BACE1 inhibitory activity of Crataegus pinnatifida fruits

ZHOU YAJUAN 부경대학교 대학원 2019 국내석사

산사는 중국에서는 여러가지 대사장애, 혈관장애 그리고 신경퇴행성 질환과 더불어 항암과 항바이러스 효과로 인해 전통적인 한방 약재로 쓰여진다. 산사 열매의 항알츠하이머 질환 활성에 관한 여러가지 연구가 보고되었음에도 불구하고 추출물과 그 구성성분의 BACE1 저해 활성에 관한 연구는 보고된 바가 없다. 이번 연구에서 산사 열매의 메탄올 추출물과 용매별 분획물의 BACE1 저해에 관한 예비 평가가 이루어 졌으며 에틸아세테이트 분획물의 활성이 가장 높았고 디클로로메탄 분획물과 부탄올 분획물이 그 뒤를 이어 활성을 보였다. 우리는 생리활성에 따른 활성 물질 분리법을 통해 두 개의 triterpenoid [corosolic acid (1), euscaphic acid (2)], β-sitosterol glucoside (3), 네 개의flavonoid [astragalin (4), hyperoside (5), isovitexin (6), isoorientin (7)], 그리고 다섯 개의 phenolic acid 유도체 [gentisic acid (8), protocatechuric acid (9), p-hydroxybenzoic acid (10), p-coumaric acid (11), hymenoside X (12)]를 에틸아세테이트 분획물로부터 얻었다. 디클로로메탄 분획물에서도 두 개의 triterpenoids [3-epicorosolic acid (13), ursolic acid (14)] 그리고 β-sitosterol (15)을 분리 및 동정 하였다. 화합물 2,7,8 그리고 11은 산사 열매로부터 처음으로 분리되었으며 화합물12는 Crataegus 종에서 처음으로 분리되었다. 화합물 13은 40.12 ± 1.72 µM의 반수저해농도 (IC50) 를 가짐으로써 가장 높은 BACE1 저해 활성을 보였고 화합물 5는 98.50 ± 4.20 µM의 IC50을 가지는 보통의 저해 활성을 보였다. 하지만, 화합물 1과 4는 낮은 BACE1 저해 활성을 나타내었고 (각각의 IC50 = 179.55 ± 1.57 µM과 192.30 ± 5.14 µM), 화합물 14는 이들보다 더 낮은 저해 활성을 보였다 (IC50 = 236.46 ± 3.90 µM). 다른 화합물들은 400 µM의 농도까지 유의적인 저해 효과를 나타내지 않았다. 게다가 우리는 in vitro와 in silico에 근거한 ursane-type triterpenoid들의 구조적 활성 상관관계를 연구했으며 triterpene의 수산기의 위치가 효능에 큰 영향을 미치는 것을 밝혀냈다. C-2와 C-3 위치에서 alpha-수산기의 존재와 C-19 위치에서 alpha-수산기의 부재는 BACE1 저해 활성에 필수적인 것으로 사료된다. 이번 연구는 ursane-type triterpene 골격에서 수산기들의 배열과 위치의 중요성 그리고 그것이 BACE1 저해 활성에 미치는 영향을 밝혀 냈으며, 잠재적인 항알츠하이머 질환 약물의 치료 전략으로 활용될 수 있을 것이다.

