Na Young Yoon

Acetyl- and butyrylcholinesterase inhibitory activities of sterols and phlorotannins from Ecklonia stolonifera

As part of this study on the isolation of cholinesterase inhibitors from natural marine products, the bioactivity of the ethanolic extracts from 27 Korean seaweeds were screened using acetylcholinesterase (AChE) and butyrylcholine sterase (BChE) inhibitory assays. Ecklonia stolonifera exhibited promising inhibitory properties against both AChE and BChE. Bioassay-guided fractionation of the active n-hexane and ethyl acetate (EtOAc) soluble fractions, obtained from the ethanolic extract of E. stolonifera, resulted in the isolation of the sterols; fucosterol (1) and 24-hydroperoxy 24-vinylcholesterol (2), from the n-hexane fraction and the phlorotannins; phloroglucinol (3), ecks-tolonol (4), eckol (5), phlorofucofuroeckol-A (6), dieckol (7), triphlorethol-A (8), 2-phloroeckol (9) and 7-phloroeckol (10), from the EtOAc fraction. Of these, compounds 2, 9 and 10 were isolated from E. stolonifera for the first time. Compounds 4–7, 9 and 10 exhibited inhibitory potential against AChE, with 50% inhibition concentration (IC50) values of 42.66±8.48, 20.56±5,61, 4.89±2.28, 17.11±3.24, 38.13±4.95 and 21.11±4.16 μM, respectively; whereas, compounds 1, 2, 4 and 6 were found to be active against BChE, with IC50 values of 421.72±1.43, 176.46±2.51, 230.27±3.52 and 136.71±3.33 μM, respectively. It has been suggested that the inhibition of these enzymes by the sterols and phlorotannins derived from marine brown algae could be a useful approach for the treatment of Alzheimer’s disease.

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.

Hepatoprotective constituents of the edible brown alga Ecklonia stolonifera on tacrine-induced cytotoxicity in Hep G2 cells

In this study, ethanolic extracts from 18 seaweed variants were assessed for hepatoprotective activity against tacrine-induced cytotoxicity in Hep G2 cells. Only one of these,Ecklonia stolonifera Okamura (Laminariaceae), a member of the brown algae, exhibited promising hepatoprotective activity. Bioassay-guided fractionation of the active ethyl acetate (EtOAc) soluble fraction obtained from the ethanolic extract ofE. stolonifera, resulted in the isolation of several phlorotannins [phloroglucinol (1), eckstolonol (2), eckol (3), phlorofucofuroeckol A (4), and dieckol (5)]. Compounds 2 and 4 were determined to protect Hep G2 cells against the cytotoxic effects of tacrine, with EC50 values of 62.0 and 79.2 μg/mL, respectively. Silybin, a well characterized hepatoprotective agent, was used as a positive control, and exhibited an EC50 value of 50.0 μg/mL. It has been suggested that the phlorotannins derived from marine brown algae might prove useful sources in the development of novel hepatoprotective agents.

Anti-hyperlipidemic Effect of an Edible Brown Algae, Ecklonia stolonifera, and its Constituents on Poloxamer 407-Induced Hyperlipidemic and Cholesterol-fed Rats

We conducted this study to isolate novel anti-hyperlipidemic agents derived from natural marine products. To accomplish this, we investigated the effects of ethanolic (EtOH) extracts of Ecklonia stolonifera and its phlorotannin constituents, eckol and dieckol, on serum lipid levels in rats with hyperlipidemia that was induced by a high-cholesterol diet or poloxamer 407. Treatment with the EtOH extracts of E. stolonifera and its phlorotannin-rich ethyl acetate (EtOAc) and n-butanol (n-BuOH) fractions induced a significant reduction in triglycerides (TG), total cholesterol (TC), and low-density lipoprotein-cholesterol (LDL-C) levels, as well as a significant increase in the high-density lipoprotein-cholesterol (HDLC) level in hyperlipidemic rats. However, treatment with the water (H2O) fraction did not exert any significant effects on the serum levels of hyperlipidemic rats. In addition, eckol and dieckol isolated from the active EtOAc fraction induced a significant reduction in serum TG, TC, and LDL-C levels, as well as in the atherogenic index (A.I.). Furthermore, treatment with dieckol induced a greater decrease in the serum TG, TC, and LDL-C levels of hyperlipidemic rats than eckol or lovastatin, as well as an increase in the serum HDL-C levels. Taken together, these results suggest that phlorotannins such as eckol and dieckol have the potential for use for the prevention of hyperlipidemic atherosclerosis.

