D,L-Methadone causes leukemic cell apoptosis via an OPRM1-triggered increase in IP3R-mediated ER Ca2+ release and decrease in Ca2+ efflux, elevating [Ca2+]i.

The search continues for improved therapy for acute lymphoblastic leukemia (aLL), the most common malignancy in children. Recently, D,L-methadone was put forth as sensitizer for aLL chemotherapy. However, the specific target of D,L-methadone in leukemic cells and the mechanism by which it induces leukemic cell apoptosis remain to be defined. Here, we demonstrate that D,L-methadone induces leukemic cell apoptosis through activation of the mu1 subtype of opioid receptors (OPRM1). D,L-Methadone evokes IP3R-mediated ER Ca2+ release that is inhibited by OPRM1 loss. In addition, the rate of Ca2+ extrusion following D,L-methadone treatment is reduced, but is accelerated by loss of OPRM1. These D,L-methadone effects cause a lethal rise in [Ca2+]i that is again inhibited by OPRM1 loss, which then prevents D,L-methadone-induced apoptosis that is associated with activation of calpain-1, truncation of Bid, cytochrome C release, and proteolysis of caspase-3/12. Chelating intracellular Ca2+ with BAPTA-AM reverses D,L-methadone-induced apoptosis, establishing a link between the rise in [Ca2+]i and D,L-methadone-induced apoptosis. Altogether, our findings point to OPRM1 as a specific target of D,L-methadone in leukemic cells, and that OPRM1 activation by D,L-methadone disrupts IP3R-mediated ER Ca2+ release and rate of Ca2+ efflux, causing a rise in [Ca2+]i that upregulates the calpain-1-Bid-cytochrome C-caspase-3/12 apoptotic pathway.

Neuroprotective Effect of ß-secretase Inhibitory Peptide from Pacific Hake (Merluccius productus) Fish Protein Hydrolysate.

BACKGROUND: Various methodologies have been employed for the therapeutic interpolation of the progressive brain disorder Alzheimer's disease. Thus, ß-secretase inhibition is significant to prevent disease progression in the early stages. OBJECTIVE: This study seeks to purify and characterize a novel ß-secretase inhibitory peptide from Pacific hake enzymatic hydrolysate. METHODS: A potent ß-secretase inhibitory peptide was isolated by sequential purifications using Sephadex G-25 column chromatography and octadecylsilane (ODS) C18 reversed-phase HPLC. A total of seven peptides were synthesized using the isolated peptide sequences. SH-SY5Y cells stably transfected with the human ''Swedish'' amyloid precursor protein (APP) mutation APP695 (SH-SY5YAPP695swe) were used as an in-vitro model system to investigate the effect of Leu-Asn peptide on APP processing. RESULTS: The ß-secretase inhibitory activity (IC50) of the purified peptide (Ser-Leu-Ala-Phe-Val-Asp- Asp-Val-Leu-Asn) from fish protein hydrolysate was 18.65 µM and dipeptide Leu-Asn was the most potent ß-secretase inhibitor (IC50 value = 8.82 µM). When comparing all the seven peptides, the inhibition pattern of Leu-Asn dipeptide was found to be competitive by Lineweaver-Burk plot and Dixon plot (Ki value = 4.24 µM). The 24 h treatment with Leu-Asn peptide in SH-SY5Y cells resulted in reducing the ß-amyloid (Aß) production in a dose-dependent manner. CONCLUSION: Therefore, the results of this study suggest that ß-secretase inhibitory peptides derived from marine organisms could be potential candidates to develop nutraceuticals or pharmaceuticals as antidementia agents.

Anti-Alzheimer's Materials Isolated from Marine Bio-resources: A Review.

The most common type of dementia found in the elderly population is Alzheimer's disease. The disease not only impacts the patients and their families but also the society therefore, the main focus of researchers is to search new bioactive materials for treating AD. The marine environment is a rich source of functional ingredients and to date, we can find sufficient research relating to anti- Alzheimer's compounds isolated from marine environment. Therefore, this review focuses on the anti- Alzheimer's material from marine bio-resources and then expounds on the anti-Alzheimer's compounds from marine seaweed, marine animal and marine microorganisms. Moreover, because of the complexity of the disease, different hypothesizes have been elaborated and active compounds have been isolated to inhibit different stages of pathophysiological mechanisms. Sulfated polysaccharides, glycoprotein, and enzymatic hydrolysates from marine seaweeds, peptides, dietary omega-3 polyunsaturated fatty acids and skeletal polysaccharide from marine animals and secondary metabolites from marine microorganism are summarized in this review under the anti-Alzheimer's compounds from the marine.

