Hyun Ah Jung

Inhibitory phlorotannins from the edible brown algaecklonia stolonifera on total reactive oxygen species (ROS) generation

Reactive oxygen species (ROS) play an important role in the pathogenesis of many human degenerative diseases such as cancer, aging, arteriosclerosis, and rheumatism. Much attention has been focused on the development of safe and effective antioxidants. To discover sources of antioxidative activity in marine algae, extracts from 17 kinds of seaweed were screened for their inhibitory effect on total ROS generation in kidney homogenate using 2′,7′-dichlorofluorescein diacetate (DCFH-DA). ROS inhibition was seen in three species:Ulva pertusa, Symphyocladia latiuscula, andEcklonia stolonifera. At a final concentration of 25 μg/mL,U. pertusa inhibited 85.65±20.28% of total ROS generation,S. latiscula caused 50.63±0.09% inhibitory, and theEcklonia species was 44.30±7.33% inhibition.E. stolonifera Okamura (Laminariaceae), which belongs to the brown algae, has been further investigated because it is commonly used as a foodstuff in Korea. Five compounds, phloroglucinol (1), eckstolonol (2), eckol (3), phlorofucofuroeckol A (4), and dieckol (5), isolated from the ethyl acetate soluble fraction of the methanolic extract ofE. stolonifera inhibited total ROS generation.

Antioxidant flavonoids and chlorogenic acid from the leaves ofEriobotrya japonica

The antioxidant activity ofEriobotrya japonica was determined by measuring the radical scavenging effect on DPPH (1,1-diphenyl-2-picrylhydrazyl) radical and lipid peroxidation produced when mouse liver homogenate was exposed to the air at 37°C, using 2-thiobarbituric acid (TBA). The methanol extract and its fractions ofEriobotrya japonica leaves showed strong antioxidant activity. The antioxidant activity of EtOAc andn-BuOH soluble fractions were stronger than the others, and were further purified by repeated silica gel, MCI gel CHP-20P, and Sephadex LH-20 column chromatography. Antioxidant chlorogenic acid, quercetin-3-sambubioside fromn-BuOH fraction, and methyl chlorogenate, kaempferol- and quercetin-3-rhamnosides, together with the inactive ursolic acid and 2α-hydroxyursolic acid from EtOAc fraction were isolated. Antioxidant flavonoids and chlorogenic acid also showed prominent inhibitory activity against free radical generation in dichlorofluorescein (DCF) method.

Angiotensin-converting enzyme I inhibitory activity of phlorotannins from Ecklonia stolonifera

As part of our study of the isolation of antihypertensive agents derived from natural marine products, the bioactivity of 10 edible Korean seaweeds were screened by angiotensin converting enzyme (ACE) inhibitory and peroxynitrite assays. Among the crude extracts of selected sea-weeds, including five Phaeophyta (Ecklonia stolonifera, E. cava, Pelvetia siliquosa, Hizikia fusiforme, and Undaria pinnatifida), four Rhodophyta (Gigartina tenella, Gelidium amansii, Chondria crassicaulis, and Porphyra tenera) and one Chlorophyta (Capsosiphon fulvescens), the ethanol extracts of E. stolonifera, E. cava, P. siliquosa, U. pinnatifida, and G. tenella exhibited significant inhibitory properties against ACE at more than 50% inhibition at a concentration of 163.93 μg/mL. Phloroglucinol 1, eckstolonol 2, eckol 3, phlorofucofuroeckol A 4, and dieckol 5 had been isolated previously, and triphlorethol-A 6 and fucosterol 7 were isolated for the first time from E. stolonifera. Also, the ACE inhibitory and peroxynitrite scavenging properties of phlorotannins 1–6 were evaluated, along with fucosterol 7 obtained from E. stolonifera. Among profound peroxynitrite scavenging compounds 1–6, phlorotannins 3, 4 and 5 were also determined to manifest marked inhibitory activity against ACE, with 50% inhibition concentration (IC50) values of 70.82±0.25, 12.74¯0.15, and 34.25±3.56 μM, respectively.

