Antioxidant and Anti-photoaging Activities of Agastache rugosa Seeds in Human Dermal Fibroblasts

Lee, Mina 충북대학교 2022 국내석사

수수가루 첨가에 따른 만두피의 생리활성 및 품질특성

박재희 충북대학교 2021 국내석사

This study was conducted to evaluate the quality characteristics and biological activities of dumpling shell prepared by adding sorghum powder at 0%, 10%, 15%, and 20%. Their water-binding capacity, weight and volume decreased after cooking. The turbidity of cooked dumpling shell was the lowest in the control group and the turbidity as the increasing addition amount of sorghum powder. As a result of measuring color of the cooked dumpling shell, the L value of the brightness and b value of yewllowness was the highest in control group and decreased with the addition of sorghum powder. The value of redness was the lowest in the control group and increased as the addition amount of the sorghum powder increased. For the cooked dumpling shell texture, the measurement results of hardness were highest in the control group and decreased with the addition of sorghum powder in the addition group. On the contrary adhesiveness, springiness, chewiness, gumminess and cohesiveness were the lowest in the control group and increased with the addition of sorghum powder. This suggests that the general dumpling shell control is the lowest and the addition of the appropriate sorghum powder has a synergistic effect on the preference of color and texture. However, It is considered that the addition of too much amount may be an obstacle to the increase of preference degree. As a result of measuring antioxidant compounds of dumpling shells, the control group had lowest total phenol compound contents and flavonoid contents significantly increased as the sorghum powder content increased. The results of DPPH and ABTS free radical scavenging activity was lowest in the control group and the antioxidant activity significantly increased as the sorghum powder content increased. α-glucosidase inhibitory activity showed the lowest value in control group(3.12%), and increased with increasing amounts of sorghum powder. The findings of this study suggest the feasibility of sorghum added dumpling shell as a health food with physiological benefits and provide evidence for introducing various health foods by adding sorghum.

Anti-hypertensive Activities and Underlying Mechanisms of Cereal Grains and Phenolic Acids

유명남 충북대학교 2021 국내석사

산화적 스트레스는 고혈압 발병의 핵심적 요소이다. 이러한 산화스트레스를 줄이기 위해 사용되는 항산화제는 고혈압 예방 및 정상적인 혈관기능의 유지와 2차적인 질병의 위험을 감소시킬 수 있어 최근 이러한 항산화제를 천연물에서 찾으려는 시도가 많아지고 있다. 잡곡은 항산화, 항암 활성과 당뇨, 고혈압 예방 등의 다양한 생리활성이 알려지면서 기능성 식품원료로 주목받고 있다. 잡곡은 비타민, 식이섬유 등이 풍부하고 폴리페놀 화합물이 다량 함유되어 있어 항산화 및 항고혈압 활성을 나타내며 혈관기능을 유지하는데 도움을 줄 수 있다고 알려져 있다. 여러 연구에서 잡곡과 고혈압에 관한 동물실험이 많이 연구되어 있지만 혈관내피세포 및 활성성분의 핵심적인 작용기작에 대한 연구는 부족하다. 따라서 본 연구에서는 국내산 잡곡 (조, 수수, 기장, 귀리, 검은콩, 팥)과 잡곡에 다량 함유되어 있는 phenolic acids의 항 고혈압 활성을 검토하여 잡곡을 이용한 다양한 식품소재 및 가공식품의 제조에 필요한 기초자료를 제공하고자 하였다. 모든 잡곡과 phenolic acids 중, 수수와 sinapic acid가 가장 높은 항 고혈압 활성을 나타내었다. 수수와 sinapic acid는 강력한 세포보호효과 및 ROS 생성 억제효과를 나타내었으며 NO의 생성을 유도하고 주요 항산화 물질인 GSH의 손실을 억제하였다. 이러한 수수와 sinapic acid의 혈관내피세포 보호효과는 혈관 이완 기능을 하는 NO를 생성하는 Akt/eNOS와 항산화 작용을 하는 phase 2 enzyme의 전사인자인 Nrf-2의 활성화에 의한 것으로 판단된다. 수수와 sinapic acid를 처리하였을 때 Akt와 eNOS가 인산화되고 Nrf-2가 Keap-1 단백질로부터 떨어져 세포 핵으로 이동하여 HO-1, NQO-1, GCLC 등의 발현을 증가시켰다. 본 연구를 통해 다양한 잡곡과 phenolic acids의 항 고혈압 활성과 핵심 작용기작을 분석하였다. 따라서 가장 수수와 sinapic acid가 함유된 소재를 이용하여 고혈압 예방과 치료를 위한 기능성 원료로 활용될 수 있을 것으로 사료된다. Oxidative stress is a key player in the pathogenesis of hypertension. Dietary antioxidants may have beneficial effects on hypertension and cardiovascular risk factors. Cereal grains are a good source of bioactive phenolic acids, which play significant roles in many physiological and metabolic processes. This study examined the anti-hypertensive activities and underlying mechanisms of cereal grains and their phenolic acids. Among the cereal grains, sorghums had the highest antioxidant activity. The angiotensin-converting-enzyme inhibitory activity was the highest in the sorghum extracts (51∼65%), followed by adzuki bean (35%). In EA.hy926 endothelial cells, the protective activity against H2O2-induced oxidative stress was the highest in the sorghum extracts. Pretreatment with the sorghum extracts significantly decreased H2O2-induced reactive oxygen species (ROS) production and significantly increased nitric oxide (NO) level in EA.hy926 cells. A strong correlation was observed between antioxidant and anti-hypertensive activities (r2=0.356∼0.774). In addition, among all phenolic acids, sinapic acid shwoed the highest ACE inhibitory activity (88%). In EA.hy926 cells, pretreatment of sinapic acid dramatically alleviated oxidative stress and increased NO level. Sinapic acid also attenuated the expression of malondialdehyde (MDA) and glutathione (GSH) depletion. Furthermore, the underlying mechanisms of the sorghum and sinapic acid were more extensively examined by targeting the stimulation of Akt/eNOS pathways, expressions of phase II enzymes and modulations of Nrf-2 translocation into nucleus in EA.hy926 cells. As a results, sorghum and sinapic acid significantly activated Akt/eNOS pathways and phase II enzymes including HO-1, NQO-1 and γ-GCLC. In addition, sorghum and sinapic acid markedly increased Nrf-2 translocation into nucleus. These results indicate that sorghum and sinapic acid may prove to be a promising functional food and a compound for the prevention and treatment of hypertension.

