Evaluation of Economic Aspects and Antioxidant Activity of Botanical Extracts

Bae, hyemin 국민대학교 일반대학원 2021 국내석사

현재 인구의 연령이 전 세계적으로 고령화 사회에 진입함에 따라 각종 문제로 한국인의 인구가 급속도로 고령화되면서 노인문제가 사회문제로 대두되고 있다. 그 결과 고령화 소비자의 분포가 증가하여 고령화 친화적인 산업의 필요성이 높아졌다. 이러한 노화는 신체 구조와 생리 기능에 퇴행성 변화를 가져와 신체 민감도를 높이고 각종 질병의 발생률을 높인다. 산화스트레스에 의한 세포 손상은 노화 이론의 가장 중요한 메커니즘 중 하나이다. 현재 항산화물질로 알려진 물질은 대부분 식물성 물질이며, 활동을 나타내는 주성분은 2차 대사물 페놀화 화합물인 것으로 밝혀졌다. 식물에 대한 항산화제 연구는 오래전부터 진행되어 왔으며, 생물학적인 방어와 면역 조절을 통해 질병을 예방하거나 노화를 지연시키는 효과가 있는 것으로 알려져 있다. 본 연구는 항노화 기능성 물질을 발굴, 선정하기 위해 마련된 것으로, 15가지 약용식물 추출물의 항산화 활성이 조사되었다. 경제적 측면을 비교하고 15개 식물 추출물의 물과 에탄올 추출물의 항산화 활동을 조사하여 총 폴리페놀 함량과 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, 2,2'-azino-di-2-ethyl-benzthiazoline sulphonate (ABTS) radical scavenging activity 를 측정하였다. 15가지 식물 추출물의 경제성 평가에서는 측백, 하수오, 두충, 산사, 황정 순으로 다음과 같은 5가지의 물질이 가장 경제적으로 우수하였다. 추출 수율에서는 천문동 (38.6%, 물 추출물), 현삼 (10.5%, 물 추출물), 천문동 (10.4%, 에탄올 추출물), 황정 (9.0%, 에탄올 추출물), and 고본 (8.6%, 물 추출물) 순으로 수율이 좋은 것으로 나타났다. 또한 물 추출물보다 에탄올 추출물에서의 항산화능이 더 높게 평가되었다. 에탄올로 추출한 15가지 식물추출물을 동일한 농도인 0.5 mg/ml 로 폴리페놀 실험을 진행해 다음과 같이 측백 (21.94 mg/g extract), 정공등 (20.93 mg/g extract), 산사 (15.73 mg/g extract), 가시오갈피 (15.41 mg/g extract), 부처손 (12.47 mg/g extract) 순으로 폴리페놀 함량이 높은 5가지 물질을 나타내었다. 에탄올로 추출한 15가지 식물추출물의 DPPH 실험에서는 측백 (0.03 mg/ml, SC50), 정공등 (0.04 mg/ml, SC50), 산사 (0.12 mg/ml, SC50), 가시오갈피 (0.14 mg/ml, SC50), 부처손 (0.55 mg/ml, SC50) 순으로 다음과 같은 5가지 추출물이 가장 항산화능이 높다고 평가되었다. 마지막으로 에탄올로 추출한 15가지 식물추출물의 ABTS 실험에서는 측백 (0.04 mg/ml, SC50), 정공등 (0.05 mg/ml, SC50), 산사 (0.12 mg/ml, SC50), 가시오갈피 (0.24 mg/ml, SC50), 부처손 (1.00 mg/ml, SC50) 순으로 다음과 같은 5가지 추출물이 가장 항산화능이 높다고 평가되었다. 결론적으로, 경제성 평가와 항산화 실험을 통해 항산화능이 가장 뛰어난 측백, 정공등, 산사, 가시오갈피, 부처손 순으로 위와 같은 5가지 물질을 선정하였다. 따라서 본 연구에서는 이러한 5가지 물질이 앞으로 항산화 및 항노화 기능성 소재로 기대됨을 시사한다. The growing population around the world has entered an aging society. As the Korean’s population ages rapidly due to various problems, the problem of the elderly has emerged as a social issue. As a result, the distribution of elderly consumers has increased, raising the need for senior-friendly industries. Aging brings degenerative changes to body structure and physiological function, increasing body sensitivity, and incidence of various diseases. Therefore, the most important risk factor that causes chronic diseases is aging. The aging theory begins with the reactive oxygen species (ROS) produced by metabolism, accumulating damage throughout one’s lifetime. Cell damage by oxidative stress is one of the most important mechanisms in the aging theory. Thus, the effective defense system against oxidative stress is thought to play an important role in cell damage caused by ROS continuously produced in energy metabolism, and the factors involved in the defense mechanism for oxidative stress are of great interest. Currently, most of the substances known as antioxidants are plant materials, and the main ingredient with activity are the secondary metabolites called phenolic compounds. Antioxidant research on plants has long been underway, and it is known to prevent diseases or delay aging by controlling the biological defense and immune function. This study was designed to find out and select anti-aging functional materials. The antioxidant activities of 15 medicinal plants extracts were evaluated. Economic aspects were compared and antioxidant activities of water and ethanol extract of 15 botanical extracts were investigated by measuring their total polyphenol content (TPC), 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, and 2,2'-azino-di-2-ethyl-benzthiazoline sulphonate (ABTS) radical scavenging activity. The results showed that Platycladus orientalis, Pleuropterus multiflorus, Eucommia ulmoides, Crataegus pinnatifide, and Polygonatum sibiricum were assessed to be appropriate. The botanical extracts in order of extraction yield were Asparagus cochinchinensis (38.6%, water solvent), Scrophularia buergeriana (10.5%, water solvent), Asparagus cochinchinensis (10.4%, ethanol solvent), Polyonatum sibiricum (9.0%, ethanol solvent), and Ligusticum tenuissimum (8.6%, water solvent). In addition, the antioxidant activities of ethanol extracts were higher than that of the water extracts. Thus, the antioxidant properties of ethanol-extracted botanical samples were compared by experiment. The TPC of the ethanol extract at 0.5 mg/mL was the highest in Platycladus orientalis (21.94 mg/g extract), followed by Erycibe obtusifolia (20.93 mg/g extract), Crataegus pinnatifide (15.73 mg/g extract), Eleutherococcus senticosus (15.41 mg/g extract), and Selaginella tamartscine (12.47 mg/g extract). Among the 15 botanical extracts obtained by ethanol extraction, the plant with the highest DPPH antioxidant levels was Platycladus orientalis (0.03 mg/mL, SC50), followed by Erycibe obtusifolia (0.04 mg/mL, SC50), Crataegus pinnatifide (0.12 mg/mL, SC50), Eleutherococcus senticosus (0.14 mg/mL, SC50), and Selaginella tamartscine (0.55 mg/mL, SC50). Based on the ABTS of 15 botanical extracts obtained by ethanol extraction, the order of plants with high antioxidant levels was Platycladus orientalis (0.04 mg/mL, SC50), Erycibe obtusifolia (0.05 mg/mL, SC50), Crataegus pinnatifide (0.12 mg/mL, SC50), Eleutherococcus senticosus (0.24 mg/mL, SC50), and Selaginella tamartscine (1.00 mg/mL, SC50). In conclusion, five extracts, such as Platycladus orientalis, Erycibe obtusifolia, Crataegus pinnatifide, Eleutherococcus senticosus, and Selaginella tamartscine, were finally selected as substances that are expected to be antioxidant and anti-aging functional materials through economic evaluation and antioxidant experiments.

Effects of fermented soybean extracts on differentiation of 3T3-L1 preadipocytes and glucose utilization : 3T3-L1지방세포 모델에서 발효 대두 추출물이 지방세포 분화와 당 이용능력에 미치는 영향 연구

Hwang, Ji Won 고려대학교 일반대학원 2015 국내석사

Obesity is a serious public health problem worldwide for continuously increasing the morbidity and mortality of a variety of acute and chronic diseases. This study aimed to examine the antiobesity effect of soybean extracts fermented by Bacillus subtilis MORI and to elucidate the mechanisms underlying such effects using 3T3-L1 preadipocytes. The 3T3-L1 preadipocytes were induced to differentiate in the presence of fermented soybean extracts for 7 days and the cells were treated with either DW (Distilled water) or fermented soybean extracts at various concentrations (0-100 g/ml) during adipogenesis. Oil red O staining, triglyceride (TG) contents, glycerol release and glucose uptake level were measured on differentiated adipocytes. Levels of adipogenesis-related protein expression were evaluated using immunoblotting. Lipid accumulations as measured by Oil Red O staining were significantly inhibited by fermented soybean extracts treatment in a dose dependent manner. In addition, intracellular TG contents and glycerol release level were dramatically decreased by fermented soybean extracts treatment, which were accompanied by the decreased expression of CCAAT element binding protein α (C/EBPα) protein expression and the increased phosphorylation of acetyl-CoA carboxylase (ACC) protein expression. Also, fermented soybean extracts treatment increased the expression of glucose transporter 4 (GLUT4) along with a significant increase in glucose uptake into the adipocytes based on results of a 2-deoxy-D-[3H] glucose uptake assay. Several studies showed that C/EBPα and peroxisome proliferator-activated receptor γ (PPARγ) are known as a cross-regulator on adipogenesis, which partly differs with our results showing that fermented soybean extracts act as an agonistic ligand of PPARγ, thereby PPARγ contributing to glucose uptake through improving insulin sensitivity. Our findings suggest that fermented soybean extracts may suppress the differentiation of 3T3-L1 adipocytes and greatly facilitated glucose uptake into the adipocytes. Based on these results, fermented soybean extracts may have potential to develop therapeutic agent for the prevention or treatment of obesity and related metabolic disorders.