PC12 세포에서 아밀로이드 베타 펩타이드로 유도된 독성에 대한 식용 갈조류 대황의 신경보호 효과

안보라 부경대학교 대학원 2012 국내석사

Alzheimer’s disease (AD) is the most general neurodegenerative disease in humans that is characterized by neuronal degeneration and senile plaques. Amyloid beta peptide (Aβ) induced neurocytotoxicity is the important pathway of neuronal cell death which might play a crucial role in the development of AD. The mechanism by which Aβ induces cell death is not clear yet. However, it has been suggested that one of the mechanisms is oxidative stress produced by reactive oxygen species (ROS), reactive nitrogen species (RNS). Aβ induced toxicity is also related with the disturbance of calcium (Ca2+) homeostasis. Eisenia bicyclis (Kjellman) Setchell is a perennial brown alga, belonging to the family Laminariaceae. It is widely distributed in Korea and Japan and especially abundantly produced in Ulleung and Dok Island in Korea. This alga is frequently used as a dietary alternative of Laminaria japonica, along with Ecklonia stolonifera and Ecklonia cava. This species has a lot of bioactive components, including phlorotannins, polysaccharides, pyropheophytin, lipids, tripeptides, and oxylipins. Particularly phlorotannins, produced by polymerization of phloroglucinol (1, 3, 5-trihydroxybenzene), have been reported for various biological activities such as antioxidant, anti-diabetic complication, antitumor, hepatoprotective, anti-plasmin inhibitory, tyrosinase inhibitory, anti-inflammatory, nitrite-scavenging, anti-skin aging, anti-cholinesterase, anti-hyperlipidemic, anti-allergic, angiotensin converting enzyme-I inhibitory, β-secretase1 inhibitory, protein tyrosine phosphatase 1B, and α-glucosidase inhibitory activities. However, the neuroprotective effects of E. bicyclis on Aβ induced neurocytotoxicity had not been investigated. In the present study, the neuroprotective effects of methanolic (MeOH) extract and its solvent soluble fractions including dichloromethane (CH2Cl2), ethyl acetate (EtOAc), n-butanol (n-BuOH) and water (H2O) from E. bicyclis were investigated against Aβ induced neurocytotoxicity, as well as inhibitory activities of intracellular ROS, peroxynitrite (ONOO¯) and Ca2+ in PC12 cells. Exposure of Aβ to PC12 cells caused significant cell death and increased the number of apoptotic cells. Treatment of 2.5 μM Aβ alone induced about 50% cell death compared with control. However, the addition of 20 μg/ml MeOH extract and its two subfractions, EtOAc and n-BuOH fractions from E. bicyclis increased cell viability to 75.06, 104.30 and 82.76%, respectively. On the other hand, the CH2Cl2 and H2O fractions had no obvious effects. In particular, the EtOAc fraction restored cell viability to almost the normal level, indicating that the EtOAc fraction showed the highest neuroprotective effects against Aβ induced neurocytotoxicity among the tested fractions and has neuroprotective constituents. Therefore, the EtOAc fraction was selected and chromatographed over a silica, RP-18, and Sephadex LH-20 column to yield active compounds. Six phlorotannins, including phloroglucinol (1), dioxinodehydroeckol (2), eckol (3), phlorofucofuroeckol-A (4), dieckol (5), and 7-phloroeckol (6) were isolated from the EtOAc fraction of E. bicyclis and their chemical structures were elucidated by spectral analysis. Among six phlorotannins, 4, 5, and 6 significantly decreased Aβ induced cell death. 4, 5, and 6 with no obvious toxic concentration (5, 20, and 30 μM, respectively) increased cell viability by 15, 20, and 40%, respectively. The MeOH extract and its fractions from E. bicyclis, mainly EtOAc fraction, and isolated phlorotannins from active EtOAc fraction also inhibited the level of intracellular ROS and ONOO¯, indicating the neuroprotective effects may be mediated through reduced intracellular ROS and ONOO¯ generation. The EtOAc fraction manifested potent t-BHP-induced intracellular ROS and ONOO¯ generation inhibitory activity with IC50 values of 1.85 ± 0.03 and 3.93 ± 0.51 μg/ml, respectively, followed by the n-BuOH fraction with IC50 values of 2.25 ± 0.11 and 7.49 ± 0.58 μg/ml, respectively. Among the compounds from EtOAc fraction, 4, 5, and 6 significantly decreased t-BHP-induced intracellular ROS generation. 5 and 6 also significantly decreased t-BHP-induced intracellular ONOO¯ generation, while 4 had cytotoxicity in tested concentrations. Incubation of PC12 cells with the fractions and the compounds from E. bicyclis which have relatively high neuroprotective effects also significantly inhibited Aβ-induced ROS generation. Furthermore, since the mechanism of toxicity of Aβ may be mediated by elevations of Ca2+, the study examined whether the phlorotannins affected the Aβ-induced increase of Ca2+. 3, 4, and 6 decreased the elevations of Ca2+ in a dose dependent manner, and especially pretreatment with 6 (30 μM) reduced intracellular Ca2+ to the normal level. Thus, the results of the present study imply that E. bicyclis and its active components attenuated the oxidative stress and reduced neuronal cell death, suggesting that it may be used as a dietary neuroprotective agent in AD.