MicroRNA Modulation by Sorghum (Sorghum bicolor L.) in Type 2 Diabetes

이하나 충북대학교 2021 국내박사

식생활 패턴과 생활환경의 변화로 인해 당뇨, 심혈관계질환 등을 포함한 대사질환의 발병률이 높아지고 있다. 마이크로 RNA는 단일가닥 RNA 분자로서 mRNA의 3'-untranslated region에 상보적으로 결합하여 유전자 발현을 제어하는 새로운 조절물질이다. 마이크로 RNA는 세포의 발달, 분화, 증식, 사멸 등 기본적인 생명 현상에서 중요한 역할을 하고 있다. 최근 파이토케미컬들이 마이크로 RNA발현 조절을 통해 대사질환을 완화시킨다고 알려져 관련 기작 구명에 관한 연구가 활발히 진행되고 있다. 파이토케미컬, 섬유질, 단백질, 비타민 등을 풍부하게 함유하고 있는 잡곡은 항균, 항암, 항산화뿐만 아니라 다양한 생리활성을 가지고 있어 관심이 높아지고 있다. 본 연구에서는 잡곡의 항산화 활성과 항당뇨 활성 및 상관관계를 알아보고자 하였다. 잡곡 에탄올 추출물의 총 폴리페놀 함량은 6.57~242.30 mg GAE/g residue, ABTS라디칼 소거능은 5.03~408.88 mg TEAC/g residue, DPPH라디칼 소거능은 1.50~313.09 mg TEAC/g residue, 환원력은 0.07~1.14로 측정되었으며, 가장 높은 활성을 가진 것은 수수와 팥이었다. α-Glucosidase 저해 활성을 분석한 결과 수수는 10 mg/mL의 농도에서 모든 품종이 100%에 가까운 수치를 보였으며, 기장이 29.1%, 팥이 27.5%로 측정되었다. 간세포의 포도당 흡수율도 α-glucosidase 저해 활성과 유사하게 수수 10 μg/mL의 농도에서 약 55~64%로 가장 높았으며 양성 대조군인 메트포민(100 μM)과 비슷한 활성을 보였다. 기장은 약 37~39%, 팥은 35%로 측정되었다. 항산화 활성과 항당뇨 활성은 유의적인 양의 상관관계(R2=0.628~0.956)를 보였다. 결론적으로 항산화 활성과 항당뇨 활성이 가장 우수한 수수 에탄올 추출물은 제2형 당뇨병을 효과적으로 개선하는 천연 소재로 이용될 수 있을 것으로 기대된다. 수수 추출물에는 페놀산 뿐만 아니라 탁시폴린과 같은 플라보노이드가 존재하며 탁시폴린은 항고혈압, 항산화 등의 활성이 보고되어 있다. 한편 마이크로 RNA-195는 인슐린 저항성에 관여한다고 알려져있어 본 연구에서는 수수 에탄올 추출물과 이에 함유되어있는 성분인 탁시폴린이 마이크로 RNA-195의 발현 조절을 통해 인슐린 저항성을 완화시키는지 알아보고자 하였다. 유리지방산으로 인슐린 저항성을 유도한 간세포군은 대조군과 비교하였을 때 포도당 흡수가 약 16% 감소하였지만 탁시폴린(25, 50 μM)과 수수 에탄올 추출물(5, 10 μg/mL)은 유리지방산 처리군보다 포도당 흡수를 각각 16%, 24%, 23%, 28% 증가시켰다. 인슐린 신호전달과 관련된 단백질들의 발현을 확인한 결과, 간세포에 유리지방산처리를 하였을 때 p-IRS1, p-PI3K, p-AKT, p-AMPK, p-ACC의 단백질 발현이 현저하게 감소하였으나 탁시폴린과 수수 에탄올 추출물 처리 시 이 단백질들의 발현이 유의적으로 증가하였다. 인슐린 신호전달과 관련된 단백질들의 발현 변화가 마이크로 RNA-195발현 조절을 통한 것인지 알아보기 위하여 유사체 및 저해제를 처리하여 확인하였다. 마이크로 RNA-195 유사체를 형질주입시킨 간세포에서p-IRS1, p-PI3K, p-AKT, p-AMPK, p-ACC의 단백질 발현량은 유리지방산 처리군보다 유의적으로 감소되었다. 그러나 탁시폴린과 수수 에탄올 추출물의 처리는 유사체에 의한 마이크로 RNA-195의 발현 증가를 억제시켜 p-IRS1, p-PI3K, p-AKT, p-AMPK, p-ACC의 단백질 발현량을 유의적으로 증가시켰다. 또한 마이크로RNA-195 저해제를 형질주입시킨 간세포에서p-IRS1, p-PI3K, p-AKT, p-AMPK, p-ACC의 단백질 발현량은 유리지방산 처리군보다 유의적으로 증가되었으며, 탁시폴린과 수수 에탄올 추출물의 처리군에서는 저해제 처리군보다도 마이크로 RNA-195의 발현을 더욱 감소시켜 인슐린 신호전달과 관련있는 단백질들의 발현량을 증가시켰다. 따라서, 탁시폴린과 수수 에탄올 추출물이 마이크로 RNA-195의 억제를 통해 인슐린 저항성을 개선했다는 것을 알 수 있으며, 이러한 항당뇨 기작연구를 통해 마이크로 RNA의 조절은 효과적인 당뇨 개선 방법이 될 수 있을 것으로 판단된다. 수수 추출물에 함유되어 있는 페놀산은 하이드록시 벤조산 유도체와 하이드록시 시나믹산 유도체로 분류된다. 하이드록시 벤조산 유도체에는 벤조산, 하이드록시 벤조산, 프로토카테츄익산, 갈산 등이 있다. 이 유도체들은 하이드록실 그룹의 개수와 위치에 따라 항산화 활성이 달라질 수 있으며, 3개의 하이드록실 그룹을 가진 갈산은 가장 우수한 항산화 활성을 가지고 있다고 보고되어 있다. 본 연구에서는 하이드록시 벤조산 유도체인 벤조산, 하이드록시 벤조산, 프로토카테츄익산, 갈산의 항당뇨 활성을 알아보고 관련 마이크로 RNA를 탐색하여 그 작용기작에 대해 알아보고자 하였다. 차세대 염기서열 분석을 이용하여 마이크로 RNA 서열을 시퀀싱하였고 인슐린 저항성과 관련있는 마이크로 RNA-1271을 도출하였다. 갈산은 α-glucosidase 저해 활성, 라디컬 소거능과 환원력 측정시 가장 우수한 활성을 보였다. 유리지방산으로 간세포에 인슐린 저항성을 유도하고 하이드록시 벤조산 유도체를 처리하였을 때 포도당 흡수 증가와 활성산소종 생성 감소에 있어서 갈산의 효과가 가장 우수하였다. 인슐린 신호전달과 관련된 단백질들의 발현을 확인한 결과, 간세포에 유리지방산처리를 하였을 때 p-IRS1, p-PI3K, p-AKT, p-FOXO1의 단백질 발현량이 현저하게 감소하였으나 갈산 처리시 이 단백질들의 발현이 유의적으로 증가하였다. 인슐린 신호전달과 관련된 단백질들의 발현변화가 마이크로 RNA-1271발현 조절을 통한 것인지 알아보기 위하여 유사체 및 저해제를 처리하여 확인하였다. 마이크로 RNA-1271유사체를 형질주입시킨 간세포에서p-IRS1, p-PI3K, p-AKT, p-FOXO1의 단백질 발현량은 유리지방산 처리군보다 유의적으로 감소되었다. 그러나 갈산의 처리는 유사체에 의한 마이크로 RNA-1271의 발현 증가를 억제시켜 p-IRS1, p-PI3K, p-AKT, p-FOXO1의 단백질 발현량을 유의적으로 증가시켰다. 또한 마이크로 RNA-1271 저해제를 형질주입시킨 간세포에서p-IRS1, p-PI3K, p-AKT, p-FOXO1의 발현량은 유리지방산 처리군보다 유의적으로 증가되었으며, 갈산 처리군에서는 저해제 처리군보다 마이크로 RNA-1271의 발현을 더욱 감소시켜 인슐린 신호전달과 관련있는 단백질들의 발현량을 증가시켰다. 또한 갈산은 포도당 대사와 관련된 PEPCK, G6Pase, PGC-1α, GLUT2의 mRNA 발현을 마이크로 RNA-1271억제를 통해 긍정적으로 조절하였다. 따라서, 갈산은 효과적인 항당뇨 소재로서 사용할 수 있을 것이라 생각한다. 따라서, 본 연구를 통해 수수 에탄올 추출물과 수수에 함유되어 있는 다양한 페놀화합물들은 마이크로 RNA를 효과적으로 조절하고, 인슐린 신호전달 및 포도당대사에 관련된 단백질이나 mRNA의 발현에 관여함을 알 수 있다. 