붉은 찹쌀 (자광찰벼) 추출물의 산화적 스트레스 방어 효과 및 세포사멸 유도에 관한 연구

지희연 건국대학교 대학원 2005 국내박사

한글초록:자광찰벼 (붉은색 찰벼), 화선찰벼 (하양색 찰벼), 및 일품벼 (하양색 메벼) 에탄올 분획의 산화적 스트레스 보호효과와 간암 세포의 세포사멸에 대한 효과를 검정하기 위한 몇 가지 실험을 수행하였다. 1. 쌀 추출물의 에탄올 분획 중에 함유된 성분 분석 및 함량측정을 위한 실험을 수행하였다. TLC를 사용하여 에탄올 분획 중의 페놀성분을 확인 후, 폴린-데니스 방법으로 총 페놀 함량을 측정하였다. HPLC를 사용하여 시료 중의 페놀성 물질을 동정하였다. 그 결과로부터, 자광찰벼는 화선찰벼와 일품벼에 비해 페놀 성분을 월등히 많이 함유함을 규명하였다.2. DPPH에 의한 자유라디칼 소거능 평가 결과로부터, 페놀성 화합물 표준품과 화선찰벼 및 일품벼 추출물에 비하여 자광찰벼 추출물의 항산화 활성이 월등히 높게 관찰되었다. 3. 과산화수소에 의하여 생성된 자유라디칼은 H4ⅡE 세포에 산화적 손상을 초래하며, 이러한 손상에 기인된 세포사멸에 대하여 화선찰벼와 일품벼 추출물은 방어효과가 없었으나 자광찰벼 추출물은 효과적인 방어작용을 보여 주었다. 또한, FACS를 사용한 항산화 효과를 검정한 결과, 자광찰벼 혹은 화선찰벼 추출물 처치는 과산화수소에 의하여 유도된 DCF 형광 발생 강도를 감소시켰다. 이는 자광찰벼 혹은 화선찰벼 추출물이 항산화 효과를 나타내고 있음을 제시한다.4. 쌀 시료 추출물의 세포 사멸에 미치는 영향을 평가한 결과, 자광찰벼, 화선찰벼, 및 일품벼 추출물 처치는 농도 및 시간 의존적으로 간암 세포인 H4ⅡE 세포 사멸을 촉진하였으며, 특히 자광찰벼 추출물이 가장 강한 세포 사멸 효과를 나타내었다. 즉, 100 μg/mL에서 24, 48, 및 72 시간 처치 시, 각각 69.5%, 57.2%, 및 46.1% 정도의 세포 생존률을 보였다. 5. 세포 사멸 양상을 조사하기 위하여 FACS 분석을 시행한 결과, 자광찰벼 추출물은 화선찰벼와 일품벼 추출물과는 달리 24, 48, 및 72 시간 동안의 시료 처치 시 42.0%, 59.6%, 및 70.7% 정도로 아포토시스에 의한 세포 사멸 효과를 보였다. 6. 세포 사멸 양상을 보다 자세히 확인하기 위하여 아포토시스에 의한 세포 사멸을 반영하는 생체 지표인 PARP 분해정도를 웨스턴 불로팅 방법을 사용하여 검정하였다. 그 결과 화선찰벼와 일품벼 추출물과는 달리 자광찰벼 추출물 처치에 의하여 24, 48, 및 72 시간 후에 생성된 분자량 89 kDa의 PARP 분해산물의 농도가 증가되었다. 그러므로 자광찰벼 추출물은 아포토시스에 의한 세포 사멸 효과를 발휘하는 것으로 사료된다. 7. 자광찰벼, 화선찰벼, 및 일품벼 추출물을 에탄올과 병용 투여한 생체 실험에서 쌀 추출물의 성장에 대한 촉진 혹은 억제 작용을 평가하기 위하여 실험동물의 체중변화에 대한 영향을 측정한 결과, 에탄올 투여군에 비하여 자광찰벼 추출물 투여군에서 현저한 체중증가가 관찰되었다. 이는 자광찰벼 추출물 처치는 에탄올에 의하여 유도된 실험동물의 성장억제를 반전시키는 경향이 있는 것으로 사료된다. 8. 혈액 중의 GOT, GPT를 측정한 결과, 쌀 추출물 투여가 에탄올에 의하여 유도된 간 손상을 막지는 못하는 것으로 사료된다. 또한 혈액 생화학 지표 중, 혈중 총 콜레스테롤 수치, 고밀도 지질단백 수치 및 저밀도 지질단백 수치는 에탄올 처치군과 시료 추출물 처치군 간에 유의적인 차이는 없었다. 따라서 본 실험에 사용한 시료 추출물 투여 용량은 에탄올에 의한 간 손상을 보호함에 효과가 없는 것으로 사료된다. 9. 간, 뇌 및 이자 조직에서의 지질 과산화물 생성을 측정한 결과, 에탄올 처치 군에 비해 쌀 추출물 처치 군에서의 뇌조직 중의 MDA 함량이 다소 감소되었다. 또한 쌀 추출물 처치는 간 조직 중의 LOOH 생성을 어느 정도 감소시켰다. 따라서 시료 쌀 추출물은 일부 생체조직에서의 에탄올에 의하여 유발된 산화적 스트레스에 대하여 방어작용이 다소 있는 것으로 사료된다. 10. 간, 뇌 및 이자 조직의 GST 활성도를 측정한 결과, 간에서는 화선찰벼 추출물 처리가, 뇌에서는 일품벼 추출물 처리가, 이자에서는 자광찰벼 추출물 처리가 에탄올에 의해 감소된 GST 활성도를 상당 수준 정상화 시켰다. 또한, 간 조직의 GPx 활성도는 쌀 추출물 처치에 의하여 약간 증가하였다.결론적으로 본 실험에 사용된 자광찰벼, 화선찰벼 및 일품벼 추출물의 에탄올 분획이 가지는 항산화 효과와 세포 사멸 효과를 H4ⅡE 세포에서 검정한 결과, 적색을 띤 자광찰벼 추출물의 에탄올 분획이 과산화수소에 의하여 유도된 산화적 스트레스를 가장 현저하게 억제하였으며, 아포토시스에 의한 세포 사멸을 가장 효과적으로 유도하였다. 또한, in vivo 실험에서 평가한 쌀 추출물의 항산화효과는 실험에 사용된 조직과 측정한 항산화 지표의 종류에 따라 다소 가변적이기는 하나, 개괄적으로 고려할 때 뇌 및 간에서의 산화적 스트레스에 대한 쌀 추출물의 보호효과가 있는 것으로 사료된다. 향 후, 시료의 분획 방법, 유효 주성분 분석 및 동정, 투여 용량, 실험동물의 종차 등을 고려하여 보다 광범위하고 집중적인 후속 연구를 수행한다면 쌀 함유 유효성분들의 생리활성에 관한 보다 명확한 효과를 규명할 수 있을 것으로 사료된다 영문초록:Experiments were performed to investigate the effects of ethanol fraction of three different rice (Oryza sativa L. var. japonica) grain (Jakwangchalbyeo, red-pericarp glutinous rice; Hwasunchalbyeo, white-pericarp glutinous rice; Ilpumbyeo, white- pericarp non-glutinous rice) extracts on the protection of oxidative stress and the apoptotic cell death. Various phenolic substances were identified to explain the biological activity of rice extracts. For this, the amounts of total phenolic substances and several phenolic compounds in the ethanol fraction of three different rice grains were measured by TLC and HPLC methods. Among them, Jakwangchalbyeo extract contained the highest amount of total phenolic substances. Antioxidant activities (radical scavenging ability) in ethanol fraction assessed by DPPH method were 87.5% (Jakwangchalbyeo), 45.0% (Hwasunchalbyeo) and 50.0% (Ilpum- byeo), suggesting that the Jakwangchalbyeo extract has the highest radical scavenging activity. In addition, Jakwangchalbyeo extract has better radical scavenging activity than various phenolic standard compounds. Further assessment of antioxidant activities of ethanol fraction of rice grains extracts were made with MTT cell viability assay and FACS analysis in H4ⅡE cell that is challenged with hydrogen peroxide. Hwasunchalbyeo extract and Ilpumbyeo extract did not show any significant protective effects on the H2O2-induced oxidative stress in H4ⅡE cells, and Jakwangchal- byeo extract improved the cell viability up to 82% and 74% at concentration of 100 μg/mL for 5 h and 24 h treatment, respectively. DCF fluorescence intensity curve was left-shifted by application of Jakwangchalbyeo extract or Hwasunchalbyeo extract in the presence of hydrogen peroxide as compared with the curve obtained by treatment with Jakwangchalbyeo or Hwasunchalbyeo extract alone. On the other hand, DCF fluorescence was right- shifted by treating the cell with Ilpumbyeo extract in the presence of H2O2 as compared with that treated with Ilpumbyeo extract alone. These results suggested that Jakwangchalbyeo or Hwasunchalbyeo extracts have an antioxidant activity to a certain extent. Effects of rice grain extracts on cell death was evaluated with MTT cell viability assay. Treatment of cells with ethanol fraction of rice grain extracts induced cell death in a concentration- and time- dependent manner. Especially, Jakwangchalbyeo extract, at concentration of 100 μg/mL, significantly reduced the cell viability up to 69.5%, 57.2% and 46.1% within 24 h, 48 h and 72 h, respectively. Hwasunchal- byeo extract slightly decreased the cell viability with time (48 h and 72 h) at concentration of 25 μg/mL, 50 μg/mL, and 100 μg/ mL. As for Ilpumbyeo extract, slight reduction in the cell viability was observed with time (48 h and 72 h) at concentration of 10 μg/mL, 25 μg/mL, and 50 μg/mL, respectively. Moreover, cell viability decreased continuously up to 70.1%, 67.8% and 60.9% upon treating with 100 μg/mL for 24 h, 48 h and 72 h, respectively. FACS analysis was done to characterize the cell death pattern caused by the treatment of rice grain extract. Flow cytometric diagrams showed an apoptotic cell death profile. Particularly, apoptotic cell death was clearly observed with time after treating the cell with the Jakwangchalbyeo extract, representing the percentage of cell death of 42.0% at 24 h, 59.6% at 48 h, and 70.7% at 72 h, respectively. However, other extracts did not cause any effects on the apoptotic cell death. PARP degradation pattern was considered as an important biomarker of the apoptosis. In western blotting analysis, degradation of 116 kDa PARP molecule was observed with concomitant formation of 89 kDa product at 24 h, 48 h, and 72h after treating the cells with Jakwangchalbyeo extract, indicating the that cell death is undergone by apoptotic process.Separate experiments were also performed to evaluate antioxidant activities of each extract of Jakwangchalbyeo, Hwasunchalbyeo, and Ilpumbyeo in the ethanol-induced oxidative stress model in vivo. Each group of 12 male Sprague Dawley rats were treated orally with physiological saline (C), glucose (G), ethanol (E), ethanol-dissolved Jakwangchalbyeo extract (JE), ethanol-dissolved Hwasunchalbyeo extract (HE), and ethanol- dissolved Ilpumbyeo extract (IE) for 2 weeks, respectively. Results were as follows; significant weight loss was observed in the group E as compared with the group C. Significant gain in body weight was seen in group JE as compared with group E. But there was no significant increment in body weight in groups HE and IE as compared with group E. With regards to the effects on the changes in blood biochemical index, the ethanol-induced liver damages was not prevented from the application of rice extracts, and the levels of total cholesterol, TG, HDL and LDL were not affected by the treatment of rice extracts. Considering the lipid peroxide generation profiles in the liver, pancreas, and brain tissue, levels of MDA in brain tissue was reduced by the application of rice extracts. Levels of hepatic LOOH formation apparently decreased with the treatment of rice extracts. These results are supposed to indicate that rice extracts are able to prevent the ethanol-induced oxidative stress in brain and liver to a certain degree. The ethanol-induced decrement in tissue GST activity was slightly restored and hepatic GPx activity increased slightly by adminstration of rice extracts. In conclusion, red-pericarp Jakwangchalbyeo extract as compared with other rice extracts exerted significant inhibitory effects on the hydrogen peroxide-induced oxidative stress and also induced apoptotic cell death in the H4ⅡE cells. Although antioxidant activities of rice extracts was not resolved throughly in vivo, our results suggested that in general rice extracts have certain protective effects on the ethanol-induced oxidative stress in brain and liver. Further studies should be performed more throughly to evaluate the biological activity of rice extracts. For this, changes in the route of administration, the way of sample preparation, dosage adjustment, and selection of animal species should be considered in detail