후코스테롤과 후코잔틴의 항당뇨 효과

이찬미 부경대학교 2013 국내석사

Fucosterol, the predominant sterol in brown seaweeds, constitutes 83-97% of the content. There have been many studies on the biological activities including antioxidant, hepatoprotective, anti-inflammatory, anticancer, anti-fungal, antihistaminic, anti-diabetic and anticholinergic. Fucoxanthin is a marine carotenoid that is characteristically present in edible brown seaweeds such as Eisenia bicyclis (Arame), Undaria pinnatifida (Wakame) and Hijikia fusiformis (Hijiki). Fucoxanthin, one of the most abundant carotenoids, accounts for more than 10% of the estimated total natural production of carotenoids and has a unique structure with an unusual allenic bond and a 5,6-monoepoxide. Fucoxanthin has received considerable attention because of its wide array of beneficial functions in human health, including anti-inflammatory, antioxidant, antimutagenecity, and anticancer activities. In this study, the anti-diabetic activities of fucosterol and fucoxanthin were investigated by evaluating the ability of these compounds to inhibit α-glucosidase, PTP1B, rat lens AR (RLAR), human recombinant AR (HRAR), AGE formation. Since there is no detailed information on the mode of inhibition, or the molecular interactions of fucosterol and fucoxanthin with the corresponding enzymes, this study was also designed to identify an approach to develop potent anti-diabetic or anti diabetic complications drugs using molecular docking predictions and enzyme kinetics of these compounds. Fucosterol exhibited moderate RLAR and HRAR inhibitory activity with IC50 values of 18.94 ± 1.90 μM and 143.88 ± 3.76 μM, respectively, as compared with the positive control, quercetin with respective IC50 values of 1.34 ± 0.05 μM and 7.63 ± 0.05 μM. On the other hand, fucosterol exhibited marginal inhibitory activity on PTP1B and α-glucosidase, and AGE formation. By means of Lineweaver-Burk double reciprocal plots, the various concentration lines of fucosterol intersected in the left side, indicating mixed type inhibitors on RLAR and HRAR with respection IC50 values of 7.0 and 99.50 μM, while the lines show the same point on the x-intercept representing noncompetitive inhibitors against PTP1B with a Ki value of 77.13 μM in Dixon plots. Due to the presence of hydrophobic ring nucleus and hydrophobic hydrocarbon side chain of fucosterol, it was observed that this compound interacted with human AR and RLAR through well-known active sites, such as hydrophobic specificity residues. In particular, the hydrophobic ring system of fucosterol is bound tightly in a specificity pocket through apolar amino acid residues, including Trp80, His111, Phe122/Phe123, and Trp112. In addition, the interaction of AR and the aliphatic side chain in fucosterol with Val48, Trp80, Trp122, Phe122/Phe123, Cys299, and Leu301 residues while a polar site with Trp21 and Tyr49 residues on AR interacts with 3-hydroxyl group and double bond in side chain of fucosterol. Results of the docking simulation of fucosterol demonstrated negative binding energies (–8.2 kcal/mol for RLAR and –8.5 kcal/mol for HRAR), indicating higher affinity and tighter binding capacity of fucosterol for the active site of the enzyme. Fucoxanthin exhibited potent AGE inhibitory activity with an IC50 value of 86.48 ± 2.16 μM as compared with the positive control, aminoguanidine, with an IC50 value of 530.37 ± 6.52 μM. Notably, fucoxanthin showed six times stronger inhibition of AGE formation than aminoguanidine, a well-known inhibitor of AGE formation. On the other hand, fucoxanthin exhibited marginal AR inhibitory activities with IC50 values of 108.31 ± 3.99 μM for HRAR and 264.67 ± 13.76 μM for RLAR. Also, fucoxanthin exerted significant PTP1B inhibitory activity with an IC50 value of 4.80 ± 0.49 μM, where the positive control ursolic acid had an IC50 value of 2.56 ± 0.07 μM. On the other hand, fucoxanthin did not exhibit α-glucosidase inhibitory activity up to a concentration of 200 μM. Using Lineweaver-Burk plots, the various concentration lines of fucoxanthin had the same y-intercept, representing its ability to act as a competitive RLAR inhibitor, while the lines of fucoxanthin intersected the axis on the left side of the zero point, indicating its ability to act as a mixed-type PTP1B inhibitor. The Ki values obtained from the Dixon plotting were 67.67 μM for RLAR inhibition and 1.53 μM for PTP1B inhibition. The corresponding ligand-interactions of fucoxanthin in the active site of PTP1B are the three hydrogen bonding interactions between Phe30, Phe52 and Gly183 residues of the enzyme and the two hydroxyl groups of fucoxanthin, while the five residues, Tyr20, Lys116, Arg24, Arg254, and Gln262, of the enzyme participated in hydrogen bonding interactions with the carboxylate anions of compound 23. In addition, there were additional hydrophobic interactions between long hydrocarbon chains harboring a conjugated double bond of fucoxanthin and Ile219, Tyr46, Val49, and Ala217 residues on PTP1B. Moreover, the binding energies of both compounds were negative values of -7.66 kcal/mol for fucoxanthin and -10.18 kcal/mol for compound 23. In the present study, fucosterol and fucoxanthin showed inhibitory potential against AR, PTP1B, and AGE formation thus holds promise for its use as a therapeutic agent for the treatment of diabetes as well as related complications.

