조에서 배양하였다. 배양된 정소조직은 D-MEM 배양액(10% FCS와 antibiotics 첨가)으로 2~3회 세척한 후 세포 수를 1$\times$$10^{6}$ cells/$m\ell$/well로 분주하여, F(20, 40, 80, 160ng), IGF-I(50, 100ng) 와 LH(10, 100ng) 용량별 각각 첨가하여 배양하였다. 각 처리 후, 배양시간(3, 6, 12, 24, 36시간)에 따라 배양액을 회수하여 COAT-A-COUNT(DPC, USA) kit를 이용하여 RIA방법에 의해 Testosterone을 분석한 결과는 다음과 같다. 대조구 Leydig 세포를 36시간까지 체외 배양하여 Testosterone의 농도를 조사한 결과, 24시간 배양구가 가장 높은 농도를 나타내었다. F를 단독 첨가하여 12시간 배양한 실험에서 F 80ng 첨가구에서 유의적으로 높은 농도를 보였다. LH 10ng 및 100ng 첨가구의 시간별 변화로는, LH 10ng 첨가구에서는 6~12시간 이후, LH 100ng 첨가구에서는 3~6시간 사이에서 유의적인 증가를 보였다. 또한, LH 10ng 첨가 실험에서는 LH 10ng+F 40ng에서 12시간 배양 시, 유의적으로 높은 Testosterone 분비를 확인하였으며, LH 100ng을 첨가하여 3시간 배양 시는 각 처리구 마다 높은 유의적 차이를 보였다. 한편, GH에 관여하는 IGF-I 첨가효과를 비교 검토한 결과, LH 100ng+IGF-I 100ng, 6시간 배양구에서 유의적으로 높게 나타내었다. 이상의 결과로, F 단독 처리 시의 적정량은 약 80ng이며, 흰쥐 정소 Leydig 세포의 Testosterone분비를 촉진하는 작용을 하는 것으로 사료되며, 특히 LH+F구에서 Testosterone분비를 더욱 향상시킴으로써 정소 Leydig 세포의 Androgen 생성을 촉진시키는 역할이 있는 것으로 보여진다 따라서 옻나무 유래 F는 포유동물의 생식기능에 중요하게 작용하는 것으로 사료된다.

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Cooperation of $G{\beta}$ and $G_{\alpha}q$ Protein in Contractile Response of Cat Lower Esophageal Sphincter (LES)

  • Sohn, Uy-Dong;Lee, Tai-Sang
    • The Korean Journal of Physiology and Pharmacology
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    • v.7 no.6
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    • pp.349-355
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    • 2003
  • We previously shown that LES contraction depends on $M_3$ receptors linked to PTX insensitive $G_q$ protein and activation of PLC. This results in production of $IP_3$, which mediates calcium release, and contraction through a CaM dependent pathway. In the esophagus ACh activates $M_2$ receptors linked to PTX sensitive $G_{i3}$ protein, resulting in activation of PLD, presumably, production of DAG. We investigated the role of PLC isozymes which can be activated by $G_q$ or $G{\beta}$ protein on ACh-induced contraction in LES and esophagus. Immunoblot analysis showed the presence of 3 types of PLC isozymes, $PLC-{\beta}1$, $PLC-{\beta}3$, and $PLC-{\gamma}1$, but not $PLC-{\beta}2$, $PLC-{\beta}4$, $PLC-{\gamma}2$, $PLC-{\delta}1$, and $PLC-{\delta}2$ from both LES and esophageal muscle. ACh produced contraction in a dose dependent manner in LES and esophageal muscle cells obtained by enzymatic digestion with collagenase. $PLC-{\beta}1$ or $PLC-{\beta}3$ antibody incubation reduced contraction in response to ACh in LES but not in esophageal permeabilized cells, but $PLC-{\gamma}1$ antibody incubation did not have an inhibitory effect. The inhibition by $PLC-{\beta}1$ or $PLC-{\beta}3$ antibody on Ach-induced contraction was antibody concentration dependent. The combination with $PLC-{\beta}_1$ and $PLC-{\beta}_3$ antibody completely abolished the contraction, suggesting that $PLC-{\beta}1$ and $PLC-{\beta}3$ have a synergism to inhibit the contraction in LES. $PLC-{\beta}1$, -${\beta}3$ or -${\gamma}1$ antibody did not reduce the contraction of LES cells in response to DAG ($10^{-6}$ M), suggesting that this isozyme of PLC may not activate PKC. When $G_{q/11}$ antibody was incubated, the inhibitory effect of the incubation of PLC ${\beta}3$, but not of PLC ${\beta}_1$ was additive (Fig. 6). In contrast, when $G_{\beta}$ antibody was incubated, the inhibitory effect of the incubation of PLC ${\beta}_1$, but not of PLC ${\beta}_3$ was additive. This data suggest that $G_{q/11}$/11 or $G{\beta}$ may activate cooperatively different PLC isozyme, $PLC{\beta}_1$ or $PLC{\beta}_3$ respectively.