Antioxidant principles of Nelumbo nucifera stamens.

In our ongoing study to identity antioxidants from natural sources, the antioxidant activity ofNelumbo nucifera stamens was evaluated for their potential to scavenge stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals, inhibit total reactive oxygen species (ROS) generation, in kidney homogenates using 2v,7’-dichlorodihydrofluorescein diacetate (DCHF-DA), and scavenge authentic peroxynitrites (ONOO-). A methanol (MeOH) extract of the stamens ofN. nucifera showed strong antioxidant activity in the ONOO system, and marginal activity in the DPPH and total ROS systems, so were therefore fractionated with several organic solvents, such as dichloromethane (CH2CI2), ethyl acetate (EtOAc) and n-butanol (n-BuOH). The EtOAc soluble fraction, which exhibited strong antioxidant activity in all the model systems tested, was further purified by repeated silica gel and Sephadex LH-20 column chromatographies. Seven known flavonoids [kaempferol (1), kaempferol 3-O-β-D-glucuronopyranosyl methylester (2), kaempferol 3-O-β-D-glucopyranoside (3), kaempferol 3-0-β-D-galactopyranoside (4), myricetin 3’,5’-dimethylether 3-O-β-D-glucopyranoside (5), kaempferol 3-0-α-L-rhamnopyranosyl-(1-6)-β-D-glucopyranoside (6) and kaempferol 3-O-β-D-glucuronopyranoside (7)], along with β-sitosterol glucopyranoside (8), were isolated. Compound 1 possessed good activities in all the model systems tested. Compounds 2 and 7 showed scavenging activities in the DPPH and ONOO tests, while compounds 3 and 4 were only active in the ONOO test. Conversely, compound 8 showed no activities in any of the model systems tested.

Inhibitory effects of Nelumbo nucifera leaves on rat lens aldose reductase, advanced glycation endproducts formation, and oxidative stress

The preventive and therapeutic potency against oxidative stress and diabetic complications of Nelumbo nucifera were evaluated via the 1,1-diphenyl-2-picrylhydrazyl (DPPH), Trolox equivalent antioxidant capacity (TEAC), and total reactive oxygen species (ROS) assays, as well as the rat lens aldose reductase (RLAR) and advanced glycation endproducts (AGE) assays. The leaf extract of N. nucifera exerted potent antioxidant effects as well as marked inhibitory effects for RLAR and AGE formation, corresponding to high values for total phenolic content (TPC) and total flavonoid content (TFC). Among several solvent fractions, the EtOAc and n-BuOH fractions, having prominent TPC and TFC values, showed significant antioxidant effects in the DPPH and TEAC assays. Moreover, the EtOAc fraction exhibited superior inhibitory effects in the total ROS, RLAR, and AGE assays, with IC(50) values of 9.4, 2.4, and 28.2microg/ml, respectively. Also, the HPLC profiles of the active EtOAc fraction indicated that quercetin 3-O-beta-d-glucopyranoside (Qc-3-Glc) and Qc 3-O-beta-d-glucuronopyranoside (Qc-3-Gln) were two of its major components, as well as Qc 3-O-beta-d-galactopyranoside (Qc-3-Gal) as a minor compound. Therefore, the results suggest that two key antioxidant flavonoids, Qc-3-Glc and Qc-3-Gln, may play important roles in the antioxidant and RLAR inhibitory effects of N. nucifera leaves. Also, the leaves, and the flavonoids contained within them, would clearly have potential uses in the development of therapeutic or preventive agents for diabetic complications and oxidative stress-related diseases.

