본 연구는 Diode Array Detector (DAD)가 장착된 고성능 액체 크로마토그래피(HPLC)를 이용하여 산초나무 잎에 함유된 chlorogenic acid와 hesperidin을 동시에 분석할 수 있는 표준 정량법 개발을 확립하고자 실시하였다. 산초나무 잎의 chlorogenic acid와 hesperidin은 MS/MS 분석 및 표준물질과의 유지시간(RT), UV 흡광도 비교를 통해 확인하였고, 산초나무 잎의 지표물질로 선정하였다. 개발한 분석방법은 지표물질을 대상으로 특이성, 정확성, 정밀도, 검출한계, 정량한계, 선형성에 대해 최적화하고 검증하였다. HPLC 분석으로 두 화합물에 대한 검량선은 각각 0.99% 이상의 높은 상관계수(r2)를 보였다. Chlorogenic acid의 전체 회수율은 106.82%이었고, hesperidin의 전체 회수율은 107.37%였다. Chlorogenic acid의 검출한계(LOD)는 0.271μg/ml, Hesperidin의 검출한계(LOD)는 0.005μg/ml였다. Chlorogenic acid의 일간정밀도는 0.102% (RSD%), Hesperidin의 일간정밀도는 1.038% (RSD%)였다. 본 연구에서 개발한 HPLC-DAD 분석방법은 기능성 원료 인정을 위한 분석기준을 충족하였다. 따라서 본 연구에서 확립한 HPLC 분석법은 향후 산초나무 잎에서 유래한 생리활성물질의 대량추출 및 관련제품을 개발할 때 원료 표준화에 적용가능할 것이다.

구절초(Chrysanthemum zawadskii var. latilobum (Maxim.) Kitam.)는 linarin, chlorogenic acid 등의 유용성분들을 함유하고 있으며, 산업적으 로 이용가치가 있는 식물이다. 구절초의 산업화를 위해 유용성분인 linarin과 chlorogenic acid의 최적 추출 조건을 구하기 위해 반응표면분석을 수행하였으며, Linarin의 최적 추출 조건은 에탄올 농도 100%, 추출 시간 30h, 추출 온도 75℃였다. 최적 추출 조건에서 예상된 linarin의 값은 33.36 mg/g였으며 실험값은 38.33±2.66 mg/g였다. Chlorogenic acid의 최적 추출 조건은 에탄올 농도 48%, 추출 시간 30h, 추출 온도 65℃로 예상된 chlorogenic acid의 함량은 42.19 mg/g였으며 실험값은 44.34±2.09 mg/g이었다. 반응표면분석법으로 도출된 최적 추출 조건을 통해 구절초에서 linarin과 chlorogenic acid의 효율적인 추출이 가능함을 확인하였다.

Ischemic stroke is a high mortality disease that causes irreversible damage. Chlorogenic acid is a polyphenolic substance with neuroprotective properties. Bcl-2 family proteins perform a critical role in apoptosis process. Bcl-2 and Bcl-xL are anti-apoptotic proteins that prevent cell death, and Bax and Bad are pro-apoptotic proteins that promote apoptosis. We investigated whether chlorogenic acid modulates Bcl-2 family proteins during focal cerebral ischemia. We made a rat model of ischemic stroke by performing middle cerebral artery occlusion (MCAO). Chlorogenic acid (30 mg/kg) or phosphate-buffered saline was treated via intraperitoneal injection 2 hr before MCAO. Neurological behavioral tests were performed 24 hr after MCAO damage and cortical tissues were collected. Reverse transcription-PCR, Western blot, and immunofluorescence staining were performed to observe changes in Bcl-2 family proteins expression. MCAO-damage induced neurobehavioral disorders and chlorogenic acid alleviate these deficits. Bcl-2 and Bcl-xL expressions were decreased and Bax and Bad expressions were increased in MCAO animals. However, chlorogenic acid treatment attenuated the decrease of Bcl-2 and Bcl-xL and the increase of Bad and Bax due to MCAO surgery. Moreover, chlorogenic acid treatment attenuated MCAO-induced upregulation of caspase-3. These findings suggest that chlorogenic acid exerts neuroprotective effects against MCAO damage by regulating Bcl-2 family proteins including Bcl-2, Bcl-xL, Bax, and Bad.