마이크로 RNA가 관여된 항당뇨 기작을 밝힘으로써 대사질환 예방 및 치료에 대한 새로운 방법을 제시하였고, 식품을 소재로 사용함으로써 보다 안전한 기능성 소재 개발에 도움이 될 수 있을 것이라 생각한다. Cereal grains have recently increased interest because of their potential health benefits. Phenolic compounds are widely found in daily foods such as cereals, legumes, fruits, and vegetables and they provide medicinal and biological properties. Type 2 diabetes is associated with the generation of an oxidative stress, which may damage the action of insulin. The liver is an important insulin target organ and plays a crucial role in the regulation of glucose metabolism. MicroRNAs (miRs) play vital roles in many biological processes. The miRs targeting insulin signaling molecules participate actively in the pathogenesis of hepatic insulin resistance. Therefore, the regulation of the insulin signaling pathway and miR expression is important for the alleviation of type 2 diabetes. In this study, the effect of cereal grains on anti-diabetic activity in relation to antioxidant activity was investigated. Three variety of sorghum was the highest in total phenolic content (TPC). ABTS radical scavenging activity, DPPH radical scavenging activity, and reducing power showed similar results. Sorghum ethanol extracts were evaluated higher inhibitory activities against α-glucosidase than acarbose. Sorghum extracts strongly enhanced glucose consumption, whereas extracts from italian millets, black bean, and oat exhibited no visible effect on glucose consumption. Antioxidant and anti-diabetic activities showed high correlation. Our data indicate that sorghum ethanol extract may be a useful anti-diabetic and antioxidant material. Taxifolin in sorghum ethanol extract was identified by UPLC-ESI-Q-TOF/MS. Based on the MS/MS spectra and the comparisons of the proposed fragment pattern in the previous studies, the compound was identified as taxifolin. Glucose uptake in HepG2 cells was measured to investigate the effects of taxifolin and sorghum ethanol extract on free fatty acid (FFA)-induced hepatic insulin resistance. FFA treatment decreased glucose uptake by 16.2% compared to the control; however, taxifolin and sorghum ethanol extract increased the glucose uptake. The expression of p-IRS1, p-PI3K, p-AKT, p-AMPK, and p-ACC levels was increased by taxifolin and sorghum ethanol extract in FFA-induced hepatocytes. The expression level of miR-195 increased by FFA. However, treatment of taxifolin and sorghum ethanol extract dose-dependently decreased the miR-195 expression, compared to FFA treatment group cells. Taxifolin and sorghum ethanol extract enhanced p-IRS1, p-PI3K, p-AKT, p-AMPK, and p-ACC protein expression levels via suppressing of miR-195 level in miR-195 mimic or inhibitor transfected cells. These data indicate that taxifolin and sorghum ethanol extract attenuated the insulin resistance by regulation of the miR-195, suggesting that taxifolin and sorghum ethanol extract may be a useful anti-diabetic ingredient. This study analyzed the role of miRs, particularly miR-1271, in insulin resistance and, changes in miR expression in hepatocytes using hydroxybenzoic acid derivatives. The results showed that gallic acid (GA) improved insulin resistance, increased glucose consumption, and decreased reactive oxygen species FFA-induced HepG2 cells. GA inhibited the upregulation of miR-1271 induced by FFA and upregulated its targets such as p-IRS1, p-PI3K, p-AKT, and p-FOXO1, accompanied by the regulation of glucose metabolism genes. GA activated the insulin signaling pathway by directly reducing of miR-1271 expression in miR-1271 mimic-treated HepG2 cells. In miR-1271 inhibitor-treated cells, hepatic insulin resistance was improved by GA. Our results suggest that GA attenuates insulin resistance via the miR-1271/IRS1/PI3K/AKT/FOXO1 pathways and thus could be considered for the management of insulin resistance. Therefore, our study suggests that sorghum and its phenolics may be safe and effective functional ingredients for alleviation of miR-mediated insulin resistance.