Innovative approaches for bioactive metabolite-rich green tea extracts: Metabolite profiling and bioactivities

프로티바 라니 다스 전남대학교 2019 국내박사

식물 기반의 천연 식이 활성 대사물질은 그들의 건강 증진 효과 기능을 이용한 식품 개발 방향으로 많은 관심을 받고 있습니다. 녹차를 추출하는 방법으론 Ultrasonication extraction (UE)와 agitation extraction (AE)을 사용하였고 추출 조건을 다르게 하여 이들이 녹차 추출물의 생체 대사물질 함량에 미치는 영향을 조사하였습니다. 녹차 추출물은 분말 형태의 녹차를 물로 추출하며, 추출 방법은 UE와 AE를 이용하였고 3가지 온도 (60, 70, 80 ℃)에서 5-30분간 추출하여 제조되었으며 대조군으로는 일반 추출법 (conventional extraction method-CE)을 이용한 추출물을 사용하였다. 이후, 녹차 추출물의 화학적 조성 및 물리적 특성을 조사하였다. UE 및 AE 으로 추출한 녹차 추출물 모두에서 대사산물을 얻을 수 있는 가장 효율적인 조건은 80 ℃에서 20 분 동안 추출하는 것으로 확인되었다. UE와 AE를 이용한 녹차 추출물은 CE를 이용한 추출물과 비교했을 때 polyphenols (3배), catechins (2배), flavonoids (2배) 및 항산화 활성이 유의적으로 (p <0.05) 높았다. 뿐만 아니라 총 카페인, 총 유리 아미노산, 총 비타민 C 및 총 가용성 고형분 함량은 CE를 이용한 추출물에 비해 나머지 두 방법을 이용한 추출물 내의 함량이 유의적으로 (p <0.05) 높았다. 다음으로, 최적 추출 조건을 찾기 위해 녹차의 입자 크기, 이용하여 녹차 분말의 particle size, constant thermal treatment, 추출물 여과 후 필터에 잔류한 입자의 미세 구조를 분석했다. 녹차 추출물은 UE, AE, 열수 추출 (hot water extraction-HWE) 및 CE를 사용하여 분쇄된 녹차 잎 (green tea ground leaf-GTGL) 및 녹차 잎 (green tea whole leaf-GTWL) 2가지 시료를 이용하여 제조되었다. 대사 물질의 추출량은 GTGL에서 GTWL보다 유의하게 높았으며 (p <0.05), 추출 방법에 따른 대사물질 추출량은 UE ≈ AE> HWE> CE의 순서로 높게 나타났다. Scanning electron microscopy(SEM)을 이용한 여과 후 녹차 잔류물의 미세 구조 분석 결과, UE 및 AE 추출법은 녹차 잎 셀 구조의 파괴에 지대한 영향을 미치고, 이는 용매 - 매트릭스 간의 상호 작용을 증가시켰다. 다음으로, 최적으로 판단된 추출 조건을 이용하여 UE, AE 및 CE에 의해 얻어진 유리, 에스테르화, 글리코실 및 세포벽 결합 형태의 녹차 페놀 대사산물을 UPLC-DAD-QToF/MS를 이용해 측정하였다. 녹차 추출물에서 총 22개의 플라보노이드와 11개의 페놀산을 포함한 33개의 페놀릭 화합물이 나타났다. 플라보노이드는 flavanols 및 flavonols로 분류되었으며, 페놀산은 hydroxybenzoic acid, hydroxybenzoylquinic acid, hydroxycinnamic acid 및 hydroxycinnamoylquinic acid으로 분류되었다. 주요 플라보노이드는 catechins과 같은 flavanols였고 주요 페놀산은 gallic acid였다. CE (3283 mg/100 g)에서 얻은 것보다 UE (7261 mg / 100 g)와 AE (6765 mg / 100 g)에서 얻은 녹차 추출물의 총 플라보노이드의 total free forms (모든 화합물의 합)이 유의적으로 높았다. 대조적으로, 플라보노이드의 총 세포벽 결합형은 CE (1860 mg / 100 g)에서 UE (131 mg / 100 g) 및 AE (338 mg / 100 g)에 비해 더 높았다. Gallic acid는 세포벽 결합 형태로 주로 확인되었는데, 이는 CE에서 유의적으로 높았으며 (유리 지방산과 에스테르화 형태는 UE와 AE에서 더 높았다) 또한 UE 및 AE 추출법으로 얻은 녹차 추출물은 항산화 활성이 높았으며 대사산물 함량과 양의 상관관계를 보였다. 그리하여, 최적의 추출 조건을 이용하여 UE, AE, HWE 및 CE에서 얻은 녹차 추출물의 휘발성 화합물 및 비 페놀 대사산물 (아미노산, 유기산 및 당 화합물)을 gas chromatography와 결합 된 SPME-GC-MS 및 HPLC 분석을 수행하였다. HWE 및 CE보다 UE 및 AE에서 휘발성 물질 및 비 페놀 대사산물의 수율이 유의적으로 더 높게 관찰되었다 (p <0.05). 휘발성 물질의 경우 UE, AE, HWE 및 CE가 각각 212, 201, 103 및 65개의 화합물을 나타냈다. 총 아미노산 (합계)은 54.57, 54.35, 27.11 및 12.67 (mg / 100g)으로 확인되었고 총 유기산은 각각 UE, AE, HWE 및 CE에서 5.96, 6.19, 3.81 및 1.68 (mg / 100g)으로 측정되었다. 질소 함유 화합물을 제외한 휘발성 물질은 L-theanine, sucrose, malic acid, and catechins의 수율과 높은 상관관계를 보였다. 그 후, 체외 α-amylase 및 α-glucosidase 억제에 대한 항 당뇨병 활성 및 UE 및 AE 추출법에 의해 얻어진 대사산물이 풍부한 녹차 추출물의 항산화 활성을 CE와 비교 하였다. α-amylase 저해를 통한 항 당뇨병 활성은 CE, UE 및 AE에 의해 각각 44, 92 및 93 (%)로 확인되었고, α-glucosidase 억제는 48, 95 및 94 (%)로 측정되었다. 유사하게, DPPH, ABTS, FRAP, ORAC, H2O2 억제 및 metal chelating activities을 이용하여 평가 된 항산화활성은 CE에 비해 UE 및 AE에서 높은 활성을 보였다. catechins과 페놀산의 차이점을 분석하기 위해 표준 EGCG, ECG, EGC 및 GA를 같은 과정으로 평가했다. 표준 화합물의 항산화 및 항 당뇨병 가능성은 ECG ≈ EGCG> GA ≈ EGC로 측정되었다. 마지막으로, UE로 추출한 생체 대사 물질이 풍부한 bioactive metabolite-rich functional green tea extracts (BMF-GTE)의 세포 에너지 및 포도당 대사에 미치는 영향을 immortialized brown adipocytes (BAC)와 differentiated C3H/10T1/2 cells (10T)인 adipocytes 및 C2C12 myoblast에서 분화된 골격근 세포와 고지방식 쥐를 이용하여 평가했다. The thermoregulatory marker인 Ucp1 및 Fgf21 유전자 발현은 분화 된 BAC 및 10T 세포에서 BMF-GTE 처리에 의해 증가되었다. 열 생성 신호 분자인 p-PKA-substrate 및 p-p38, the lipolysis marker인 p-HSL은 분화 된 BAC 세포의 BMF-GTE에 의해 활성화되었다. 또한, 분화 된 C2C12 세포에서 Glut4, Ppargc1-α 및 Ctrp-15 유전자 발현과 같은 the glucose uptake marker는 BMF-GTE에 의해 증가되었다. 쥐를 이용한 실험에서 BMF-GTE 처리 이후 고지방식으로 유도된 인슐린 저항성과 체중이 유의적으로 (p <0.05) 감소하였다. 결과적으로, 이 연구는 녹차 추출물의 bioactivities와 phenolic 및 non-phenolic metabolite profiles를 고려한 유용한 데이터베이스를 제공한다. UE와 AE는 일반적인 추출 방법과 비교하여 녹차 대사 물질의 최대 함량을 추출 할 가능성은 비슷했다. 또한 증가된 생체 활성은 세포 주 및 쥐를 이용한 실험에서 대사산물 수율에 의해 확인되었으며 이러한 추출법이 녹차 추출물의 기능을 향상시키는 데 중요한 역할을 한다는 것을 시사한다. 이 발견은 기능성 음료, 식품, 화장품, 의약품, 부가가치 제품을 개발하거나 인간의 건강과 웰빙을 위한 표적 생체 활성 화합물을 추출하기 위해 식품, 제약, 화장품 산업에 활용될 것이다. Plant-based natural dietary bioactive metabolites are gaining utmost research interest for the development of functional foods because of their health-promoting effects. The effects of ultrasonication extraction (UE) and agitation extraction (AE) techniques on green tea metabolite yields under different extraction conditions were investigated for preparing bioactive-metabolite rich functional green tea extracts. First, aqueous green tea leaf powder extracts were prepared by UE and AE at different temperatures (60, 70, and 80°C) and times (5–30 min), where the conventional extraction method (CE) served as the control. The chemical compositions and physical properties of the green tea extracts were investigated. For both UE and AE techniques, the highest extraction efficiency to release green tea metabolites was observed at 80°C for 20 min. The significantly (p < 0.05) higher yields of polyphenols (three-fold), catechins (two-fold), and flavonoids (two-fold) along with higher antioxidant activity were observed with UE and AE than that of CE. In contrast, total caffeine, total free amino acids, total vitamin C, and total soluble solid contents were also significantly (p < 0.05) increased using either technique compared to those with CE. Next, the extraction strategy of green tea metabolites was evaluated with respect to particle size of samples, constant thermal treatment, and microstructural analysis of solid residues using the optimum