Antioxidant Activities of Viviparus Contectus Extract Against Tert-Butylhydroperoxide-Induced Oxidative Stress.

In this study, the antioxidant properties of Viviparus contectus (V. contectus) extract were evaluated for various radical scavenging activities, ferric reducing antioxidant power (FRAP), ABTS radical scavenging activity and oxygen radical absorbance capacity (ORAC). In addition, inhibition effect of the V. contectus extract against DNA scission induced by hydroxyl radical was measured. We also studied the protective effect of V. contectus extract against oxidative damage through measurements of intracellular reactive oxygen species (ROS) in Chang cells and zebrafish embryo. We found that V. contectus extract contains strong radical scavenging activities and antioxidant properties, which prevent tert-butylhydroperoxide (t-BHP)-induced oxidative stress, enhance cell viability, reduce ROS production, inhibit oxidative damage and improve mitochondrial function in Chang cells. Also, we determined that the V. contectus extract reduced ROS production mediated by t-BHP induced oxidative stress on zebrafish embryo.

Antioxidant and Protective Effects of Atrina Pectinata Extract.

Atrina pectinata (A. pectinata), called pen shell, is an edible shellfish that adheres to the seabed pointed downward and has a triangular shell reaching about 40 cm in length.In this study, we examined the antioxidant effect of an A. pectinata extract exhibiting various radical scavenging activities. These scavenging activities were evaluated using electron spin resonance. Anti-oxidant activities were also determined using the ferric reducing antioxidant power (FRAP) and the ABTS radical scavenging assays. Lipid peroxidation inhibitory activity was confirmed using ferric thiocyanate and thiobarbituric acid assays. Furthermore, the protective effect of the A. pectinata extract against t-BHP-induced oxidative stress on Chang cells were evaluated using MTT assay and the measurement of reactive oxygen species (ROS). These results showed that the A. pectinata extract have strong radical scavenging activities, and exerts protective effect against oxidative stress through reducing intracellular ROS content of Chang cells.

HAP1 loss confers l-asparaginase resistance in ALL by downregulating the calpain-1-Bid-caspase-3/12 pathway.

l-Asparaginase (l-ASNase) is a strategic component of treatment protocols for acute lymphoblastic leukemia (ALL). It causes asparagine deficit, resulting in protein synthesis inhibition and subsequent leukemic cell death and ALL remission. However, patients often relapse because of the development of resistance, but the underlying mechanism of ALL cell resistance to l-asparaginase remains unknown. Through unbiased genome-wide RNA interference screening, we identified huntingtin associated protein 1 (HAP1) as an ALL biomarker for l-asparaginase resistance. Knocking down HAP1 induces l-asparaginase resistance. HAP1 interacts with huntingtin and the intracellular Ca2+ channel, inositol 1,4,5-triphosphate receptor to form a ternary complex that mediates endoplasmic reticulum (ER) Ca2+ release upon stimulation with inositol 1,4,5-triphosphate3 Loss of HAP1 prevents the formation of the ternary complex and thus l-asparaginase-mediated ER Ca2+ release. HAP1 loss also inhibits external Ca2+ entry, blocking an excessive rise in [Ca2+]i, and reduces activation of the Ca2+-dependent calpain-1, Bid, and caspase-3 and caspase-12, leading to reduced number of apoptotic cells. These findings indicate that HAP1 loss prevents l-asparaginase-induced apoptosis through downregulation of the Ca2+-mediated calpain-1-Bid-caspase-3/12 apoptotic pathway. Treatment with BAPTA-AM [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester)] reverses the l-asparaginase apoptotic effect in control cells, supporting a link between l-asparaginase-induced [Ca2+]i increase and apoptotic cell death. Consistent with these findings, ALL patient leukemic cells with lower HAP1 levels showed resistance to l-asparaginase, indicating the clinical relevance of HAP1 loss in the development of l-asparaginase resistance, and pointing to HAP1 as a functional l-asparaginase resistance biomarker that may be used for the design of effective treatment of l-asparaginase-resistant ALL.