본 연구는 그린커피빈추출물이 「건강기능식품의 기준 및 규격」에 추가로 등재될 경우를 대비하여 표준화된 클 로로겐산 시험법을 설정하고, 카페인이 동시 분석되도록 최적화하는 연구를 진행하였다. 최적화된 시험법을 마련 하기 위해 기기분석 및 전처리 조건을 비교·분석하여 클 로로겐산과 카페인을 30% 메탄올 추출하여 인산용액과 인산 함유 아세토니트릴으로 액체크로마토그래프를 통해 330 nm, 280 nm에서 분석하도록 시험법을 설정하였다. 시 험법 밸리데이션 결과, 직선성 정량범위 내에서 상관계수 (R2) 0.999 이상의 유의수준을 보였고, 클로로겐산과 카페 인 검출한계는 0.5와 0.2 μg/mL, 정량한계는 1.4와 0.4 μg/ mL로 나타났다. 정밀도와 정확도 결과는 AOAC 밸리데 이션 가이드라인를 통해 적합함을 확인하였고, 클로로겐 산 및 카페인 동시분석법을 최종적으로 마련하였다. 또한, 시제품과 유통제품을 통해 제형별 적용성 검토하여 클 로로겐산과 카페인을 동시에 정량 가능한 시험법임을 재확인하였다. 최적화된 시험법은 클로로겐산을 함유한 건강기능식품 품질관리에 대한 신뢰성을 더 높일 것으 로 본다.

Ischemic stroke is caused by a blockage of the cerebral artery, which leads to a severe neurological disorder. Chlorogenic acid is a phenolic acid found mainly in plants such as coffee beans, eggplants, and carrots. It exerts a neuroprotective effect against cerebral ischemic damage. Bcl-2 family protein is a representative apoptosis regulatory protein. Bcl-2 and Bcl-xL act as apoptosis inhibitors, while Bax and Bad act as apoptosis inducers.The interaction of Bcl-2 family protein plays an important role in determining cell fate. The aim of this study was to investigate whether chlorogenic acid modulates the interaction of Bcl-2 family proteins during ischemic injury. Middle cerebral artery occlusion (MCAO) surgery was performed to induce cerebral ischemia. Chlorogenic acid (30 mg/kg) or phosphate buffered saline was intraperitoneally injected to adult male rats 2 h after MCAO surgery. Neurobehavioral tests were performed to confirm the neuroprotective effect of chlorogenic acid 24 h after MCAO injury, and immunoprecipitation analysis was performed to investigate the interaction of Bcl-2 family protein. MCAO damage showed signs of severe neurological disorders, while chlorogenic acid improved these disorders. Results of immunoprecipitation analysis were as follows. Interaction between Bax and Bcl-2 or Bcl-xL was decreased in MCAO injury, chlorogenic acid prevents these decreases. In contrast to Bax, Interaction between Bad and Bcl-2 or Bcl-xL was increased in MCAO injury, chlorogenic acid prevents these increases. Furthermore, chlorogenic acid attenuated MCAO-induced increase of capase-9. In conclusion, our findings demonstrate that chlorogenic acid exerts a neuroprotective effect against cerebral ischemic injury by modulating interaction of Bcl-2 family proteins.

Ischemic stroke leads to severe brain damage and high mortality. Chlorogenic acid is a phenolic compound known to have neuroprotective properties. Bcl-2 family protein plays an important role in the regulation of apoptosis. We investigated whether chlorogenic acid exerts neuroprotective effects against ischemic injury by modulating Bcl-2 and Bax proteins. Middle cerebral artery occlusion (MCAO) was performed to induce cerebral ischemia and rats were injected intraperitoneally with phosphate buffered saline or chlorogenic acid (30 mg/kg) for 2 h after MCAO. Cortical tissues were collected 24 h after MCAO injury and reverse transcription-quantitative real time polymerase chain reaction and Western blot analyses were performed to investigate the expression of Bcl-2 and Bax. The regulation of Bcl-2 and Bax proteins by chlorogenic acid during glutamateinduced cell damage were examined. Cells were collected at 24 h after administration of glutamate (5 mM) and chlorogenic acid (10, 30, 50 μM). These results showed a decrease in Bcl-2 expression and an increase in Bax expression in MCAO animals, but chlorogenic acid treatment alleviated these changes by MCAO damage. Glutamate significantly reduced cell viability, and chlorogenic acid treatment alleviated this reduction in a dose-dependent manner. Glutamate induced a decrease in Bcl-2 expression and an increase in Bax expression, but chlorogenic acid treatment alleviated these changes. We found that chlorogenic acid alleviates changes in the expression of Bcl-2 and Bax proteins induced by brain injury. Therefore, our findings provide an evidence that chlorogenic acid has neuroprotective effects against MCAO damage by modulating Bcl-2 and Bax proteins.