extraction condition (80°C, 20 min). The aqueous green tea extracts were prepared from green tea ground leaf (GTGL) and green tea whole leaf (GTWL) using UE, AE, hot water extraction (HWE) and CE. The yield of all metabolites was significantly higher (p < 0.05) in GTGL than that of GTWL and were in the following order: UE ≈ AE > HWE > CE. The microstructural analysis of green tea solid residues by scanning electron microscopy revealed that the UE and AE techniques had potential effects on the remarkable destruction of cell structure, which increased the solvent-matrix interactions. Following, green tea phenolic metabolites in their free, esterified, glycosylated, and cell wall-bound forms obtained by UE, AE, and CE using optimum extraction condition were characterized using UPLC-DAD-QToF/MS analysis. In total, thirty-three phenolic compounds were characterized in green tea extracts, including twenty-two flavonoids and eleven phenolic acids. The flavonoids were characterized by their sub-classifications such as flavanols and flavonols, and phenolic acids were sub-classified as hydroxybenzoic acid, hydroxybenzoylquinic acid, hydroxycinnamic acid, and hydroxycinnamoylquinic acid. Flavanols such as catechins were the dominant flavonoids and gallic acid was the major phenolic acid. Total free forms (sum of all compounds) of flavonoids were significantly higher (p < 0.05) in green tea extracts obtained by UE (7261 mg/100 g) and AE (6765 mg/100 g) compared to those obtained by CE (3283 mg/100 g). In contrast, total cell wall-bound forms of flavonoids were higher in CE (1860 mg/100 g) compared to those by UE (131 mg/100 g) and AE (338 mg/100 g). Gallic acid was mainly quantified in its cell wall-bound form, which was significantly higher (p < 0.05) in CE, whereas its free and esterified forms were higher in UE and AE. Furthermore, green tea extracts obtained by UE and AE techniques had higher antioxidant activities, showing a positive correlation with their metabolite contents. Therefore, volatile compounds and non-phenolic metabolites (amino acids, organic acids, and sugar compounds) of aqueous green tea extracts obtained by UE, AE, HWE, and CE using optimum extraction condition, were investigated by solid-phase microextraction coupled to a gas chromatography-mass spectrometry (SPME-GC-MS) and high performance liquid chromatography (HPLC) analysis, respectively. Significantly higher (p < 0.05) volatiles and non-phenolic metabolite yields were also observed in UE and AE than HWE and CE. For volatiles, UE, AE, HWE, and CE released 212, 201, 103, and 65 compounds, respectively. Total amino acids (sum) were determined as 54.57, 54.35, 27.11, and 12.67 (mg/100g); total organic acids were determined as 5.96, 6.19, 3.81, and 1.68 (mg/100g) in UE, AE, HWE, and CE, respectively. Volatiles except nitrogen-containing compounds had higher positive correlations with L-theanine, sucrose, malic acid, and catechins yields. Then, the antidiabetic activity against in-vitro α-amylase and α-glucosidase inhibition and the antioxidant activities of metabolite-rich green tea extracts obtained by UE and AE techniques were compared with CE. The anti-diabetic activity through α-amylase inhibition was determined as 44, 92, and 93 (%) and through α-glucosidase inhibition was determined as 48, 95, and 94 (%) by CE, UE, and AE, respectively. Similarly, antioxidant activities assessed using DPPH, ABTS, FRAP, ORAC, H2O2 inhibition, and metal chelating activities showed significant potential by UE and AE than that by CE. To analyze the apparent relationship between catechins and phenolic acids, standard EGCG, ECG, EGC, and GA were assessed for similar activities. The antioxidant and antidiabetic potential of standard compounds was determined as ECG ≈ EGCG > GA ≈ EGC. Finally, we evaluated the effects of bioactive metabolite-rich functional green tea extracts (BMF-GTE) obtained by UE on cellular energy and glucose metabolism in adipocytes which are immortialized brown adipocytes (BAC) and differentiated C3H/10T1/2 cells (10T) and skeletal muscle cells differentiated from C2C12 myoblast and high-fat-diet mice. The thermoregulatory marker Ucp1 and Fgf21 gene expressions were stimulated by BMF-GTE treatment in differentiated BAC and 10T cells. The thermogenesis signaling molecules including p-PKA-substrate and p-p38 and the lipolysis marker p-HSL were also activated by BMF-GTE in differentiated BAC cells. In differentiated C2C12 cells, the glucose uptake marker as Glut4, Ppargc1-α and Ctrp-15 gene expression were increased by BMF-GTE as well. The significant decreases (p < 0.05) of high fat-diet induced insulin resistance and body weight were observed with BMF-GTE treatment in mice study. Taken together, this study provides a useful database considering the phenolic and non-phenolic metabolite profiles of aqueous green tea extracts with their bioactivities. The UE and AE had the similar potentiality to extract maximal content of green tea metabolites than conventional extraction methods. In addition, the increased bioactivities were confirmed with their metabolite yields in both cell lines and mice study, suggesting that a potential extraction strategy plays crucial role to improve the functionalities of green tea infusion. This finding will be utilized by the food industry/pharmaceutical industry/cosmetic industry to develop any functional/nutraceutical beverages, foods, cosmetics, drugs, value-added products and/or to extract the target bioactive compounds for human good health and well-being.

