Hydroxyproline의 안면부 주름 개선 효과에 관한 연구

서기석 ( Kee Suck Suh ),최수영 ( Soo Young Choi ),김상태 ( Sang Tae Kim ) 대한피부과학회 2007 대한피부과학회지 Vol.45 No.2

Background: Many treatment modalities for wrinkle reduction have been developed in the field of dermatology. Hydroxyproline plays a critical role in stabilizing the triple helix of collagen. The triple-helical conformation gives collagen most of its unique properties and is essential for normal fibrillogenesis. Objective: This study was performed to investigate the effect of hydroxyproline on the improvement of wrinkles in vitro and in vivo. Methods: Cultured fibroblasts and 25 human volunteers were used for in vitro and in vivo studies respectively. Hydroxyproline was administered to a fibroblast culture system and was also applied to periorbital wrinkle lesions twice daily for 12 weeks. The effect of hydroxyproline was examined by using an image analysis system with skin replica, and subjective and objective assessment of wrinkles were evaluated. Results: With the in vitro study, the number of cultured fibroblasts did not increase in the 10(-4), 10(-6) and 10(-8)ug/ml hydroxyproline groups compared to the control groups. Procollagen Type 1 C-peptide of cultured fibroblasts did increase in the 10(-8)ug/ml hydroxyproline group compared to the control group (p<0.05). With the in vivo study, there was a significant decrease in wrinkles at 8 and 12 weeks after application in the 0.5% hydroxyproline applied group compared to the control group (p<0.001). In the subjective visual assessment by the investigator and the subjects at 8 and 12 weeks after application, the 0.5% hydroxyproline treated group showed a significant improvement of wrinkles and more personal satisfaction than the control group (p<0.05). Conclusion: 0.5% hydroxyproline had an effect in vivo and in vitro on wrinkle improvement. It is suggested that hydroxyproline might be a candidate compound for treatment or reduction of wrinkles. (Korean J Dermatol 2007;45(2):140~148)

자외선에 의해 피부가 손상된 hairless mouse에서의 hydroxyproline, Pro-Hyp 경구반복투여시 피부 상태 개선 효과

이지해(Ji-Hae Lee),서정혜(Jeong-Hye Seo),박영호(Young-Ho Park),김완기(Wan-Gi Kim),임경민(Kyung-Min Lim),이상준(Sang-Jun Lee) 한국식품과학회 2008 한국식품과학회지 Vol.40 No.4