Chlorella ethanol extract induced phase II enzyme through NFE2L2 (nuclear factor [erythroid-derived] 2-like 2, NRF2) activation and protected ethanol-induced hepatoxicity.

In this study, we investigated the hepatoprotective effects of ethanol extracts from Chlorella vulgaris (CH) on animals. We measured its effect on the quinone reductase (QR) activity in Hepa1c1c7 cells, finding that CH induced a significantly higher QR activity in these cells. We isolated the active fraction (CH F4-2) from CH using chromatography methods. CH F4-2 may activate cellular antioxidant enzymes through upregulation of the Nrf2 pathway in hepatocarcinoma cells with CH F4-2 (25.0-200 µg/mL) for 48 h. Furthermore, CH F4-2 increased the expression of NQO1 [ NAD(P)H: quinone oxidoreductase, also known as QR], heme oxygenase-1, and glutathione-S-transferase P. Moreover, we found that ethanol-induced hepatic pathological changes-elevations in glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, γ-glutamyltransferase, and lactate dehydrogenase-were significantly decreased. The inhibitory effect of CH on alcohol-induced liver injury was associated with the suppression of alcohol-induced increases in intestinal permeability. The ethanol extract from CH was found to induce QR activation, making it a potentially good candidate for a hepatoprotection agent.

Angiotensin-I converting enzyme inhibitory peptides from antihypertensive skate (Okamejei kenojei) skin gelatin hydrolysate in spontaneously hypertensive rats.

The aim of this study was to investigate antihypertensive effect of bioactive peptides from skate (Okamejei kenojei) skin gelatin. The Alcalase/protease gelatin hydrolysate below 1 kDa (SAP) exhibited the highest angiotensin-I converting enzyme (ACE) inhibition compared to other hydrolysates. SAP can decrease systolic blood pressure significantly in spontaneously hypertensive rats. SAP inhibited vasoconstriction via PPAR-γ expression, activation and phosphorylation of eNOS in lungs. Moreover, the expression levels of endothelin-1, RhoA, α-smooth muscle actin, cleaved caspase 3 and MAPK were decreased by SAP in lungs. Vascularity, muscularization and cellular proliferation in lungs were detected by immunohistochemical staining. Finally, two purified peptides (LGPLGHQ, 720Da and MVGSAPGVL, 829Da) showed potent ACE inhibition with IC50 values of 4.22 and 3.09 µM, respectively. These results indicate that bioactive peptides isolated from skate skin gelatin may serve as candidates against hypertension and could be used as functional food ingredients.

Inhibitory effect of the carnosine-gallic acid synthetic peptide on MMP-2 and MMP-9 in human fibrosarcoma HT1080 cells.

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that degrade extracellular matrix components and play important roles in a variety of biological and pathological processes such as malignant tumor metastasis and invasion. In this study, we constructed carnosine-gallic acid peptide (CGP) to identify a better MMP inhibitor than carnosine. The inhibitory effects of CGP on MMP-2 and MMP-9 were investigated in the human fibrosarcoma (HT1080) cell line. As a result, CGP significantly decreased MMP-2 and MMP-9 expression levels without a cytotoxic effect. Moreover, CGP may inhibit migration and invasion in HT1080 cells through the urokinase plasminogen activator (uPA)-uPA receptor signaling pathways to inhibit MMP-2 and MMP-9. Based on these results, it appears that CGP may play an important role in preventing and treating several MMP-2 and MMP-9-mediated health problems such as metastasis.

ß-secretase inhibitory activity of phenolic acid conjugated chitooligosaccharides.

Eight kinds of phenolic acid conjugated chitooligosaccharides (COSs) were synthesized using hydroxyl benzoic acid and hydroxyl cinnamic acid. These phenolic acid conjugated-COSs with different substitution groups, including p-hydroxyl, 3,4-dihydroxyl, 3-methoxyl-4-hydroxyl and 3,5-dimethoxyl-4-hydroxy groups, were evaluated for their inhibitory activities against ß-site amyloid precursor protein (APP)-cleaving enzyme (BACE) and inhibited BACE with a ratio of 50.8%, 74.8%, 62.1%, 64.8% and 42.6%, respectively at the concentration of 1,000 µg/mL. BACE is a critical component to reduce the levels of Aß amyloid peptide in Alzheimer's disease (AD) which is based on the amyloid cascade theory in the brain, as this protease initiates the first step in Aß production. Among them, Caffeic acid conjugated-COS (CFA-COS) was further analysed to determine mode of inhibition of BACE and it showed non-competitive inhibition. Hence in this study, we suggest that CFA-COS derivatives have potential to be used as novel BACE inhibitors to reduce the risk of AD.