Ischemic stroke causes severe neuronal damage. Chlorogenic acid is a phenolic substance present in fruits and coffee. It also exerts neuroprotective effects against various brain injuries. The 14-3-3 family protein perform a variety of functions including metabolism, signal transduction, cell differentiation, and apoptosis. The purpose of this study is to investigate whether chlorogenic acid regulates the expression of 14-3-3 protein in stroke animal models. Ischemic stroke was induced by middle cerebral artery occlusion (MCAO) surgery. Phosphate buffered saline (PBS) or chlorogenic acid (30 mg/kg) were intraperitoneally injected to adult male rats 2 h before MCAO surgery. Adhesive-removal test was performed 24 h after MCAO surgery and cerebral cortical tissues were collected for further study. MCAO damage caused severe neurological impairment and chlorogenic acid treatment ameliorated this disorder. Our proteomic approach showed a decrease in 14-3-3 expression in MCAO animals with PBS. The decrease in 14-3-3 expression alleviated in MCAO animal with chlorogenic acid. We confirmed changes in various 14-3-3 protein isoforms, including beta/alpha, zeta/delta, gamma, epsilon, eta, and tau through reverse transcription-PCR. These results explained that chlorogenic acid regulates the expression of 14-3-3 protein in MCAO-induced cerebral ischemia. 14-3-3 is considered to be an important protein for cell survival through binding to pro-apoptotic proteins. The maintenance of 14-3-3 levels is an important event in neuroprotection against ischemic injury. Therefore, we can demonstrate that the 14-3-3 protein contributes to the neuroprotective effect of chlorogenic acid in stroke animal models.

본 연구에서는 chlorogenic acid 이성질체 9종과 arbutin을 분석하기 위하여, 전처리 과정과 UHPLC-MS/MS 동시 분석 조건을 확립하였다. UHPLC-MS/MS에서 C18 column을 사용하여 15분 동안 분석할 수 있는 용매 조성을 만들고, 10가지 물질에 대한 정량, 정성 이온을 선택하여 negative 모드에서 분석하는 동시분석법을 확립하였고 과일류를 대상으로 추출, 진탕 추출, 초음파 추출, 원심분리, 농축 등의 과정을 거치는 전처리법을 개발하였다. 기기분석과 전처리법에 대한 유효성은 특이성, 직선성, 정확성, 정밀성 그리고 검출한계 및 정량한계 등을 통하여 확인하였다. 회수율은 48.1-120.3%, intraday precision (RSD)는 0.4-7.9%, interday precision (RSD)는 0.0-7.2%로 확인되었다. 따라서, 본 연구에서 확립한 동시분석방법은 과일류 중 chlorogenic acid와 arbutin을 효과적으로 분석하는데 유용할 것으로 사료된다.

In all living organisms, respiration may lead to oxidative stress, a state where increased formation of reactive oxygen species overwhelms host protection and subsequently induces DNA damage, lipid peroxidation, and protein denaturation. As a phenolic acid, chlorogenic acid occurs ubiquitously in food. It has been proven to have a number of biological effects in vitro and in vivo, including antioxidant, anti-inflammatory, and anticarcinogenic properties. Therefore, to some extent, chlorogenic acid can promote human health, and hopefully provide new methods for treatment of chronic disease. Recent studies have focused on the antioxidant properties of dietary polyphenols. The in vitro data often conflict with results obtained from in vivo studies on the antioxidant capacity of plasma or the resistance of plasma and lipoproteins to oxidation ex vivo after consumption of polyphenol-rich foods by human subjects. These inconsistencies are likely explained by the limited bioavailability of dietary polyphenols and their extensive metabolism in humans. Polyphenols exert multifaceted actions, and any clinical application using these substances should be based on a precise understanding of the physiologically relevant mechanisms.