본 연구를 통하여 피부 미용 소재로 알려져 있는 콜라겐 펩타이드를 섭취한 후 혈중에 나타나는 소화분해산물 중 콜라겐 특유의 아미노산인 hydroxyproline과 이것의 dipeptide 형태인 Pro-Hyp이 피부에 미치는 영향을 알아보고자 하였다. 피부 미용 소재를 경구 섭취하는 사람의 피부는 광노화 및 자연노화가 어느 정도 진행된 상태이므로, 이와 유사한 조건을 조성하기 위해 경구 투여를 시작하기 전에 hairless mouse에 7주간 주 3회 UV를 조사하여 주름을 선유발시켰다. 군 분리를 시킨 후 7주간 경구로 실험물질을 주 5회 반복 투여하면서, 주 3회 UV 조사를 병행하였으며, 시험 기간 중 임상증상, 체중변화를 관찰하였고, 3회(0, 4, 7주차) 피부주형을 채취하여 영상 분석을 실시하였으며, 2회(0, 7주차)의 피부탄력측정 및 1회(7주차)의 TEWL측정을 병행하였다. 실험종료 후 피부조직의 병리조직학적 검사(H&E, Masson-Trichrome stain) 및 피부두께 측정을 실시하였다. 실험기간 중 이상 임상증상을 보이거나 사망한 개체는 없었으며, 체중에 대한 통계적으로 유의한 변화는 관찰할 수 없었다. 피부 육안관찰 시 NC군에 비해 UC군의 피부 주름 증가가 뚜렷하였으며, 시험군들은 UC군에 비해 주름 증가가 적었다. 경구 투여 0, 4, 7주차에 제작된 replica의 주름 지표를 통계 처리한 결과, UV/H군과 UV/P군은 UC군에 비해 감소경향은 보였으나 유의적인 차이는 없었다. 탄력측정검사 결과 UV를 쬐지 않은 NC군은 UC군에 비해 UA/UF, Ur/Ue, Ur/Uf 지표에서 유의적인 증가를 보였다. UV/H군과 UV/P군은 모두 0, 7주차 모두 유의적인 차이를 보이지 않았다. 피부 보습력 측정 결과 NC군 및 모든 실험군에서 UC군에 비해 유의적인 TEWL 감소를 관찰할 수 있었다. 피부 두께의 경우 NC군, UV/H군, UV/P군은 UC군에 비해 피부 두께가 유의적으로 감소하였다. 진피층에서의 교원질 확인을 위해 실시한 Masson-Trichrome stain의 경우 UC군에서는 상부 진피층에서의 염색상 소실이 관찰되었으나, 모든 실험군에서 진피층 손상이 거의 관찰되지 않아 실험물질에 의한 회복을 확인할 수 있었다. 이상의 실험 결과 hydroxyproline, Pro-Hyp의 경구반복투여시 자외선에 의해 광노화가 유발된 피부에 보습 효과, 피부 두께 감소 효과, 진피층 손상 회복 효과를 유의적으로 확인할 수 있었으며, 단, 피부 주름 개선 정도는 통계적 유의성은 없이 경향성만을 확인할 수 있었다. Hydroxyproline과 이를 함유하는 dipeptide 형태의 Pro-Hyp를 경구 투여한 시험으로부터 확인한 피부 보습 및 진피층 회복 효과는 이 물질들이 유래된 콜라겐을 도포 또는 섭취했을 때 피부에 나타날 수 있는 작용들과 상통하며, 단독 미용 소재로도 활용될 수 있는 가능성을 보여주었다. 본 연구에서는 콜라겐 펩타이드를 섭취 시 체내에 흡수되어 혈액을 통해 각 조직에 운반되는 분해산물이 피부에 미치는 영향을 동물 시험을 통해 살펴보았는데, 향후 연구에서는 관절, 혈관과 같은 다른 생체 조직들에 미치는 영향에 대해서도 검증해 볼 필요가 있다. Hydroxyproline and Pro-Hyp dipeptide are the digestive products of collagen hydrolysate called collagen peptide. Some suggested that collagen peptides could improve aged or damaged skins, however, the effects of collagen peptides on the skin have not been known. In this study, we investigated the effects of digestive products of collagen peptides, hydroxyproline and Pro-Hyp dipeptide on skin quality using the UV-damaged dorsal skin of hairless mouse as a model system. Female SKH hairless mice were pre-irradiated with UV for 7 weeks, and then hydroxyproline, Pro-Hyp dipeptide were orally administered for 7 weeks with UV irradiation. Wrinkle formation (by replica image), skin elasticity, barrier status (by TEWL, transepidermal water loss), epidermis thickness, and biophysical changes in the stratum corneum (by hematoxylin & eosin staining) were examined. With the oral peptide treatment, effects such as skin barrier maintenance, anti-skin thickening, and recovery of the stratum corneum were observed. These results indicate that oral intake of collagen peptides may have beneficial effects on damaged skin cells.