Antioxidant and anti-inflammatory activities of Hizikia fusiformis

YuRanHan 부경대학교 대학원 2014 국내석사

다양한 프리 라디컬들에 의해 생성되는 산화적 스트레스는 염증질환 및 노화, 암, 당뇨등의 다양한 질병들을 일으키는 주요원인으로 작용한다. 체내의 항산화 방어시스템의 불균형으로 인해 Reactive oxygen species (ROS), reactive nitrogen species (RNS), superoxide (•O2), hydrogen peroxide (H2O2) 와 같은 프리 라디컬들이 과다하게 생성되면 면역체계는 무너지게 되고 이에 따라 각종 염증 사이토카인과 염증 관련 효소 및 유전자들이 발현되며 결국 염증 질환 및 염증 관련 질병들을 일으키게 된다. 또한 증가된 프리 라디컬은 nitrite oxide (NO)와 연쇄반응을 일으켜 peroxynitrite (ONOO-) 와 nitro tyrosine 같은 반응생성물을 생성한다. 특히 ONOO-는 반응성이 높아 세포독성뿐 아니라 세포막의 지질과산화를 일으며 세포죽음과 조직손상을 야기시키게 된다. 따라서 질병들의 근원이되는 산화적 스트레스를 감소시키고 예방하는 것은 중요한 의미를 가질 것이다. 톳은 한국, 일본 그리고 중국 등지의 해안에서 풍부하게 서식하고 있는 식용 갈조류이다. 아시아 지역에서 건강기능 식품으로서 널리 이용되고 있으며 다양한 건강적 이점들 때문에 그 활성들에 대한 집중적 연구가 진행되어 오고있다. 이번 연구에서는 톳의 메탄올 추출물과 그 분획물을 이용하여 총 페놀함량 측정, peroxynitrite (ONOO-), 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2’-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) 라디컬, 그리고 ONOO-를 매개로 하여 생성되는 tyrosine nitration의 소거 및 억제에 대한 효과를 in vitro assay를 통하여 항산화 활성을 평가하였다. 뿐만 아니라 마우스의 대식세포로부터 얻어지는 RAW 264.7 세포주를 사용하여 lipopolysaccharide (LPS) 에 의해 유도되는 NO의 생성, tert-butylhydroperoxide (t-BHP) 로 유도되는 세포내 ROS의 생성, 그리고 LPS의 유도로 발현되는 inducible nitric oxide synthase (iNOS) 와 cyclooxygenase-2 (COX-2) 단백질 발현의 억제효과를 평가함으로서 항염증 활성을 조사하였다. 그 결과, 실험에 사용된 톳의 메탄올 추출물 및 그 분획물들 중에서 ethyl acetate (EtOAc) 분획물이 55.86 GAE, mg/g of fraction으로 가장 높은 페놀함량을 가지고 있었고 ONOO-, DPPH, ABTS 라디컬에 대해 가장 높은 프리 라디컬 소거활성을 보였으며 그들의 IC50 값은 각각 9.54, 216.75, 그리고 55.71 μg/ml 이였다. 게다가 EtOAc 분획물은 3.12, 6.25, 12.5, 그리고 25 μg/ml의 농도범위에서 ONOO-에 의해 생성되는 tyrosine nitration을 효과적으로 억제하였다. 그리고 dichloromethane (CH2Cl2) 분획물 또한 ONOO-에 의해 생성되는 tyrosine nitration을 12.5 에서 100 μg/ml 농도범위 에서억제활성을 보였다. 그리고 RAW 264.7 세포에서 LPS로 유도되는 NO의 생성을 가장 효과적으로 억제하였으며 그의 IC50 값은 4.46 μg/ml로 나타났고 CH2Cl2 분획물의 처리는 LPS에 의해 발현량이 높아지는 iNOS 단백질을 2.5, 5, 그리고 10 μg/ml 농도에서 상당히 억제하였다. t-BHP로 유도되는 세포 내 ROS의 생성 실험의 경우에서는 CH2Cl2와 EtOAc 분획물 모두 세포독성을 띄지 않는 12.5 μg/ml 그리고 12.5 에서 100 μg/ml 처리 농도 사이에서 각각 ROS의 생성을 감소 시켰다. 이번 연구결과를 통해 톳의 CH2Cl2와 EtOAc 분획물이 상당한 항산화 및 항염증 활성을 가짐을 확인하였으며 이들 분획물에서 생리활성물질을 찾아 그 활성을 연구함으로서 앞으로 산화적스트레스와 연관되는 염증적 질환에 대해 효과적인 예방 및 치료제가 될 수 있을 것이라 기대된다.

곰피의 항당뇨 및 항동맥경화 효과

문혜은 부경대학교 대학원 2012 국내석사

Ecklonia stolonifera OKAMURA belonging to the perennial brown alga, is a member of the family Laminariaceae. It usually grows well in subtidal zones at 2-10 m depth and commonly distributed in the mid-Pacific coast around Korea and Japan. It is frequently consumed as a foodstuff, together with Undaria pinnatifida and Laminaria japonica. This brown alga has been also used as phlorotannin-rich raw materials and phlorotannins are secondary metabolites polymerizing phloroglucinol (1,3,5-trihydroxybenzene) through an ether, phenyl, or 1,4-dibenzodioxin linkage. Phlorotannins were found to have a variety of bioactivities, such as anti-diabetic complications, antioxidant, tyrosinase inhibitory, angiotensin-converting enzyme inhibitory, antimutagenic, nitrite-scavenging, algicidal, anti-inflammatory, nitrite-scavenging, anti-skin aging, and anti-allergic. However, protein tyrosine phosphatase 1B (PTP1B) and α-glucosidase inhibitory activities of phlorotannins isolated from E. stolonifera and their antioxidative effects on human low density lipoprotein (human LDL) have not been investigated yet. Increased PTP1B enzyme related insulin resistance and α-glucosidase that catalyze the final step in the digestive process of carbohydrates have been implicated in the pathogenesis of many diabetic complications containing atherosclerosis, cardiac dysfunction, retinopathy, and nephropathy. The diabetic patients have a higher risk of developing atherosclerotic diseases than non-diabetic. Oxidation of LDL has been represented as one of the main reason for atherosclerosis and has been reported that dietary antioxidants and free radical scavenger are able to prevent LDL oxidation which can reduce the risk of atherosclerosis in diabetes. The present work investigated the anti-diabetic and anti-atherosclerotic effects of the methanol (MeOH) extract and different solvent soluble fractions including dichloromethane (CH2Cl2), ethyl acetate (EtOAc), n-butanol (n-BuOH), and water (H2O) of the edible brown alga, E. stolonifera together with its isolated phlorotannins via the inhibition of α-glucosidase, protein tyrosine phosphatase 1B (PTP1B), the inhibition of human low density lipoprotein (LDL) oxidation and the conjugated diene formation in human LDL under in vitro conditions, respectively. The MeOH extract showed promising inhibitory activities of both PTP1B and α-glucosidase, with IC50 values of 6.39 ± 0.18 μg/ml against PTP1B and 2.82 ± 0.63 μg/ml against α-glucosidase, respectively. Among several fractions, the EtOAc fraction and n-BuOH fraction exhibited potential inhibitory activities in PTP1B with IC50 values of 0.26 ± 0.06 μg/ml and 0.23 ± 0.07 μg/ml and in human LDL oxidation with IC50 values of 3.04 ± 0.13 μg/ml and 4.54 ± 0.02 μg/ml, respectively. On the other hand n-BuOH fraction showed weak α-glucosidase inhibitory activities with IC50 value of 4.59 ± 0.57 μg/ml than EtOAc fraction with IC50 value of 1.15 ± 4.59 μg/ml. Since the EtOAc fraction showed significant inhibitory activities in PTP1B, α-glucosidase, human LDL oxidation, it was selected for chromatographic separation of active compounds using silica gel, Sephadex LH-20, RP-18 column chromatographies to isolate six phlorotannins, phloroglucinol (1), dioxinodehydroeckol (2), eckol (3), phlorofurofucoeckol–A (4), dieckol (5), and 7-phloroeckol (6). Phlorotannins 3-6 were found as the potent and non-competitive PTP1B inhibitors with respective Ki values of 2.86, 1.43, 1.22, and 2.90 µM and with IC50 values ranging from 0.56 to 2.64 µM. Phlorotannins 4-6 exhibited the most potent α-glucosidase inhibitory activity with IC50 values ranging from 1.37 to 6.13 µM. Interestingly, phlorotannins 4 and 6 were non-competitive with Ki values of 0.45 and 0.56 µM, while phlorotannins 5 exhibited competitive inhibition with a Ki value of 0.27 µM against α-glucosidase enzyme. Furthermore, phlorotannins 3-5 showed the strongest inhibitory activity against human LDL oxidation with IC50 value of 7.47 ± 0.05 μM, 4.34 ± 0.07 μM, 3.10 ± 0.24 μM and significantly reduced the lag time (117.48 ± 0.15 min, 105.42 ± 0.59 min, 137.61 ± 0.39 min at concentration 10 μM) of conjugated diene formation, respectively. These results demonstrated that the MeOH extract and individual fractions of E. stolonifera as well as its isolated phlorotannins exhibited the potent anti-diabetic and anti-atherosclerotic effects via the inhibition of PTP1B, α-glucosidase, and human LDL oxidation, conjugated diene formation. Therefore, the extract and individual fractions of E. stolonifera as well as its isolated phlorotannins have anti-diabetic and anti-atherosclerotic effects in vitro conditions, which can be explored further for developing pharmaceutical drugs or functional foods to treat diabetes and atherosclerosis.