Inhibitory activities of the alkaloids from Coptidis Rhizoma against aldose reductase

As part of our ongoing search of natural sources for therapeutic and preventive agents for diabetic complications, the rat lens aldose reductase (RLAR) inhibitory effect of Coptidis Rhizoma (the rhizome of Coptis chinensis Franch) was evaluated. Its extract and fractions exhibited broad and moderate RLAR inhibitory activities of 38.9∼67.5 μg/mL. In an attempt to identify bioactive components, six quaternary protoberberine-type alkaloids (berberine, palmatine, jateorrhizine, epiberberine, coptisine, and groenlandicine) and one quaternary aporphine-type alkaloid (magnoflorine) were isolated from the most active n-BuOH fraction, and the chemical structures therein were elucidated on the basis of spectroscopic evidence and comparison with published data. The anti-diabetic complications capacities of seven C. chinensis-derived alkaloids were evaluated via RLAR and human recombinant AR (HRAR) inhibitory assays. Although berberine and palmatine were previously reported as prime contributors to AR inhibition, these two major components exhibited no AR inhibitory effects at a higher concentration of 50 μg/ml in the present study. Conversely, epiberberine, coptisine, and groenlandicine exhibited moderate inhibitory effects with IC50 values of 100.1, 118.4, 140.1 μM for RLAR and 168.1, 187.3, 154.2 μM for HRAR. The results clearly indicated that the presence of the dioxymethylene group in the D ring and the oxidized form of the dioxymethylene group in the A ring were partly responsible for the AR inhibitory activities of protoberberine-type alkaloids. Therefore, Coptidis Rhizoma, and the alkaloids contained therein, would clearly have beneficial uses in the development of therapeutic and preventive agents for diabetic complications and diabetes mellitus.

Effects of C-glycosylation on anti-diabetic, anti-Alzheimer’s disease and anti-inflammatory potential of apigenin

Apigenin has gained particular interests in recent years as a beneficial and health promoting agent because of its low intrinsic toxicity. Vitexin and isovitexin, naturally occurring C-glycosylated derivatives of apigenin, have been known to possess potent anti-diabetic, anti-Alzheimers disease (anti-AD), and anti-inflammatory activities. The present study was designed to investigate the anti-diabetic, anti-AD, and anti-inflammatory potential of apigenin and its two C-glycosylated derivatives, vitexin and isovitexin by in vitro assays including rat lens aldose reductase (RLAR), human recombinant aldose reductase (HRAR), advanced glycation endproducts (AGEs), protein tyrosine phosphatase 1B (PTP1B), acetylcholinesterase (AChE), butyrylcholinesterase (BChE), β-site amyloid precursor (APP) cleaving enzyme 1 (BACE1), and nitric oxide (NO), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in lipopolysaccharide (LPS)-induced RAW 264.7 cells. Among them, isovitexin was found as the most potent inhibitor against RLAR, HRAR, AGE, AChE, and BChE while vitexin showed the most potent PTP1B inhibitory activity. Despite the relatively weak anti-diabetic and anti-AD potentials, apigenin showed powerful antiinflammatory activity by inhibiting NO production and iNOS and COX-2 expression while vitexin and isovitexin were inactive. Therefore, it could be speculated that C-glycosylation of apigenin at different positions might be closely linked to relative intensity of anti-diabetic, anti-AD, and anti-inflammatory potentials.

Anti-inflammatory Activity of Edible Brown Alga Eisenia bicyclis and Its Constituents Fucosterol and Phlorotannins in LPS-stimulated RAW264.7 Macrophages

Although individual phlorotannins contained in the edible brown algae have been reported to possess strong anti-inflammatory activity, the responsible components of Eisenia bicyclis have yet to be fully studied. Thus, we evaluated their anti-inflammatory activity via inhibition against production of lipopolysaccharide (LPS)-induced nitric oxide (NO) and tert-butylhydroperoxide (t-BHP)-induced reactive oxygen species (ROS), along with suppression against expression of inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), in RAW 264.7 cells. The anti-inflammatory activity potential of the methanolic extract and its fractions of E. bicyclis was in the order of dichloromethane>methanol>ethyl acetate>n-butanol. The strong anti-inflammatory dichloromethane fraction was further purified to yield fucosterol. From the ethyl acetate fraction, six known phlorotannins were isolated: phloroglucinol, eckol, dieckol, 7-phloroeckol, phlorofucofuroeckol A and dioxinodehydroeckol. We found that these compounds, at non-toxic concentrations, dose-dependently inhibited LPS-induced NO production. Fucosterol also inhibited t-BHP-induced ROS generation and suppressed the expression of iNOS and COX-2. These results indicate that E. bicyclis and its constituents exhibited anti-inflammatory activity which might attribute to inhibition of NO and ROS generation and suppression of the NF-κB pathway and can therefore be considered as a useful therapeutic and preventive approach to various inflammatory and oxidative stress-related diseases.