加減茵蔯蒿湯 水鍼液이 膽道結紮로 유도된 肝損傷에 미치는 영향

成樂箕,金元信,田炳薰,李建穆 대한동의병리학회 1993 동의생리병리학회지 Vol.8 No.-

연구배경 : 肝癌 및 肝硬變은 韓國人의 疾病으로 인한 중요한 喪亡原因의 하나이며, 특히 우리나라 B型肝炎의 流行地域으로 急慢性 肝炎이나 肝經辨證, 肝癌의 發病率이 높다. 그리하여 肝硬變症을 寢具治療法중 水鍼을 이용하여 肝의 募穴인 期門穴에 投與하면 治療效果가 있는지에 대하여, 그리고 藥量에 따라 治療效果가 달라지는 지에 대하여 알아보기 위하여 본 연구를 시행하였다. 방법 : 加減茵蔯蒿湯 水鍼液을 膽道를 結紮한 白鼠에 26guage의 1ml 주사기를 이용하여 투여하였다. 經穴은 人體의 期門에 相應하는 左石部位와 腹部內의 任意의 部位를 設定하여 매일 1회 7주간 수침하였다. 결과 : 體重과 Total Bilirubin은 實驗群 A, C에서 有意性 있는 增加와 減少를 보였고, WBC·ALT·AST·ALT 및 Cholesterole은 全實驗群에서 有意性 있는 減少를, Albumin은 全實驗群에서 有意性 있는 增加를 각각 나타내었으며, 組織學的 觀察도 全實驗群에서 有意性을 나타내었다. 또한 Hydroxyproline도 實驗群 A, B에서 有意性있는 減少를 보였다. 결론 : 加減茵蔯蒿湯 水鍼液을 膽道를 結紮한 白鼠의 期門穴에 投與한 缺課 有意한 효과를 보였으나 水鍼量에 따라 有意性의 차이가 있어 敵意한 水鍼量을 투여하는 것이 治療시 重要하다고 思料되며, 또한 水鍼에 대한 安全性 問題에 대하여서도 더욱 硏究할 필요가 있다. In order to study the effects of the water extract of Gagaminjinhotang in the Meridian points Liv 14 on the liver damage indued by bile duct ligation during 7 weeks after the operation of bile duct ligation, blood was taken from the heart and body weight, WBC, RBC, hemoglobin, hematocrit, total bilirubin, ALT, AST, ALP, cholesterol, albumin and hydroxyproline were in each experimental apparatus. The results of this study were as follows; 1. Body weight of EXA, C were significantly increased by the water extract of Gagaminjinhotang. 2. WBC of EXA,B,C were significantly decreased by the water extract of Gagaminjinhotang. 3. RBC, hemoglobin and hematocrit of EXA,B,C were not significantly influenced by the water extract of Gagaminjinhotang. 4. Total bilirubin of EXA,C and direct bilirubin of EXA,B,C were significantly decreased by the water extract of Gagaminjinhotang. 5. ALT, AST,ALP of EXA,B,C were significantly decreased by the water extract of Gagaminjinhotang. 6. Cholesterol of EXA,B,C, were significantly decreased by the water extract of Gagaminjinhotang. 7. Albumin of EXA,B,C, were significantly increased by the water extract Gagaminjinhotang. 8. Hydroxyproline of EXA,B were significantly decreased by the water extract Gagaminjinhtang. 9. On the histological observation, the results of the experimental groups showed significant hepatoprotective effects compared with control group from 3 weeks after the administration of the water extract of Gagaminjinhotang. According to the above result, it is suggested that the water extract Gagaminjinhotang was significantly effective in WBC, bilirubin, ALT, AST, ALP, cholesterol, albumin and hydroxyproline level and then it seems to be applicable to the fibrosis or cirrhosis of liver.

Therapeutic Effects of Lipoic Acid Complex on CCI₄-Induced Hepatic Fibrosis in Sprague-Dawley Rats

Nam-Jin Lee,Geun Soo Kim,Jong-Hun You,Yu-Ri Jung,Chun Mei Lin,Bum-Hee Yang,Jong-Koo Kang 한국실험동물학회 2007 Laboratory Animal Research Vol.23 No.1

The hepato-protective activity of the lipoic acid complex (LAC) has been studied in the rats against carbon tetrachloride (CCI₄)-ethanol-induced liver toxicity. Rats were fed 10% ethanol in drinking water for 8 weeks. From 1 week after the start of ethanol feeding, the animals were subcutaneously treated with single dose of 1 ㎖/㎏ CCI₄ (2 ㎖/㎏ as 50% in corn oil), and one week later, followed by 0.5 ㎖/㎏ CCI₄ twice per week for 7 weeks. After 8-week induction of liver dysfunction, the rats were orally administered with LAC (200 or 800 ㎎/㎏) or silymarin (80 ㎎/㎏), or their vehicle (0.5% carboxymethyl cellulose, CMC) for 4 weeks, along with additional 4-week treatment of ethanol and CCI₄ (0.5 ㎎/㎏). CCI₄ induced hepatocellular degeneration and necrosis, leading to a great increase in serum aspartate aminotransferase (AST) and alanine aminotrasferase (ALT). Also, lipid peroxidation and fibrosis of liver tissues were confirmed by malondialdehyde (MDA) and hydroxyproline. In addition to AST and ALT, increased TSARS and hydroxyproline contents caused by CCI₄ were significantly attenuated by LAC in a dose-dependent manner. In histopathological results, severe vacoulations and inflammatory cell infiltrations around portal triad were observed in CCI₄-treated rats, which were greatly attenuated by LAC treatment. These results support the effectiveness of LAC for the treatment of hepatic fibrosis.