조리방법에 따른 병풀의 항산화 활성과 기능성 성분 변화

박주형 충북대학교 2021 국내석사

This study compared and analyzed the cooking method of Centella asiatica (L.) with the cooking method of boiling, steaming, and the changes in antioxidant activity and functional components according to cooking time of 1 min, 5 min, 10 min, 30 min, and 60 min. As a result of analyzing triterpene, the main indicator of Centella asiatica (L.), the content of asiaticoside was high in boiling and steaming samples according to the cooking method, followed by madecassoside. The production of Madecassic acid and asiatic acid did not appear. The total polyphenol content of Centella asiatica (L.) was 175.7-277.0 mg GAE/100 g CW range according to the cooking method of Centella asiatica (L.) and the significance of boiling and steaming was recognized (p<0.05). The boiling method decreased and the steaming method did not show a significant difference in the content due to the increase in cooking time. The total flavonoid content was 22.7-120.9mg CE/100 g CW range, and the significance of boiling and steaming was recognized (p<0.05). The flavonoid content was decreased in boiling method, and the difference in content was not observed in steaming method according to the increase of cooking time. chlorophyll content of Centella asiatica (L.) increased in a boiling and in steaming, it decreased with increasing cooking time. B did not show any difference in content even when cooking time was increased in boiling, and it was sharply decreased when cooking time was 30 min in steaming. The protective effects of fibroblasts (Hs68 cells) induced by boiling and steaming were reduced from 10min in steaming and the protective effects were overall in boiling. Experimental results on endovascular cells (Ea.hy926 cells) and muscle cells (C2C12 cells) showed that the overall protective effect was protected from oxidative stress in boiling and steaming. The antioxidant activity of ABTS was 60.6-275.4 mg of Trolox Eq./100 g CW, which showed excellent ABTS radical removal ability compared to steaming and boiling. In addition, the antioxidant activity of DPPH was 16.7-186.7 mg of Trolox Eq./100 g CW range, and the steaming and boiling like ABTS showed a tendency to decrease from 10 min in boiling, and there was no significant difference in the cooking time in steaming. Reducing power of the yield was 30.9-123.9%, and the results of boiling and Steamin showed that the boiling tended to decrease compared to steaming, and the difference in the amount of steaming was not shown. Based on the results of the above study, both boiling and steaming, the cooking method of Centella asiatica (L.) can minimize the loss rate by 5 to 10 minutes depending on the cooking time, and use Centella asiatica (L.) between antioxidants and activities. 본 연구는 병풀의 boiling, steaming조리방법에 따른 시간대별의 Triterpene 성분 분석, 세포독성 및 보호효과, 항산화 활성 변화를 비교 분석하였다. 병풀의 Triterpene 성분 분석 결과 steaming 시료의 조리방법에서는 병풀의 주요 함유성분 중에서도 asiaticosside의 함량이 다른 성분들에 비해 높은 것으로 확인되었으며, 다음으로는 madecassoside 함량 순으로 확인하였으며, madecassic acid 및 asiatic acid 성분은 나타나지 않았다. 또한 boiling 조리방법에서도 steaming 과 동일하게 asiaticosside함량이 제일 높게 나타났으며, 다음으로 madecassoside 함량 순으로 확인되었으며 steaming 시료와 동일하게 madecassic acid 및 asiatic acid 성분은 나타나지 않았다. 총 폴리페놀 함량은 175.7-277.0 mg GAE/100 g CW 범위로 boiling 및 steaming의 유의성이 인정되었다. 조리방법에 따른 대조군인 raw(not cooking)와 폴리페놀 함량을 비교하면, boiling 방법에서는 감소하고 steaming 에서는 함량차이는 보이지 않았다. 또한 총 플라보노이드 함량은 22.7-120.9mg CE/100 g CW 범위로 boiling 및 steaming 의 유의성이 인정되었다. 플라보노이드 함량은 폴리페놀 함량 결과와 유사하게 boiling 방법에서 함량이 감소하였고, steaming에서는 함량차이는 보이지 않았다. 반면에 클로로필 함량은 클로로필 a의 boiling방법에서 증가하였고, steaming 에서는 조리시간이 증가함에 따라 감소하는 경향을 나타내었다. 클로로필 b는 boiling 방법에서는 조리시간이 증가하여도 함량차이는 보이지 않았고, steaming 에서 조리시간 30분 일 때 급격하게 감소하는 경향을 나타내었다. 세포독성 실험결과, boiling, steaming 방법에서 UVB에 의해 유도된 섬유아세포(Hs68 cells)의 자극에 대한 보호 효과는 steaming조리 방법 에서 조리시간 10분 부터는 보호효과가 감소하는 것으로 나타났으며, boiling 에서는 전체적으로 보호 효과가 나타냈다. 내혈관 세포(Ea. hy926 cells) 실험결과 및 근육세포(C2C12 cells)에 실험결과는 조리방법 인 boiling 와 steaming 시료에서는 전체적으로 산화 스트레스로 부터 보호하는 효과를 나타내었다. ABTS에 의한 항산화 활성은 60.6-275.4 mg of Trolox Eq./100 g CW 범위로 steaming 및 boiling 은 비교하여 ABTS 라디칼 소거능이 뛰어난 것으로 나타났다. 또한, DPPH에 의한 항산화 활성은 16.7-186.7 mg of Trolox Eq./100 g CW 범위로 ABTS와 같이 steaming 및 boiling 은 비교하였을 때 boiling에서 10 min 부터 감소경향을 나타내었고, steaming에서는 조리시간 증가하여도 함량 차이는 보이지 않았다. 환원력은 30.9-123.9% 범위로 boiling 및 steaming 조리방법의 실험결과로 boiling에 비해 steaming 에서 감소하는 경향을 보였으며, steaming 에서 조리 시간 증가하여도 함량 차이는 보이지 않았다. 이상의 연구 결과를 종합하여 검토하였을 때 boiling, steaming 조리 방법 중에서는 steaming 방법이 boiling 보다 효과적인 것을 알 수 있었으며, 두 조리 방법 모두 5~10분 조리시간을 이용하여 항산화 물질의 손실을 최소화 시킬 수 있을 것으로 판단된다.