Isolation, identification and quantitative analysis of the BACE1 active compounds from the flowers of Cirsium maackii

BHATTARAI GRISHMA 부경대학교 대학원 2019 국내석사

알츠하이머 질환은 만성 진행성 퇴행성 질환이며 주요 건강 문제 중 하나로 대두되고 있다. 여러 연구들이 식물의 꽃 부위가 신경 퇴행을 예방하기 위해 사용될 수 있을 것이라고 제안하였다. 이전의 연구에서 Cirsium의 꽃 추출물이 전인지적 효과를 가지고 있다고 보고되었다. 이러한 근거들에 의거하여, 우리는 엉겅퀴 꽃의 항알츠하이머 질환 효과를 측정하고 꽃의 항알츠하이머 질환 효과가 어떤 성분에 의한 것인지 찾기 위해서 구성 성분들에 대한 정성 및 정량 분석을 실시하고자 하였다. BACE1 억제 활성 측정 결과, 꽃의 메탄올 추출물은 농도 의존적으로 효소를 억제하였으며 76.47 ± 1.66 µg/ml의 반수저해농도 (IC50)를 가지는 것으로 나타났다. 다음으로, 메탄올 추출물의 극성에 따른 용매별 분획물들에 대한 BACE1 억제 활성을 측정하였다. 그 결과, 에틸아세테이트, 디클로로메탄 그리고 부탄올 분획물 순으로 높은 억제 활성을 보였으며 각각 22.98 ± 1.45, 8.65 ± 0.63 그리고 72.47 ± 3.04 µg/ml 의 IC50를 보였다. 양성 대조군으로는 4.51 ± 3.63 µg/ml의 IC50를 가지는 퀘르세틴을 사용하였다. 오픈 컬럼 크로마토그래피를 통해서 디클로로메탄 분획물로부터 β-amyrenone, lupeol acetate, lupeol, β-sitosterol 그리고 β-sitosterol glucoside를 분리하였고, 에틸아세테이트 분획물로부터 apigenin, luteolin, apigenin 7-O-β-D-glucuronide methyl ester, tracheloside 그리고 luteolin 5-O-β-D-glucoside를 분리하였다. 또한, apigenin 5-O-β-D-glucoside 그리고 apigenin 7-O-β-D-glucuronide를 부탄올 분획물로부터 분리하였고 luteolin 7-O-β-D-glucuronide를 물 분획물로부터 분리하였다. 특히, 디클로로메탄 분획물의 lupeol과 lupeol acetate은 다른 성분들과 비교해서 상당히 많이 분리되었다. 게다가, 고속 액체 크로마토그래피 (HPLC)를 이용하여 350nm의 파장에서의 꽃 성분의 정량 및 정성분석을 실시하였다. 그 결과, apigenin, luteolin 그리고 apigenin 7-O-β-D-glucuronide가 메탄올 추출물, 에틸아세테이트 분획물 그리고 부탄올 분획물의 주요 성분인 것으로 나타났다. 특히, 에틸아세테이트 분획물에 apigenin과 luteolin이 각각 162.27 mg/g 그리고 97.17 mg/g으로 상당히 많이 함유되어 있음을 확인하였고, 부탄올 분획물에는 apigenin 7-O-β-D-glucuronide가 106.38 mg/g으로 많이 함유되어 있는 것을 HPLC를 통해 확인하였다. 이전의 연구에서 lupeol과 luteolin이 BACE1 저해제로써 보고된 바 있으므로, 엉겅퀴 꽃의 항알츠하이머 질환 효과는 이러한 주요 화합물들의 존재에 의한 것으로 사료된다. 따라서, 이번 연구를 통에서 엉겅퀴 꽃의 알츠하이머 질환에 대한 역할이 주요 triterpenoid와 flavonoid에 의한 것을 확인하였다.