Isolation and characterization of acid-soluble bluefin tuna (Thunnus orientalis) skin collagen

Tanaka, Teruyoshi,Takahashi, Kenji,Tsubaki, Kazufumi,Hirata, Maika,Yamamoto, Keiko,Biswas, Amal,Moriyama, Tatsuya,Kawamura, Yukio The Korean Society of Fisheries and Aquatic Scienc 2018 Fisheries and Aquatic Sciences Vol.21 No.4

In this study, we isolated and characterized the acid-soluble skin collagen of Pacific bluefin tuna (PBT, Thunnus orientalis). The PBT skin collagen was composed of two ${\alpha}$ chains (${\alpha}1$ and ${\alpha}2$) and one ${\beta}$ chain. The denaturation temperature of PBT collagen was low although it was rich in proline and hydroxyproline. The primary structure of PBT skin collagen was almost identical to that of calf and salmon skin collagen; however, it differed with respect to the epitope recognition of the antibody against salmon type I collagen. These results suggest that the primary structure of skin collagen was highly conserved among animal species, although partial sequences that included the epitope structure differed among collagens.

Isolation and characterization of acidsoluble bluefin tuna (Thunnus orientalis) skin collagen

Teruyoshi Tanaka,Kenji Takahashi,Kazufumi Tsubaki,Maika Hirata,Keiko Yamamoto,Amal Biswas,Tatsuya Moriyama,Yukio Kawamura 한국수산과학회 2018 Fisheries and Aquatic Sciences Vol.21 No.2

In this study, we isolated and characterized the acid-soluble skin collagen of Pacific bluefin tuna (PBT, Thunnus orientalis). The PBT skin collagen was composed of two α chains (α1 and α2) and one β chain. The denaturation temperature of PBT collagen was low although it was rich in proline and hydroxyproline. The primary structure of PBT skin collagen was almost identical to that of calf and salmon skin collagen; however, it differed with respect to the epitope recognition of the antibody against salmon type I collagen. These results suggest that the primary structure of skin collagen was highly conserved among animal species, although partial sequences that included the epitope structure differed among collagens.

간섬유화 동물에서 D-페니실라민의 항섬유화 효과 검색

김기영(Ki Young Kim),윤기중(Ki Jung Yun),문형배(Huyng Bae Moon) 대한약학회 1996 약학회지 Vol.40 No.5

One of therapeutics in liver disease (morbus wilson) is D-penicillamin (D-pen: D-3-mercapto-valin). Especially the cross-linking of collagen molecules could be inhibited by D-pen in extracellular space. In this study we investigated the antifibrotic effects of D-pen in rats that were induced the liver fibrosis by bile duct ligation and scission (BDL/S). Rats were treated for 4 weeks with D-pen after BDL/S operation or sham operation. The balance between fibrogenesis-marker (PNIIIP) and the fibrolysis-maker (PNIVP) were observed in sera by RIA (radioimmunoassay), and the parameter of collagen deposition in liver tissue (hydroxyproline: HYP) was measured by colorimetry. The weight of liver in BDL/S operated group was increased significantly in compared with sham operation group (15.2g +/- 1.1, vs 11.9g +/- 3.9: p<0.005, p<0.05). The rats group treated by D-pen showed the lower level of PNIIIP (6.7ng/ml +/- 1.5, vs 9.5ng/ml +/- 2.8) and the higher value of PIVCP (14.0ng/ml +/- 1.9, vs 7.9ng/ml +/- 1.5) in sera that compared to untreated rats. The content of HYP was decreased by 141% in BDL/S with D-pen treated group than that of it in BDL/S group. No correlation was revealed between collagen parameters in sera and HYP in liver tissue of BDL/S operated and D-pen treated rats. The group treated with D-pen showed the lower value of clinical biochemistry parameters (GOT: glutamate oxalacetate transaminase, Total-Bilirubin) in compared with only BDL/S operated rats, but the value of GPT (glutamate pyruvate transaminase) and Alkaline phosphatase in two BDL/S groups was nearly same. In the histological finding, we observed mild bile duct proliferation, weak inflammation and fibrosis in BDL/S with D-pen treated group, but BDL/S operated group showed the formation of septum (island of hepatocytes), massive bile duct proliferation. This result represents that the BDL/S operation induces liver fibrosis (cirrhosis) in 4 weeks, and D-pen inhibits the synthesis of collagen weakly and stimulates the degradation of collagen in the extracellular space. We conclude that the monitoring of PNIIIP, PIVCP in sera is useful parameter for screening of antifibrotic effect, and D-pen delay the liver fibrosis.