Nitric oxide-mediated vasorelaxation effects of anti-angiotensin I-converting enzyme (ACE) peptide from Styela clava flesh tissue and its anti-hypertensive effect in spontaneously hypertensive rats.

In our previous study, an anti-angiotensin I converting enzyme (ACE) peptide (Ala-His-Ile-Ile-Ile, MW: 565.3Da) was isolated from Styela clava flesh tissue. In this study the fractions obtained during the isolation process and the finally purified peptide were examined to see if they had vasorelaxation effects in isolated rat aortas, and then the peptide was investigated for anti-hypertensive effect in spontaneously hypertensive rats (SHRs). The induction of vasorelaxation in the rat aortas was observed with the isolated fractions and the peptide from the enzymatic hydrolysate of S. clava flesh tissue and could be markedly blocked by pretreatment with the nitric oxide synthase (NOS) inhibitor, N(G)-nitro-l-arginine methyl ester (l-NAME). In human endothelial cells, NO synthesis was found to be increased and eNOS phosphorylation was upregulated when the cells were cultured with the purified peptide. Furthermore, systolic blood pressure was reduced by administration of the potent vasorelaxation peptide in SHRs.

Characterization of bioactive peptides obtained from marine invertebrates.

Bioactive peptides as products of hydrolysis of diverse marine invertebrate (shellfish, crustacean, rotifer, etc.) proteins are the focus of current research. After much research on these muscles and by-products, some biologically active peptides were identified and applied to useful compounds for human utilization. This chapter reviews bioactive peptides from marine invertebrates in regarding to their bioactivities. Additionally, specific characteristics of antihypertensive, anti-Alzheimer, antioxidant, antimicrobial peptide enzymatic production, methods to evaluate bioactivity capacity, bioavailability, and safety concerns of peptides are reviewed.

Anti-obesity effect of carboxymethyl chitin by AMPK and aquaporin-7 pathways in 3T3-L1 adipocytes.

The aim of this study was to investigate the anti-obesity effect of carboxymethyl-chitin (CM-chitin), a water-soluble derivative of chitin, by measuring lipid accumulation and adipogenesis related factors in 3T3-L1 adipocytes. CM-chitin was synthesized by means of carboxymethylation reaction. Its inhibitory effect on lipid accumulation was investigated by measuring triglyceride content and glycerol release level. The gene and protein levels associated with adipogenesis were determined using reverse transcriptase-polymerase chain reaction and Western blot analysis. Treatment with CM-chitin reduced triglyceride content and enhanced glycerol secretion in a dose-dependent manner. CM-chitin induced the down-regulation of adipogenesis related transcriptional factors and adipocyte specific gene promoters. Moreover, the specific mechanism by CM-chitin was confirmed by transcriptional activations of the phosphorylated adenosine monophosphate-activated protein kinase (AMPK) and aquaporin-7. These results suggest that CM-chitin exerts anti-adipogenic effect on lipid accumulation through modulations of AMPK and aquaporin-7 signal pathways.

Angiotensin I converting enzyme inhibitory peptide extracted from freshwater zooplankton.

In this study, hydrolysates obtained from the freshwater rotifer Brachionus calyciflonus were investigated for angiotensin I converting enzyme (ACE) inhibitory peptides. Freshwater rotifer protein was hydrolyzed using six separate enzymes in a batch reactor. The peptic hydrolysate had the highest ACE inhibitory activity compared to the other hydrolysates. The highest ACE inhibitory peptide was separated using Sephadex G-25 column chromatography and high-performance liquid chromatography on a C18 column. The 50% inhibitory concentration (IC(50)) value of purified ACE inhibitory peptide was 40.01 microg/mL. ACE inhibitory peptide was identified as being seven amino acid residues of Ala-Gln-Gly-Glu-Arg-His-Arg by N-terminal amino acid sequence analysis. The IC(50) value of purified ACE inhibitory peptide was 47.1 microM, and Lineweaver-Burk plots suggested that the peptide purified from rotifer protein acts as a competitive inhibitor against ACE. The results of this study suggest that peptides derived from freshwater rotifers may be beneficial as antihypertension compounds in functional foods or as pharmaceuticals.