Inhibitory activities of prenylated flavonoids from Sophora flavescens against aldose reductase and generation of advanced glycation endproducts

Important targets for the prevention and treatment of diabetic complications include aldose reductase (AR) inhibitors (ARIs) and inhibitors of advanced glycation endproduct (AGE) formation. Here we evaluate the inhibitory activities of prenylated flavonoids isolated from Sophora flavescens, a traditional herbal medicine, on rat lens AR (RLAR), human recombinant AR (HRAR) and AGE formation. Among the tested compounds, two prenylated chalcones — desmethylanhydroicaritin (1) and 8‐lavandulylkaempferol (2) — along with five prenylated flavanones — kurarinol (8), kurarinone (9), (2S)‐2′‐methoxykurarinone (10), (2S)‐3β,7,4′‐trihydroxy‐5‐methoxy‐8‐(γ,γ‐dimethylally)‐flavanone (11), and kushenol E (13) were potent inhibitors of RLAR, with IC50 values of 0.95, 3.80, 2.13, 2.99, 3.77, 3.63 and 7.74 μM, respectively, compared with quercetin (IC50 7.73 μM). In the HRAR assay, most of the prenylated flavonoids tested showed marked inhibitory activity compared with quercetin (IC50 2.54 μM). In particular, all tested prenylated flavonols, such as desmethylanhydroicaritin (1, IC50 0.45 μM), 8‐lavandulylkaempferol (2, IC50 0.79 μM) and kushenol C (3, IC50 0.85 μM), as well as a prenylated chalcone, kuraridin (5, IC50 0.27 μM), and a prenylated flavanone, (2S)‐7,4′‐dihydroxy‐5‐methoxy‐8‐(γ,γ‐dimethylally)‐flavanone (12, IC50 0.37 μM), showed significant inhibitory activities compared with the potent AR inhibitor epalrestat (IC50 0.28 μM). Interestingly, prenylated flavonoids 1 (IC50 104.3 μgmL−1), 2 (IC50 132.1 μgmL−1), 3 (IC50 84.6 μgmL−1) and 11 (IC50 261.0 μgmL−1), which harbour a 3‐hydroxyl group, also possessed good inhibitory activity toward AGE formation compared with the positive control aminoguanidine (IC50 115.7 μgmL−1). Thus, S. flavescens and its prenylated flavonoids inhibit the processes that underlie diabetic complications and related diseases and may therefore have therapeutic benefit.

Inhibitory activities of extracts from several kinds of seaweeds and phlorotannins from the brown alga Ecklonia stolonifera on glucose-mediated protein damage and rat lens aldose reductase

Diabetic complications, including retinopathy, neuropathy, nephropathy and cataracts, are the leading causes of death in diabetics. Also, an increasing population of diabetic patients has triggered the increasing publicity for treatments and prevention of diabetic complications throughout the world. There are direct evidences indicating the relevance of advanced glycation endproducts (AGE) and aldose reductase (AR) in diabetic complications, both of which are highly implicated in hyperglycemia and oxidative stress. In chronic hyperglycemia, there are evidences of increasing AGE and overexpression of AGE receptors (RAGE), as well as AR-related polyol pathway flux. Glycation, a non-enzymatic Maillard reaction between reducing sugars and proteins, is related to the pathogenesis of diabetic complications. In the later stages of the Maillard reaction, the products undergo conversion to reactive dicarbonyl intermediates to form AGE, which have properties such as cross-linking fluorescence that include pentosidine and crosslines. Excess glucose is metabolized into sorbitol by the NADP+-AR pathway, followed by NAD+-sorbitol dehydrogenase induced fructose formation, which is highly related to AGE formation. In other pathways, glyceraldehyde-3phosphate is degenerated by transition metal ions and oxygen (autoxidation and glycoxidation). Due to the different steps to form the end-products, the development of AGE inhibitors has approached several targets, such as AR inhibition and antioxidant activity. There is growing interest in the benefits of seaweeds as nutraceuticals, which have been promoted by the present work on the inhibitory effects of seaweeds on diabetic complications. The objectives of this present study were to investigate the inhibitory effects of several seaweeds, as well as Ecklonia stolonifera and isolated phlorotannins, on AGE and RLAR. Furthermore, the study took into account the possible-mechanism involved, such as an oxidative stress-mediated pathway, as well as the development of therapeutic or preventing agents for diabetic complications and related diseases. The assay for the inhibition of AGE formation was as follows: To prepare the AGE reaction solution, 10 mg/mL of bovine serum albumin (Sigma, St. Louis, MO, USA) in 50 mM sodium phosphate buffer (pH 7.4), with 0.02% sodium azide to prevent bacterial growth, was added to 0.2 M fructose and 0.2 M glucose. The reaction mixture (950 mL) was then mixed with various concentrations of the samples (50 mL, f.c. 200, 100, 50 mg/mL) dissolved in 10% dimethyl sulfoxide. After incubating at 37°C for 7 days, the fluorescence intensity of the reaction products was determined on a spectrofluorometric detector (FLx800 microplate fluorescence reader; Bio-Tek Instrument Inc., Winooski, VT, USA) with excitation and emission wavelengths at 350 nm and 450 nm, respectively. The inhibition percentage of AGE formation *Corresponding author: Tel: 82-51-620-6335. Fax: 82-51-620-6330. Email: choijs@pknu.ac.kr Received 18 September 2007. Accepted 15 January 2008. FISHERIES SCIENCE 2008; 74: 1363–1365