Intragastrically Applicated CCl₄-Thiopental Sodium Enhanced Lipid Peroxidation and Liver Fibrosis (Cirrhosis) in Rat: Malonedialdehyde as a Parameter of Lipid Peroxidation Correlated with Hydroxyproline as a Parameter of Collagen Synthesis (De

Kim, Ki-Young,Cho, Syung-Eun,Yu, Byung-Soo Korean Society of ToxicologyKorea Environmental Mu 2009 Toxicological Research Vol.26 No.2

We investigated the pathogenesis of liver tissue damage during the lipid peroxidation and fibrogenesis with the observation of correlations between the parameters of collagen synthesis (and deposition) and lipid peroxidation in liver fibrosis (cirrhosis) rats. Rats were randomly divided into two groups, normal and $CCl_4$-thiopental sod. intoxicated group. And the one group was treated intragastrically with the mixture of $CCl_4$-thiopental sod. 3 times per week for 3 weeks. The liver tissue and sera were used for the measurement of hydroxyproline (HYP), malonedialdehyde (MDA) and superoxide dismutase (SOD). Biochemical parameters such as aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), total-bilirubin and blood urea nitrogen (BUN) were measured. Additionally, the expression of collagen ${\alpha}1$(III) and $\beta$-actin mRNA was observed by RTPCR. The histological change in liver tissue was also observed by Masson's trichrome and H&E staining. Correlation analysis was carried by Spearman's rho method. All biochemical parameters except total-bilirubin were significantly higher in the $CCl_4$-thiopental sod. treated group than that of the normal group (p < 0.01). In the $CCl_4$-thiopental sod. treated group, Hyp as a parameter of collagen synthesis (deposition) and MDA as a metabolite of lipid peroxidation, were significantly elevated by 1.98 and 2.11 times higher than that of the normal group (p < 0.001) respectively. The activity of SOD in the $CCl_4$-thiopental sod. treated group is decreased significantly by 44.8% (p < 0.001). And collagen ${\alpha}1$(III) mRNA was more expressed in the $CCl_4$-thiopental sod. treated group than that of the normal group. However, the expression of $\beta$-actin mRNA is showed similar in both of groups. A good correlation was observed between the content of hyp and MDA concentration (r = 0.70, n = 40) in the two groups. And the correlation between the levels of hyp and SOD (r = -0.71, n = 25) is also reliable. However, no correlation were observed between MDA concentration and SOD (r = -0.40, n = 25) in the two groups. Elevated levels of MDA in $CCl_4$-thiopental sod. treated rats indicated enhancement of lipid peroxidation, which is accompanied by a decrease in SOD activity. Moreover, we could confirm that the parameters of collagen synthesis (and deposition) is in good correlation with the metabolite of lipid peroxidation (MDA) and the lipid peroxidation antagonizing enzyme (SOD). Hence, we propose that (1) lipid peroxidation and collagen synthesis (and deposition) could be enhanced by intragastrically application of $CCl_4$-thiopental sod. during a short terms. And (2) the intoxication of $CCl_4$-thiopental sod. could be used for monitoring of lipid peroxidation and collagen synthesis (and deposition) for test of antioxidant and antifibrotic agent.