감꼭지의 다양한 생리 활성

박정환 충북대학교 2022 국내박사

Diospyros kaki Thunberg has been mainly cultivated in Korea, Japan, and China. The calyx of Diospyros kaki Thunberg has been known to be effective for the treatment of obstinate hiccups for a long time in Korea medicine. In this review, we provide a summary of the latest pharmacological efficacy and clinical study of the calyx of Diospyros kaki Thunberg. Seven electronic databases, including those in English (PubMed), Korean (KMbase, KoreaMED, NDSL, OASIS), Chinese (CNKI), and Japanese (J-STAGE), were systematically searched. Twenty-five studies, including 21 pharmacological efficacy studies and 4 clinical study, were evaluated. The calyx of Diospyros kaki Thunberg showed antioxidant, anti-inflammatory, anticoagulant, anti-thrombotic, anti-cancer, antimicrobial, anti-genotoxic, tyrosinase inhibitory, elastase inhibitory, and diaphragm contraction inhibitory effect. Antioxidant components of the calyx of Diospyros kaki Thunberg were identified tannin, quercetin, catechin, gallic acid, and rutin. Also, the calyx of Diospyros kaki Thunberg was effective for treatment of omphalitis of newborn and intractable hiccups with serious diseases such as cancer in clinical studies. Alcohol consumption is customary in most cultures and alcohol abuse has a negative effect on the liver. In this study, the calyx of Diospyros kaki Thunberg was evaluated for antioxidative activity using HepG2 cells treated with alcohol-induced oxidative stress and quantitatively analyzed for phenolic acids and flavonoid concentration by LC-MS/MS. The extract of the calyx of Diospyros kaki Thunberg showed no significant toxicity to HepG2 cells till a concentration of 10 μg/mL was reached and displayed increased cell viability compared to the 3% ethanol-treated group. The extract inhibited the production of reactive oxygen species (ROS) at all concentrations. Besides, the extract inhibited malondialdehyde (MDA) production at all concentrations and the inhibitory effect was higher than the positive control (esculetin 100 μM). Also, the extract increased the concentration of glutathione (GSH). The major phenolic acids and flavonoid in the extract were vanillic acid (729.01±42.48 μg/g) and catechin (1,894.89±105.10 μg/g), respectively. This study shows that the calyx of Diospyros kaki Thunberg can be used as a functional material that exhibits antioxidative and hepatoprotective effects. H2O2 can diffuse across cellular membranes and increase the intracellular ROS levels, especially in muscles. This study investigated the protective effects of Diospyros kaki Thunberg using C2C12 myoblasts of H2O2-induced oxidative stress. Cell viability and cytotoxicity were determined by MTT assay. The level of ROS, MDA, and GSH were measured by using optical properties. The calyx of Diospyros kaki Thunberg extract showed no toxicity to C2C12 myoblasts until 20 μg/mL concentration and increased cell viability compared to the H2O2 treated group. The calyx of Diospyros kaki Thunberg extract inhibited the production of ROS and MDA at all concentrations. In addition, the calyx of Diospyros kaki Thunberg extract increased the concentration of GSH. This study provides that the calyx of Diospyros kaki Thunberg can be used as a potential material that exhibit antioxidative and protective effects on H2O2-induced oxidative stress in C2C12 Myoblasts. Ultraviolet exposure causes photoaging such as pigmentation, dryness, and wrinkles of the skin. This study investigated the protective effects of the calyx of Diospyros kaki Thunberg using Human foreskin fibroblast (Hs68) cells against UVB-induced photoaging. The calyx of Diospyros kaki extract was not toxic to Hs68 cells until 20 μg/mL concentration and noticeably increased cell viability compared to the UVB (30 mJ/cm2) treated group. The calyx of Diospyros kaki Thunberg extract had very strong activity in inhibiting ROS production. In addition, the extract significantly reduced the production of MMP-1 and MMP-3. In particular, at 10 and 20 μg/mL concentration, the extract showed a very strong inhibitory effect than the 100 μM of ascorbic acid, which is a positive control. The collagen levels tended to increase markedly by the extract. The calyx of Diospyros kaki Thunberg can be used as herbal material that exhibits antioxidant activity and protective effects against photoaging. The oxidative stress causes damage to vascular endothelial cells, which is an important mediator in the pathogenesis of hypertension. The antioxidant and antihypertensive activity the calyx of Diospyros kaki Thunberg extract was measured in this study. At concentrations of 2.5, 5, and 10 μg/mL of the calyx of Diospyros kaki Thunberg extract, cytotoxicity was not observed, and cell viability was increased compared to EA.hy926 cells treated with H2O2. The calyx of Diospyros kaki Thunberg extract significantly inhibit the production of ROS. In addition, the extract increased nitric oxide production and showed angiotensin converting enzyme inhibitory activity. Our results provided experimental evidence that the calyx of Diospyros kaki Thunberg extract exhibit antioxidant activity and antihypertensive activity in vascular endothelial cells. Therefore, this study suggests that the calyx of Diospyros kaki is potentially functional bioactive candidates for human health.

Protective Effect of Edible Insects against H₂O₂-induced Oxidative Damages in Skeletal Muscle Cells

정택영 충북대학교 2021 국내석사

Oxidative stress caused by the excessive production of reactive oxygen species (ROS) is an important initiating factor in various chronic diseases. ROS are generated by muscle contraction and physical exercise in skeletals. Continuous high-intensity exercises generate high levels of ROS, which promote a skeletal muscle contractile dysfunction resulting in muscle damages and/or muscle fatigues. Edible insects contatin abundant protein of a high biological value with good amino acid profiles and high levels of digestibility. Furthermore, edible insects are a potential source of a variety of micronutrients such as minerals and vitamins. Edible insects are known to exhibit antioxidant, hepatoprotective, and antidiabetic activities. However, few investigations have reported the protective effect of edible insects on H₂O₂-induced oxidative stress in skeletal muscle cells. The aim of this study was to investigate the protective effect of edible insects on H₂O₂ induced oxidative stress in C2C12 muscle cells. The contents of total polyphenolics and flavonoids in edible insects were significantly higher in Oxya chinensis sinuosa. Edible insects showed significant protective effect on H₂O₂-induced cell damages in C2C12 cells. Furthermore, H₂O₂-induced ROS generation was markedly attenuated by pretreatment of edible insects, demonstrating that edible insects are to be a potent ROS scavenger. Edible insects significantly decreased lactate dehydrogenase and creatine kinase activity in media. Moreover, edible insects increased the protein and mRNA levels of MyHC, a marker of myscle differentiation, and increased the size of the diameter of myotubes, thereby affecting the the increase of muscle hypertrophy. These results indicate that edible insects protect skeletal muscle cells from H₂O₂-induced oxidative damages and therefore have the potential for use in health functional foods.