Extraction and identification of three major aldose reductase inhibitors from Artemisia montana

Aldose reductase inhibitors (ARIs) provide an important therapeutic and preventive opportunity against hyperglycemia associated diabetic complications. The methanolic extracts of 12 species from the genus Artemisia exhibited significant in vitro rat lens AR (RLAR) inhibitory activities with IC(50) values ranging from 0.51 to 13.45 μg/mL (quercetin, 0.64 μg/mL). Since the whole plant of Artemisia montana showed the highest RLAR inhibitory activity, bioassay-guided fractionation was performed to obtain ethyl acetate and n-butanol fractions. Repeated column chromatography of two active fractions, yielded fifteen compounds, including four chlorogenic acids (3,5-di-O-caffeoylquinic acid, chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid), six flavonoids (apigenin, luteolin, quercetin, isoquercitrin, hyperoside, luteolin 7-rutinoside), and five coumarins (umbelliferone, scoparone, scopoletin, esculetin, and scopolin); their structures were confirmed by spectroscopic methods. 3,5-Di-O-caffeoylquinic acid and chlorogenic acid, as well as test flavonoids, displayed the most potent RLAR inhibitory activities with IC(50) values ranging from 0.19 to 5.37 μM. Furthermore, the HPLC profiles of the ethyl acetate and n-butanol fractions indicated that 3,5-di-O-caffeoylquinic acid, chlorogenic acid, and hyperoside, as major compounds, might play crucial roles in RLAR inhibition. The results suggest that A. montana and three key AR inhibitors therein would clearly be potential candidates as therapeutic or preventive agents for diabetic complications.

Antioxidant constituents and a new triterpenoid glycoside fromFlos Lonicerae

As a component of our continuing investigations into herb-derived antioxidant agents, we have evaluated the antioxidant effects ofFlos Lonicerae (Lonicera japonica flowers),via 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, total reactive oxygen species (ROS), hydroxyl radical (OH), and peroxynitrite (ONOO-) assays. Among the methanolic extract and the dichloromethane, ethyl acetate,n-butanol, and water fractions, the EtOAc fraction ofFlos Lonicerae exhibited marked scavenging/inhibitory activities, as follows: IC50 values of 4.37, 27.58 ± 0.71, 0.47 ± 0.05, and 12.13 ± 0.79 μg/mL in the DPPH, total ROS, ONOO-, and OH assays, respectively.Via a bioactivity-guided fractionation approach, a new triterpenoid glycoside, oleanolic acid 28-O-α-L-rhamnopyranosyl-(1→2)-[β-D-xylopyranosyl(1→6)]-β-D-glucopyranosyl ester (12), along with eleven known compounds, including chrysoeriol (1), luteolin (2), 5-hydroxymethyl-2-furfural (3), caffeic acid (4), protocatechuic acid (5), chrysoeriol 7-O-β-D-glucopyranoside (6), isorhamnetin 3-O-α-D-glucopyranoside (7), kaempferol 3-O-β-D-glucopyranoside (8), quercetin 3-O-α-D-glucopyranoside (9), hederagenin 3-O-α-L-arabinopyranoside (10), and luteolin 7-O-α-D-glucopyranoside (11), were isolated from the EtOAc fraction. The structures of isolated compounds 1–12 were elucidatedvia spectroscopic analyses. Compound12 was isolated from a natural source for the first time. Compounds2,4,5,7,9, and11 evidenced marked scavenging activities, with IC50 values of 2.08-11.76 μM for DPPH radicals, and 1.47-6.98 μM for ONOO-.