백장미 꽃잎 페놀화합물의 추출 조건 최적화 및 근아세포 보호 효과 평가

최재권 충북대학교 2020 국내박사

우리 사회가 경제적으로 발전하고 고령화됨에 따라, 노화 및 관련 질병에 관한 연구 또한 증가하고 있다. 근육의 감소는 노화 증상 중 가장 눈에 띄게 나타나는 현상으로, 나이가 들어감에 따라 자연스럽게 발생한다. 선행 연구에 따르면, 백장미(Rosa spp.) 꽃잎 추출물은 강력한 항산화 능력과 항염증 효과뿐만 아니라 추출물에 다량 함유된 gallic acid(3,4,5-trihydroxybenzoic acid)가 근아세포(C2C12)의 분화와 증식을 촉진한다고 보고 되었다. 따라서 백장미 꽃잎 유래의 추출물이 근아세포에 대한 잠재적 항노화 효능이 있을 것으로 기대된다. 그러나 백장미 꽃잎에서의 경제적 추출 조건 및 효능을 극대화 할 수 있는 추출방법, 추출물에 함유된 생리활성 성분을 분석하는 연구는 현재까지 매우 미비한 실정이다. 특히, 근아세포내에서 항산화, 항염증, 근육관련 단백질의 발현에 대한 연구는 아직까지 수행되지 않았다. 따라서 본 연구의 목적은, 백장미 꽃잎에서 페놀화합물의 추출조건을 최적화하고, 생리활성 성분을 분리 및 동정하며, 산화 및 염증이 유도된 쥐 C2C12 근아세포에서 추출물의 세포 보호효과를 평가하는 것이다. 백장미 꽃잎으로부터 자유라디칼(DPPH)에 대한 높은 항산화 효능이 있는 추출물의 추출조건을 최적화하기 위해 반응표면 분석법(Response surface methodology)을 사용하여, 에탄올 농도(X1), 추출 온도(X2) 및 추출 시간(X3)을 세 가지 독립 변수로 설정하고 종속변수의 효과를 예측했다. 항산화 활성을 갖는 페놀화합물의 추출조건 최적화 연구결과, 에탄올 농도 42 %(X1), 추출 시간 80분(X3) 및 추출 온도 75 ℃(X2)의 독립변수 조건을 확인 할 수 있었다. 반면 플라보노이드 화합물의 최적 추출조건은 에탄올 농도 41 %(X1), 추출 시간 119 분(X3) 및 추출 온도 75 ℃(X2) 였다. 이러한 추출조건 아래에서, 페놀화합물 및 플라보노이드 화합물의 예측된 함량은 각각 243.5 mg GAE/g 및 19.93 mg CE/g이었다. 백장미 꽃잎에서 잘 알려지지 않은 생리활성 성분을 분리 및 동정하기 위해, 극성이 다른 유기용매로 분획을 실시하고, 분취용 고속액체크로마토그래피(Preparative high-performance liquid chromatography, Prep HPLC)로 정제하여 DPPH 라디칼 소거능, 산화질소(NO) 생성 억제 활성을 평가하였다. 분석 결과, 에틸아세테이트(EA) 분획물이 가장 뛰어난 DPPH 라디칼 소거능과 산화질소(NO) 생성 억제 활성을 보였고, 에탄올(EtOH) 추출물 또한 우수한 생리활성을 나타냈다. 에틸아세테이트(EA) 분획물과 에탄올(EtOH) 추출물에서 각각 분리, 정제된 2가지 화합물(F4, F11)의 구조를 핵자기공명기(1H-NMR, 13C-NMR) 및 질량분석기(Gas chromatography–mass spectrometry, GC-MS)로 분석하여 확인한 결과, 에탄올(EtOH) 추출물에서 분리, 정제된 화합물(F4)은 methyl gallate (methyl 3,4,5-trihydroxy benzoate)이었고, 에틸아세테이트(EA)에서 분리, 정제된 화합물(F11)은 gallic acid(3,4,5-tri-hydroxybenzoic acid)로 확인되었다. 백장미 꽃잎 추출물(WRPE)은 강력한 항산화 및 항염증 효과를 갖는 것으로 알려져 있지만, 생리활성 특성은 추출방법 및 적용된 추출조건의 다양한 매개변수에 영향을 받는다. 백장미 꽃잎 에탄올 추출물(WRPE-EtOH)은 고온가압 추출물(WRPE-HTHP) 및 효소분해 추출물(WRPE-enzyme)과 비교하였을 때, 가장 효과적인 항산화 (DPPH소거능, TBARS생성억제)효과 및 항염증 활성(NO 생성억제, SOD2와 iNOS 발현 억제)을 나타내었다. 골격근량과 강도를 감소시키는 근육 손실의 주요 원인은 반응성 산소종(reactive oxygen species, ROS)에 의한 세포 내인성 산화스트레스 및 사이토카인(cytokine)에 의해 유발된 염증으로 알려져있다. 그러므로, 내인성 산화스트레스 및 염증으로부터 WRPE-EtOH가 C2C12근아세포의 생존력, 지질산화 및 근손실 지표 단백질의 생성 억제 효과가 있는지 확인하기 위해MTT검정과 TBARS(Thiobarbituric acid reactive substances) 분석, 근육파괴단백질인MuRF-1(muscle RING-finger protein-1) 및 Atrogin-1의 발현 정도를 평가하였다. WRPE-EtOH는 세포독성(cytotoxicity) 없이 종양괴사인자(tumor necrosis factor alpha, TNF-α)에 의한 세포사멸을 농도 의존적으로 감소시켰다. 유사하게, 반응성 산소종(reactive oxygen species, ROS)에 의한 세포 손상 또한 추출물의 농도가 증가함에 따라 유의적으로 회복되었다. 이러한 결과는 WRPE-EtOH가 항산화 및 항염증 활성과 관련된 TNF-α 및 ROS에 의한 손상에 대해 보호 효과가 있음을 뒷받침한다. 또한, 유비퀴틴리가제인 MuRF-1 및 Atrogin-1 단백질의 발현은 추출물 처리 후 현저하게 감소하였는데, 이는WRPE-EtOH가 항산화 및 항염증 기작을 통해 근손실 억제 효과가 있음을 시사한다. 이번 연구를 통해 페놀화합물의 추출 특성과 최적 조건이 확인되었고, 페놀화합물을 함유한 추출 수율은 이전 연구(15%)보다 약2.5 배 높은 40%로 증가되었다. 또한 그 동안 명확하게 보고되지 않았던 백장미 꽃잎 추출물의 주요 생리 활성 성분으로 gallic acid와 methyl gallate를 핵자기공명기 및 질량분석기로 최초로 확인하였다. 더불어 WRPE-EtOH는 C2C12근아세포 내에서 강력한 항산화 및 항염증 활성 기작을 통해 MuRF-1 및 Atrogin-1 단백질 발현을 억제함으로써 잠재적인 근손실 억제 물질임을 시사하였다. 결과적으로 뛰어난 항산화 활성과 근아세포에서 항노화 효과를 가진 백장미 추출물은 향후 건강기능식품 시장의 활성화와 공중보건 개선에 기여할 것으로 예상한다. Recently, researches on aging and aging-related diseases are increasing, as our society has been developed economically and has dominant aging population. As symptoms of aging, muscle loss is the most visible phenomena and it is expressed naturally with aging. Based on previous researches, white rose petal extracts (WRPE) were known to contain large amount of gallic acid that induces myoblast differentiation and promotes proliferation. Therefore, it was expected that the extracts have anti-aging potentials in muscular fiber due to their ability to exert potent antioxidative and anti-inflammatory effects. However, researches on the bioactive components and economical extraction conditions of white rose petals were very lacking. In addition, studies have not been conducted to assess their activities to inhibit inflammation and muscle-related protein degradation in cells. Therefore, the purposes of this research were to optimize the extraction conditions of phenolic compounds from white rose petals, to identify the compounds having biological activities in the extracts, and to evaluate their protective effects in mouse myoblasts (C2C12). To investigate the optimal conditions to extract anti-oxidative compounds from white rose petals, response surface methodology based on a central composite design was used by comparing the effects of three independent variables: ethanol concentration (X1), extraction temperature (X2), and extraction time (X3). The estimated optimal conditions to obtain phenolic compounds with antioxidant activities were as follows: ethanol concentration of 42 % (X1), extraction time of 80 min (X3), and extraction temperature of 75 ℃ (X2). Meanwhile, the estimated optimal conditions to obtain flavonoid compounds with antioxidant activities were an ethanol concentration of 41 % (X1), extraction time of 119 min (X3), and an extraction temperature of 75 ℃ (X2). Under these conditions, predicted response values for the phenolic and flavonoid contents were 243.5 mg gallic acid equivalent (GAE)/g dry mass and 19.93 mg catechin equivalent (CE)/g dry mass, respectively. Bioactive compounds in Rosa spp. with white petals were not well known, and thus isolation and identification of those compounds were necessary. Therefore, chromatographic techniques, solvent fractionation, and evaluations of biological activities, such as antioxidant capacity and nitric oxide scavenging activity, were applied to obtain the solvent extracts and their fractions. As result, ethyl acetate (EA) fractions from white rose petals showed the highest antioxidant capacity and nitric oxide scavenging activity. Ethanol (EtOH) extracts also showed high biological activities in all parts. The structure of the purified compound was determined by spectroscopic methods including ultraviolet, proton nuclear magnetic resonance (1H-NMR), carbon-13 nuclear magnetic resonance (13C-NMR), and mass spectrometry (MS). As results, two phenolic compounds (F4, F11) were isolated and identified from the secondary fractionation of EA fractions and EtOH extracts. Based on data obtained from 1H-NMR and 13C-NMR with molecular weight (MW) of MS, the two compounds isolated from EtOH extracts and EA fractions were determined as methyl gallate (methyl 3,4,5-trihydroxy benzoate) and gallic acid (3,4,5-trihydroxybenzoic acid), respectively. WRPE have potent antioxidant and anti-inflammatory activities, meanwhile, the biological properties are known to be influenced by various parameters of employed extraction techniques. It was confirmed WRPE-EtOH is the extracts with the most effective antioxidant and anti-inflammatory activities compared to WRPE-high temperatures-high pressures (HTHP) or WRPE-enzyme. Both endogenous oxidative stress by reactive oxygen species (ROS) and inflammation induced by cytokines have been accepted as the major causes of muscle loss that include loss of skeletal muscle mass and strength. Therefore, the protective effects of WRPE-EtOH were examined in mouse myoblast (C2C12) by using various methods such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, thiobarbituric acid reactive substances (TBARS) levels, viability of C2C12 myotubes, and expression levels of muscle RING-finger protein-1 (MuRF-1) and Atrogin-1 protein. According to the result of evaluating the antioxidant, anti-inflammatory and protective effects of WRPE-EtOH in C2C12 myoblasts, WRPE-EtOH decreased tumor necrosis factor-α (TNF-α) triggered muscle atrophy without cytotoxicity in a dose-dependent manner. Similarly, ROS-induced cell damage was recovered significantly with increasing concentrations of WRPE-EtOH. Also, WRPE-EtOH decreased TNF-α-induced oxidative damage. These results support that WRPE-EtOH has protective effect against the damages by TNF-α and ROS, which is related to its antioxidant and anti-inflammatory activity. The total protein content was increased in a dose-dependent manner of WRPE-EtOH treatment. In addition, the expression of ubiquitin ligases MuRF-1 and Atrogin-1 proteins significantly decreased after WRPE-EtOH treatment. These results suggest that the ubiquitin ligase inhibition effect of WRPE-EtOH is associated with its protective effect in C2C12 cells. Taken together, in this study, the optimum extraction conditions of white rose petals for polyphenol compounds and flavonoids were determined; the yield of extracting solid contents containing polyphenols was increased to 40%, which was 2.5 times higher compared to 15% of previous studies. In addition, the main biologically active compounds of WRPE were newly identified as gallic acid and methyl gallate. Furthermore, the WRPE-EtOH was effective in protecting C2C12 cells from muscle loss with its strong antioxidative and anti-inflammatory activity. The results of this study demonstrate that WRPE are strong antioxidative and anti-aging substance to be used as helath promoting materials in the future.