Promising anti-diabetic potential of Artemisia capillaris and its constituents

Md. Nurul Islam 부경대학교 대학원 2014 국내박사

Artemisia, a diverse genus of the family Asteraceae, has been used as anti-pyretic, anti-diabetic, anti-hypertensive, anti-hepatotoxic, anti-inflammatory, eczema, jaundice, and anti-hemorrhoid. Recently, Artemisia genus has emerged as a source of naturally occurring therapeutic agents for the treatment of diabetes and related complications due to the presence of wide varieties of bioactive substances including caffeoyl quinic acids, flavonoids, and coumarins. As a part of our ongoing search for anti-diabetic constituents from natural sources, we selected methanol (MeOH) extracts of 12 species of the genus Artemisia based on their frequent use in traditional medicines and investigated their anti-diabetic potential via α-glucosidase, protein tyrosine phosphatase 1B (PTP1B), and advanced glycation end products (AGE) formation inhibitory assays. The MeOH extracts of different species exhibited promising inhibitory activities ranging from 34.12 to 64.17% at a concentration of 250 μg/mL against α-glucosidase and 27.40 to 98.60% at a concentration of 10 μg/mL against PTP1B while their AGE formation inhibitory activities ranging from 21.40 to 87.47% at a concentration of 10 μg/mL. Since the MeOH extract of A. capillaris exhibited the highest α-glucosidase and AGE formation inhibitory activities together with significant PTP1B inhibitory activity, it was selected for detailed investigation. Repeated column chromatography of different fractions yielded 46 compounds including 13 coumarins (esculetin, esculin, scopolin, isoscopolin, scoparone, scopoletin, isoscopoletin, artemicapin A, fraxidin, 5,6,7-trimethoxy coumarin, 6-methoxy artemicapin C, tomerin, umbelliferone) and 19 flavonoids (cirsilineol, cirsimaritin, arcapillin, linarin, quercetin, hyperoside, isorhamnetin 3-O-robinobioside, quercetin 5-O-glucoside, quercetin 3-O-glucoside, quercetin 3-O-robinobioside, chrysoeriol-7-O-rutinoside, quercetin 7-O-galatopyranosyl 7-O-rhamnoside, isorhamnetin 3-O-galactoside, isorhamnetin 3-O-glucoside, acacetin-7-O-(6″-O-acetyl)-β-D-glucopyranosyl-(1→2)[α-L-rhamnopyranosyl]-(1→6)-β-D-glucopyranoside, acacetin-7-O-β-D-glucopyranosyl-(1→2)[α-L-rhamnopyranosyl]-(1→6)-β-D-glucopyranoside, isorhamnetin-3-O-arabinopyranoside, vicenin 2, apigenin), 6 caffeoylquinic acid derivatives (4,5 di-O-caffeoyl quinic acid, 3,5 di-O-caffeoyl quinic acid, 3,4 di-O-caffeoyl quinic acid, chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid), 2 chromones (capillarisin and 7-methyl capillarisin), 2 essential oils (capillin and capllinol), and 4 phenolic compounds (paramethoxy phenyl glucoside, eugenol glucoside, koaburaside and 4-hydroxy-3-O-glucopyranosyl benzoic acid). Among them, acacetin-7-O-(6″-O-acetyl)-β-D-glucopyranosyl-(1→2)[α-L-rhamnopyranosyl]-(1→6)-β-D-glucopyranoside is a new acetylated flavonoid glycoside isolated for the first time. Among the isolated compounds esculetin, quercetin, scopoletin, isorhamnetin, vicenin 2, cirsilineol, 3,5-O-dicaffeoylquinic acid, 1,5-O-dicaffeoylquinic acid, 3,4-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid, exhibited potent inhibitory activities in α-glucosidase and PTP1B inhibitory assays with IC50 values of 82.92, 58.93, 159.16, 141.17, 270.53, 351.71, 217.40, 146.06, 128.07, and 229.94 μM, respectively, compared to the positive control acarbose wth IC50 value of 130.52 μM against α-glucosidase and 10.08, 20.35, 227.28, 43.88, 139.75, 206.78, 2.02, 16.05, 2.60, and 3.21 μM, respectively compared to the positive control ursolic acid with an IC50 value of 4.05 μM against PTP1B. Interestingly, 3-O-caffeoylquinic acid, 6-methoxy artemicapin C and capillarisin exhibited potent inhibitory activity against PTP1B while hyperoside exhibited potent inhibitory activity against α-glucosidase rather than PTP1B. In addition, two essential oils, capillin and capillinol were also found to be potent inhibitors of α-glucosidase, PTP1B and RLAR. Kinetic analysis revealed that capllin noncompetitively inhibited both α-glucosidase and RLAR, while it showed mixed type inhibition against PTP1B. On the other hand, capllinol showed mixed type inhibiton against both α-glucosidase and PTP1B. In case of AGE formation inhibitory assay, esculetin, scopoletin, isoscopoletin, isoscopolin, scopolin, quercetin, hyperoside, vicenin 2, 4,5 di-O-caffeoyl quinic acid, and chlorogenic acid showed remarkable inhibitory potential against AGE formation with IC50 values of 5.02, 3.90, 18.78, 41.04, 60.10, 24.35, 48.20, 54.43, 6.48 and 21.08 μM, respectively compared to the positive control aminoguanidine with IC50 an value of 932.66 μM. Vcenin 2 also strongly inhibited RLAR with an IC50 value of 7.85 μM compared to the positive control quercetin with an IC50 value of 11.64 μM. Further study with vicenin 2 revealed that it can inhibit the formation of fluorescent AGE and non-fluorescent AGE such as (Nε-(carboxymethyl) lysine (CML). Vicenin 2 also inhibited protein oxidation indicated by decreasing protein carbonyl formation and thiol modification in BSA-glucose-fructose system. Moreover, vicenin 2 also suppressed the formation of β-cross amyloid structures of BSA. Therefore, the results of the present study clearly demonstrate the promising α-glucosidase, PTP1B, and AGE formation inhibitory potential of A. capillaris as well as its isolated constituents, which could be further explored to develop therapeutic or preventive agents for the treatment of diabetes and related complications.