소변 중 4-hydroxyproline 분석에 관한 연구

이규원,조영봉,이경종,Lee, Keou-Weon,Cho, Young-Bong,Lee, Kyung-Jong 대한예방의학회 2002 Journal of Preventive Medicine and Public Health Vol.35 No.4

Objectives : The level of 4-hydroxyproline (4-Hyp) in human urine was measured using high performance liquid chromatography (HPLC) with a fluorescence detector. This method is useful for medical examinations and investigating the radicals induced by physical, chemical, mental stresses. This method is superior to many published several methods in terms of its low cost and ability to analyze many samples. Methods : The urine from workers in a tire manufacturing company (22 male pre- and post-shift workers) and 18 office-workers as controls were analyzed. Data concerning age, the cumulative drinking amount and the cumulative smoking amount was collected with a questionnaire. The optimum applied amount of dansyl-Cl, the optimum reaction temperature and time, the recoveries and the optimum pH of the eluent and buffer were determined.4-Hyp from human urine was derivatized with dansyl-Cl (dimethylamino-naphthalene-1-sulfonyl chloride) after removing the a-amino acid by a treatment with phthalic dicarboxaldehyde (OPA) and cleaned with Bond Elut C18 column. The 4-Hyp derivatives were separated on a reversed phase column by gradient elution with a phosphate buffer (5 mmol, pH 8.0) and acetonitrile, and detected by fluorescence measurements at 340 nm (excitation) and 538 nm (emission). Results : The detection limit for the urinary free 4-Hyp was $0.364{\mu}mol/l$. The recovery rate of 4-Hyp was 99.7%, and the effective pH of the phosphate buffer and borate buffer were 3.0 and 8.0, respectively. From statistical analysis, age, drinking and smoking did not affect the urinary free 4-Hyp in both the controls and workers. The range of urinary 4-Hyp in the controls, pre-shift, and post-shift workers were 0.33-16.44, N.D-49.06, and $0.32-56.27{\mu}mol/l$. From the pared-sample t-test, the urinary 4-Hyp levels in post-shift workers ($11.82{\pm}6.73\;nmmol/mg\;Cre$) were 2-fold higher than in pre-shift workers ($5.36{\pm}5.53\;nmol;/mg\;Cre$) and controls ($4.91{\pm}4.89\;nmol;/mg\;Cre$). Conclusions : This method was developed with high sensitivity, accuracy, and precision. The present method was effectively applied to analyze the urinary free 4-Hyp in both controls and workers.

Investigation into the Distribution of Total, Free, Peptide-bound, Protein-bound, Soluble-and Insoluble-Collagen Hydroxyproline in Various Bovine Tissues

Siddiqi, Nikhat J.,Alhomida, Abdullah S. Korean Society for Biochemistry and Molecular Biol 2003 Journal of biochemistry and molecular biology Vol.36 No.2

Collagen is a family of proteins which consists of several genetically distinct molecular species and is intimately involved in tissue organization, function, differentiation and development. The purpose of this study was to investigate the concentration of different hydroxyproline (Hyp) fractions viz., total, free, peptide-bound, protein-bound, soluble- and insoluble-collagen hydroxyproline (Hyp) in various bovine tissues. Results showed that liver had the highest concentration of free Hyp followed by kidney, brain, spleen, lungs, muscle and heart. Liver also had the highest concentration of peptide-bound collagen Hyp followed by kidney, heart, spleen, lungs, brain and muscle. The concentration of protein-bound collagen Hyp was highest in the liver, followed by kidney, spleen, lungs, muscle, brain and heart. Total Hyp was highest in the liver, followed by kidney, spleen, brain, heart, muscle and lungs. Liver also had significantly high concentration of collagen as compared to other tissues examined (P<0.001). Spleen had the significantly higher concentration of soluble-collagen Hyp when compared to other tissues (P<0.001). This was followed by heart, muscle, lungs, brain, kidney and liver. Heart had the highest concentration of insoluble-collagen Hyp followed by lungs, kidney, liver, muscle, spleen and brain. The variation among the insoluble-collagen Hyp concentration of heart and muscle, spleen and brain was significant (P<0.001). We speculate that these differences could be due to the variation in turn over of rate of collagen metabolism in this species.