抽出方式에 따른 防風通聖散이 脂肪細胞 代謝에 미치는 影響

李相旼 尙志大學校 大學院 2008 국내박사

Objectives : The purpose of this study is to investigate the effects of Bangpungtongsung-san extracts on the preadipocytes proliferation, of 3T3-L1 cell line. lipolysis of adipocytes in rat's epididymis and localized fat accumulation of porcine by extraction methods(alcohol and water). Methods : Diminish 3T3-L1 proliferation and lipogenesis do primary role to reduce obesity. So, 3T3-L1 preadipocyte and adipocytes were performed on cell cultures, and using Sprague-Dawley rats for the lipogenesis, and treated with 0.01-1㎎/㎖ Bangpungtongsung-san Extracts depend on concentrations. Porcine skin including fat tissue after treated Bangpungtongsung-san Extracts by means of the dosage dependent variation are investigated the histologic changes after injection of these extracts. Results : Following results were obtained from the 3T3-L1 preadipocyte proliferation and lipolysis of adipocyte in rats and histologic investigation of fat tissue. 1. Bangpungtongsung-san extracts were showed the effect of decreased preadipocyte proliferation on the high dosage(1.0 ㎎/㎖). 2. Bangpungtongsung-san extracts were showed the effect of decreased the activity of glycerol-3-phosphate dehydrogenase(GPDH) on the high dosage(1.0 ㎎/㎖) and Specially, alcohol extract of Bangpungtongsung -san was clear as time goes by high concentration. 3. Bangpungtongsung-san extracts were showed tries to compare the effect of lipolysis, alcohol extract of Bangpungtongsung-san on the high dosage(1.0 ㎎/㎖) was observed the effect is higher than water extract. 4. Investigated the histological changes in porcine fat tissue after treated Bangpungtongsung-san extracts, we knew that water extract of Bangpungtongsung-san was showed the effect of lipolysis on the high dosage(10.0 ㎎/㎖) and alcohol extract of Bangpungtongsung-san was showed significant activity to the lysis of cell membranes in all concentration. Conclusion : These results suggest that Bangpungtongsung-san extracts efficiently induces diminish proliferation of preadipocyte and lipolysis in adipose tissue. Keywords in context: Bangpungtongsung-san, alcohol extract, Water extracts, 3T3-L1 preadipocyte, adipocytes, lipolysis, Fat tissues.

Green synthesis and characterization of silver nanoparticles using pig skin extracts