황색 및 자색 양파 껍질의 항당뇨 및 항산화 활성 비교

양수진 부경대학교 2017 국내석사

The aim of the present study was to investigate comparative anti-diabetic activities of both water and ethanol extracts derived from yellow and red onion (Allium cepa L.) peels via human recombinant protein tyrosine phosphatase 1B (PTP1B), α-glucosidase and advanced glycation end-products (AGEs) formation inhibitory assays, as well as antioxidant activities by evaluating the ability of extracts to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), respectively. In addition, total phenolic (TP) and total flavonoid (TF) contents were measured to assess phytochemical characteristics. Higher TP and TF contents derived from both yellow and red peel ethanol extracts (YE and RE) than both yellow and red water peel extracts (YW and RW) were correlated to the corresponding higher antioxidant effects in DPPH and ABTS scavenging assays as well as anti-diabetic effects in α-glucosidase and AGEs formation assays. Between the two cultivars, yellow onion peel showed higher antioxidant activity than red onion peel. Considering the anti-diabetic potential, RE, YE, RW, and YW exhibited significant PTP1B inhibitory activities with corresponding IC50 values of 0.76 ± 0.17, 0.86 ± 0.04, 0.33 ± 0.01, and 0.30 ± 0.08 µg/mL, as compared with the positive control, ursolic acid with IC50 value of 3.40 ± 0.34 µg/mL. In particular, water extracts were found to be more effective against PTP1B inhibition than the ethanol extracts. On the other hand, yellow onion peel (YE and YW) exhibited higher α-glucosidase inhibitory activities than red onion peel (RE and RW) with the IC50 values of 3.90 ± 0.08, 5.44 ± 0.06, 5.76 ± 0.03, and 8.99 ± 0.56 µg/mL, respectively. Moreover, ethanol extracts (RE and YE) exhibited higher AGEs formation inhibitory activity than water extracts (RW and YW) with the respective IC50 values of 12.79 ± 0.22, 12.25 ± 1.35, 51.91 ± 1.53, and 25.17 ± 1.75 µg/mL. Interestingly, yellow and red onion peel extracts exhibited higher anti-diabetic activity than positive control in the respective assays. In conclusion, both yellow and red onion peel revealed significant in vitro antioxidant and anti-diabetic potential. Therefore, these results suggest that inhibitory activities of onion peel depends upon cultivar color and extraction method which could be a promising strategy to prevent diabetes and diabetic complications.

Bioactivity-guided isolation of anti-inflammatory constituents from Angelica decursiva