병풀 유래 주요 Triterpenes의 생리활성 비교

임진영 충북대학교 2022 국내석사

Centella asiatica L. (C. asiatica), a perennial plant belonging to the Apiaceae, has been widely reported about its potential antioxidant, antimicrobial, cytotoxic, neuroprotective and other activities: especially skin calming, wound healing and anti-inflammatory. Biological activities of C. asiatica are very much related to its properties and mechanism of action of the plant’s bioactive constituents, pentacyclic triterpenes, namely the triterpenic acid (asiatic acid and madecassic acid) and the triterpenic saponin (asiaticoside and madecassoside), The objective of this study is to compare various biological activities of asiatic acid (AA), asiaticoside (AD), madecassic acid (MA) and madecassoside (MD) in skin fibroblasts, macrophages, hepatocytes and endothelial cells induced different stimulations. In UVB-induced Hs68 cells, AA had the highest protective effect against UVB-irradiation. Pretreatment with triterpenes attenuated the level of reactive oxygen species (ROS), the expression of malondialdehyde (MDA) and glutathione (GSH) depletion induced by UVB. In addition, Pretreatment with triterpenes effectively inhibited the generation of MMPs induced by UVB, thereby increasing the intracellular collagen level. In LPS-induced RAW264.7 cells, the production of NO, TNF-α, and IL-6, which are indicators of LPS-induced inflammation, were significantly reduced by treatment with triterpenes. In t-BHP-induced HepG2 cells, pretreatment with triterpenes significantly improved the cell viability reduced by t-BHP and attenuated the level of ROS and ALT. In EA.hy926 cells, AA had the highest protective effect against H2O2. Pretreatment with triterpenes attenuated the level of ROS, the expression of MDA and GSH depletion. Also, the production of NO was significantly increased only in AD and MD. As a result of statistical analysis of the biological activities with principal component analysis (PCA) and hierarchical clustering analysis (HCA), it was found that AA was a variable with the greatest influence on overall variable and was a component showing remarkable activities in Hs68 and HepG2 cells. 병풀은 만병통치약으로 사용되어졌던 약용식물로, 피부 진정, 항염증, 항산화 등의 활성이 잘 알려져 있으며, 이러한 활성의 주요 active compound는 pentacyclic triterpenes으로 알려져 있다. 본 연구는 triterpene의 4가지 성분인 asiatic acid(AA), asiaticoside(AD), madecassic acid(MA) 및 madecassoside(MD)의 피부세포, 대식세포, 간세포, 혈관내피세포에서의 활성을 비교하고자 하였다. 다양한 세포에서 triterpene의 보호효과를 보기위해 세포 모델을 설정하였다. Hs68 cell에 UVB 자극을 주어 피부세포에서 자외선에 대한 보호효과를 보았고, Raw264.7 cell에 LPS 자극을 주어 염증에 대한 효과를 보았다. HepG2 cell에 tBHP 자극을 주어 간세포에서 산화적 스트레스에 대한 보호효과를 보았으며, EA.hy926 cell에서 H2O2 자극을 주어 혈관내피세포에서 산화적 스트레스에 대한 보호효과를 보았다. 주성분분석을 실시한 결과, 대부분의 항목들이 제1주성분에 크게 기여하고 있고, 이들은 서로 가까이 분포하여 두 성분 간의 상관관계가 높은 것으로 여겨진다. 계층적 군집분석 결과, A 그룹에서는 네 가지 성분들이 모두 효과가 우수했던 혈관내피세포의 GSH 실험이 해당되었으며, C그룹은 AA의 높은 활성이 뚜렷하게 보이는 그룹으로, 대부분 피부세포와 간세포에서의 측정항목들이 해당되는 것으로 나타났다.

Effect of Sucrose Treatment on Functional Compounds and Bioactivities in Mung Bean (Vigna radiata L.) Sprouts