A potent anti-complementary acylated sterol glucoside fromOrostachys japonicus

In order to isolate substances that inhibit the hemolytic activity of human serum against erythrocytes, we have evaluated whole plants of theOrostachys japonicus species with regard to its anti-complement activity, and have identified its active principles following activity-guided isolation. A methanol extract of theO. japonicus, as well as itsn-hexane soluble fraction, exhibited significant anti-complement activity on the complement system, which was expressed as total hemolytic activity. A bioassay-guided chromatographic separation of the constituents resulted in the isolation of three known compounds1–3 from the activen-hexane fraction. The structure of these compounds were analyzed, and they were identified as hydroxyhopanone (1), β-sitosteryl-3-O-β-d-glucopyranosyl-6′-O-palmitate (2), and β-sitosteryl-3-O-β-d-glucopyranoside (3), respectively. Of these compounds, compound2 exhibited potent anti-complement activity (IC50=1.0±0.1 μM) on the classical pathway of the complement, as compared to tiliroside (IC50=76.5±1.1 μM), which was used as a positive control. However, compounds1 and3 exhibited no activity in this system.

Quinone Reductase Induction Activity of Phlorotannins Derived from Eisenia bicyclis in Hepa1c1c7 Cells

To assess the feasibility of phlorotannins from Eisenia bicyclis as cancer chemopreventative agents, we tested whether they induced quinone reductase (QR) in Hepa1c1c7 cells. The ethyl acetate (EtOAc) soluble fraction obtained from E. bicyclis exhibited a QR induction activity in Hepa1c1c7 cells. Successive column chromatography of the active EtOAc fraction resulted in the isolation of four phlorotannins. Their structures were elucidated using one- and two-dimensional nuclear magnetic resonance spectroscopic techniques and characterized as phloroglucinol (1), dioxinodehydroeckol (2), dieckol (3), and fucofuroeckol-A (4). Among these compounds, fucofuroeckol-A (4) showed moderate QR induction activity, and dioxinodehydroeckol (2) exhibited potent QR induction potency with 2.05 ± 0.04 fold induction at a concentration of 50 µM compared to the dimethyl sulfoxide solvent-treated control cells. However, phloroglucinol (1) and dieckol (3) exerted no detectable QR induction activity in Hepa1c1c7 cells. These results suggest that dioxinodehydroeckol could serve as a useful cancer chemopreventive chemical.

Relationship of pH, Glycogen, Soluble Protein, and Turbidity between Freshness of Raw Oyster Crassostrea gigas

We examined chemical changes in oysters Crassostrea gigas and packing water that were sold after storage at 5, 10, and 20°C. The pH of oysters stored at 5°C dropped to 5.81 after 10 days of storage, while that of oysters at 10°C and 20°C dropped to 5.37 after 8 days and to 5.04 after 4 days, respectively. The glycogen content of oysters stored at 5°C decreased from 718.89 to 421.85 mg/100g during storage, while that of oysters at 10°C decreased to 351.49 mg/100 g after 4 days. The turbidity and soluble protein in packing water increased slightly. The viable cell count of oysters did not exceed 6 log CFU/g after 10 days of storage at 5°C, but that of oysters at 10°C did so after 8 days. Additionally, the viable cell count of packing water was lower than that of oysters. We performed a principal component analysis, where the first principal component (55.03%-57.24%) and second principal component (42.76%-44.97%) described most variation. The first principal component included the pH of oysters and packing water, and the glycogen content of oysters. A Pearson correlation between the first two principal components had a higher R value than that between other components. Freshness was evaluated using the pH of oysters and packing water, and glycogen. We found that soluble protein content was significantly associated with a lower pH and glycogen content.