ISHRATJAHANISHITA 부경대학교 대학원 2014 국내석사

Angelica, a circumboreal genus comprises of more than 60 different species, is by far the largest genus of Apeaceae in the Korean flora, and many species of this genus have long been used in traditional medicine for the treatment of many diseases. Because of the high therapeutic value of the traditionally used Angelica species, extensive research has been carried out on different species of this genus which reported wide range of bioactivities including anti-inflammatory, anti-oxidant, anti-diabetic, anticancer, neuroprotective effects. Among them, Angelica decursiva has been long used in Korean traditional medicine as an antitussive, analgesic, antipyretic, and cough remedy. In a recent study, the potential antioxidant and anti-inflammatory activities of methanol (MeOH) extract of A. decursiva and its different solvent soluble fractions have been reported. Among the fractions, the ethyl acetate (EtOAc) fraction showed strong antioxidant and anti-inflammatory activities, while the dichloromethane (CH2Cl2) fraction-derived 90% MeOH soluble fraction showed the most promising anti-inflammatory activity. Although the 90% MeOH soluble fraction was found as the most active anti-inflammatory fraction, compounds responsible for anti-inflammatory activity were not identified yet. Therefore, the present study was designed to isolate active components from 90% MeOH soluble fraction and to evaluate their anti-inflammatory activity via inhibition of nitric oxide (NO) production, tumor necrosis factor-α (TNF-α) production as well as iNOS and COX-2 expression in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Repeated column chromatography of the 90% MeOH soluble fraction yielded 9 coumarins, including edulisin II (1) decursidin (2), Pd-C-III (3), 3'(S)-angeloyloxy-4'(R)-hydroxy-3',4'-dihydroxanthyletin (4),Pd-C-I (5), Pd-C-II (6), (+)-trans-decursidinol (7), umbelliferone (8), and nodakenetin (9). Among them, compound 1 was isolated for the first time from A. decursiva, while compound 4 was isolated as a new compound from natural sources. Since nodakenetin and umbelliferone showed poor NO production inhibitory activity in previous study, we have selected compounds 1 ~ 7 in the present study. Among the tested compounds, compound 1 exhibited the highest NO production inhibitory activity in a dose-dependent manner in LPS-induced NO production in RAW 264.7 cells with an IC50 value of 2.50 ± 0.16 μM. Compound 2 also showed very strong NO production inhibitory effect in LPS-stimulated RAW 264.7 cells with an IC50 value of 4.08 ± 0.09 μM, which is nearly similar to that of compound 1. In addition, compounds 3 ~ 5 showed moderate NO production inhibitory activity compared to compounds 1 and 2 with IC50 values of 15.60 ± 0.80, 26.00 ± 1.00, and 31.83 ± 0.36 μM, respectively. On the other hand, compound 6 showed weaker NO production inhibitory activity compared to compounds 1 and 2 with an IC50 value of 62.70 ± 1.86 μM. In contrast, compound 7 did not exhibit any suppressive effect on LPS-stimulated RAW 264.7 cells at the concentrations tested. Therefore, considering the NO production inhibitory activity of tested coumarins (compounds 1 ~ 7), it can be speculated that esterification of –OH at 3' or 4' position of compound 7 with senecioyl or angeloyl or acetyl group is essential for exhibiting NO production inhibitory activity by these coumarins derivatives, and the position and number of senecioyl/angeoloyl/acetyl group on these coumarins largely affects their potency. All six active coumarins (compounds 1 ~ 6) also inhibited TNF-α production and iNOS protein expression, while compounds 1 ~ 4 inhibited COX-2 protein expression in LPS-stimulated RAW 264.7 cells. These results suggest that coumarins isolated from A. decursiva might be used as potential leads for the development of therapeutic remedies for inflammation associated disorders. However, further research is needed to clarify the mechanisms of action behind this anti-inflammatory activity.

HEK293-APP695 세포에서 coptisine이 아밀로이드 베타 펩타이드 발현에 미치는 영향

변정수 부경대학교 대학원 2014 국내석사

Alzheimer's disease (AD) is regarded as the main cause of dementia and characterized by neurodegenerative disorder. Amyloid-beta (Aβ) appears in the brain of AD, it's hallmarks in AD pathology and is typically deposited as senile plaques in cerebral. Aβ is generated from amyloid precursor protein (APP) by proteases, β-secretase and γ-secretase. β-site amyloid precursor protein cleaving enzyme 1, called BACE1, it cleaves at the N-terminal domain of APP to release sAPPβ and C99, and C99 converted by γ-secretase to release diverse forms of Aβ. On the other hand, α-secretase, cleaves APP in the center of the Aβ domain, thus it prevents the generation of Aβ. Although the exact mechanism is unknown, Aβ is strongly involved in the etiology of all forms of AD. Thus, compounds that enhance α-secretase, but inhibit β- or γ-secretase activity, have therapeutic potential in the treatment of AD. Coptidis rhizoma (C. rhizoma) and its isolated alkaloids such as berberine, coptisine, palmatine, jateorrhizine, epiberberine, and groenlandicine has been reported to exhibit anti-AD and antioxidant activity through multiple pharmacological effects including cholinesterase inhibitory activity, as well as ONOO-, and ROS scavenging activity. It has also been demonstrated that berberine, a major alkaloid from C. rhizoma can reduce the production of Aβ, which plays a critical role in the pathogenesis of AD. Also, berberine, the potent alkaloid on Aβ production has similar chemical structure with other minor alkaloids from C. rhizoma. However, despite these chemical structure similarity, the inhibitory effect on Aβ secretion of minor alkaloids have not been investigated. Therefore the effect of six alkaloids of Aβ expression were evaluated using HEK293 cells stably transfected with APP695 and its activity was compared with their structure activity relationship. Among these alkaloids, berberine, epiberberine, groenlandicine, and coptisine significantly exhibited Aβ inhibitory effect and also has common structure with dioxymethylene group. On the other hand, palmatine and jateorrhizine showed low inhibition of Aβ peptide on HEK293-APP695 cells and the absence of dioxymethylene group in their structure further helps to predict the structure activity relationship. This result suggests that the presence of dioxymethylene group may attribute for the inhibitory activity of Aβ expression. Among these minor alkaloids, coptisine exhibited strong inhibition of Aβ expression. So, the focus of our investigation was the effect of coptisine on Aβ expression as well as on enzymes responsible in its cleavage using HEK293-APP695 cells. As a result, coptisine dose-, and time- dependently reduced the Aβ and the expression of β-, and γ-secretase in HEK293-APP695 cells. Therefore, coptisine from C. rhizoma seems to have a BACE inhibitor as well as PS1 inhibitor-like activity that β-, and γ-secretase induced APP processing and Aβ secretion. These results indicate that minor alkaloids such as epiberberine, groenlandicine, and coptisine have strong potential of inhibition and prevention of AD. In particular, coptisine may be a promising anti-AD agent due to its potent inhibitory activity of Aβ by inhibiting the expression of BACE1 and PS1. Therefore, coptisine would clearly have beneficial effect in the development of therapeutic and preventive agents for AD.