Isolation and Characterization of Antioxidative Peptides from Enzymatic Hydrolysates of Yellowfin Sole Skin Gelatin (가자미피 젤라틴 가수분해물로부터 항산화성 펩티드의 분리${\cdot}$정제 및 특성)

  • KIM Se-Kwon;LEE Hyun-Chel;BYUN He-Guk;JEON Yon-Jin
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.29 no.2
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    • pp.246-255
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    • 1996
  • To develop a natural antioxidative peptide, the gelatin was extracted from fish (Yellowfin sole) skin by hot $water(50^{\circ}C)$ extraction method and hydrolyzed with Alcalase, pronase and collagenase through a continuous 3-step membrane reactor. Each step enzymatic hydrolysates were determined the antioxidative activity and their synergistic effects, compared with $\alpha-tocopherol$ and butylated hydroxytoluene (BHT). Also, we tried to investigate the antioxidative disposition of peptide which was successfully separated by gel filtration, ion-exchange chromatography, and HPIC in cultured rat hepatocytes intoxicated with tert-butyl hydroperoxide (TBHP). Second step enzymatic hydrolysate (SSEH) among all hydrolysates and $\alpha-tocoperol$ was showed the strongest antioxidative activity. The optimum concentration of antioxidative activity for SSEH was $1\%(w/w)$ in linoleic acid. The synergistic effects were increased in using the hydrolysate with tocopherol and BHT. In the presence of the peptide isolated from SSEH, supplemented hepatocytes exposed to TBHP showed that delayed cell killing and decreased significantly the lipid peroxidation, compared with hepatocytes not cultured with isolated peptide.

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Phenotypic Characterization of Cementum-Derived Cells in Human (사람 백악질 유래 세포의 형질 특성에 관한 연구)

  • Kim, Su-Hwan;Yang, Byung-Kun;Ku, Young;Rhyu, In-Chul;Chung, Chong-Pyoung;Han, Soo-Boo;Lee, Yong-Moo
    • Journal of Periodontal and Implant Science
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    • v.34 no.2
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    • pp.269-279
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    • 2004
  • 백악질 세포의 분리 및 배양방법을 확립하고, 이를 이용하여 백악질 세포의 형질특성을 알아보고자 하였다. 교정목적으로 발거된 소구치를 이용하여, 치은섬유아세포, 치주인대 세포 및 백악질 유래세포를 분리, 배양하였다. 백악질 유래 세포 배양시에는 백악질을 절제한 후 Collagenase P를 이용하여 백악질 유래 세포 외의 다른 세포의 개제를 배제하였고, 기질을 분해하여 세포의 분리 및 배양이 용이하도록 하였다. 분리 및 배양시기의 세포의 형태를 광화현미정을 이용하여 관찰하였다. 조골세포의 특성을 가지는 SaOs-2 세포를 대조군으로 이용하여 분리 및 배양된 세포군들을 동일한 조건으로 배양하였다. 3일 및 7일째에 세포증식도를 측정하였고 7일째에 ALPase 효소 활성도를 측정하였다. 각 세포의 형질 특성을 알아보기 위해 RT-PCR을 실시하여 조골세포 분화 표식자와 연관된 osteopontin(OPN), Alkaline phosphatase(ALP), type I collagen(COL-I), Bone sialoprotein(BSP), BMP-2 및 osteocalcin(OC)의 발현을 비교 관찰하였다. 백악질 유래 세포의 분리 및 배양을 시도한 5명의 치아 중에서 3명의 치아에서 세포군을 배양해 낼 수 있었다. 배양한 백악질 유래 세포는 섬유아세포와 유사한 형태와 증식을 보였다. ALPase 효소 활성도 검사 결과 백악질 유래 세포는 SaOs-2 세포보다 낮은 활성도를 나타내었으며, 배양된 세포의 RT-PCR 결과 백악질 유래 세포군에서는 ALPase의 발현이 나타나지 않았고, 다른 조골세포 표식자의 발현도 낮게 나타났다. 이는 백악질 유래 세포가 조골세포 및 다른 대조군의 세포와는 다른 형질 특성을 가지고 있다는 것을 시사한다. 이상의 관찰결과로 사람의 백악질 유래 세포롤 백악질의 절제 및 효소처리 방법으로 효과적인 분리 및 배양이 가능하며, 이는 향후 백악질 세포의 형질 특성 및 백악질 형성의 분자적 기전을 파악하는 중요한 연구자료로 활용 될 수 있을 것으로 사료된다.

Establishment of Purification and Incubation Conditions of Leydig Cells for Screen Endocrine Disruptors Altering Steroidogenesis (스테로이드 합성을 교란하는 내분비계장애물질 검색을 위한 라이디히 세포 분리 및 배양조건 확립)