LEE, Hwan Jin 고려대학교 대학원 2022 국내석사

Silver nanoparticle은 나노의학 뿐만 아니라 여러 분야에서 많이 활용되고 있다. AgNPs는 항균 효과가 있는 것으로 밝혀졌으며 관련된 연구는 지금도 활발히 진행되고 있다. Green synthesis는 식물 등 천연물에서 추출한 물질로 합성되어 친환경 합성 방법 중 하나로 떠오르고 있다. 은 나노입자를 만들기 위해 인체와 환경에 해로운 영향을 미치는 환원제가 사용되고 있다. 이러한 문제점을 개선하기 위해 천연물 유래 추출물로 나노입자를 합성하는 방법이 개발되었다. 이것은 green synthesis이라고 하며 Green synthesis은 starch, leaves, roots, flowers, fruits, honey, bacteria, fungi, algae, microbial enzyme 등을 이용하여 metallic nanoparticles를 합성하는 방법이다. 친환경적인 합성법이기 때문에 인체에 치명적이지 않고 환경오염에도 영향이 미치지 않아서 차세대 합성 방법으로 많이 연구되고 있다. 본 연구의 목적은 pig skin extracts에서 추출한 물질을 이용하여 AgNPs를 합성하고 AgNPs에 대한 항균 효과를 입증하는 것이다. 본 논문에서는 최초로 동물 조직 추출물을 이용하여 green synthesis AgNPs를 합성하는데 성공하였다. silver nanoparticles의 특성화를 위해 UV-vis spectrophotometer, zeta-sizer, SEM, FT-IR 장비를 사용하였다. UV-vis spectrophotometer는 Surface plasmon resonance (SPR) peak를 이용하여 400~500nm파장에서 silver nanoparticles의 파장을 확인하였다. 동적 광산란(DLS)의 원리를 이용한 측정기기인 zeta-sizer는 paper filter와 syringe filter로 여과한 pig skin extracts와 AgNO3로 합성된 silver nanoparticle의 크기를 측정한 결과 각각 212.3d.nm, 162.1d.nm로 관찰되었다. Paper filter로 여과한 pig skin extracts보다 syringe filter로 여과한 pig skin extracts에서 더 높은 흡광도를 확인하여 실험에는 syringe filter를 이용한 pig skin extracts를 사용하였다. 1mM AgNO3 5ml와 syringe filter로 여과한 pig skin extracts를 0.25mL. 0.5mL, 1mL, 3mL, 5mL 비율로 혼합하여 UV 노출(1~5분)과 48시간 실온 방지하였을 때 UV 5분 노출 시, syringe filter로 여과한 pig skin extracts 0.25mL와 1mL에서 가장 높은 흡광도가 관찰되었다. 또한 푸리에 변환 스펙트럼을 이용한 FT-IR 장비를 사용하여 AgNPs의 파장을 확인하고 선행연구 된 토마토 추출물로 합성된 AgNPs와 비교 분석하였다. syringe filter로 여과한 Pig skin extracts와 tomato extracts는 Ag+가 Ag0에 영향을 미치는 -OH와 -C=O- 파장에서는 유사한 형태를 보였지만 -C-O-와 관련된 파장에서는 차이점을 관찰하였다. FT-IR 측정 결과는 pig skin extracts가 AgNPs의 환원제와 안전화에 관여하는 캡핑제로 작용한다는 것을 확인하였다. AgNPs의 항균 효과를 증명하기 위해 Candida species와 gram positive bacteria와 gram negative bacteria로 나뉘어 실험하였으며 미생물의 생물학적 활성과 MIC50은 OD600 분석법을 통해 확인하였다. Fungus는 Candida albicans, Candida guilliermondii와 fluconazole resistant Candida albicans, Candida tropicalis로 사용하였으며 Candida albicans의 경우 fluconazole resistant 관계없이 MIC50이 8 µg/mL 농도에서 측정되었다. Candida guilliermondii와 fluconazole resistant Candida tropicalis는 16 µg/mL 농도에서 MIC50를 확인하였다. Gram positive bacteria는 Staphylococcus aureus (ATCC 29213), Staphylococcus haemolyticus (ATCC 29970), Enterococcus faecalis (ATCC 29212), methicillin-resistant Staphylococcus aureus (ATCC 19434), vancomysin-resistant Enerococcus faecalis (ATCC 49573), vancomysin-resistant Enerococcus gallinarum (ATCC 49573)로 실험하였다. Gram positive bacteria는 모두 8 µg/mL의 AgNPs 농도에서 MIC50을 확인하였다. Gram negative bacteria는 Salmonella typhi (ATCC 6539), Salmonella choleraesuis (ATCC 35640), Esherichia coli (ATCC 25922), Stenotrophomonas maltophilia (ATCC 13637), Serratia marcescens (ATCC 27853), Pneumoniae auroginosa (ATCC 27853), Acinetonater baumannii (ATCC BAA1605)Multidrug-resistant Pseudomonas aeruginosa (CCARM 2092), Multidrug-resistant Acinetobacter baumannii (ATCC BBA 1605)를 실험하였으며 Gram negative bacteria는 모두 8 µg/mL 농도에서 MIC50을 확인하였다. 전세계적으로 항생제 내성 미생물의 억제 방법과 관련된 연구가 활발히 이루어지고 있다. Green sythesis는 천연물을 통한 친환경적인 방법이다. 또한 합성 방법이 간편하고, 합성된 입자의 생체적합성이 높다는 장점을 가지고 있다. Pig skin을 이용한 green synthesis는 기존에 식물 추출물 중점으로 합성하던 방법에서 새로운 합성 방법을 제시할 뿐만 아니라 항생제 내성 미생물의 억제 연구에 대한 새로운 방향을 제시하고 향후 연구를 통해 다양한 분야에 적용될 수 있을 것으로 기대한다. Silver nanoparticles (AgNps) are known to have an antibacterial effect, and research on the same is being actively conducted. In addition, AgNps are being developed for use in public places and life. However, reducing agents that are harmful to the human body and the environment are used in the synthesis of AgNps. To overcome this problem, a method for synthesizing nanoparticles using a natural product-derived extract has been developed. This method is known as green synthesis. In this study, using the green synthesis method, we successfully synthesized AgNPs using pig skin extracts for the first time. The environmentally synthesized AgNps were characterized by zetasizer measurement, Fourier-transform infrared spectroscopy, UV-vis spectrophotometry, scanning electron microscopy, and dynamic light scattering and filtered with a syringe filter. Our pig skin extract was confirmed to effectively act as a reducing agent and capping agent for AgNPs. Because of climate changes and environmental pollution, microorganisms are becoming more diverse and toxic. Consequently, the treatment of antimicrobial-resistant microorganisms is becoming challenging. The treatment of antimicrobial-resistant microorganisms requires more powerful antimicrobial agents, placing physical and economic burdens on the patient. Therefore, there is a need to develop a novel and powerful antimicrobial agent that is environmentally friendly. In this study, green synthesis of AgNPs was performed using pig skin extracts filtered with a syringe filter. The MIC50 and antimicrobial effects (against fungi, gram-positive bacteria, and gram-negative bacteria) were confirmed at OD600.

양파열수추출물에 대한 소비실태조사 및 관능평가

김수렴 창원대학교 2009 국내석사

First study conducted a survey about consumer perception and satisfaction on onion hot-water extracts. Second study conducted a study about sensory characteristics and preference test on onion hot-water extracts in market. 1. Among the study subjects, the numbers of females(53.3%) were more than those of males and people in their 40s(35.1%) were the largest group. Most respondents(84.5%) knew the efficacy of onion hot-water extracts concerning the detailed efficacy of onion hot-water extracts, and females recognized the efficacy of onion hot-water extracts more than males(p<0.001). 2. Most consumers(67.3%) purchased onion hot-water extracts from ‘health food store using double boiler’ and many consumers(47.4%) got information from families and relatives. 51.8% of respondents said that they purchased 'quantities for 1~3 months' at one time and 33.1% said that the price of hot water extracts from onions was expensive. They considered 'health' the most when purchasing, preferred 'pouch pack(60.3%)' and considered 'easiness to open, convenience to drink and safety(42.0%)' the most. 62.8% of respondents ate onion hot-water extracts and Many of them drank it 1~3 times a week, '70mL' as one dose and drank it 'regardless of time'. 3. The lists health effects of onion hot-water extracts were satisfied, but 'taste', 'smell' and 'color' were not. Concerning advertisements for the efficacy of onion hot-water extracts, 72.5% of them replied 'I trust it a little'. Concerning the expanding onion hot-water extracts market, many respondents said 'it is difficult to choose a onion hot-water extracts because of many similar products in the market and requested to improve the taste and flavor. 4. Descriptive ananlysis of onion hot-water extracts come out difference. A, B and C comercial products showed higher intensity for flavor and onion taste. Intensity of oriental medicinal herbs flavor and sweetness of C and E comercial products were high than other products. A and B comercial products showed higher intensity for sourness and bitterness. Preference about color, taste, flavor, viscosity, ovarall acceptability were high according to priority A=B, D<C<E In sensory characteristics, C and E commercial products had low intensity for onion taste and onion flavor, had high intensity for oriental medicinal herbs flavor and sweetness. In the preference test, so, preference and purchasing intention of C and E commercial products were high. This study had limitation because the range of survey was restricted to residents in Kyungnam region and there are many non-intake respondents. Therefore, nation wide research on commercial products to generalize the study results was needed in the future.

Pharmacological effects of Vitis vinifera extracts on trastuzumab-resistant breast cancer cells

김성아 성균관대학교 일반대학원 2022 국내석사

Fibronectin (FN) expression plays an important role on cell metastasis and cell invasion in a variety of cancers. We previously reported that HER2 over-expression and EGFR ligands induce FN expression which is one of pro-oncogenic genes. However, HER2-induced FN expression was not affected by trastuzumab. Here, we confirmed that FN and EGF expression were significantly increased in trastuzumab-resistant (TR) breast cancer cells when compared to parental cells. In addition, Aberrant FN expression was related with poor prognosis in HER2-posistive breast cancer and improved cell adhesion and migration abilities through FN treatment. Interestingly, the level of FN expression was induced by EGFR over-expression in HCC1419 breast cancer cells. In the present study, new pharmacological effects of Vitis vinifera extracts were investigated. Our results showed that FN expression was significantly decreased by Vitis vinifera extracts treatment in TR breast cancer cells. EGF/EGFR signaling axis also dramatically suppressed by Vitis vinifera extracts treatment. Finally, the effect of Vitis vinifera extracts on FN-induced tumorigenecity was confirmed. As expected, our results showed that the tumorigenecity by FN was completely reduced by Vitis vinifera extracts. Therefore, we suggest that Vitis vinifera extracts will be promising agent to overcome trastuzumab resistance through inhibiting EGFR signaling pathway. 피브로넥틴 발현은 다양한 암에서 세포 전이와 세포 침입에 중요한 역할을 한다. 이전에 유방암 세포에서 HER2 과발현과 EGFR 리간드가 프로 발암성 유전자 중 하나인 피브로넥틴 발현을 유도한다고 보고했다. 그러나 HER2에 의한 피브로넥틴 발현은 트라스투주맙의 영향을 받지 않았다. 본 연구에서 트라스투주맙 저항성 BT-474 세포에서 피브로넥틴과 EGF 발현이 유의하게 증가했음을 확인하여 트라스투주맙 저항성에서 중요한 치료적 대상임을 밝혔다. 또한 피브로넥틴 발현 증가는 HER2 양성 유방암의 나쁜 예후와 관련이 있었고 피브로넥틴 처리를 통해 세포 유착과 이동 능력이 향상되었다. HCC1419 유방암 세포에서 EGFR 과다발현으로 인해 피브로넥틴 발현 수치가 증가했고 이를 통해 EGFR 신호 경로가 피브로넥틴을 조절하는데 중요하다는 것을 확인했다. 본 연구에서는 비티스 비니페라 추출물이 피브로넥틴 발현에 미치는 약리학적 영향을 탐구하였다. 우리의 결과는 트라스트주맙 저항성 유방암 세포에서 비티스 비니페라 추출물 치료에 의해 EGF 유도 피브로넥틴 발현이 크게 감소했음을 보여주었다. 또한 EGF/EGFR 신호 축은 비티스 비니페라 추출물 처리에 의해 극적으로 억제된다. 마지막으로, 비티스 비니페라 추출물이 피브로넥틴 유도 종양 유전성에 미치는 영향을 조사했다. 예상대로, 우리의 결과는 비티스 비니페라 추출물에 의해 피브로넥틴 유도 종양 유전성이 완전히 감소되었음을 보여주었다. 따라서, 우리는 비티스 비니페라 추출물이 EGFR 신호 경로를 통해 피브로넥틴을 억제함으로써 본래의 부종 제거 효과 외에도 트라스투주맙 저항성을 극복하는 치료효능을 나타냄으로써 유망한 작용제가 될 것을 제안한다.