유재은 충북대학교 2021 국내석사

Sprouts are attracting a lot of attention due to their various health effects. Especially, mung bean sprouts are known to be rich in bioactive compounds such as vitamins and flavonoids. Elicitation is good strategy for induction and enhancement of secondary metabolite biosynthesis in plants. To enhance bioactive compounds in plants, the treatment of elicitor has been proven to be a useful method. Therefore, this study was investgated the effects of sucrose treatments with different concentrations on the growth profiles, nutritional quality, antioxidant and antidiabetic activities of germinated mung bean sprouts. Mung bean seeds were germinated at 25°C in darkness for 4 days and sprayed with solutions containing different concentrations of sucrose. The results showed that sucrose treatments inhibited growth of mung bean sprouts. Treatments with sucrose resulted in an increases in the amount of functional components such as vitamins, four C-glycosylflavones, and rutin and phytosterols. In addition, polyphenolic and flavonoid contents of sprouts treated with sucrose were five times higher than those of controls, respectively. Sucrose treatments increased tyrosine aminotransferase, phenylalanine ammonia-lyase and glutamate dehydrogenase activities resulting in increases in the flavonoid and γ-aminobutyric acid contents. HepG2 cells were pretreated with mung bean sprouts with different concentrations sucrose and then hepatotoxicity was stimulated by 3% ethanol. Mung bean sprout extracts pretreatment significantly inhibited the leakage of AST and ALT in HepG2 cells without appreciable cytotoxic effects. We also found that mung bean sprouts extracts pretreatment significantly decreased free fatty acid-induced tumor necrosis factor-α (TNF-α) in HepG2 cells. The mung bean sprout extracts also increased glucose uptake. These results suggest that 3% sucrose treatment had a better influence on improving hepatoprotective effect, antidiabetic activities and food quality of mung bean sprouts than distilled water. 두류에 대한 기능성과 이용성에 대한 국민적 관심이 증대되면서 두과작물 종자를 이용한 연구가 활발히 진행되고 있다. 다양한 기능성과 영양성분이 풍부한 새싹 채소에 대한 관심 역시 증가하고 있다. 특히 녹두 새싹은 비타민, 무기질, 미네랄 등의 영양성분과 기능성 성분인 플라보노이드가 풍부하여 세계적으로 많은 소비가 이루어지고 있다. 녹두 새싹의 추출물은 녹두 종자 추출물에 비해 총 페놀 함량, 총 플라보노이드 함량, DPPH 라디컬 소거능 및 폐암 세포주와 위암 세포주에 대한 항암활성이 유의적으로 더 높다고 보고되어져있다. 식물면역활성제 (Elicitor)는 식물에서 이차대사산물의 생합성을 유도하고 증가시킬 수 있는 좋은 전략으로 최근 많은 연구가 이루어 지고 있다. 따라서 본 연구에서는 비생물학적 elicitor인 sucrose의 처리가 녹두 새싹의 성장과 영양학적 품질 및 항산화 활성에 미치는 영향을 확인하고자 하였다. 녹두 새싹은 씨앗을 25℃에서 4일 동안 암 발아 시켰으며 재배기에서 증류수, 1%, 2%, 3% sucrose를 함유한 용액을 분무하며 암발아 시켜주었다. 그 결과 sucrose의 처리는 성장을 저해하는 경향을 보였으나 기능성 성분의 함량은 총 폴리페놀의 경우 3% sucrose 처리군에서 control에 비해 1.29 배 높았으며 총 플라보노이드는 1.53 배 높았다. 또한 sucrose 처리 시 비타민, 플라보노이드, 카로티노이드 및 phytosterol과 같은 기능성 성분의 증가를 확인하였다. Sucrose의 농도가 증가 할수록 osmoprotectant인 proline과 soluble sugar의 함량이 증가 되었는데 이는 삼투 스트레스에 대항하기 위해 생성된 물질이라고 판단된다. 이러한 elicitor처리에 의한 플라보노이드의 함량 증가는 합성 경로의 Key enzyme인 Tyrosine ammonia-lyase(TAL) 와 Phenylalanine ammonia-lyase(PAL)의 활성 증가에 따른 것임을 확인하였다. 또한, γ-aminobutyric acid (GABA)함량의 증가는 glutamate dehydrogenase (GAD)활성 증가에 따른 것임을 확인하였다. 녹두 새싹 추출물의 항당뇨 효과를 알아보기 위해 α-amylase 및 α-glucosidase 저해 활성을 평가한 결과 sucrose 농도가 증가된 녹두 새싹 일수록 저해능이 증가되었다. HepG2세포에서 지방산 자극으로 인슐린 저항성을 유도한 뒤 glucose consumption과 TNF- α의 발현량을 측정하였다. 그 결과 3% sucrose를 처리하여 재배한 녹두 새싹 추출물 처리군에서 가장 높은 glucose consumption과 가장 낮은 TNF- α의 발현량을 보여 항 당뇨에 효과를 나타내었다. 녹두 새싹 추출물의 간보호 효과를 알아보기 위해 alcohol 자극을 준 뒤 녹두 새싹 추출물을 처리하여 ALT, AST 발현량을 측정하였다. Sucrose처리하여 재배한 녹두 새싹 추출물에서 sucrose의 농도의존적으로 보호효과를 나타내었으며 3% sucrose 처리 재배군에서 가장 높은 활성을 보였다. 연구의 결과 3% sucrose 처리는 성장을 저해 하지만 기능성은 향상시킬 수 있는 편리한 수단으로 판단되며, elicitor가 처리된 녹두 새싹은 다양한 분야의 소재로 활용될 수 있을 것으로 생각된다.