Purification and characterization of angiotensin I converting enzyme inhibitory peptides from the rotifer, Brachionus rotundiformis.

Angiotensin I converting enzyme (ACE) inhibitory peptide was isolated from the marine rotifer, Brachionus rotundiformis. ACE inhibitory peptides were separated from rotifer hydrolysate prepared by Alcalase, alpha-chymotrypsin, Neutrase, papain, and trypsin. The Alcalase hydrolysate had the highest ACE inhibitory activity compared to the other hydrolysates. The IC(50) value of Alcalase hydrolysate for ACE inhibitory activity was 0.63 mg/ml. We attempted to isolate ACE inhibitory peptides from Alcalase prepared rotifer hydrolysate using gel filtration on a Sephadex G-25 column and high performance liquid chromatography on an ODS column. The IC(50) value of purified ACE inhibitory peptide was 9.64 microM, and Lineweaver-Burk plots suggest that the peptide purified from rotifer protein acts as a competitive inhibitor against ACE. Amino acid sequence of the peptide was identified as Asp-Asp-Thr-Gly-His-Asp-Phe-Glu-Asp-Thr-Gly-Glu-Ala-Met, with a molecular weight 1538 Da. The results of this study suggest that peptides derived from rotifers may be beneficial as anti-hypertension compounds in functional foods resource.

Inhibitory effects and molecular mechanism of dieckol isolated from marine brown alga on COX-2 and iNOS in microglial cells.

To identify the neuroprotective effect of dieckol, a hexameric compound of phloroglucinol isolated from marine brown alga, Ecklonia cava , this study investigated the anti-inflammatory effect of dieckol on lipopolysaccharide (LPS)-stimulated murine BV2 microglia and elucidated the molecular mechanism. The results showed that dieckol suppresses LPS-induced production of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) and expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in a dose-dependent manner, without causing cytotoxicity. It also significantly reduced the generation of proinflammatory cytokines, such as interleukin (IL)-1ß and tumor necrosis factor (TNF)-α. Moreover, dieckol significantly reduced LPS-induced nuclear factor κB (NF-κB) and p38 mitogen-activated protein kinases (MAPKs) activation, as well as reactive oxygen species (ROS) production. Taken together, the inhibition of LPS-induced NO and PGE(2) production might be due to the suppression of NF-κB and p38 MAPK signal pathway and, at least in part, by inhibiting the generation of ROS. Hence, these effects of dieckol might assist therapeutic treatment for neurodegenerative diseases that are accompanied by microglial activation.

Purification and antioxidant properties of bigeye tuna (Thunnus obesus) dark muscle peptide on free radical-mediated oxidative systems.

To produce bioactive peptides from by-products of fish processing, bigeye tuna dark muscle was hydrolyzed using various enzymes (alcalase, alpha-chymotrypsin, neutrase, papain, pepsin, and trypsin), and the hydrolysates were evaluated for antioxidant activity. Considering the results of degree of hydrolysis and antioxidant activities, peptic hydrolysate was used for further studies to identify a potent antioxidant peptide. Antioxidant peptide was purified using consecutive chromatographic methods and was identified as being H-Leu-Asn-Leu-Pro-Thr-Ala-Val-Tyr-Met-Val-Thr-OH (MW 1,222 Da) by quantitative time-of-flight electrospray ionization mass spectrometry. Purified antioxidant peptide from bigeye tuna dark muscle (APTDM) was investigated for its antioxidant activities using both free radical scavenging effects and polyunsaturated fatty acid (PUFA) peroxidation inhibitory activity. The results showed that APTDM effectively quenched with low 50% inhibitory concentration values compared to vitamin C as a positive control against four different free radicals: 1,1-diphenyl-2-picrylhydrazyl, hydroxyl, superoxide, and alkyl radical. APTDM also inhibited PUFA peroxidation in a linoleic acid emulsion system, and the activity was similar to that of alpha-tocopherol. We further investigated its antioxidant activities on cellular systems, and the results showed that APTDM significantly scavenged cellular radicals and enhanced the viability of tert-butyl hydroperoxide-induced cytotoxicity. These results indicate that APTDM or a peptide fraction containing APTDM would be a beneficial ingredient for functional food and/or pharmaceuticals.