Effects of marine macroalgae on in vitro ruminal fermentation characteristics and methane emission

신년학 Gyeongsang National University 2016 국내박사

Experiment Ⅰ. Effect of Phaeophyta Extracts on In vitro Ruminal Fermentation Characteristics, Methanogenesis and Microbial populations Algae have become as a source of intensive research in many fields of study. Areas of application are becoming increasingly diverse with the advent of technologies particularly in the mass production of algae biomass. Algae contain complex bioactive compounds and these are gaining importance in emerging technologies with nutritional and environmental applications. The aim of this work was to investigate the effect of Phaeophyta extracts on in vitro rumen fermentation and rumen microbial diversity in view of methane emission. A cannulted Holstein cow was used as the rumen fluid donor. The 15 ml of rumen fluid:buffer (1:2) was incubated for up to 72 h with six possible treatments: control (no addition of algae) and five kinds of Phaeophyta extracts (Ecklonia stolonifera Okamura, Eisenia bicyclis (Kjellman) Setchell, Sargarssum fulvellum (Turner) C. Agardh, Undaria pinnatifida (Harvey) Suringar, Sargassum fusiformis (Harvey) Okamura), respectively. Our results indicate that Phaeophyta extracts can be used as a possible viable alternative to assist in ruminant for improved growth performance (increased total gas production and glucose concentration and decreased A/P ratio) and methane abatement as compared to control. In particular, Real-time PCR indicated that the ciliate-associated methanogens, Ruminococcus albus and Ruminococcus albus flavefaciens populations significantly decreased, while the Fibrobacter succinogens population significantly increased at 24 h, when supplemented with Phaeophyta extracts as compared with control, respectively. As expected, all species of Phaeophyta tested in the study have greater effect on gas production and microbial growth, expecially ciliate-associated methanogens. It has been demonstrated in this study that all classes of algae have candidate members with potential to assist in ruminant feeding for improved gas production, fermentation management, and methane abatement. Ecklonia stolonifera Okamura significantly reduced methane production and had a moderate effect on total gas as compared with control, suggesting that Phaeophyta extracts can mitigate variable effects on overall in vitro rumen fermentation. However, more research is required to demonstrate and elucidate what various Phaeophyta extracts can improve feed intake and utilization efficiency, growth performance and methane abatement. Experiment Ⅱ. Effect of Rhodophyta Extracts on In vitro Ruminal Fermentation Characteristics, Methanogenesis and Microbial Populations Research on feed additives is currently ongoing to determine their potential effect in reducing anthropogenic greenhouse gas emissions, especially from ruminants. This study investigated the effect of dietary supplementation of Rhodophyta extracts on in vitro rumen fermentation and rumen microbial diversity. A cannulated Holstein cow was used as the rumen fluid donor. The 15 ml of rumen fluid:buffer (1:2) was incubated for up to 72 h with six possible treatments: control with only timothy and five kinds of Rhodophyta extracts 5% as basis of substrate (Grateloupia lanceolate (Okamura) Kawaguchi, Hypnea japonica Tanake, Pterocladia capillacea (Gmelin) Bornet, Chondria crassicaulis Harvey, or Gelidium amansii (Lam.) Lamouroux, respectively. Our results indicated that Rhodophyta extracts increased cumulative gas production at 24 and 72 h (p = 0.0297 and p = 0.0047) as compared with control. Rhodophyta extracts reduced methane emission at 12 and 24 h (p = 0.0077 and p = 0.0008 and p = 0.0183), in particular Real-Time PCR, indicated that ciliate-associated methanogens, Ruminococcus albus and Ruminococcus flavefaciens populations decreased at 24 h (p = 0.0002, p < 0.0001 and p < 0.0001), while the Fibrobacter succinogens population significantly increased at 24 h (p = 0.0004) as compared with control, respectively. Additionally, Rhodophyta extracts improved acetate concentration at 12 and 24 h (p = 0.0766 and p = 0.0132) and A/P ratio at 6 and 12 h (p = 0.0106 and p = 0.0278) as compared with control, respectively. In conclusion, Rhodophyta extracts can be used as a possible viable alternative to assist in ruminant for improved growth performance (increased total gas production and decreased A/P ratio) and methane abatement (reduced ciliate-associated methanogens, Ruminococcus albus and Ruminococcus flavefaciens population and increased Fibrobacter succinogenes population) as compared to control. Experiment Ⅲ. Impact of Ecklonia Stolonifera Extracts on In vitro Ruminal Fermentation Characteristics, Methanogenesis and Microbial Populations Ecklonia stolonifera is a brown algae belonging to the Laminariaceae family and is found commonly in the sea forests, off the coasts of Korea and Japan. E. stolonifera has traditionally been utilized as an edible sea weed and contains high levels of diverse phlorotannins, which have diverse biological activities. This study investigated the effect of dietary supplementation of E. stolonifera extracts on in vitro rumen fermentation by assessing pH, total gas, volatile fatty acid production, gas profile (methane, carbon dioxide, hydrogen and ammonia) and rumenal microbial diversity as compared to basal diet with timothy. A cannulated Holstein cow was used as the rumen fluid donor. The 15ml of rumen fluid:buffer (1:2) was incubated for up to 72 h with four possible treatments and divided into four treatments with three replicates. The doses were: control with only timothy, basal diet with E. stolonifera extract 1%, basial diet with E. stolonifera extract 2% and basial diet with E. stolonifera extract 3%. Our results indicate that E. stolonifera extracts can be used as a possible viable alternative to assist in improved growth performance (significantly increased total gas production) in ruminants, suggesting that E. stolonifera extracts could mitigate variable effects throughout the whole period of in vitro rumen fermentation. Unexpectedly, E. stolonifera extracts did not appear a noticeable effect on methane and hydrogen abatement, which is different from previous observations with brown algae extracts use under in vitro fermentation conditions. Interestingly, Real-time PCR indicated that the ciliate-associated methanogen and Fibrobacter succinogenes population significantly decreased, whereas the Ruminococcus flavefaciens population increased as a result of from E. stolonifera extract supplementation as compared with control. More research is required to demonstrate and elucidate what E. stolonifera extracts can do to improve growth performance and affect methane production in ruminants. Experiment Ⅳ. Effect of Gelidium Amansii Extracts on In vitro Ruminal Fermentation Characteristics, Methanogenesis and Microbial Populations Gelidium amansii (Lamouroux) Lamouroux is a red algae belonging to the family Gelidaceae and is commonly found in the shallow coasts of many east Asian countries, including Korea, China and Japan. G. amansii has traditionally been utilized as an edible sea weed for a long time and has various biological activities. The objective of this study was to investigate and determine whether dietary supplementation of G. amansii could be useful for improving ruminal fermentation, as assessed by in vitro fermentation parameters, such as pH, total gas, volatile fatty acid (VFA) production, gas profile (methane, carbon dioxide, hydrogen and ammonia) and microbial growth rate as compared to basal diet with timothy. Cannulated Holstein cows were used as rumen fluid donors and 15 ml of rumen fluid:buffer (1:2) was incubated for up to 72 h with four possible treatments with three replicates. The treatments were: control (timothy only), basal diet with 1% G. amansii extract, basal diet with 2% G. amansii extract and basal diet with 3% G. amansii extract. Overall, the results of our study indicate that G. amansii supplementation is potentially useful, improving ruminant growth performance, via increased total gas and decreased methane and hydrogen emission; but does come with some undesirable effects, such as decreasing total volatile fatty acids and unaffected microbial growth performance. In particular, Real-time PCR indicated that the methanogenic archaea and Fibrobacter succinogens populations were significantly reduced, while the Ruminococcus flavefaciens population significantly increased at 24 h, when supplemented with G. amansii extracts as compared with control.