The Protective Effects of GABA and Germinated Brown Rice Treated with CaCl2 against H2O2 Induced Oxidative Damage in Myoblasts

최현정 충북대학교 2020 국내석사

Brown rice, containing endosperm, embryo and bran, is nutritionally more complete and provides phytochemicals with health-promoting relevance as γ-oryzanol, γ-aminobutyric acid (GABA). GABA is a well-known non-protein amino acid which exists widely in both plants and animals. Several health benefits of GABA have been reported e.g. lower blood pressure and blood cholesterol, greater kidney and liver activity, inhibition of cancer cell proliferation and stimulation of cancer cell apoptosis. Given their health benefits and commercial value, the production of germinated brown rice with enhanced nutritional and functional compounds is desirable. Elicitation is a good strategy to induce physiological changes and stimulates defense or stress-induced responses in plants. The elicitor treatments trigger the synthesis of phytochemical compounds in vegetables. However, few investigations has reported the effect of GABA against oxidative stress in myoblasts and the effect of calcium chloride on the GABA contents and biological activities of germinated brown rice. Therefore, this study investigated the protective effect of GABA against H2O2 induced oxidative stress in myoblasts and the effect of calcium chloride as an abiotic elicitor on the nutritional and functional composition as well as antioxidant activities in brown rice during germination. The results revealed that GABA markedly inhibited oxidative stress in C2C12 cells. GABA attenuated the expression of reactive oxygen species (ROS) and glutathione (GSH) depletion. Moreover, GABA significantly developed NADPH quinone oxidoreductase (NQO-1) and heme oxygenase 1 (HO-1). Also, GABA observably increased nuclear factor erythroid 2-related factor (Nrf2) in nucleus. And treatment with various concentrations of calcium chloride resulted in an increase in the amounts of nutritional components such as GABA, total phenolic contents, and total flavonoid contents. Calcium chloride treatment increased glutamate decarboxylase and phenylalanine ammonia-lyase activities resulting in an increase in the levels of phenolic compounds. Moreover, treatment of C2C12 cells with germinated brown rice protected against oxidative damage by modulating reactive oxygen species production and glutathione generation. These results suggest that calcium chloride treatment could improve the nutritional composition and antioxidant effects of germinated brown rice. In conclusions, the GABA may attenuate the H2O2 induced oxidative stress in myoblasts through regulation of Nrf2 signaling pathway, and germinated brown rice with calcium chloride treatment could be used as an ingredient as well as dietary supplement in the formulation of convenience and functional food.