Protective effect of an antioxidative peptide purified from gastrointestinal digests of oyster, Crassostrea gigas against free radical induced DNA damage.

In this study, in vitro gastrointestinal digestion was employed to obtain potent antioxidative peptide from protein of oyster, Crassostrea gias. The protein was subjected to hydrolysate using consecutive chromatographic methods, on a Hiprep 16/10 diethylaminoethyl fast flow (DEAE FF) anion exchange column and octadecylsilane (ODS) C18 reversed phase column. Finally, the amino acid sequence of the peptide was determined. The peptide, having the amino acid sequence Leu-Lys-Gln-Glu-Leu-Glu-Asp-Leu-Leu-Glu-Lys-Gln-Glu (1.60 kDa), exhibited the higher activity against polyunsaturated fatty acid (PUFA) peroxidation than that of native antioxidant, alpha-tocopherol. The free radical scavenging assay conducted using electron spin resonance (ESR) spectroscopy, clearly exhibited that it scavenged hydroxyl radical and superoxide radical at IC50 values of 28.76 microM and 78.97 microM, respectively. Further, we investigated its antioxidant activities on cellular system, and the results showed that purified peptide significantly scavenged cellular radicals and protective effect on DNA damage caused by hydroxyl radicals generated. Furthermore (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) MTT assay showed no cytotoxicity on human embryonic lung fibroblasts cell line (MRC-5) and mouse macrophages cell (RAW264.7), respectively. These results indicate that this peptide shows potent antioxidant.

Free radical scavenging activity of a novel antioxidative peptide isolated from in vitro gastrointestinal digests of Mytilus coruscus.

A low-molecular-weight peptide with potent antioxidative activity was obtained from Mytilus coruscus muscle protein using an in vitro gastrointestinal digestion system. The potent antioxidant peptide, which was identified as Leu-Val-Gly-Asp-Glu-Gln-Ala-Val-Pro-Ala-Val-Cys-Val-Pro (1.59 kDa), exhibited higher protective activity against polyunsaturated fatty acid (PUFA) peroxidation than the native antioxidants, ascorbic acid and alpha-tocopherol. In a free radical scavenging assay using electron spin resonance spectroscopy, hydroxyl radical formation was quenched by 75.04% in the presence of M. coruscus peptide (50 microg/mL), which was similar to ascorbic acid. In addition, the purified peptide could also quench super-oxide and carbon-centered radicals, but those activities were weaker than for ascorbic acid. This study showed that the low-molecular-weight peptide released from in vitro gastrointestinal digestion of mussel exhibited potent antioxidant potential by inhibiting the formation of reactive oxygen species formed by the peroxidation of PUFAs.

Phlorotannins in Ecklonia cava extract inhibit matrix metalloproteinase activity.

Matrix metalloproteinase (MMP) inhibitors have been identified as potential therapeutic candidates for metastasis, arthritis, chronic inflammation and wrinkle formation. For the first time here we report a detailed study on the inhibitory effects of phlorotannins in brown algae, Ecklonia cava (EC) on MMP activities in cultured human cell lines. A novel gelatin digestion assay could visualize complete inhibition of bacterial collagenase-1 activity at 20 microg/ml of EC extract during preliminary screening studies. Sensitive fluorometric assay revealed that EC extract can specifically inhibit both MMP-2 and MMP-9 activities significantly (P < 0.001) at 10 microg/ml. In addition, artificially induced activities of MMP-2 and MMP-9 in human dermal fibroblasts and HT1080 cells were inhibited by EC extract in a more or less similar manner to the positive control doxycycline. Even though the expression levels of MMPs differ from one cell type to the other, gelatin zymography clearly revealed that both MMP expression and activity in cells can be inhibited by EC extract. More interestingly, EC extract did not exert any cytotoxic effect even at 100 microg/ml anticipating its potential use as a safe MMP inhibitor.