Background : Campanula takesimana is a herb(Jabanpungnyeoncho) used traditionally in the korea private and we tried the development on a medicinal material. It was known that it has chlorogenic acid, as a immunoadjuvant activity. In this study we investigated the contents of the chlorogenic acid in Companula takesimana at different harversting time by the high performance liquid chromatographic(HPLC)-PDA. Campanula takesimana was collected on May maddle (Flower buds farmed), June middle (flowers opened) and July middle (seeds were mature). Methods and Results : HPLC analysis was carried out using X-bridge C18 column (5um, 250*4.6mm) and a mobile phase consisting of acetonitrile and water containing 0.1% formic acid at a flow rate of 1.0 ml/min. The optimum wavelength for the detection of the Chlorogenic acid was 330 nm using Photo Diode Array Detector. The contents of the chlorogenic acid in the Campanula takesimana extrat were 1.00% (May middle), 0.84% (June middle), 0.05% (July middle), respectively. As the different parts of Campanula takesimana, chlorogenic acid contents in May middle extrat were 2.73% (aerial part), 0.05% (root) and in June middle extrat were 1.61% (aerial part), 0.03% (root), 0.70% (flower) and in July middle were 0.13% (aerial part), 0.03% (root), 0.00% (seed), respectively. Conclusion : From the above results, the highes content of chlorogenic acid was observed in aerial part and May middle extract of Campanula takesimana.

Chlorogenic acid, formed of an ester of caffeic acid and quinic acid, which is naturally abundant in many plant species, was used as a model O-dihydoxy phenolic compound. In the previous study, we have reported that the isolated constituent from Apocynum venetum leaves has an inhibitory effect on Cu2+-induced oxidative modification of low-density lipoprotein (LDL). Among them, chlorogenic acid showed the most potent anti-LDL oxidative activity than other compounds. For the reason, we investigated the inhibitory effect of the chlorogenic acid on Cu2+-induced oxidative modification of LDL, monitored a lag time in the conjugated-diene formation and TBARS formation, and measured TNBS free amino acid group, and form cell formation in vitro system. The TBARS- and diene- formation were strongly inhibited by chlorogenic acid (0~100 μg/ml) with dose dependent manner. On the other hand, TNBS reactive lysine amino groups on LDL oxidation were protected by chlorogenic acid- treated cell group. Therefore, chlorogenic acid inhibited to cholesterol accumulation in the isolated peritoneal macrophage.

A series of aqueous or methanol extracts from four different Korean salad plants were assayed to determine their allelopathy and antioxidant activity. The extracts applied on filter paper in a Petri-dish bioassay significantly inhibited root growth of against alfalfa (Medicago sativa) seedlings. Leaf extracts from 40 g dry tissue L-1 of Aster yomena was most phytotoxic to alfalfa root growth, and followed by that of Cirsium japonicum, Taraxacum officinale, and Ixeris dentate. Methanol extracts of plants dose-dependently increased DPPH free radical scavenging activity in vitro. Antioxidant activity of methanol extracts from the same plant species was investigated, and the result showed high DPPH free radical scavenging activity in Cirsium japonicum, Aster yomena, and Ixeris dentate, however, in Taraxacum officinale was least activity. By means of HPLC analysis, chlorogenic acid, p-coumaric acid, and total phenolics with 7.68, 17.47 and 18.64 mg, 100g-1, respectively, showed the highest amounts in methanol extracts from Cirsium japonicum leaves. These results suggest that Compositae salad plants contain water-soluble substances with allelopathic potential as well as antioxidant activity.

Lettuce (Lactuca sativa L.) is known to contain water-soluble substances that improve antioxidant status due to the richness in antioxidants. Greenhouse experiment was carried out under different shading conditions during spring lettuce growing season. Shade significantly reduced shoot weight, number of leaves and chlorophyll content, while it increased shoot length of lettuce plants. The antioxidant potential of the individual fraction was in order of n-butanol 〉 ethyl acetate 〉 water 〉 n-hexane fraction, although was less than that of commonly used antioxidants, BHT and ascorbic acid. Fractions from lettuce plants dose-dependently increased DPPH free radical scavenging activity, in vitro test. By means of HPLC analysis, BuOH fraction of cultivar 'Hwahyang' (57.93 mg 100g-1) had the highest amount of antioxidant chi orogenic acid. Shading treatment increased average amount of chlorogenic acid of all cultivars in BuoH, EtOAc, hexane and water fractions by 33, 120, 144, and 58%, respectively. These results suggest that lettuce plants had potent antioxidant activity, and their activities were differently exhibited depending on cultivar and fraction.