  • Kang Il-Hyun;Kang Tae-Seok;Kang Ho-Il;Moon Hyun-Ju;Kim Tae-Sung;Ki Ho-Hyun;Ryu Hye-Won;Sin Jae-Ho;Dong Mi-Sook;Han Soon-Young;Kim Seung-Hee;Hong Jin-Hwan
    • Environmental Mutagens and Carcinogens
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    • v.26 no.2
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    • pp.53-58
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    • 2006
  • Normally, environmental toxicants are classified as endocrine disruptors if they interfere with regulation of cellular function by endogeneous steroids through inhibition of receptor binding and/or transcriptional activation. So, many studies have been performed about agonist/antagonist of hormone receptor to study mechanisms of endocrine disruptors. If toxicants affect steroid biosynthesis and/or degradation and alter hormone homeostasis, these also are classified as endocrine disruptors. But there are not many studies of the mechanisms of endocrine disruptors on the basis of alteration of steroid biosynthesis and/or degradation. Isolation and culture of Leydig cells from testis is one of methods for the steroidogenesis screening assays to evaluate a substance for altering steroidogenesis. Leydig cells were harvested using the method described by Klinefelter with modifications. Leydig cells were purified by perfusion of testis and incubation ($34^{\circ}C$, 80cycles/minute, 20 minutes) with collagenase (0.25 mg/kg), centrifugal elutriation, percoll gradient centrifugation and BSA multidensity gradient centrifugation. To confirm if this method is one of appropriate tools to evaluate a substance for altering steroidogenesis, ketoconazole, positive control was administered to purified Leydig cells. Ketoconazole ($10^{-8}M$ and above) significantly reduced testosterone production in purified Leydig cells. From above results, we suggest that this method for steroidogenesis screening assay appears to be a appropriate tool to detect suspected compounds for altering steroidogenesis.

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Disulfiram Suppresses Invasive Ability of Osteosarcoma Cells Via the Inhibition of MMP-2 and MMP-9 Expression

  • Cho, Hyun-Ji;Lee, Tae-Sung;Park, Jae-Bok;Park, Kwan-Kyu;Choe, Jung-Yoon;Sin, Doo-Il;Park, Yoon-Yub;Moon, Yong-Suk;Lee, Kwang-Gill;Yeo, Joo-Hong;Han, Sang-Mi;Cho, Young-Su;Choi, Myeong-Rak;Park, Nam-Gyu;Lee, Yun-Sik;Chang, Young-Chae
    • BMB Reports
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    • v.40 no.6
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    • pp.1069-1076
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    • 2007
  • Cancer cells, characterized by local invasion and distant metastasis, are very much dependant on the extracellular matrix. The expression of matrix metalloproteinases (MMPs) has been implicated in the invasion and metastasis of cancer cells. In this study, we reported the effects of disulfiram, a clinically used anti-alcoholism drug, on tumor invasion suppression, as well as its effects on the activity of MMP-2 and MMP-9 in human osteosarcoma cells (U2OS). Disulfiram has been used for alcohol aversion therapy. However, recent reports have shown that disulfiram may have potential in the treatment of human cancers. Herewith, we showed that the anti-tumor effects of disulfiram, in an invasion assay using U2OS cells and that disulfiram has a type IV collagenase inhibitory activity that inhibits expression of genes and proteins responsible for both cell and non-cell mediated invasion on pathways. In conclusion, disulfiram inhibited expression of MMP-2 and MMP-9 and it regulated the invasion of human osteosarcoma cells. These observations raise the possibility of disulfiram being used clinical for the inhibition of cancer invasion.

3,9-Diferuloyl-6-oxopterocarpen (Tensolin-F$^\circledR$): a Novel Anti-wrinkle Agent for Cosmeceuticals (새로운 주름개선 소재: 3,9-diferuloyl-6-oxopterocarpen (Tensolin-F$^\circledR$)의 개발과 화장품 응용)

  • 이범천;이정재;박성민;김철배;심관섭;김진화;이근수;이천일;표형배
    • Journal of the Society of Cosmetic Scientists of Korea
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    • v.30 no.1
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    • pp.7-13
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    • 2004
  • As a novel anti-wrinkle agent, 3,9-diferuloyl-6-oxopterocarpen (Tensolin-F$^{(R)}$) has been synthesized and its anti-aging effects have been investigated. In the present study, to investigate the relationship between aging and Tensolin-F$^{(R)}$, we examined its effect on scavenging activities of radicals and reactive oxygen species (ROS), in vitro inhibition activity of matrix-metalloproteinase (MMP) and expression of UVA-induced MMPs in human dermal fibroblasts (HDF). Tensolin-F$^{(R)}$ was found to show activities of scavenging radicals and ROS with the $IC_{50}$/ values of 0.2 mM and 0.95 mM against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and superoxide radicals, respectively, in the xanthine/xanthine oxidase system. Fluorometric assays for the proteolytic activities of MMP-l (collagenase) were performed using fluorescent collagen substrates. Tensolin-F$^{(R)}$ inhibited the activities of MMP-l in a dose-dependent manner and the $IC_{50}$/ values calculated from semi-log plots were 0.025 mM. Also, UVA induced MMP-1 expression was reduced 85% by treatment with Tensolin-F$^{(R)}$ at 0.8 uM, which was reduced dose-dependent manner. The results of clinical study showed that 4.8 mM Tensolin-F$^{(R)}$ treated group reduced wrinkle significantly compared with placebo treated group (P 〈 0.05). Taken together, these result suggest that Tensolin-F$^{(R)}$ act as an anti-wrinkle agent by taking effects to antioxidation and reducing UVA-induced MMP-l production.-l production.